MOLECULAR PROFILING FOR CANCER

Abstract
Provided herein are methods and systems of molecular profiling of diseases, such as cancer. In some embodiments, the molecular profiling can be used to identify treatments that have likely benefit for a cancer, such as treatments that were not initially identified as a treatment for the disease or not expected to be a treatment for a particular disease. The molecular profiling can be used to identify likely have lack of benefit for treating the cancer.
Description
BACKGROUND

Disease states in patients are typically treated with treatment regimens or therapies that are selected based on clinical based criteria; that is, a treatment therapy or regimen is selected for a patient based on the determination that the patient has been diagnosed with a particular disease (which diagnosis has been made from classical diagnostic assays). Although the molecular mechanisms behind various disease states have been the subject of studies for years, the specific application of a diseased individual's molecular profile in determining treatment regimens and therapies for that individual has been disease specific and not widely pursued.


Some treatment regimens have been determined using molecular profiling in combination with clinical characterization of a patient such as observations made by a physician (such as a code from the International Classification of Diseases, for example, and the dates such codes were determined), laboratory test results, x-rays, biopsy results, statements made by the patient, and any other medical information typically relied upon by a physician to make a diagnosis in a specific disease. However, using a combination of selection material based on molecular profiling and clinical characterizations (such as the diagnosis of a particular type of cancer) to determine a treatment regimen or therapy presents a risk that an effective treatment regimen may be overlooked for a particular individual since some treatment regimens may work well for different disease states even though they are associated with treating a particular type of disease state.


Patients with refractory or metastatic cancer are of particular concern for treating physicians. The majority of patients with metastatic or refractory cancer eventually run out of treatment options or may suffer a cancer type with no real treatment options. For example, some patients have very limited options after their tumor has progressed in spite of front line, second line and sometimes third line and beyond) therapies. For these patients, molecular profiling of their cancer may provide the only viable option for prolonging life.


More particularly, additional targets or specific therapeutic agents can be identified assessment of a comprehensive number of targets or molecular findings examining molecular mechanisms, genes, gene expressed proteins, and/or combinations of such in a patient's tumor. Identifying multiple agents that can treat multiple targets or underlying mechanisms would provide cancer patients with a viable therapeutic alternative on a personalized basis so as to avoid standar therapies, which may simply not work or identify therapies that would not otherwise be considered by the treating physician.


There remains a need for better theranostic assessment of cancer vicitims, including molecular profiling analysis that identifies one or more individual profiles to provide more informed and effective personalized treatment options, resulting in improved patient care and enhanced treatment outcomes. The present invention provides methods and systems for identifying treatments for these individuals by molecular profiling a sample from the individual.


SUMMARY OF THE INVENTION

The present invention provides methods and system for molecular profiling, using the results from molecular profiling to identify treatments for individuals. In some embodiments, the treatments were not identified initially as a treatment for the disease or disease lineage.


In an aspect, the invention provides a method of identifying one or more candidate treatment for a cancer in a subject in need thereof, comprising: (a) determining a molecular profile for a sample from the subject by assessing a panel of gene or gene products, wherein the panel of gene or gene products are assessed as indicated in Table 21, FIG. 33A or FIG. 33B; and (b) identifying one or more treatment that is beneficially associated with the molecular profile of the subject, and optionally one or more treatment associated with lack of benefit, according to the determining in (a) and one or more rules in Table 22, thereby identifying the one or more candidate treatment. The panel of gene or gene products may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 or 58, of: ABL1, AKT1, ALK, APC, AR, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ER, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HER2, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MGMT, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PGP, PIK3CA, PR, PTEN, PTPN11, RB1, RET, RRM1, SMAD4, SMARCB1, SMO, SPARC, STK11, TLE3, TOP2A, TOPO1, TP53, TS, TUBB3 and VHL. Assessing the panel of gene or gene products may comprise using ISH to assess 1 or 2 of cMET and HER2. Assessing the panel of gene or gene products may comprise using IHC to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 of AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3. Assessing the panel of gene or gene products may comprise using sequence analysis to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 of: ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL. Assessing the panel of gene or gene products may comprise using ISH to assess 1 or 2 of cMET and HER2; using IHC to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 of AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3; and/or comprises using sequence analysis to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 of: ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL. Assessing the panel of gene or gene products may comprise using sequence analysis to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1, PTPN11, RB1, SMAD4, SMARCB1 and STK11. The sequence analysis can be performed using Next Generation Sequencing.


In some embodiments, the panel of gene or gene products comprises the androgen receptor (AR). In such cases, the one or more candidate treatment can be an antiandrogen. The antiandrogen may suppress androgen production and/or inhibits androgens from binding to AR. The antiandrogen can be one or more of abarelix, bicalutamide, flutamide, gonadorelin, goserelin, leuprolide, nilutamide, a 5-alpha-reductase inhibitor, finasteride, dutasteride, bexlosteride, izonsteride, turosteride, and epristeride. The cancer can be androgen independent. In embodiments, the one or more candidate treatment comprises one or more of a CYP17 inhibitor, CYP17A1 inhibitor, chemotherapeutic agent, antiandrogen, an endocrine disruptor, immunotherapy, and bone-targeting radiopharmaceutical.


The methods of the invention can be used to profile any cancer. For example, the cancer may comprise an acute lymphoblastic leukemia; acute myeloid leukemia; adrenocortical carcinoma; AIDS-related cancer; AIDS-related lymphoma; anal cancer; appendix cancer; astrocytomas; atypical teratoid/rhabdoid tumor; basal cell carcinoma; bladder cancer; brain stem glioma; brain tumor, brain stem glioma, central nervous system atypical teratoid/rhabdoid tumor, central nervous system embryonal tumors, astrocytomas, craniopharyngioma, ependymoblastoma, ependymoma, medulloblastoma, medulloepithelioma, pineal parenchymal tumors of intermediate differentiation, supratentorial primitive neuroectodermal tumors and pineoblastoma; breast cancer; bronchial tumors; Burkitt lymphoma; cancer of unknown primary site (CUP); carcinoid tumor; carcinoma of unknown primary site; central nervous system atypical teratoid/rhabdoid tumor; central nervous system embryonal tumors; cervical cancer; childhood cancers; chordoma; chronic lymphocytic leukemia; chronic myelogenous leukemia; chronic myeloproliferative disorders; colon cancer; colorectal cancer; craniopharyngioma; cutaneous T-cell lymphoma; endocrine pancreas islet cell tumors; endometrial cancer; ependymoblastoma; ependymoma; esophageal cancer; esthesioneuroblastoma; Ewing sarcoma; extracranial germ cell tumor; extragonadal germ cell tumor; extrahepatic bile duct cancer; gallbladder cancer; gastric (stomach) cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal cell tumor; gastrointestinal stromal tumor (GIST); gestational trophoblastic tumor; glioma; hairy cell leukemia; head and neck cancer; heart cancer; Hodgkin lymphoma; hypopharyngeal cancer; intraocular melanoma; islet cell tumors; Kaposi sarcoma; kidney cancer; Langerhans cell histiocytosis; laryngeal cancer; lip cancer; liver cancer; malignant fibrous histiocytoma bone cancer; medulloblastoma; medulloepithelioma; melanoma; Merkel cell carcinoma; Merkel cell skin carcinoma; mesothelioma; metastatic squamous neck cancer with occult primary; mouth cancer; multiple endocrine neoplasia syndromes; multiple myeloma; multiple myeloma/plasma cell neoplasm; mycosis fungoides; myelodysplastic syndromes; myeloproliferative neoplasms; nasal cavity cancer; nasopharyngeal cancer; neuroblastoma; Non-Hodgkin lymphoma; nonmelanoma skin cancer; non-small cell lung cancer; oral cancer; oral cavity cancer; oropharyngeal cancer; osteosarcoma; other brain and spinal cord tumors; ovarian cancer; ovarian epithelial cancer; ovarian germ cell tumor; ovarian low malignant potential tumor; pancreatic cancer; papillomatosis; paranasal sinus cancer; parathyroid cancer; pelvic cancer; penile cancer; pharyngeal cancer; pineal parenchymal tumors of intermediate differentiation; pineoblastoma; pituitary tumor; plasma cell neoplasm/multiple myeloma; pleuropulmonary blastoma; primary central nervous system (CNS) lymphoma; primary hepatocellular liver cancer; prostate cancer; rectal cancer; renal cancer; renal cell (kidney) cancer; renal cell cancer; respiratory tract cancer; retinoblastoma; rhabdomyosarcoma; salivary gland cancer; Sézary syndrome; small cell lung cancer; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma; squamous neck cancer; stomach (gastric) cancer; supratentorial primitive neuroectodermal tumors; T-cell lymphoma; testicular cancer; throat cancer; thymic carcinoma; thymoma; thyroid cancer; transitional cell cancer; transitional cell cancer of the renal pelvis and ureter; trophoblastic tumor; ureter cancer; urethral cancer; uterine cancer; uterine sarcoma; vaginal cancer; vulvar cancer; Waldenstrim macroglobulinemia; or Wilm's tumor. The cancer can be an acute myeloid leukemia (AML), breast carcinoma, cholangiocarcinoma, colorectal adenocarcinoma, extrahepatic bile duct adenocarcinoma, female genital tract malignancy, gastric adenocarcinoma, gastroesophageal adenocarcinoma, gastrointestinal stromal tumor (GIST), glioblastoma, head and neck squamous carcinoma, leukemia, liver hepatocellular carcinoma, low grade glioma, lung bronchioloalveolar carcinoma (BAC), non-small cell lung cancer (NSCLC), lung small cell cancer (SCLC), lymphoma, male genital tract malignancy, malignant solitary fibrous tumor of the pleura (MSFT), melanoma, multiple myeloma, neuroendocrine tumor, nodal diffuse large B-cell lymphoma, non epithelial ovarian cancer (non-EOC), ovarian surface epithelial carcinoma, pancreatic adenocarcinoma, pituitary carcinomas, oligodendroglioma, prostatic adenocarcinoma, retroperitoneal or peritoneal carcinoma, retroperitoneal or peritoneal sarcoma, small intestinal malignancy, soft tissue tumor, thymic carcinoma, thyroid carcinoma, or uveal melanoma. In some embodiments, the cancer comprises a prostate, bladder, kidney, lung, breast, or liver cancer.


In an aspect, the invention provides a method of identifying one or more candidate treatment for an ovarian cancer in a subject in need thereof, comprising: (a) determining a molecular profile for a sample from the subject by assessing a panel of gene or gene products, wherein the panel of gene or gene products are assessed as indicated in Table 7, FIG. 33C or FIG. 33D; and (b) identifying one or more treatment that is beneficially associated with the molecular profile of the subject, and optionally one or more treatment associated with lack of benefit, according to the determining in (a) and one or more rules in Table 8, thereby identifying the one or more candidate treatment. The panel of gene or gene products can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 or 58, of: ABL1, AKT1, ALK, APC, AR, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ER, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HER2, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MGMT, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PGP, PIK3CA, PR, PTEN, PTPN11, RB1, RET, RRM1, SMAD4, SMARCB1, SMO, SPARC, STK11, TLE3, TOP2A, TOPO1, TP53, TS, TUBB3, VHL. Assessing the panel of gene or gene products may comprise using ISH to assess 1 or 2 of cMET and HER2. Assessing the panel of gene or gene products may comprise using IHC to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 of: AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3. Assessing the panel of gene or gene products may comprise using sequence analysis to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 of: ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL. Assessing the panel of gene or gene products may comprise using ISH to assess 1 or 2 ofcMET and HER2; using IHC to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 of: AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3; and/or using sequence analysis to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 of: ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL. Assessing the panel of gene or gene products may comprise using sequence analysis to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1, PTPN11, RB1, SMAD4, SMARCB1 and STK11. In some embodiments, the sequence analysis comprises Next Generation Sequencing.


In an aspect, the invention provides a method of identifying one or more candidate treatment for a breast cancer in a subject in need thereof, comprising: (a) determining a molecular profile for a sample from the subject by assessing a panel of gene or gene products, wherein the panel of gene or gene products are assessed as indicated in Table 9, FIG. 33K or FIG. 33L; and (b) identifying one or more treatment that is beneficially associated with the molecular profile of the subject, and optionally one or more treatment associated with lack of benefit, according to the determining in (a) and one or more rules in Table 10, thereby identifying the one or more candidate treatment. The panel of gene or gene products can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 or 58, of: ABL1, AKT1, ALK, APC, AR, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ER, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HER2, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MGMT, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PGP, PIK3CA, PR, PTEN, PTPN11, RB1, RET, RRM1, SMAD4, SMARCB1, SMO, SPARC, STK11, TLE3, TOP2A, TOPO1, TP53, TS, TUBB3, VHL. Assessing the panel of gene or gene products may comprise using ISH to assess 1, 2 or 3, of: cMET, HER2, TOP2A. Assessing the panel of gene or gene products may comprise using IHC to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 of: AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOPO1, TS, TUBB3. Assessing the panel of gene or gene products may comprise using sequence analysis to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 of: ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL. Assessing the panel of gene or gene products may comprise using ISH to assess 1, 2 or 3, of: cMET, HER2, TOP2A; using IHC to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 of: AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOPO1, TS, TUBB3; and/or using sequence analysis to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 of: ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL. Assessing the panel of gene or gene products may comprise using sequence analysis to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1, PTPN11, RB1, SMAD4, SMARCB1 and STK11. In some embodiments, the sequence analysis comprises Next Generation Sequencing.


In an aspect, the invention provides a method of identifying one or more candidate treatment for a skin cancer (melanoma) in a subject in need thereof, comprising: (a) determining a molecular profile for a sample from the subject by assessing a panel of gene or gene products, wherein the panel of gene or gene products are assessed as indicated in Table 11, FIG. 33E or FIG. 33F; and (b) identifying one or more treatment that is beneficially associated with the molecular profile of the subject, and optionally one or more treatment associated with lack of benefit, according to the determining in (a) and one or more rules in Table 12, thereby identifying the one or more candidate treatment. The panel of gene or gene products can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 or 58, of: ABL1, AKT1, ALK, APC, AR, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ER, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HER2, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MGMT, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PGP, PIK3CA, PR, PTEN, PTPN11, RB1, RET, RRM1, SMAD4, SMARCB1, SMO, SPARC, STK11, TLE3, TOP2A, TOPO1, TP53, TS, TUBB3, VHL. Assessing the panel of gene or gene products may comprise using ISH to assess 1 or 2 of: cMET, HER2. Assessing the panel of gene or gene products may comprise using IHC to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 of: AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3. Assessing the panel of gene or gene products may comprise using sequence analysis to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 of: ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL. Assessing the panel of gene or gene products may comprise using ISH to assess 1 or 2 of: cMET, HER2; using IHC to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 of: AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3; and/or using sequence analysis to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 of: ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL. Assessing the panel of gene or gene products may comprise using sequence analysis to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1, PTPN11, RB1, SMAD4, SMARCB1 and STK11. In some embodiments, the sequence analysis comprises Next Generation Sequencing. In various embodiments, the sequence analysis of BRAF comprises PCR, e.g., the FDA approved cobas PCR assay.


In an aspect, the invention provides a method of identifying one or more candidate treatment for a uveal melanoma cancer in a subject in need thereof, comprising: (a) determining a molecular profile for a sample from the subject by assessing a panel of gene or gene products, wherein the panel of gene or gene products are assessed as indicated in Table 13, FIG. 33G or FIG. 33H; and (b) identifying one or more treatment that is beneficially associated with the molecular profile of the subject, and optionally one or more treatment associated with lack of benefit, according to the determining in (a) and one or more rules in Table 14, thereby identifying the one or more candidate treatment. The panel of gene or gene products can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 or 58, of: ABL1, AKT1, ALK, APC, AR, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ER, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HER2, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MGMT, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PGP, PIK3CA, PR, PTEN, PTPN11, RB1, RET, RRM1, SMAD4, SMARCB1, SMO, SPARC, STK11, TLE3, TOP2A, TOPO1, TP53, TS, TUBB3, VHL. Assessing the panel of gene or gene products may comprise using ISH to assess 1 or 2, of: cMET, HER2. Assessing the panel of gene or gene products may comprise using IHC to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 of: AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3. Assessing the panel of gene or gene products may comprise using sequence analysis to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 of: ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL. Assessing the panel of gene or gene products may comprise using ISH to assess 1 or 2, of: cMET, HER2; using IHC to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 of: AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3; and/or using sequence analysis to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 of: ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL. Assessing the panel of gene or gene products may comprise using sequence analysis to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1, PTPN11, RB1, SMAD4, SMARCB1 and STK11. In some embodiments, the sequence analysis comprises Next Generation Sequencing. In various embodiments, the sequence analysis of BRAF comprises PCR, e.g., the FDA approved cobas PCR assay.


In an aspect, the invention provides a method of identifying one or more candidate treatment for a colorectal cancer in a subject in need thereof, comprising: (a) determining a molecular profile for a sample from the subject by assessing a panel of gene or gene products, wherein the panel of gene or gene products are assessed as indicated in Table 15, FIG. 33M or FIG. 33N; and (b) identifying one or more treatment that is beneficially associated with the molecular profile of the subject, and optionally one or more treatment associated with lack of benefit, according to the determining in (a) and one or more rules in Table 16, thereby identifying the one or more candidate treatment. The panel of gene or gene products can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 or 58, of: ABL1, AKT1, ALK, APC, AR, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ER, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HER2, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MGMT, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PGP, PIK3CA, PR, PTEN, PTPN11, RB1, RET, RRM1, SMAD4, SMARCB1, SMO, SPARC, STK11, TLE3, TOP2A, TOPO1, TP53, TS, TUBB3, VHL. Assessing the panel of gene or gene products may comprise using ISH to assess 1 or 2 of: cMET, HER2. Assessing the panel of gene or gene products may comprise using IHC to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 of: AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3. Assessing the panel of gene or gene products may comprise using sequence analysis to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 of: ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL. Assessing the panel of gene or gene products may comprise using ISH to assess 1 or 2 of: cMET, HER2; using IHC to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 of: AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3; and/or using sequence analysis to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 of: ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL. Assessing the panel of gene or gene products may comprise using sequence analysis to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1, PTPN11, RB1, SMAD4, SMARCB1 and STK11. In some embodiments, the sequence analysis comprises Next Generation Sequencing.


In an aspect, the invention provides a method of identifying one or more candidate treatment for a lung cancer in a subject in need thereof, comprising: (a) determining a molecular profile for a sample from the subject by assessing a panel of gene or gene products, wherein the panel of gene or gene products are assessed as indicated in Table 17, FIG. 33I or FIG. 33J; and (b) identifying one or more treatment that is beneficially associated with the molecular profile of the subject, and optionally one or more treatment associated with lack of benefit, according to the determining in (a) and one or more rules in Table 18, thereby identifying the one or more candidate treatment. The panel of gene or gene products can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 or 59 of: ABL1, AKT1, ALK, APC, AR, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ER, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HER2, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MGMT, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PGP, PIK3CA, PR, PTEN, PTPN11, RB1, RET, ROS1, RRM1, SMAD4, SMARCB1, SMO, SPARC, STK11, TLE3, TOP2A, TOPO1, TP53, TS, TUBB3, VHL. Assessing the panel of gene or gene products may comprise using ISH to assess 1, 2, 3 or 4, of: ALK, cMET, HER2, ROS1. Assessing the panel of gene or gene products may comprise using IHC to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 of: AR, cMET, EGFR (H-score), ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3. Assessing the panel of gene or gene products may comprise using sequence analysis to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 of: ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL. Assessing the panel of gene or gene products may comprise using ISH to assess 1, 2, 3 or 4, of: ALK, cMET, HER2, ROS1; using IHC to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 of: AR, cMET, EGFR (H-score), ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3; and/or using sequence analysis to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 of: ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL. Assessing the panel of gene or gene products may comprise using sequence analysis to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1, PTPN11, RB1, SMAD4, SMARCB1 and STK11. In some embodiments, the sequence analysis comprises Next Generation Sequencing. The lung cancer can include without limitation a non-small cell lung cancer (NSCLC) or a bronchioloalveolar cancer (BAC).


In an aspect, the invention provides a method of identifying one or more candidate treatment for a glioma brain cancer in a subject in need thereof, comprising: (a) determining a molecular profile for a sample from the subject by assessing a panel of gene or gene products, wherein the panel of gene or gene products are assessed as indicated in Table 21, FIG. 33O or FIG. 33P; and (b) identifying one or more treatment that is beneficially associated with the molecular profile of the subject, and optionally one or more treatment associated with lack of benefit, according to the determining in (a) and one or more rules in Table 19, thereby identifying the one or more candidate treatment. The panel of gene or gene products can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or 61, of: ABL1, AKT1, ALK, APC, AR, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, EGFRvIII, ER, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HER2, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR (VEGFR2), KRAS, MGMT-Me, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PGP, PIK3CA, PR, PTEN, PTPN11, RB1, RET, RRM1, SMAD4, SMARCB1, SMO, SPARCm, SPARCp, STK11, TLE3, TOP2A, TOPO1, TP53, TS, TUBB3, VHL. Assessing the panel of gene or gene products may comprise using ISH to assess 1 or 2 of: cMET, HER2. Assessing the panel of gene or gene products may comprise using IHC to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, of: AR, cMET, ER, HER2, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3. Assessing the panel of gene or gene products may comprise assessing methylation of the MGMT promoter region. Assessing methylation of the MGMT promoter region can be performed using pyrosequencing and/or methylation specific PCR (MS-PCR). Assessing the panel of gene or gene products may comprise sequence analysis of IDH2. Sequence analysis of IDH2 can be performed using Sanger sequencing or Next Generation Sequencing. Assessing the panel of gene or gene products may comprise detection of the EGFRvIII variant. The EGFRvIII variant can be detected by fragment analysis. Assessing the panel of gene or gene products may comprise using sequence analysis to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 of: ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL. Assessing the panel of gene or gene products may comprise using ISH to assess 1 or 2 of: cMET, HER2; using IHC to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, of: AR, cMET, ER, HER2, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3; using pyrosequencing to detect methylation of the MGMT promoter; using Sanger sequencing to assess the sequence of IDH2; using fragment analysis to detect the EGFRvIII variant; and/or using sequence analysis to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 of: ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL. Assessing the panel of gene or gene products may comprise using sequence analysis to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1, PTPN11, RB1, SMAD4, SMARCB1 and STK11. In some embodiments, the sequence analysis comprises Next Generation Sequencing.


In an aspect, the invention provides a method of identifying one or more candidate treatment for a gastrointestinal stromal tumor (GIST) cancer in a subject in need thereof, comprising: (a) determining a molecular profile for a sample from the subject by assessing a panel of gene or gene products, wherein the panel of gene or gene products are assessed as indicated in Table 21; and (b) identifying one or more treatment that is beneficially associated with the molecular profile of the subject, and optionally one or more treatment associated with lack of benefit, according to the determining in (a) and one or more rules in Table 20, thereby identifying the one or more candidate treatment. The panel of gene or gene products can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 or 58, of: ABL1, AKT1, ALK, APC, AR, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ER, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HER2, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MGMT, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PGP, PIK3CA, PR, PTEN, PTPN11, RB1, RET, RRM1, SMAD4, SMARCB1, SMO, SPARC, STK11, TLE3, TOP2A, TOPO1, TP53, TS, TUBB3, VHL. Assessing the panel of gene or gene products may comprise using ISH to assess 1 or 2 of: cMET, HER2. Assessing the panel of gene or gene products may comprise using IHC to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 of: AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3. Assessing the panel of gene or gene products may comprise using sequence analysis to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 of: ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL. Assessing the panel of gene or gene products may comprise using ISH to assess 1 or 2 of: cMET, HER2; using IHC to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 of: AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3; and/or using sequence analysis to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 of: ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL. Assessing the panel of gene or gene products may comprise using sequence analysis to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1, PTPN11, RB1, SMAD4, SMARCB1 and STK11. In some embodiments, the sequence analysis comprises Next Generation Sequencing.


In an aspect, the invention provides a method of identifying one or more candidate treatment for a cancer in a subject in need thereof, comprising: (a) determining a molecular profile for a sample from the subject by assessing a panel of gene or gene products, wherein the panel of gene or gene products are assessed using IHC for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 of AR, cMET, EGFR (including H-score for NSCLC), ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOPO1, TOP2A, TS, TUBB3; FISH or CISH for 1, 2, 3, 4, or 5 of ALK, cMET, HER2, ROS1, TOP2A; Mutational Analysis of 1, 2, 3 or 4 of BRAF (e.g., Cobas® PCR), IDH2 (e.g., Sanger Sequencing), MGMT promoter methylation (e.g., by PyroSequencing), EGFR (e.g., fragment analysis to detect EGFRvIII); and/or Mutational Analysis (e.g., by Next-Generation Sequencing) of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 of ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CSF1R, CTNNB1, EGFR, ERBB2 (HER2), ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, STK11, TP53, VHL; and (b) identifying one or more treatment that is beneficially associated with the molecular profile of the subject, and optionally one or more treatment associated with lack of benefit, according to the determining in (a) and one or more rules in any of Tables 7-22, thereby identifying the one or more candidate treatment.


In the method of identifying one or more candidate treatment provided by the invention, the methods may further comprising additional molecular profiling according to FIG. 33Q.


In an aspect, the invention provides a method of identifying one or more candidate treatment for a prostate cancer in a subject in need thereof, comprising: (a) determining a molecular profile for a sample from the subject on a panel of gene or gene products, wherein the panel of gene or gene products comprises immunohistochemistry (IHC) of AR, MRP1, TOPO1, TLE3, EGFR, TS, PGP, TUBB3, RRM1, PTEN and/or MGMT; in situ hybridization (ISH) of EGFR and/or cMYC; and/or sequencing of TP53, PTEN, CTNNB1, PIK3CA, RB1, ATM, cMET, K/HRAS, ERBB4, ALK, BRAF and/or cKIT; and (b) identifying one or more treatment that is beneficially associated with the molecular profile of the subject, and optionally one or more treatment associated with lack of benefit, according to the determining in (a) and one or more rules in Table 22, thereby identifying the one or more candidate treatment. The rules can include one or more of: imatinib for patients with high cKIT or PDGFRA; cetuximab for patients with EGFR positivity; cabozantinib for patients with cMET aberrations; PAM pathway inhibitors (e.g., BEZ234, everolimus) for patients with PIK3CA pathway activation; HDAC inhibitors for patients with cMYC amplification; 5-FU for patients with low TS; gemcitabine for patients with low RRM1; temozolomide for patients with low MGMT; cabazitaxel for patients with low TUBB3 or PGP, or high TLE3; and anti-androgen agents (e.g., enzalutamide) for patients with high AR.


In an aspect, the invention provides a method of identifying one or more candidate treatment for a cancer in a subject in need thereof, comprising: a) determining a molecular profile for a sample from the subject by sequencing a panel of gene or gene products, wherein the panel of gene or gene products comprises one or more gene in Table 24; and b) identifying one or more treatment that is beneficially associated with the molecular profile of the subject, and optionally one or more treatment associated with lack of benefit, according to the determining in (a) and one or more rules in Table 25 or any of Tables 7-22, thereby identifying the one or more candidate treatment. Assessing the panel of gene or gene products may comprise using sequence analysis to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 of ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CSF1R, CTNNB1, EGFR, ERBB2 (HER2), ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA1, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, STK11, TP53, VHL. Assessing the panel of gene or gene products may comprise using sequence analysis to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 of: ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL. Assessing the panel of gene or gene products may comprise using sequence analysis to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 of ABL1, APC, BRAF, EGFR, FLT3, GNAQ, IDH1, JAK2, cKIT, KRAS, MPL, NPM1, NRAS, PDGFRA, VHL. Assessing the panel of gene or gene products may comprise using sequence analysis to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 of ABL1, APC, BRAF, EGFR, FLT3, GNAQ, IDH1, JAK2, cKIT, KRAS, MPL, NRAS, PDGFRA, VHL.


In the methods of the invention above, identifying the one or more treatment that is beneficially associated with the molecular profile of the subject, and optionally the one or more treatment associated with lack of benefit, can comprise: a) correlating the molecular profile with the one or more rules, wherein the one or more rules comprise a mapping of treatments whose efficacy has been previously determined in individuals having cancers that have different levels of, overexpress, underexpress, and/or have mutations in one or more members of the panel of gene or gene products; and b) identifying one or more treatment that is associated with treatment benefit based on the correlating in (a); and c) optionally identifying one or more treatment that is associated with lack of treatment benefit based on the correlating in (a). The mapping of treatments can be any of those included in Tables 3-5, 7-23, FIGS. 33A-Q, FIGS. 35A-I, or FIGS. 36A-F.


The methods of the invention above may further comprise identifying one or more candidate clinical trial for the subject based on the molecular profiling.


In an aspect, the invention provides a method of identifying one or more candidate clinical trial for a subject having a cancer, comprising: (a) determining a molecular profile for a sample from the subject on a panel of gene or gene products; and (b) identifying one or more clinical trial associated with the molecular profile of the subject according to the determining in (a) and one or more biomarker-clinical trial association rules, thereby identifying the one or more candidate clinical trial. The molecular profile can include IHC for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 ofAR, cMET, EGFR (including H-score for NSCLC), ER, HER2, MGMT, Pgp, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOPO1, TOP2A, TS, TUBB3; FISH or CISH for 1, 2, 3, 4, or 5 of ALK, cMET, HER2, ROS1, TOP2A; Mutational Analysis of 1, 2, 3 or 4 of BRAF (e.g., Cobas® PCR), IDH2 (e.g., Sanger Sequencing), MGMT promoter methylation (e.g., by PyroSequencing), EGFR (e.g., fragment analysis to detect EGFRvIII); and/or Mutational Analysis (e.g., by Next-Generation Sequencing) of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 of ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CSF1R, CTNNB1, EGFR, ERBB2 (HER2), ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, STK11, TP53, VHL.


Identifying the one or more clinical trial associated with the molecular profile of the subject according to the methods above can comprise: 1) matching to clinical trials for non-standard of care treatments for the patient's cancer (e.g., offNCCN compendium treatments) indicated as potentially beneficial according to the biomarker—drug association rules herein; 2) matching to clinical trials based on biomarker eligibility requirements of the trial; and/or 3) matching to clinical trials based on the molecular profile of the patient, biology of the disease and/or associated signaling pathways. In some embodiments, matching to clinical trials based on the molecular profile of the patient, biology of the disease and/or associated signaling pathways comprises: 1) matching trials with therapeutic agents directly targeting a gene and/or gene product in the molecular profile; 2) matching trials with therapeutic agents that target another gene or gene product in a biological pathway that directly target a gene and/or gene product in the molecular profile; 3) matching trials with therapeutic agents that target another gene or gene product in a biological pathway that indirectly target a gene and/or gene product in the molecular profile. Identifying the one or more candidate clinical trial can be performed according to one or more biomarker-clinical trial association rules in Tables 28-29.


As desired, additional genes and/or gene products may be assessed according to the methods of the invention. For example, the molecular profiles above may comprise one or more additional gene or gene product listed in Table 2, Table 6 or Table 25. Additional genes and/or gene products can be assessed as evidence becomes available linking such genes and/or gene products to a therapeutic efficacy. The one or more additional gene or gene product listed in Table 2, Table 6 or Table 25 can be assessed by any appropriate laboratory technique such as described herein, including without limitation next generation sequencing.


The sample used to perform molecular profiling in the methods of the invention can include one or more of a formalin-fixed paraffin-embedded (FFPE) tissue, fixed tissue, core needle biopsy, fine needle aspirate, unstained slides, fresh frozen (FF) tissue, formalin samples, tissue comprised in a solution that preserves nucleic acid or protein molecules, and/or a bodily fluid sample. In some embodiments, the sample comprises cells from a solid tumor. In some embodiments, the sample comprises a bodily fluid. The bodily fluid can be a malignant fluid. The bodily fluid can be a pleural or peritoneal fluid. In various embodiments, the bodily fluid comprises peripheral blood, sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, or umbilical cord blood. The sample may comprise a microvesicle population. In such cases, one or more members of the panel of gene or gene products may be associated with the microvesicle population.


The one or more candidate treatment can be selected from those listed in any of Tables 3-5, 7-22, 28, 29, 33, 36 or 37 herein. The methods of the invention may provide a prioritized list of one or more candidate treatment.


The cancer that is profiled according to the methods of the invention can be of any stage or progression. In some embodiments, the subject has not previously been treated with the one or more candidate treatment associated with treatment benefit. In some embodiments, the cancer comprises a metastatic cancer. In some embodiments, the cancer comprises a recurrent cancer. In some embodiments, the cancer is refractory to a prior treatment. The prior treatment can be the standard of care for the cancer, e.g., as based on the available evidence and/or guidelines such as the NCCN compendium. The cancer may be refractory to all known standard of care treatments. Alternately, the subject has not previously been treated for the cancer.


The one or more candidate treatment can be administered to the subject. In some embodiments of the methods herein, progression free survival (PFS) or disease free survival (DFS) for the subject is extended by administration of the one or more candidate treatment to the subject. The subject's lifespan can be extended by administration of the one or more candidate treatment to the subject.


In the methods of the invention above, the molecular profile can be compared to the one or more rules using a computer. The one or more rules may be comprised within a computer database.


In another aspect, the invention provides a method of generating a molecular profiling report comprising preparing a report comprising results of the molecular profile determined by any of the methods of the invention, e.g., as described above. Illustrative reports are shown in FIGS. 37A-37Y, FIGS. 38A-38AA and FIGS. 39A-39Y. In some embodiments, the report further comprises a list of the one or more candidate treatment that is associated with benefit for treating the cancer. The report may further comprise identification of the one or more candidate treatment as standard of care or not for the cancer lineage. The report can also comprise a list of one or more treatment that is associated with lack of benefit for treating the cancer. The report can also comprise a list of one or more treatment that is associated with indeterminate benefit for treating the cancer. In some embodiments, the report comprises a listing of members of the panel of genes or gene products assessed with description of each. In some embodiments, the report comprises a listing of members of the panel of genes or gene products assessed by one or more of ISH, IHC, Next Generation sequencing, Sanger sequencing, PCR, pyrosequencing and fragment analysis. In some embodiments, the report comprises a list of clinical trials for which the subject is eligible based on the molecular profile. In some embodiments, the report comprises a list of evidence supporting the identification of certain treatments as likely to benefit the patient, not benefit the patient, or having indeterminate benefit. The report may comprise: 1) a list of the genes and/or gene products in the molecular profile; 2) a description of the molecular profile of the genes and/or gene products as determined for the subject; 3) a treatment associated with one or more of the genes and/or gene products in the molecular profile; and 4) and an indication whether each treatment is likely to benefit the patient, not benefit the patient, or has indeterminate benefit. The description of the molecular profile of the genes and/or gene products as determined for the subject can comprise the technique used to assess the gene and/or gene products and the results of the assessment.


In an aspect, the invention provides a method of generating a molecular profiling report comprising preparing a report comprising results of the molecular profile determined by the methods for identifying one or more candidate clinical trial as provided herein, e.g., as provided above. The report can include a list of the one or more identified candidate clinical trial.


The molecular profile reports of the invention can be computer generated reports. Such reports may be provided as a printed report and/or as a computer file. The molecular profile report can be made accessible via a web portal. The reports can be transmitted over a network. In some embodiments, the results of some or all of the molecular profiling are transmitted over a network before the report is compiled.


In an aspect, the invention contemplates use of a reagent in carrying out the methods of the invention. In a related aspect, the invention contemplates use of a reagent in the manufacture of a reagent or kit for carrying out the method of the invention. In still another related aspect, the invention provides a kit comprising a reagent for carrying out the method of the invention. The reagent can be any reagent useful for carrying out one or more of the molecular profiling methods provided herein. For example, the reagent can include without limitation one or more of a reagent for extracting nucleic acid from a sample, a reagent for performing ISH, a reagent for performing IHC, a reagent for performing PCR, a reagent for performing Sanger sequencing, a reagent for performing next generation sequencing, a reagent for a DNA microarray, a reagent for performing pyrosequencing, a nucleic acid probe, a nucleic acid primer, an antibody, a reagent for performing bisulfite treatment of nucleic acid.


In a related aspect, the invention provides a report generated by the methods of report generation as described herein, e.g., as described above. Illustrative reports are shown in FIGS. 37A-37Y, FIGS. 38A-38AA and FIGS. 39A-39Y.


In an aspect, the invention provides a computer system for generating the report provided by the invention.


In a related aspect, the invention provides a system for identifying one or more candidate treatment for a cancer comprising: a host server; a user interface for accessing the host server to access and input data; a processor for processing the inputted data; a memory coupled to the processor for storing the processed data and instructions for: i) accessing a molecular profile generated by the method of the invention, e.g., as described above; ii) identifying one or more candidate treatment that is associated with likely treatment benefit by comparing the molecular profiling results to the one or more rules; iii) optionally identifying one or more treatment that is associated with likely lack of treatment benefit by comparing the molecular profiling results to the one or more rules; and iv) optionally identifying one or more treatment that is associated with indeterminate treatment benefit by comparing the molecular profiling results to the one or more rules; and a display for displaying the identified one or more candidate treatment that is associated with likely treatment benefit and the optional one or more treatment that is associated with likely lack of treatment benefit and one or more treatment that is associated with indeterminate treatment benefit. The display may comprise a report as described above. The systems of the invention may further comprise instructions for identifying one or more clinical trial that is associated with likely treatment benefit by comparing the molecular profiling results to one or more biomarker-clinical trial association rules.


In an aspect, the invention provides a system for identifying one or more candidate clinical trial for a cancer comprising: a host server; a user interface for accessing the host server to access and input data; a processor for processing the inputted data; a memory coupled to the processor for storing the processed data and instructions for: accessing a molecular profile generated by the methods of identifying one or more candidate clinical trial provided by the invention; and identifying one or more candidate candidate clinical trial by comparing the molecular profiling results to the one or more rules; and a display for displaying the identified one or more candidate candidate clinical trial. The display may comprise a report as described above.


In an aspect, the invention provides a computer medium comprising one or more rules from any of Tables 7, 9, 11, 13, 15, 17, 21 and 28. In an embodiment, the computer medium comprises one or more rules selected from: performing IHC on RRM1 to determine likely benefit or lack of benefit from an antimetabolite and/or gemcitabine; performing IHC on TS to determine likely benefit or lack of benefit from a TOPO1 inhibitor, irinotecan and/or topotecan; performing IHC on TS to determine likely benefit or lack of benefit from an antimetabolite, fluorouracil, capecitabine, and/or pemetrexed; performing IHC on MGMT to determine likely benefit or lack of benefit from an alkylating agent, temozolomide, and/or dacarbazine; performing IHC on AR to determine likely benefit or lack of benefit from an anti-androgen, bicalutamide, flutamide, and/or abiraterone; performing IHC on ER to determine likely benefit or lack of benefit from a hormonal agent, tamoxifen, fulvestrant, letrozole, and/or anastrozole; performing IHC on one or more of ER and PR to determine likely benefit or lack of benefit from a hormonal agent, tamoxifen, toremifene, fulvestrant, letrozole, anastrozole, exemestane, megestrol acetate, leuprolide, and/or goserelin; performing one or more of IHC on HER2 and ISH on HER2 to determine likely benefit or lack of benefit from a tyrosine kinase inhibitor and/or lapatinib; performing one or more of IHC on HER2 and ISH on HER2 to determine likely benefit or lack of benefit from an antibody therapy, trastuzumab, pertuzumab, and/or ado-trastuzumab emtansine (T-DM1); performing one or more of ISH on TOP2A, ISH on HER2, IHC on TOP2A and IHC on PGP to determine likely benefit or lack of benefit from an anthracyclines, doxorubicin, liposomal-doxorubicin, and/or epirubicin; performing sequencing on one or more of cKIT and PDGFRA to determine likely benefit or lack of benefit from a tyrosine kinase inhibitor and/or imatinib; performing one or more of ISH on ALK and ISH on ROS1 to determine likely benefit or lack of benefit from a tyrosine kinase inhibitor and/or crizotinib; performing sequencing on PIK3CA to determine likely benefit or lack of benefit from an mTOR inhibitor, everolimus, and/or temsirolimus; performing sequencing on RET to determine likely benefit or lack of benefit from a tyrosine kinase inhibitor, and/or vandetanib; performing IHC on one or more of SPARC, TUBB3 and PGP to determine likely benefit or lack of benefit from a taxane, paclitaxel, docetaxel, nab-paclitaxel; performing IHC on one or more of SPARC, TLE3, TUBB3 and PGP to determine likely benefit or lack of benefit from a taxane, paclitaxel, docetaxel, nab-paclitaxel; performing one or more of PCR and sequencing on BRAF to determine likely benefit or lack of benefit from a tyrosine kinase inhibitor, vemurafenib, dabrafenib, and/or trametinib; performing one or more of sequencing on KRAS, sequencing on BRAF, sequencing on NRAS, sequencing on PIK3CA and IHC on PTEN to determine likely benefit or lack of benefit from an EGFR-targeted antibody, cetuximab, and/or panitumumab; performing one or more of sequencing on EGFR, sequencing on KRAS, ISH on cMET, sequencing on PIK3CA and IHC onn PTEN to determine likely benefit or lack of benefit from a tyrosine kinase inhibitor, erlotinib, and/or gefitinib; performing sequencing on EGFR to determine likely benefit or lack of benefit from a tyrosine kinase inhibitor, and/or afatinib; and performing sequencing on cKIT to determine likely benefit or lack of benefit from a tyrosine kinase inhibitor, and/or sunitinib. The computer medium can comprise one or more rules selected from Table 28. The computer medium may comprise a partial set of rules provided in any of Tables 7, 9, 11, 13, 15, 17, 21 and 28. The computer medium may comprise the full set of rules provided in any of Tables 7, 9, 11, 13, 15, 17, 21 and 28.


INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.





BRIEF DESCRIPTION OF THE DRAWINGS

A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are used, and the accompanying drawings of which:



FIG. 1 illustrates a block diagram of an exemplary embodiment of a system for determining individualized medical intervention for a particular disease state that utilizes molecular profiling of a patient's biological specimen that is non disease specific.



FIG. 2 is a flowchart of an exemplary embodiment of a method for determining individualized medical intervention for a particular disease state that utilizes molecular profiling of a patient's biological specimen that is non disease specific.



FIGS. 3A through 3D illustrate an exemplary patient profile report in accordance with step 80 of FIG. 2.



FIG. 4 is a flowchart of an exemplary embodiment of a method for identifying a drug therapy/agent capable of interacting with a target.



FIGS. 5-14 are flowcharts and diagrams illustrating various parts of an information-based personalized medicine drug discovery system and method in accordance with the present invention.



FIGS. 15-25 are computer screen print outs associated with various parts of the information-based personalized medicine drug discovery system and method shown in FIGS. 5-14.



FIGS. 26-31 herein are incorporated by reference from FIGS. 26-31, respectively, from International Patent Application PCT/US2009/060630, filed 14 Oct. 2009 and entitled “GENE AND GENE EXPRESSED PROTEIN TARGETS DEPICTING BIOMARKER PATTERNS AND SIGNATURE SETS BY TUMOR TYPE,” which application is hereby incorporated by reference in its entirety.



FIGS. 32A-B illustrate a diagram showing a biomarker centric (FIG. 32A) and therapeutic centric (FIG. 32B) approach to identifying a therapeutic agent.



FIGS. 33A-33Q illustrate molecular intelligence (MI) profiles comprising biomarkers and associated therapeutic agents that can be assessed to identify candidate therapeutic agents. The indicated MI Plus profiles include additional cancer markers to be assessed by mutational analysis for diagnostic, prognostic and related purposes. NextGen refers to Next Generation Sequencing. PyroSeq refers to pyrosequencing. SangerSeq refers to Sanger dye termination sequencing. FIG. 33A and FIG. 33B illustrate an MI profile and and MI PLUS profile, respectively, for any solid tumor. FIG. 33C and FIG. 33D illustrate an MI profile and and MI PLUS profile, respectively, for an ovarian cancer. FIG. 33E and FIG. 33F illustrate an MI profile and and MI PLUS profile, respectively, for a melanoma. FIG. 33G and FIG. 33H illustrate an MI profile and and MI PLUS profile, respectively, for a uveal melanoma. FIG. 33I and FIG. 33J illustrate an MI profile and and MI PLUS profile, respectively, for a non-small cell lung cancer (NSCLC). FIG. 33K and FIG. 33L illustrate an MI profile and and MI PLUS profile, respectively, for a breast cancer. FIG. 33M and FIG. 33N illustrate an MI profile and and MI PLUS profile, respectively, for a colorectal cancer (CRC). FIG. 33O and FIG. 33P illustrate an MI profile and and MI PLUS profile, respectively, for a glioma. FIG. 33Q illustrates individual marker profiling that can be added to any of the molecular profiles in FIGS. 33A-33P.



FIGS. 34A-34C illustrate biomarkers assessed using a molecular profiling approach as outlined in FIGS. FIGS. 33A-33Q, Tables 7-24, and accompanying text herein. FIG. 34A illustrates biomarkers that are assessed. The biomarkers that are assessed according to the Next Generation sequencing panel in FIG. 34A are shown in FIG. 34B. FIG. 34C illustrates sample requirements that can be used to perform molecular profiling on a patient tumor sample according to the panels in FIGS. 34A-34B.



FIGS. 35A-35I illustrate biomarkers and associated therapeutic agents that can be assessed to identify candidate therapeutic agents. NextGen refers to Next Generation Sequencing.



FIGS. 36A-F illustrate how molecular profiles for any cancer, e.g., for assessment of solid tumors, can be altered depending on sample availability. FIG. 36A illustrates a core comprehensive molecular profile for cancer. FIG. 36B illustrates lineage specific components of the comprehensive molecular profile for cancer. FIG. 36C illustrates drugs and clinical trials corresponding to the profiling shown in FIGS. 36A-B. FIG. 36D illustrates a comprehensive molecular profile that can be used instead of the profile shown in FIGS. 36A-B when insufficient sample is present to perform RT-PCR. FIG. 36E illustrates additional molecular profiling that can be performed. For example, TOP2A IHC and PGP IHC can be used instead of TOP2A FISH when the sample is insufficient for FISH testing. FIG. 36F provides illustrative biomarker tests that can be prioritized for various lineages, e.g., when insufficient sample is available for comprehensive molecular profiling.



FIGS. 37A-37Y illustrate an exemplary patient report based on molecular profiling for a patient having a history of anaplastic astrocytoma, a WHO grade III type of astrocytoma, a high grade glioma.


FIGS. 38A-38AA illustrate an exemplary patient report based on molecular intelligence molecular profiling for a patient having a history of lung adenocarcinoma.



FIGS. 39A-39Y illustrate an exemplary patient report based on molecular profiling for a non-small cell lung cancer with stand alone mutational analysis.



FIG. 40 illustrates progression free survival (PFS) using therapy selected by molecular profiling (period B) with PFS for the most recent therapy on which the patient has just progressed (period A). If PFS(B)/PFS(A) ratio ≧1.3, then molecular profiling selected therapy was defined as having benefit for patient.



FIG. 41 is a schematic of methods for identifying treatments by molecular profiling if a target is identified.



FIG. 42 illustrates the distribution of the patients in the study as performed in Example 1.



FIG. 43 is graph depicting the results of the study with patients having PFS ratio ≧1.3 was 18/66 (27%).



FIG. 44 is a waterfall plot of all the patients for maximum % change of summed diameters of target lesions with respect to baseline diameter.



FIG. 45 illustrates the relationship between what clinician selected as what she/he would use to treat the patient before knowing what the molecular profiling results suggested. There were no matches for the 18 patients with PFS ratio ≧1.3.



FIG. 46 is a schematic of the overall survival for the 18 patients with PFS ratio ≧1.3 versus all 66 patients.



FIG. 47 illustrates a molecular profiling system that performs analysis of a cancer sample using a variety of components that measure expression levels, chromosomal aberrations and mutations. The molecular “blueprint” of the cancer is used to generate a prioritized ranking of druggable targets and/or drug associated targets in tumor and their associated therapies.



FIG. 48 shows an example output of microarray profiling results and calls made using a cutoff value.



FIGS. 49A-B illustrate a workflow chart for identifying a therapeutic for an individual having breast cancer. The workflow of FIG. 49A feeds into the workflow of FIG. 49B as indicated.



FIG. 50 illustrates biomarkers used for identifying a therapeutic for an individual having breast cancer such as when following the workflow of FIGS. 49A-B. The figure illustrates a biomarker centric view of the workflow described above in different cancer settings.



FIG. 51 illustrates the percentage of HER2 positive breast cancers that are likely to respond to treatment with trastuzumab (Herceptin®), which is about 30%. Characteristics of the tumor that can be identified by molecular profiling are shown as well.





DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and systems for identifying therapeutic agents for use in treatments on an individualized basis by using molecular profiling. The molecular profiling approach provides a method for selecting a candidate treatment for an individual that could favorably change the clinical course for the individual with a condition or disease, such as cancer. The molecular profiling approach provides clinical benefit for individuals, such as identifying drug target(s) that provide a longer progression free survival (PFS), longer disease free survival (DFS), longer overall survival (OS) or extended lifespan. Methods and systems of the invention are directed to molecular profiling of cancer on an individual basis that can provide alternatives for treatment that may be convention or alternative to conventional treatment regimens. For example, alternative treatment regimes can be selected through molecular profiling methods of the invention where, a disease is refractory to current therapies, e.g., after a cancer has developed resistance to a standard-of-care treatment. Illustrative schemes for using molecular profiling to identify a treatment regime are shown in FIGS. 2, 49A-B and 50, each of which is described in further detail herein. Thus, molecular profiling provides a personalized approach to selecting candidate treatments that are likely to benefit a cancer. In embodiments, the molecular profiling method is used to identify therapies for patients with poor prognosis, such as those with metastatic disease or those whose cancer has progressed on standard front line therapies, or whose cancer has progressed on multiple chemotherapeutic or hormonal regimens.


Personalized medicine based on pharmacogenetic insights, such as those provided by molecular profiling according to the invention, is increasingly taken for granted by some practitioners and the lay press, but forms the basis of hope for improved cancer therapy. However, molecular profiling as taught herein represents a fundamental departure from the traditional approach to oncologic therapy where for the most part, patients are grouped together and treated with approaches that are based on findings from light microscopy and disease stage. Traditionally, differential response to a particular therapeutic strategy has only been determined after the treatment was given, i.e. a posteriori. The “standard” approach to disease treatment relies on what is generally true about a given cancer diagnosis and treatment response has been vetted by randomized phase III clinical trials and forms the “standard of care” in medical practice. The results of these trials have been codified in consensus statements by guidelines organizations such as the National Comprehensive Cancer Network and The American Society of Clinical Oncology. The NCCN Compendium™ contains authoritative, scientifically derived information designed to support decision-making about the appropriate use of drugs and biologics in patients with cancer. The NCCN Compendium™ is recognized by the Centers for Medicare and Medicaid Services (CMS) and United Healthcare as an authoritative reference for oncology coverage policy. On-compendium treatments are those recommended by such guides. The biostatistical methods used to validate the results of clinical trials rely on minimizing differences between patients, and are based on declaring the likelihood of error that one approach is better than another for a patient group defined only by light microscopy and stage, not by individual differences in tumors. The molecular profiling methods of the invention exploit such individual differences. The methods can provide candidate treatments that can be then selected by a physician for treating a patient. In a study of such an approach presented in Example 1 herein, the results were profound: in 66 consecutive patients, the treating oncologist never managed to identify the molecular target selected by the test, and 27% of patients whose treatment was guided by molecular profiling managed a remission 1.3× longer than their previous best response. At present, such results are virtually unheard of result in the salvage therapy setting.


Molecular profiling can be used to provide a comprehensive view of the biological state of a sample. In an embodiment, molecular profiling is used for whole tumor profiling. Accordingly, a number of molecular approaches are used to assess the state of a tumor. The whole tumor profiling can be used for selecting a candidate treatment for a tumor. Molecular profiling can be used to select candidate therapeutics on any sample for any stage of a disease. In embodiment, the methods of the invention are used to profile a newly diagnosed cancer. The candidate treatments indicated by the molecular profiling can be used to select a therapy for treating the newly diagnosed cancer. In other embodiments, the methods of the invention are used to profile a cancer that has already been treated, e.g., with one or more standard-of-care therapy. In embodiments, the cancer is refractory to the prior treatment/s. For example, the cancer may be refractory to the standard of care treatments for the cancer. The cancer can be a metastatic cancer or other recurrent cancer. The treatments can be on-compendium or off-compendium treatments.


Molecular profiling can be performed by any known means for detecting a molecule in a biological sample. Molecular profiling comprises methods that include but are not limited to, nucleic acid sequencing, such as a DNA sequencing or mRNA sequencing; immunohistochemistry (IHC); in situ hybridization (ISH); fluorescent in situ hybridization (FISH); chromogenic in situ hybridization (CISH); PCR amplification (e.g., qPCR or RT-PCR); various types of microarray (mRNA expression arrays, low density arrays, protein arrays, etc); various types of sequencing (Sanger, pyrosequencing, etc); comparative genomic hybridization (CGH); NextGen sequencing; Northern blot; Southern blot; immunoassay; and any other appropriate technique to assay the presence or quantity of a biological molecule of interest. In various embodiments of the invention, any one or more of these methods can be used concurrently or subsequent to each other for assessing target genes disclosed herein.


Molecular profiling of individual samples is used to select one or more candidate treatments for a disorder in a subject, e.g., by identifying targets for drugs that may be effective for a given cancer. For example, the candidate treatment can be a treatment known to have an effect on cells that differentially express genes as identified by molecular profiling techniques, an experimental drug, a government or regulatory approved drug or any combination of such drugs, which may have been studied and approved for a particular indication that is the same as or different from the indication of the subject from whom a biological sample is obtain and molecularly profiled.


When multiple biomarker targets are revealed by assessing target genes by molecular profiling, one or more decision rules can be put in place to prioritize the selection of certain therapeutic agent for treatment of an individual on a personalized basis. Rules of the invention aide prioritizing treatment, e.g., direct results of molecular profiling, anticipated efficacy of therapeutic agent, prior history with the same or other treatments, expected side effects, availability of therapeutic agent, cost of therapeutic agent, drug-drug interactions, and other factors considered by a treating physician. Based on the recommended and prioritized therapeutic agent targets, a physician can decide on the course of treatment for a particular individual. Accordingly, molecular profiling methods and systems of the invention can select candidate treatments based on individual characteristics of diseased cells, e.g., tumor cells, and other personalized factors in a subject in need of treatment, as opposed to relying on a traditional one-size fits all approach that is conventionally used to treat individuals suffering from a disease, especially cancer. In some cases, the recommended treatments are those not typically used to treat the disease or disorder inflicting the subject. In some cases, the recommended treatments are used after standard-of-care therapies are no longer providing adequate efficacy.


The treating physician can use the results of the molecular profiling methods to optimize a treatment regimen for a patient. The candidate treatment identified by the methods of the invention can be used to treat a patient; however, such treatment is not required of the methods. Indeed, the analysis of molecular profiling results and identification of candidate treatments based on those results can be automated and does not require physician involvement.


Biological Entities

Nucleic acids include deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, or complements thereof. Nucleic acids can contain known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). Nucleic acid sequence can encompass conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell Probes 8:91-98 (1994)). The term nucleic acid can be used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.


A particular nucleic acid sequence may implicitly encompass the particular sequence and “splice variants” and nucleic acid sequences encoding truncated forms. Similarly, a particular protein encoded by a nucleic acid can encompass any protein encoded by a splice variant or truncated form of that nucleic acid. “Splice variants,” as the name suggests, are products of alternative splicing of a gene. After transcription, an initial nucleic acid transcript may be spliced such that different (alternate) nucleic acid splice products encode different polypeptides. Mechanisms for the production of splice variants vary, but include alternate splicing of exons. Alternate polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any products of a splicing reaction, including recombinant forms of the splice products, are included in this definition. Nucleic acids can be truncated at the 5′ end or at the 3′ end. Polypeptides can be truncated at the N-terminal end or the C-terminal end. Truncated versions of nucleic acid or polypeptide sequences can be naturally occurring or created using recombinant techniques.


The terms “genetic variant” and “nucleotide variant” are used herein interchangeably to refer to changes or alterations to the reference human gene or cDNA sequence at a particular locus, including, but not limited to, nucleotide base deletions, insertions, inversions, and substitutions in the coding and non-coding regions. Deletions may be of a single nucleotide base, a portion or a region of the nucleotide sequence of the gene, or of the entire gene sequence. Insertions may be of one or more nucleotide bases. The genetic variant or nucleotide variant may occur in transcriptional regulatory regions, untranslated regions of mRNA, exons, introns, exon/intron junctions, etc. The genetic variant or nucleotide variant can potentially result in stop codons, frame shifts, deletions of amino acids, altered gene transcript splice forms or altered amino acid sequence.


An allele or gene allele comprises generally a naturally occurring gene having a reference sequence or a gene containing a specific nucleotide variant.


A haplotype refers to a combination of genetic (nucleotide) variants in a region of an mRNA or a genomic DNA on a chromosome found in an individual. Thus, a haplotype includes a number of genetically linked polymorphic variants which are typically inherited together as a unit.


As used herein, the term “amino acid variant” is used to refer to an amino acid change to a reference human protein sequence resulting from genetic variants or nucleotide variants to the reference human gene encoding the reference protein. The term “amino acid variant” is intended to encompass not only single amino acid substitutions, but also amino acid deletions, insertions, and other significant changes of amino acid sequence in the reference protein.


The term “genotype” as used herein means the nucleotide characters at a particular nucleotide variant marker (or locus) in either one allele or both alleles of a gene (or a particular chromosome region). With respect to a particular nucleotide position of a gene of interest, the nucleotide(s) at that locus or equivalent thereof in one or both alleles form the genotype of the gene at that locus. A genotype can be homozygous or heterozygous. Accordingly, “genotyping” means determining the genotype, that is, the nucleotide(s) at a particular gene locus. Genotyping can also be done by determining the amino acid variant at a particular position of a protein which can be used to deduce the corresponding nucleotide variant(s).


The term “locus” refers to a specific position or site in a gene sequence or protein. Thus, there may be one or more contiguous nucleotides in a particular gene locus, or one or more amino acids at a particular locus in a polypeptide. Moreover, a locus may refer to a particular position in a gene where one or more nucleotides have been deleted, inserted, or inverted.


Unless specified otherwise or understood by one of skill in art, the terms “polypeptide,” “protein,” and “peptide” are used interchangeably herein to refer to an amino acid chain in which the amino acid residues are linked by covalent peptide bonds. The amino acid chain can be of any length of at least two amino acids, including full-length proteins. Unless otherwise specified, polypeptide, protein, and peptide also encompass various modified forms thereof, including but not limited to glycosylated forms, phosphorylated forms, etc. A polypeptide, protein or peptide can also be referred to as a gene product.


Lists of gene and gene products that can be assayed by molecular profiling techniques are presented herein. Lists of genes may be presented in the context of molecular profiling techniques that detect a gene product (e.g., an mRNA or protein). One of skill will understand that this implies detection of the gene product of the listed genes. Similarly, lists of gene products may be presented in the context of molecular profiling techniques that detect a gene sequence or copy number. One of skill will understand that this implies detection of the gene corresponding to the gene products, including as an example DNA encoding the gene products. As will be appreciated by those skilled in the art, a “biomarker” or “marker” comprises a gene and/or gene product depending on the context.


The terms “label” and “detectable label” can refer to any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical or similar methods. Such labels include biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g., DYNABEADS™), fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g., 3H, 125I, 35S, 14C, or 32P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc) beads. Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Means of detecting such labels are well known to those of skill in the art. Thus, for example, radiolabels may be detected using photographic film or scintillation counters, fluorescent markers may be detected using a photodetector to detect emitted light. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and calorimetric labels are detected by simply visualizing the colored label. Labels can include, e.g., ligands that bind to labeled antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies which can serve as specific binding pair members for a labeled ligand. An introduction to labels, labeling procedures and detection of labels is found in Polak and Van Noorden Introduction to Immunocytochemistry, 2nd ed., Springer Verlag, N.Y. (1997); and in Haugland Handbook of Fluorescent Probes and Research Chemicals, a combined handbook and catalogue Published by Molecular Probes, Inc. (1996).


Detectable labels include, but are not limited to, nucleotides (labeled or unlabelled), compomers, sugars, peptides, proteins, antibodies, chemical compounds, conducting polymers, binding moieties such as biotin, mass tags, calorimetric agents, light emitting agents, chemiluminescent agents, light scattering agents, fluorescent tags, radioactive tags, charge tags (electrical or magnetic charge), volatile tags and hydrophobic tags, biomolecules (e.g., members of a binding pair antibody/antigen, antibody/antibody, antibody/antibody fragment, antibody/antibody receptor, antibody/protein A or protein G, hapten/anti-hapten, biotin/avidin, biotin/streptavidin, folic acid/folate binding protein, vitamin B12/intrinsic factor, chemical reactive group/complementary chemical reactive group (e.g., sulfhydryl/maleimide, sulfhydryl/haloacetyl derivative, amine/isotriocyanate, amine/succinimidyl ester, and amine/sulfonyl halides) and the like.


The term “antibody” as used herein encompasses naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, single chain antibodies, chimeric, bifunctional and humanized antibodies, as well as antigen-binding fragments thereof, (e.g., Fab′, F(ab′)2, Fab, Fv and rIgG). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.). See also, e.g., Kuby, J., Immunology, 3.sup.rd Ed., W. H. Freeman & Co., New York (1998). Such non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy chains and variable light chains as described by Huse et al., Science 246:1275-1281 (1989), which is incorporated herein by reference. These and other methods of making, for example, chimeric, humanized, CDR-grafted, single chain, and bifunctional antibodies are well known to those skilled in the art. See, e.g., Winter and Harris, Immunol. Today 14:243-246 (1993); Ward et al., Nature 341:544-546 (1989); Harlow and Lane, Antibodies, 511-52, Cold Spring Harbor Laboratory publications, New York, 1988; Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrebaeck, Antibody Engineering, 2d ed. (Oxford University Press 1995); each of which is incorporated herein by reference.


Unless otherwise specified, antibodies can include both polyclonal and monoclonal antibodies. Antibodies also include genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies). The term also refers to recombinant single chain Fv fragments (scFv). The term antibody also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies. Bivalent and bispecific molecules are described in, e.g., Kostelny et al. (1992) J Immunol 148:1547, Pack and Pluckthun (1992) Biochemistry 31:1579, Holliger et al. (1993) Proc Natl Acad Sci USA. 90:6444, Gruber et al. (1994) J Immunol:5368, Zhu et al. (1997) Protein Sci 6:781, Hu et al. (1997) Cancer Res. 56:3055, Adams et al. (1993) Cancer Res. 53:4026, and McCartney, et al. (1995) Protein Eng. 8:301.


Typically, an antibody has a heavy and light chain. Each heavy and light chain contains a constant region and a variable region, (the regions are also known as “domains”). Light and heavy chain variable regions contain four framework regions interrupted by three hyper-variable regions, also called complementarity-determining regions (CDRs). The extent of the framework regions and CDRs have been defined. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three dimensional spaces. The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. References to VH refer to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv, or Fab. References to VL refer to the variable region of an immunoglobulin light chain, including the light chain of an Fv, scFv, dsFv or Fab.


The phrase “single chain Fv” or “scFv” refers to an antibody in which the variable domains of the heavy chain and of the light chain of a traditional two chain antibody have been joined to form one chain. Typically, a linker peptide is inserted between the two chains to allow for proper folding and creation of an active binding site. A “chimeric antibody” is an immunoglobulin molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.


A “humanized antibody” is an immunoglobulin molecule that contains minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fe), typically that of a human immunoglobulin (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)). Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.


The terms “epitope” and “antigenic determinant” refer to a site on an antigen to which an antibody binds. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996).


The terms “primer”, “probe,” and “oligonucleotide” are used herein interchangeably to refer to a relatively short nucleic acid fragment or sequence. They can comprise DNA, RNA, or a hybrid thereof, or chemically modified analog or derivatives thereof. Typically, they are single-stranded. However, they can also be double-stranded having two complementing strands which can be separated by denaturation. Normally, primers, probes and oligonucleotides have a length of from about 8 nucleotides to about 200 nucleotides, preferably from about 12 nucleotides to about 100 nucleotides, and more preferably about 18 to about 50 nucleotides. They can be labeled with detectable markers or modified using conventional manners for various molecular biological applications.


The term “isolated” when used in reference to nucleic acids (e.g., genomic DNAs, cDNAs, mRNAs, or fragments thereof) is intended to mean that a nucleic acid molecule is present in a form that is substantially separated from other naturally occurring nucleic acids that are normally associated with the molecule. Because a naturally existing chromosome (or a viral equivalent thereof) includes a long nucleic acid sequence, an isolated nucleic acid can be a nucleic acid molecule having only a portion of the nucleic acid sequence in the chromosome but not one or more other portions present on the same chromosome. More specifically, an isolated nucleic acid can include naturally occurring nucleic acid sequences that flank the nucleic acid in the naturally existing chromosome (or a viral equivalent thereof). An isolated nucleic acid can be substantially separated from other naturally occurring nucleic acids that are on a different chromosome of the same organism. An isolated nucleic acid can also be a composition in which the specified nucleic acid molecule is significantly enriched so as to constitute at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or at least 99% of the total nucleic acids in the composition.


An isolated nucleic acid can be a hybrid nucleic acid having the specified nucleic acid molecule covalently linked to one or more nucleic acid molecules that are not the nucleic acids naturally flanking the specified nucleic acid. For example, an isolated nucleic acid can be in a vector. In addition, the specified nucleic acid may have a nucleotide sequence that is identical to a naturally occurring nucleic acid or a modified form or mutein thereof having one or more mutations such as nucleotide substitution, deletion/insertion, inversion, and the like.


An isolated nucleic acid can be prepared from a recombinant host cell (in which the nucleic acids have been recombinantly amplified and/or expressed), or can be a chemically synthesized nucleic acid having a naturally occurring nucleotide sequence or an artificially modified form thereof.


The term “isolated polypeptide” as used herein is defined as a polypeptide molecule that is present in a form other than that found in nature. Thus, an isolated polypeptide can be a non-naturally occurring polypeptide. For example, an isolated polypeptide can be a “hybrid polypeptide.” An isolated polypeptide can also be a polypeptide derived from a naturally occurring polypeptide by additions or deletions or substitutions of amino acids. An isolated polypeptide can also be a “purified polypeptide” which is used herein to mean a composition or preparation in which the specified polypeptide molecule is significantly enriched so as to constitute at least 10% of the total protein content in the composition. A “purified polypeptide” can be obtained from natural or recombinant host cells by standard purification techniques, or by chemically synthesis, as will be apparent to skilled artisans.


The terms “hybrid protein,” “hybrid polypeptide,” “hybrid peptide,” “fusion protein,” “fusion polypeptide,” and “fusion peptide” are used herein interchangeably to mean a non-naturally occurring polypeptide or isolated polypeptide having a specified polypeptide molecule covalently linked to one or more other polypeptide molecules that do not link to the specified polypeptide in nature. Thus, a “hybrid protein” may be two naturally occurring proteins or fragments thereof linked together by a covalent linkage. A “hybrid protein” may also be a protein formed by covalently linking two artificial polypeptides together. Typically but not necessarily, the two or more polypeptide molecules are linked or “fused” together by a peptide bond forming a single non-branched polypeptide chain.


The term “high stringency hybridization conditions,” when used in connection with nucleic acid hybridization, includes hybridization conducted overnight at 42° C. in a solution containing 50% formamide, 5×SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate, pH 7.6, 5×Denhardt's solution, 10% dextran sulfate, and 20 microgram/ml denatured and sheared salmon sperm DNA, with hybridization filters washed in 0.1×SSC at about 65° C. The term “moderate stringent hybridization conditions,” when used in connection with nucleic acid hybridization, includes hybridization conducted overnight at 37° C. in a solution containing 50% formamide, 5×SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate, pH 7.6, 5×Denhardt's solution, 10% dextran sulfate, and 20 microgram/ml denatured and sheared salmon sperm DNA, with hybridization filters washed in 1×SSC at about 50° C. It is noted that many other hybridization methods, solutions and temperatures can be used to achieve comparable stringent hybridization conditions as will be apparent to skilled artisans.


For the purpose of comparing two different nucleic acid or polypeptide sequences, one sequence (test sequence) may be described to be a specific percentage identical to another sequence (comparison sequence). The percentage identity can be determined by the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993), which is incorporated into various BLAST programs. The percentage identity can be determined by the “BLAST 2 Sequences” tool, which is available at the National Center for Biotechnology Information (NCBI) website. See Tatusova and Madden, FEMS Microbiol. Lett., 174(2):247-250 (1999). For pairwise DNA-DNA comparison, the BLASTN program is used with default parameters (e.g., Match: 1; Mismatch: −2; Open gap: 5 penalties; extension gap: 2 penalties; gap x_dropoff: 50; expect: 10; and word size: 11, with filter). For pairwise protein-protein sequence comparison, the BLASTP program can be employed using default parameters (e.g., Matrix: BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 15; expect: 10.0; and wordsize: 3, with filter). Percent identity of two sequences is calculated by aligning a test sequence with a comparison sequence using BLAST, determining the number of amino acids or nucleotides in the aligned test sequence that are identical to amino acids or nucleotides in the same position of the comparison sequence, and dividing the number of identical amino acids or nucleotides by the number of amino acids or nucleotides in the comparison sequence. When BLAST is used to compare two sequences, it aligns the sequences and yields the percent identity over defined, aligned regions. If the two sequences are aligned across their entire length, the percent identity yielded by the BLAST is the percent identity of the two sequences. If BLAST does not align the two sequences over their entire length, then the number of identical amino acids or nucleotides in the unaligned regions of the test sequence and comparison sequence is considered to be zero and the percent identity is calculated by adding the number of identical amino acids or nucleotides in the aligned regions and dividing that number by the length of the comparison sequence. Various versions of the BLAST programs can be used to compare sequences, e.g., BLAST 2.1.2 or BLAST+2.2.22.


A subject or individual can be any animal which may benefit from the methods of the invention, including, e.g., humans and non-human mammals, such as primates, rodents, horses, dogs and cats. Subjects include without limitation a eukaryotic organisms, most preferably a mammal such as a primate, e.g., chimpanzee or human, cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish. Subjects specifically intended for treatment using the methods described herein include humans. A subject may be referred to as an individual or a patient.


Treatment of a disease or individual according to the invention is an approach for obtaining beneficial or desired medical results, including clinical results, but not necessarily a cure. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment or if receiving a different treatment. A treatment can include administration of a therapeutic agent, which can be an agent that exerts a cytotoxic, cytostatic, or immunomodulatory effect on diseased cells, e.g., cancer cells, or other cells that may promote a diseased state, e.g., activated immune cells. Therapeutic agents selected by the methods of the invention are not limited. Any therapeutic agent can be selected where a link can be made between molecular profiling and potential efficacy of the agent. Therapeutic agents include without limitation drugs, pharmaceuticals, small molecules, protein therapies, antibody therapies, viral therapies, gene therapies, and the like. Cancer treatments or therapies include apoptosis-mediated and non-apoptosis mediated cancer therapies including, without limitation, chemotherapy, hormonal therapy, radiotherapy, immunotherapy, and combinations thereof. Chemotherapeutic agents comprise therapeutic agents and combinations of therapeutic agents that treat, cancer cells, e.g., by killing those cells. Examples of different types of chemotherapeutic drugs include without limitation alkylating agents (e.g., nitrogen mustard derivatives, ethylenimines, alkylsulfonates, hydrazines and triazines, nitrosureas, and metal salts), plant alkaloids (e.g., vinca alkaloids, taxanes, podophyllotoxins, and camptothecan analogs), antitumor antibiotics (e.g., anthracyclines, chromomycins, and the like), antimetabolites (e.g., folic acid antagonists, pyrimidine antagonists, purine antagonists, and adenosine deaminase inhibitors), topoisomerase I inhibitors, topoisomerase II inhibitors, and miscellaneous antineoplastics (e.g., ribonucleotide reductase inhibitors, adrenocortical steroid inhibitors, enzymes, antimicrotubule agents, and retinoids).


A biomarker refers generally to a molecule, including without limitation a gene or product thereof, nucleic acids (e.g., DNA, RNA), protein/peptide/polypeptide, carbohydrate structure, lipid, glycolipid, characteristics of which can be detected in a tissue or cell to provide information that is predictive, diagnostic, prognostic and/or theranostic for sensitivity or resistance to candidate treatment.


Biological Samples

A sample as used herein includes any relevant biological sample that can be used for molecular profiling, e.g., sections of tissues such as biopsy or tissue removed during surgical or other procedures, bodily fluids, autopsy samples, and frozen sections taken for histological purposes. Such samples include blood and blood fractions or products (e.g., serum, buffy coat, plasma, platelets, red blood cells, and the like), sputum, malignant effusion, cheek cells tissue, cultured cells (e.g., primary cultures, explants, and transformed cells), stool, urine, other biological or bodily fluids (e.g., prostatic fluid, gastric fluid, intestinal fluid, renal fluid, lung fluid, cerebrospinal fluid, and the like), etc. The sample can comprise biological material that is a fresh frozen & formalin fixed paraffin embedded (FFPE) block, formalin-fixed paraffin embedded, or is within an RNA preservative+formalin fixative. More than one sample of more than one type can be used for each patient. In a preferred embodiment, the sample comprises a fixed tumor sample.


The sample used in the methods described herein can be a formalin fixed paraffin embedded (FFPE) sample. The FFPE sample can be one or more of fixed tissue, unstained slides, bone marrow core or clot, core needle biopsy, malignant fluids and fine needle aspirate (FNA). In an embodiment, the fixed tissue comprises a tumor containing formalin fixed paraffin embedded (FFPE) block from a surgery or biopsy. In another embodiment, the unstained slides comprise unstained, charged, unbaked slides from a paraffin block. In another embodiment, bone marrow core or clot comprises a decalcified core. A formalin fixed core and/or clot can be paraffin-embedded. In still another embodiment, the core needle biopsy comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, e.g., 3-4, paraffin embedded biopsy samples. An 18 gauge needle biopsy can be used. The malignant fluid can comprise a sufficient volume of fresh pleural/ascitic fluid to produce a 5×5×2 mm cell pellet. The fluid can be formalin fixed in a paraffin block. In an embodiment, the core needle biopsy comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, e.g., 4-6, paraffin embedded aspirates.


A sample may be processed according to techniques understood by those in the art. A sample can be without limitation fresh, frozen or fixed cells or tissue. In some embodiments, a sample comprises formalin-fixed paraffin-embedded (FFPE) tissue, fresh tissue or fresh frozen (FF) tissue. A sample can comprise cultured cells, including primary or immortalized cell lines derived from a subject sample. A sample can also refer to an extract from a sample from a subject. For example, a sample can comprise DNA, RNA or protein extracted from a tissue or a bodily fluid. Many techniques and commercial kits are available for such purposes. The fresh sample from the individual can be treated with an agent to preserve RNA prior to further processing, e.g., cell lysis and extraction. Samples can include frozen samples collected for other purposes. Samples can be associated with relevant information such as age, gender, and clinical symptoms present in the subject; source of the sample; and methods of collection and storage of the sample. A sample is typically obtained from a subject.


A biopsy comprises the process of removing a tissue sample for diagnostic or prognostic evaluation, and to the tissue specimen itself. Any biopsy technique known in the art can be applied to the molecular profiling methods of the present invention. The biopsy technique applied can depend on the tissue type to be evaluated (e.g., colon, prostate, kidney, bladder, lymph node, liver, bone marrow, blood cell, lung, breast, etc.), the size and type of the tumor (e.g., solid or suspended, blood or ascites), among other factors. Representative biopsy techniques include, but are not limited to, excisional biopsy, incisional biopsy, needle biopsy, surgical biopsy, and bone marrow biopsy. An “excisional biopsy” refers to the removal of an entire tumor mass with a small margin of normal tissue surrounding it. An “incisional biopsy” refers to the removal of a wedge of tissue that includes a cross-sectional diameter of the tumor. Molecular profiling can use a “core-needle biopsy” of the tumor mass, or a “fine-needle aspiration biopsy” which generally obtains a suspension of cells from within the tumor mass. Biopsy techniques are discussed, for example, in Harrison's Principles of Internal Medicine, Kasper, et al., eds., 16th ed., 2005, Chapter 70, and throughout Part V.


Standard molecular biology techniques known in the art and not specifically described are generally followed as in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1989), and as in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989) and as in Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, New York (1988), and as in Watson et al., Recombinant DNA, Scientific American Books, New York and in Birren et al (eds) Genome Analysis: A Laboratory Manual Series, Vols. 1-4 Cold Spring Harbor Laboratory Press, New York (1998) and methodology as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057 and incorporated herein by reference. Polymerase chain reaction (PCR) can be carried out generally as in PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego, Calif. (1990).


Vesicles

The sample can comprise vesicles. Methods of the invention can include assessing one or more vesicles, including assessing vesicle populations. A vesicle, as used herein, is a membrane vesicle that is shed from cells. Vesicles or membrane vesicles include without limitation: circulating microvesicles (cMVs), microvesicle, exosome, nanovesicle, dexosome, bleb, blebby, prostasome, microparticle, intralumenal vesicle, membrane fragment, intralumenal endosomal vesicle, endosomal-like vesicle, exocytosis vehicle, endosome vesicle, endosomal vesicle, apoptotic body, multivesicular body, secretory vesicle, phospholipid vesicle, liposomal vesicle, argosome, texasome, secresome, tolerosome, melanosome, oncosome, or exocytosed vehicle. Furthermore, although vesicles may be produced by different cellular processes, the methods of the invention are not limited to or reliant on any one mechanism, insofar as such vesicles are present in a biological sample and are capable of being characterized by the methods disclosed herein. Unless otherwise specified, methods that make use of a species of vesicle can be applied to other types of vesicles. Vesicles comprise spherical structures with a lipid bilayer similar to cell membranes which surrounds an inner compartment which can contain soluble components, sometimes referred to as the payload. In some embodiments, the methods of the invention make use of exosomes, which are small secreted vesicles of about 40-100 nm in diameter. For a review of membrane vesicles, including types and characterizations, see Thery et al., Nat Rev Immunol. 2009 August; 9(8):581-93. Some properties of different types of vesicles include those in Table 1:









TABLE 1







Vesicle Properties


















Exosome-







Membrane
like
Apoptotic


Feature
Exosomes
Microvesicles
Ectosomes
particles
vesicles
vesicles






















Size
50-100
nm
100-1,000
nm
50-200
nm
50-80
nm
20-50
nm
50-500
nm




















Density in
1.13-1.19
g/ml


1.04-1.07
g/ml
1.1
g/ml
1.16-1.28
g/ml













sucrose








EM
Cup shape
Irregular
Bilamellar
Round
Irregular
Heterogeneous


appearance

shape,
round

shape





electron
structures







dense





















Sedimentation
100,000
g
10,000
g
160,000-
100,000-
175,000
g
1,200
g,

















200,000 g
200,000 g

10,000
g,








100,000
g













Lipid
Enriched in
Expose PPS
Enriched in

No lipid



composition
cholesterol,

cholesterol

rafts




sphingomyelin

and






and ceramide;

diacylglycerol;






contains lipid

expose PPS






rafts; expose








PPS







Major
Tetraspanins
Integrins,
CR1 and
CD133;
TNFRI
Histones


protein
(e.g., CD63,
selectins and
proteolytic
no CD63




markers
CD9), Alix,
CD40 ligand
enzymes;






TSG101

no CD63





Intracellular
Internal
Plasma
Plasma
Plasma




origin
compartments
membrane
membrane
membrane





(endosomes)





Abbreviations: phosphatidylserine (PPS); electron microscopy (EM)






Vesicles include shed membrane bound particles, or “microparticles,” that are derived from either the plasma membrane or an internal membrane. Vesicles can be released into the extracellular environment from cells. Cells releasing vesicles include without limitation cells that originate from, or are derived from, the ectoderm, endoderm, or mesoderm. The cells may have undergone genetic, environmental, and/or any other variations or alterations. For example, the cell can be tumor cells. A vesicle can reflect any changes in the source cell, and thereby reflect changes in the originating cells, e.g., cells having various genetic mutations. In one mechanism, a vesicle is generated intracellularly when a segment of the cell membrane spontaneously invaginates and is ultimately exocytosed (see for example, Keller et al., Immunol. Lett. 107 (2): 102-8 (2006)). Vesicles also include cell-derived structures bounded by a lipid bilayer membrane arising from both herniated evagination (blebbing) separation and sealing of portions of the plasma membrane or from the export of any intracellular membrane-bounded vesicular structure containing various membrane-associated proteins of tumor origin, including surface-bound molecules derived from the host circulation that bind selectively to the tumor-derived proteins together with molecules contained in the vesicle lumen, including but not limited to tumor-derived microRNAs or intracellular proteins. Blebs and blebbing are further described in Charras et al., Nature Reviews Molecular and Cell Biology, Vol. 9, No. 11, p. 730-736 (2008). A vesicle shed into circulation or bodily fluids from tumor cells may be referred to as a “circulating tumor-derived vesicle.” When such vesicle is an exosome, it may be referred to as a circulating-tumor derived exosome (CTE). In some instances, a vesicle can be derived from a specific cell of origin. CTE, as with a cell-of-origin specific vesicle, typically have one or more unique biomarkers that permit isolation of the CTE or cell-of-origin specific vesicle, e.g., from a bodily fluid and sometimes in a specific manner. For example, a cell or tissue specific markers are used to identify the cell of origin. Examples of such cell or tissue specific markers are disclosed herein and can further be accessed in the Tissue-specific Gene Expression and Regulation (TiGER) Database, available at bioinfo.wilmer.jhu.edu/tiger/; Liu et al. (2008) TiGER: a database for tissue-specific gene expression and regulation. BMC Bioinformatics. 9:271; TissueDistributionDBs, available at genome.dkfz-heidelberg.de/menu/tissue_db/index.html.


A vesicle can have a diameter of greater than about 10 nm, 20 nm, or 30 nm. A vesicle can have a diameter of greater than 40 nm, 50 nm, 100 nm, 200 nm, 500 nm, 1000 nm or greater than 10,000 nm. A vesicle can have a diameter of about 30-1000 nm, about 30-800 nm, about 30-200 nm, or about 30-100 nm. In some embodiments, the vesicle has a diameter of less than 10,000 nm, 1000 nm, 800 nm, 500 nm, 200 nm, 100 nm, 50 nm, 40 nm, 30 nm, 20 nm or less than 10 nm. As used herein the term “about” in reference to a numerical value means that variations of 10% above or below the numerical value are within the range ascribed to the specified value. Typical sizes for various types of vesicles are shown in Table 1. Vesicles can be assessed to measure the diameter of a single vesicle or any number of vesicles. For example, the range of diameters of a vesicle population or an average diameter of a vesicle population can be determined. Vesicle diameter can be assessed using methods known in the art, e.g., imaging technologies such as electron microscopy. In an embodiment, a diameter of one or more vesicles is determined using optical particle detection. See, e.g., U.S. Pat. No. 7,751,053, entitled “Optical Detection and Analysis of Particles” and issued Jul. 6, 2010; and U.S. Pat. No. 7,399,600, entitled “Optical Detection and Analysis of Particles” and issued Jul. 15, 2010.


In some embodiments, vesicles are directly assayed from a biological sample without prior isolation, purification, or concentration from the biological sample. For example, the amount of vesicles in the sample can by itself provide a biosignature that provides a diagnostic, prognostic or theranostic determination. Alternatively, the vesicle in the sample may be isolated, captured, purified, or concentrated from a sample prior to analysis. As noted, isolation, capture or purification as used herein comprises partial isolation, partial capture or partial purification apart from other components in the sample. Vesicle isolation can be performed using various techniques as described herein or known in the art, including without limitation size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, affinity capture, immunoassay, immunoprecipitation, microfluidic separation, flow cytometry or combinations thereof.


Vesicles can be assessed to provide a phenotypic characterization by comparing vesicle characteristics to a reference. In some embodiments, surface antigens on a vesicle are assessed. A vesicle or vesicle population carrying a specific marker can be referred to as a positive (biomarker+) vesicle or vesicle population. For example, a DLL4+ population refers to a vesicle population associated with DLL4. Conversely, a DLL4− population would not be associated with DLL4. The surface antigens can provide an indication of the anatomical origin and/or cellular of the vesicles and other phenotypic information, e.g., tumor status. For example, vesicles found in a patient sample can be assessed for surface antigens indicative of colorectal origin and the presence of cancer, thereby identifying vesicles associated with colorectal cancer cells. The surface antigens may comprise any informative biological entity that can be detected on the vesicle membrane surface, including without limitation surface proteins, lipids, carbohydrates, and other membrane components. For example, positive detection of colon derived vesicles expressing tumor antigens can indicate that the patient has colorectal cancer. As such, methods of the invention can be used to characterize any disease or condition associated with an anatomical or cellular origin, by assessing, for example, disease-specific and cell-specific biomarkers of one or more vesicles obtained from a subject.


In embodiments, one or more vesicle payloads are assessed to provide a phenotypic characterization. The payload with a vesicle comprises any informative biological entity that can be detected as encapsulated within the vesicle, including without limitation proteins and nucleic acids, e.g., genomic or cDNA, mRNA, or functional fragments thereof, as well as microRNAs (miRs). In addition, methods of the invention are directed to detecting vesicle surface antigens (in addition or exclusive to vesicle payload) to provide a phenotypic characterization. For example, vesicles can be characterized by using binding agents (e.g., antibodies or aptamers) that are specific to vesicle surface antigens, and the bound vesicles can be further assessed to identify one or more payload components disclosed therein. As described herein, the levels of vesicles with surface antigens of interest or with payload of interest can be compared to a reference to characterize a phenotype. For example, overexpression in a sample of cancer-related surface antigens or vesicle payload, e.g., a tumor associated mRNA or microRNA, as compared to a reference, can indicate the presence of cancer in the sample. The biomarkers assessed can be present or absent, increased or reduced based on the selection of the desired target sample and comparison of the target sample to the desired reference sample. Non-limiting examples of target samples include: disease; treated/not-treated; different time points, such as a in a longitudinal study; and non-limiting examples of reference sample: non-disease; normal; different time points; and sensitive or resistant to candidate treatment(s).


In an embodiment, molecular profiling of the invention comprises analysis of microvesicles, such as circulating microvesicles.


MicroRNA

Various biomarker molecules can be assessed in biological samples or vesicles obtained from such biological samples. MicroRNAs comprise one class biomarkers assessed via methods of the invention. MicroRNAs, also referred to herein as miRNAs or miRs, are short RNA strands approximately 21-23 nucleotides in length. MiRNAs are encoded by genes that are transcribed from DNA but are not translated into protein and thus comprise non-coding RNA. The miRs are processed from primary transcripts known as pri-miRNA to short stem-loop structures called pre-miRNA and finally to the resulting single strand miRNA. The pre-miRNA typically forms a structure that folds back on itself in self-complementary regions. These structures are then processed by the nuclease Dicer in animals or DCL1 in plants. Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules and can function to regulate translation of proteins. Identified sequences of miRNA can be accessed at publicly available databases, such as www.microRNA.org, www.mirbase.org, or www.mirz.unibas.ch/cgi/miRNA.cgi.


miRNAs are generally assigned a number according to the naming convention “mir-[number].” The number of a miRNA is assigned according to its order of discovery relative to previously identified miRNA species. For example, if the last published miRNA was mir-121, the next discovered miRNA will be named mir-122, etc. When a miRNA is discovered that is homologous to a known miRNA from a different organism, the name can be given an optional organism identifier, of the form [organism identifier]-mir-[number]. Identifiers include hsa for Homo sapiens and mmu for Mus Musculus. For example, a human homolog to mir-121 might be referred to as hsa-mir-121 whereas the mouse homolog can be referred to as mmu-mir-121.


Mature microRNA is commonly designated with the prefix “miR” whereas the gene or precursor miRNA is designated with the prefix “mir.” For example, mir-121 is a precursor for miR-121. When differing miRNA genes or precursors are processed into identical mature miRNAs, the genes/precursors can be delineated by a numbered suffix. For example, mir-121-1 and mir-121-2 can refer to distinct genes or precursors that are processed into miR-121. Lettered suffixes are used to indicate closely related mature sequences. For example, mir-121a and mir-121b can be processed to closely related miRNAs miR-121a and miR-121b, respectively. In the context of the invention, any microRNA (miRNA or miR) designated herein with the prefix mir-* or miR-* is understood to encompass both the precursor and/or mature species, unless otherwise explicitly stated otherwise.


Sometimes it is observed that two mature miRNA sequences originate from the same precursor. When one of the sequences is more abundant that the other, a “*” suffix can be used to designate the less common variant. For example, miR-121 would be the predominant product whereas miR-121* is the less common variant found on the opposite arm of the precursor. If the predominant variant is not identified, the miRs can be distinguished by the suffix “5p” for the variant from the 5′ arm of the precursor and the suffix “3p” for the variant from the 3′ arm. For example, miR-121-5p originates from the 5′ arm of the precursor whereas miR-121-3p originates from the 3′ arm. Less commonly, the 5p and 3p variants are referred to as the sense (“s”) and anti-sense (“as”) forms, respectively. For example, miR-121-5p may be referred to as miR-121-s whereas miR-121-3p may be referred to as miR-121-as.


The above naming conventions have evolved over time and are general guidelines rather than absolute rules. For example, the let- and lin-families of miRNAs continue to be referred to by these monikers. The mir/miR convention for precursor/mature forms is also a guideline and context should be taken into account to determine which form is referred to. Further details of miR naming can be found at www.mirbase.org or Ambros et al., A uniform system for microRNA annotation, RNA 9:277-279 (2003).


Plant miRNAs follow a different naming convention as described in Meyers et al., Plant Cell. 2008 20(12):3186-3190.


A number of miRNAs are involved in gene regulation, and miRNAs are part of a growing class of non-coding RNAs that is now recognized as a major tier of gene control. In some cases, miRNAs can interrupt translation by binding to regulatory sites embedded in the 3′-UTRs of their target mRNAs, leading to the repression of translation. Target recognition involves complementary base pairing of the target site with the miRNA's seed region (positions 2-8 at the miRNA's 5′ end), although the exact extent of seed complementarity is not precisely determined and can be modified by 3′ pairing. In other cases, miRNAs function like small interfering RNAs (siRNA) and bind to perfectly complementary mRNA sequences to destroy the target transcript.


Characterization of a number of miRNAs indicates that they influence a variety of processes, including early development, cell proliferation and cell death, apoptosis and fat metabolism. For example, some miRNAs, such as lin-4, let-7, mir-14, mir-23, and bantam, have been shown to play critical roles in cell differentiation and tissue development. Others are believed to have similarly important roles because of their differential spatial and temporal expression patterns.


The miRNA database available at miRBase (www.mirbase.org) comprises a searchable database of published miRNA sequences and annotation. Further information about miRBase can be found in the following articles, each of which is incorporated by reference in its entirety herein: Griffiths-Jones et al., miRBase: tools for microRNA genomics. NAR 2008 36 (Database Issue):D154-D158; Griffiths-Jones et al., miRBase: microRNA sequences, targets and gene nomenclature. NAR 2006 34 (Database Issue):D140-D144; and Griffiths-Jones, S. The microRNA Registry. NAR 2004 32 (Database Issue):D109-D111. Representative miRNAs contained in Release 16 of miRBase, made available September 2010.


As described herein, microRNAs are known to be involved in cancer and other diseases and can be assessed in order to characterize a phenotype in a sample. See, e.g., Ferracin et al., Micromarkers: miRNAs in cancer diagnosis and prognosis, Exp Rev Mol Diag, April 2010, Vol. 10, No. 3, Pages 297-308; Fabbri, miRNAs as molecular biomarkers of cancer, Exp Rev Mol Diag, May 2010, Vol. 10, No. 4, Pages 435-444.


In an embodiment, molecular profiling of the invention comprises analysis of microRNA.


Techniques to isolate and characterize vesicles and miRs are known to those of skill in the art. In addition to the methodology presented herein, additional methods can be found in U.S. Pat. Nos. 7,888,035, entitled “METHODS FOR ASSESSING RNA PATTERNS” and issued Feb. 15, 2011; and U.S. Pat. No. 7,897,356, entitled “METHODS AND SYSTEMS OF USING EXOSOMES FOR DETERMINING PHENOTYPES” and issued Mar. 1, 2011; and International Patent Publication Nos. WO/2011/066589, entitled “METHODS AND SYSTEMS FOR ISOLATING, STORING, AND ANALYZING VESICLES” and filed Nov. 30, 2010; WO/2011/088226, entitled “DETECTION OF GASTROINTESTINAL DISORDERS” and filed Jan. 13, 2011; WO/2011/109440, entitled “BIOMARKERS FOR THERANOSTICS” and filed Mar. 1, 2011; and WO/2011/127219, entitled “CIRCULATING BIOMARKERS FOR DISEASE” and filed Apr. 6, 2011, each of which applications are incorporated by reference herein in their entirety.


Circulating Biomarkers

Circulating biomarkers include biomarkers that are detectable in body fluids, such as blood, plasma, serum. Examples of circulating cancer biomarkers include cardiac troponin T (cTnT), prostate specific antigen (PSA) for prostate cancer and CA125 for ovarian cancer. Circulating biomarkers according to the invention include any appropriate biomarker that can be detected in bodily fluid, including without limitation protein, nucleic acids, e.g., DNA, mRNA and microRNA, lipids, carbohydrates and metabolites. Circulating biomarkers can include biomarkers that are not associated with cells, such as biomarkers that are membrane associated, embedded in membrane fragments, part of a biological complex, or free in solution. In one embodiment, circulating biomarkers are biomarkers that are associated with one or more vesicles present in the biological fluid of a subject.


Circulating biomarkers have been identified for use in characterization of various phenotypes, such as detection of a cancer. See, e.g., Ahmed N, et al., Proteomic-based identification of haptoglobin-1 precursor as a novel circulating biomarker of ovarian cancer. Br. J. Cancer 2004; Mathelin et al., Circulating proteinic biomarkers and breast cancer, Gynecol Obstet Fertil. 2006 July-August; 34(7-8):638-46. Epub 2006 Jul. 28; Ye et al., Recent technical strategies to identify diagnostic biomarkers for ovarian cancer. Expert Rev Proteomics. 2007 February; 4(1):121-31; Carney, Circulating oncoproteins HER2/neu, EGFR and CAIX (MN) as novel cancer biomarkers. Expert Rev Mol Diagn. 2007 May; 7(3):309-19; Gagnon, Discovery and application of protein biomarkers for ovarian cancer, Curr Opin Obstet Gynecol. 2008 February; 20(1):9-13; Pasterkamp et al., Immune regulatory cells: circulating biomarker factories in cardiovascular disease. Clin Sci (Lond). 2008 August; 115(4):129-31; Fabbri, miRNAs as molecular biomarkers of cancer, Exp Rev Mol Diag, May 2010, Vol. 10, No. 4, Pages 435-444; PCT Patent Publication WO/2007/088537; U.S. Pat. Nos. 7,745,150 and 7,655,479; U.S. Patent Publications 20110008808, 20100330683, 20100248290, 20100222230, 20100203566, 20100173788, 20090291932, 20090239246, 20090226937, 20090111121, 20090004687, 20080261258, 20080213907, 20060003465, 20050124071, and 20040096915, each of which publication is incorporated herein by reference in its entirety. In an embodiment, molecular profiling of the invention comprises analysis of circulating biomarkers.


Gene Expression Profiling

The methods and systems of the invention comprise expression profiling, which includes assessing differential expression of one or more target genes disclosed herein. Differential expression can include overexpression and/or underexpression of a biological product, e.g., a gene, mRNA or protein, compared to a control (or a reference). The control can include similar cells to the sample but without the disease (e.g., expression profiles obtained from samples from healthy individuals). A control can be a previously determined level that is indicative of a drug target efficacy associated with the particular disease and the particular drug target. The control can be derived from the same patient, e.g., a normal adjacent portion of the same organ as the diseased cells, the control can be derived from healthy tissues from other patients, or previously determined thresholds that are indicative of a disease responding or not-responding to a particular drug target. The control can also be a control found in the same sample, e.g. a housekeeping gene or a product thereof (e.g., mRNA or protein). For example, a control nucleic acid can be one which is known not to differ depending on the cancerous or non-cancerous state of the cell. The expression level of a control nucleic acid can be used to normalize signal levels in the test and reference populations. Illustrative control genes include, but are not limited to, e.g., (3-actin, glyceraldehyde 3-phosphate dehydrogenase and ribosomal protein P1. Multiple controls or types of controls can be used. The source of differential expression can vary. For example, a gene copy number may be increased in a cell, thereby resulting in increased expression of the gene. Alternately, transcription of the gene may be modified, e.g., by chromatin remodeling, differential methylation, differential expression or activity of transcription factors, etc. Translation may also be modified, e.g., by differential expression of factors that degrade mRNA, translate mRNA, or silence translation, e.g., microRNAs or siRNAs. In some embodiments, differential expression comprises differential activity. For example, a protein may carry a mutation that increases the activity of the protein, such as constitutive activation, thereby contributing to a diseased state. Molecular profiling that reveals changes in activity can be used to guide treatment selection.


Methods of gene expression profiling include methods based on hybridization analysis of polynucleotides, and methods based on sequencing of polynucleotides. Commonly used methods known in the art for the quantification of mRNA expression in a sample include northern blotting and in situ hybridization (Parker & Barnes (1999) Methods in Molecular Biology 106:247-283); RNAse protection assays (Hod (1992) Biotechniques 13:852-854); and reverse transcription polymerase chain reaction (RT-PCR) (Weis et al. (1992) Trends in Genetics 8:263-264). Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Representative methods for sequencing-based gene expression analysis include Serial Analysis of Gene Expression (SAGE), gene expression analysis by massively parallel signature sequencing (MPSS) and/or next generation sequencing.


RT-PCR


Reverse transcription polymerase chain reaction (RT-PCR) is a variant of polymerase chain reaction (PCR). According to this technique, a RNA strand is reverse transcribed into its DNA complement (i.e., complementary DNA, or cDNA) using the enzyme reverse transcriptase, and the resulting cDNA is amplified using PCR. Real-time polymerase chain reaction is another PCR variant, which is also referred to as quantitative PCR, Q-PCR, qRT-PCR, or sometimes as RT-PCR. Either the reverse transcription PCR method or the real-time PCR method can be used for molecular profiling according to the invention, and RT-PCR can refer to either unless otherwise specified or as understood by one of skill in the art.


RT-PCR can be used to determine RNA levels, e.g., mRNA or miRNA levels, of the biomarkers of the invention. RT-PCR can be used to compare such RNA levels of the biomarkers of the invention in different sample populations, in normal and tumor tissues, with or without drug treatment, to characterize patterns of gene expression, to discriminate between closely related RNAs, and to analyze RNA structure.


The first step is the isolation of RNA, e.g., mRNA, from a sample. The starting material can be total RNA isolated from human tumors or tumor cell lines, and corresponding normal tissues or cell lines, respectively. Thus RNA can be isolated from a sample, e.g., tumor cells or tumor cell lines, and compared with pooled DNA from healthy donors. If the source of mRNA is a primary tumor, mRNA can be extracted, for example, from frozen or archived paraffin-embedded and fixed (e.g. formalin-fixed) tissue samples.


General methods for mRNA extraction are well known in the art and are disclosed in standard textbooks of molecular biology, including Ausubel et al. (1997) Current Protocols of Molecular Biology, John Wiley and Sons. Methods for RNA extraction from paraffin embedded tissues are disclosed, for example, in Rupp & Locker (1987) Lab Invest. 56:A67, and De Andres et al., BioTechniques 18:42044 (1995). In particular, RNA isolation can be performed using purification kit, buffer set and protease from commercial manufacturers, such as Qiagen, according to the manufacturer's instructions (QIAGEN Inc., Valencia, Calif.). For example, total RNA from cells in culture can be isolated using Qiagen RNeasy mini-columns. Numerous RNA isolation kits are commercially available and can be used in the methods of the invention.


In the alternative, the first step is the isolation of miRNA from a target sample. The starting material is typically total RNA isolated from human tumors or tumor cell lines, and corresponding normal tissues or cell lines, respectively. Thus RNA can be isolated from a variety of primary tumors or tumor cell lines, with pooled DNA from healthy donors. If the source of miRNA is a primary tumor, miRNA can be extracted, for example, from frozen or archived paraffin-embedded and fixed (e.g. formalin-fixed) tissue samples.


General methods for miRNA extraction are well known in the art and are disclosed in standard textbooks of molecular biology, including Ausubel et al. (1997) Current Protocols of Molecular Biology, John Wiley and Sons. Methods for RNA extraction from paraffin embedded tissues are disclosed, for example, in Rupp & Locker (1987) Lab Invest. 56:A67, and De Andres et al., BioTechniques 18:42044 (1995). In particular, RNA isolation can be performed using purification kit, buffer set and protease from commercial manufacturers, such as Qiagen, according to the manufacturer's instructions. For example, total RNA from cells in culture can be isolated using Qiagen RNeasy mini-columns. Numerous miRNA isolation kits are commercially available and can be used in the methods of the invention.


Whether the RNA comprises mRNA, miRNA or other types of RNA, gene expression profiling by RT-PCR can include reverse transcription of the RNA template into cDNA, followed by amplification in a PCR reaction. Commonly used reverse transcriptases include, but are not limited to, avilo myeloblastosis virus reverse transcriptase (AMV-RT) and Moloney murine leukemia virus reverse transcriptase (MMLV-RT). The reverse transcription step is typically primed using specific primers, random hexamers, or oligo-dT primers, depending on the circumstances and the goal of expression profiling. For example, extracted RNA can be reverse-transcribed using a GeneAmp RNA PCR kit (Perkin Elmer, Calif., USA), following the manufacturer's instructions. The derived cDNA can then be used as a template in the subsequent PCR reaction.


Although the PCR step can use a variety of thermostable DNA-dependent DNA polymerases, it typically employs the Taq DNA polymerase, which has a 5′-3′ nuclease activity but lacks a 3′-5′ proofreading endonuclease activity. TaqMan PCR typically uses the 5′-nuclease activity of Taq or Tth polymerase to hydrolyze a hybridization probe bound to its target amplicon, but any enzyme with equivalent 5′ nuclease activity can be used. Two oligonucleotide primers are used to generate an amplicon typical of a PCR reaction. A third oligonucleotide, or probe, is designed to detect nucleotide sequence located between the two PCR primers. The probe is non-extendible by Taq DNA polymerase enzyme, and is labeled with a reporter fluorescent dye and a quencher fluorescent dye. Any laser-induced emission from the reporter dye is quenched by the quenching dye when the two dyes are located close together as they are on the probe. During the amplification reaction, the Taq DNA polymerase enzyme cleaves the probe in a template-dependent manner. The resultant probe fragments disassociate in solution, and signal from the released reporter dye is free from the quenching effect of the second fluorophore. One molecule of reporter dye is liberated for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data.


TaqMan™ RT-PCR can be performed using commercially available equipment, such as, for example, ABI PRISM 7700™ Sequence Detection System™ (Perkin-Elmer-Applied Biosystems, Foster City, Calif., USA), or LightCycler (Roche Molecular Biochemicals, Mannheim, Germany). In one specific embodiment, the 5′ nuclease procedure is run on a real-time quantitative PCR device such as the ABI PRISM 7700 Sequence Detection System. The system consists of a thermocycler, laser, charge-coupled device (CCD), camera and computer. The system amplifies samples in a 96-well format on a thermocycler. During amplification, laser-induced fluorescent signal is collected in real-time through fiber optic cables for all 96 wells, and detected at the CCD. The system includes software for running the instrument and for analyzing the data.


TaqMan data are initially expressed as Ct, or the threshold cycle. As discussed above, fluorescence values are recorded during every cycle and represent the amount of product amplified to that point in the amplification reaction. The point when the fluorescent signal is first recorded as statistically significant is the threshold cycle (Ct).


To minimize errors and the effect of sample-to-sample variation, RT-PCR is usually performed using an internal standard. The ideal internal standard is expressed at a constant level among different tissues, and is unaffected by the experimental treatment. RNAs most frequently used to normalize patterns of gene expression are mRNAs for the housekeeping genes glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and β-actin.


Real time quantitative PCR (also quantitative real time polymerase chain reaction, QRT-PCR or Q-PCR) is a more recent variation of the RT-PCR technique. Q-PCR can measure PCR product accumulation through a dual-labeled fluorigenic probe (i.e., TaqMan probe). Real time PCR is compatible both with quantitative competitive PCR, where internal competitor for each target sequence is used for normalization, and with quantitative comparative PCR using a normalization gene contained within the sample, or a housekeeping gene for RT-PCR. See, e.g. Held et al. (1996) Genome Research 6:986-994.


Protein-based detection techniques are also useful for molecular profiling, especially when the nucleotide variant causes amino acid substitutions or deletions or insertions or frame shift that affect the protein primary, secondary or tertiary structure. To detect the amino acid variations, protein sequencing techniques may be used. For example, a protein or fragment thereof corresponding to a gene can be synthesized by recombinant expression using a DNA fragment isolated from an individual to be tested. Preferably, a cDNA fragment of no more than 100 to 150 base pairs encompassing the polymorphic locus to be determined is used. The amino acid sequence of the peptide can then be determined by conventional protein sequencing methods. Alternatively, the HPLC-microscopy tandem mass spectrometry technique can be used for determining the amino acid sequence variations. In this technique, proteolytic digestion is performed on a protein, and the resulting peptide mixture is separated by reversed-phase chromatographic separation. Tandem mass spectrometry is then performed and the data collected is analyzed. See Gatlin et al., Anal. Chem., 72:757-763 (2000).


Microarray


The biomarkers of the invention can also be identified, confirmed, and/or measured using the microarray technique. Thus, the expression profile biomarkers can be measured in cancer samples using microarray technology. In this method, polynucleotide sequences of interest are plated, or arrayed, on a microchip substrate. The arrayed sequences are then hybridized with specific DNA probes from cells or tissues of interest. The source of mRNA can be total RNA isolated from a sample, e.g., human tumors or tumor cell lines and corresponding normal tissues or cell lines. Thus RNA can be isolated from a variety of primary tumors or tumor cell lines. If the source of mRNA is a primary tumor, mRNA can be extracted, for example, from frozen or archived paraffin-embedded and fixed (e.g. formalin-fixed) tissue samples, which are routinely prepared and preserved in everyday clinical practice.


The expression profile of biomarkers can be measured in either fresh or paraffin-embedded tumor tissue, or body fluids using microarray technology. In this method, polynucleotide sequences of interest are plated, or arrayed, on a microchip substrate. The arrayed sequences are then hybridized with specific DNA probes from cells or tissues of interest. As with the RT-PCR method, the source of miRNA typically is total RNA isolated from human tumors or tumor cell lines, including body fluids, such as serum, urine, tears, and exosomes and corresponding normal tissues or cell lines. Thus RNA can be isolated from a variety of sources. If the source of miRNA is a primary tumor, miRNA can be extracted, for example, from frozen tissue samples, which are routinely prepared and preserved in everyday clinical practice.


Also known as biochip, DNA chip, or gene array, cDNA microarray technology allows for identification of gene expression levels in a biologic sample. cDNAs or oligonucleotides, each representing a given gene, are immobilized on a substrate, e.g., a small chip, bead or nylon membrane, tagged, and serve as probes that will indicate whether they are expressed in biologic samples of interest. The simultaneous expression of thousands of genes can be monitored simultaneously.


In a specific embodiment of the microarray technique, PCR amplified inserts of eDNA clones are applied to a substrate in a dense array. In one aspect, at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000 or at least 50,000 nucleotide sequences are applied to the substrate. Each sequence can correspond to a different gene, or multiple sequences can be arrayed per gene. The microarrayed genes, immobilized on the microchip, are suitable for hybridization under stringent conditions. Fluorescently labeled cDNA probes may be generated through incorporation of fluorescent nucleotides by reverse transcription of RNA extracted from tissues of interest. Labeled cDNA probes applied to the chip hybridize with specificity to each spot of DNA on the array. After stringent washing to remove non-specifically bound probes, the chip is scanned by confocal laser microscopy or by another detection method, such as a CCD camera. Quantitation of hybridization of each arrayed element allows for assessment of corresponding mRNA abundance. With dual color fluorescence, separately labeled cDNA probes generated from two sources of RNA are hybridized pairwise to the array. The relative abundance of the transcripts from the two sources corresponding to each specified gene is thus determined simultaneously. The miniaturized scale of the hybridization affords a convenient and rapid evaluation of the expression pattern for large numbers of genes. Such methods have been shown to have the sensitivity required to detect rare transcripts, which are expressed at a few copies per cell, and to reproducibly detect at least approximately two-fold differences in the expression levels (Schena et al. (1996) Proc. Natl. Acad. Sci. USA 93(2):106-149). Microarray analysis can be performed by commercially available equipment following manufacturer's protocols, including without limitation the Affymetrix GeneChip technology (Affymetrix, Santa Clara, Calif.), Agilent (Agilent Technologies, Inc., Santa Clara, Calif.), or Illumina (Illumina, Inc., San Diego, Calif.) microarray technology.


The development of microarray methods for large-scale analysis of gene expression makes it possible to search systematically for molecular markers of cancer classification and outcome prediction in a variety of tumor types.


In some embodiments, the Agilent Whole Human Genome Microarray Kit (Agilent Technologies, Inc., Santa Clara, Calif.). The system can analyze more than 41,000 unique human genes and transcripts represented, all with public domain annotations. The system is used according to the manufacturer's instructions.


In some embodiments, the Illumina Whole Genome DASL assay (Illumina Inc., San Diego, Calif.) is used. The system offers a method to simultaneously profile over 24,000 transcripts from minimal RNA input, from both fresh frozen (FF) and formalin-fixed paraffin embedded (FFPE) tissue sources, in a high throughput fashion.


Microarray expression analysis comprises identifying whether a gene or gene product is up-regulated or down-regulated relative to a reference. The identification can be performed using a statistical test to determine statistical significance of any differential expression observed. In some embodiments, statistical significance is determined using a parametric statistical test. The parametric statistical test can comprise, for example, a fractional factorial design, analysis of variance (ANOVA), a t-test, least squares, a Pearson correlation, simple linear regression, nonlinear regression, multiple linear regression, or multiple nonlinear regression. Alternatively, the parametric statistical test can comprise a one-way analysis of variance, two-way analysis of variance, or repeated measures analysis of variance. In other embodiments, statistical significance is determined using a nonparametric statistical test. Examples include, but are not limited to, a Wilcoxon signed-rank test, a Mann-Whitney test, a Kruskal-Wallis test, a Friedman test, a Spearman ranked order correlation coefficient, a Kendall Tau analysis, and a nonparametric regression test. In some embodiments, statistical significance is determined at a p-value of less than about 0.05, 0.01, 0.005, 0.001, 0.0005, or 0.0001. Although the microarray systems used in the methods of the invention may assay thousands of transcripts, data analysis need only be performed on the transcripts of interest, thereby reducing the problem of multiple comparisons inherent in performing multiple statistical tests. The p-values can also be corrected for multiple comparisons, e.g., using a Bonferroni correction, a modification thereof, or other technique known to those in the art, e.g., the Hochberg correction, Holm-Bonferroni correction, Sidik correction, or Dunnett's correction. The degree of differential expression can also be taken into account. For example, a gene can be considered as differentially expressed when the fold-change in expression compared to control level is at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.5, 2.7, 3.0, 4, 5, 6, 7, 8, 9 or 10-fold different in the sample versus the control. The differential expression takes into account both overexpression and underexpression. A gene or gene product can be considered up or down-regulated if the differential expression meets a statistical threshold, a fold-change threshold, or both. For example, the criteria for identifying differential expression can comprise both a p-value of 0.001 and fold change of at least 1.5-fold (up or down). One of skill will understand that such statistical and threshold measures can be adapted to determine differential expression by any molecular profiling technique disclosed herein.


Various methods of the invention make use of many types of microarrays that detect the presence and potentially the amount of biological entities in a sample. Arrays typically contain addressable moieties that can detect the presence of the entity in the sample, e.g., via a binding event. Microarrays include without limitation DNA microarrays, such as cDNA microarrays, oligonucleotide microarrays and SNP microarrays, microRNA arrays, protein microarrays, antibody microarrays, tissue microarrays, cellular microarrays (also called transfection microarrays), chemical compound microarrays, and carbohydrate arrays (glycoarrays). DNA arrays typically comprise addressable nucleotide sequences that can bind to sequences present in a sample. MicroRNA arrays, e.g., the MMChips array from the University of Louisville or commercial systems from Agilent, can be used to detect microRNAs. Protein microarrays can be used to identify protein-protein interactions, including without limitation identifying substrates of protein kinases, transcription factor protein-activation, or to identify the targets of biologically active small molecules. Protein arrays may comprise an array of different protein molecules, commonly antibodies, or nucleotide sequences that bind to proteins of interest. Antibody microarrays comprise antibodies spotted onto the protein chip that are used as capture molecules to detect proteins or other biological materials from a sample, e.g., from cell or tissue lysate solutions. For example, antibody arrays can be used to detect biomarkers from bodily fluids, e.g., serum or urine, for diagnostic applications. Tissue microarrays comprise separate tissue cores assembled in array fashion to allow multiplex histological analysis. Cellular microarrays, also called transfection microarrays, comprise various capture agents, such as antibodies, proteins, or lipids, which can interact with cells to facilitate their capture on addressable locations. Chemical compound microarrays comprise arrays of chemical compounds and can be used to detect protein or other biological materials that bind the compounds. Carbohydrate arrays (glycoarrays) comprise arrays of carbohydrates and can detect, e.g., protein that bind sugar moieties. One of skill will appreciate that similar technologies or improvements can be used according to the methods of the invention.


Certain embodiments of the current methods comprise a multi-well reaction vessel, including without limitation, a multi-well plate or a multi-chambered microfluidic device, in which a multiplicity of amplification reactions and, in some embodiments, detection are performed, typically in parallel. In certain embodiments, one or more multiplex reactions for generating amplicons are performed in the same reaction vessel, including without limitation, a multi-well plate, such as a 96-well, a 384-well, a 1536-well plate, and so forth; or a microfluidic device, for example but not limited to, a TaqMan™ Low Density Array (Applied Biosystems, Foster City, Calif.). In some embodiments, a massively parallel amplifying step comprises a multi-well reaction vessel, including a plate comprising multiple reaction wells, for example but not limited to, a 24-well plate, a 96-well plate, a 384-well plate, or a 1536-well plate; or a multi-chamber microfluidics device, for example but not limited to a low density array wherein each chamber or well comprises an appropriate primer(s), primer set(s), and/or reporter probe(s), as appropriate. Typically such amplification steps occur in a series of parallel single-plex, two-plex, three-plex, four-plex, five-plex, or six-plex reactions, although higher levels of parallel multiplexing are also within the intended scope of the current teachings. These methods can comprise PCR methodology, such as RT-PCR, in each of the wells or chambers to amplify and/or detect nucleic acid molecules of interest.


Low density arrays can include arrays that detect 10 s or 100 s of molecules as opposed to 1000 s of molecules. These arrays can be more sensitive than high density arrays. In embodiments, a low density array such as a TaqMan™ Low Density Array is used to detect one or more gene or gene product in Table 2, Table 6 or Table 25. For example, the low density array can be used to detect at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 genes or gene products in Table 2, Table 6 or Table 25.


In some embodiments, the disclosed methods comprise a microfluidics device, “lab on a chip,” or micrototal analytical system (pTAS). In some embodiments, sample preparation is performed using a microfluidics device. In some embodiments, an amplification reaction is performed using a microfluidics device. In some embodiments, a sequencing or PCR reaction is performed using a microfluidic device. In some embodiments, the nucleotide sequence of at least a part of an amplified product is obtained using a microfluidics device. In some embodiments, detecting comprises a microfluidic device, including without limitation, a low density array, such as a TaqMan™ Low Density Array. Descriptions of exemplary microfluidic devices can be found in, among other places, Published PCT Application Nos. WO/0185341 and WO 04/011666; Kartalov and Quake, Nucl. Acids Res. 32:2873-79, 2004; and Fiorini and Chiu, Bio Techniques 38:429-46, 2005.


Any appropriate microfluidic device can be used in the methods of the invention. Examples of microfluidic devices that may be used, or adapted for use with molecular profiling, include but are not limited to those described in U.S. Pat. Nos. 7,591,936, 7,581,429, 7,579,136, 7,575,722, 7,568,399, 7,552,741, 7,544,506, 7,541,578, 7,518,726, 7,488,596, 7,485,214, 7,467,928, 7,452,713, 7,452,509, 7,449,096, 7,431,887, 7,422,725, 7,422,669, 7,419,822, 7,419,639, 7,413,709, 7,411,184, 7,402,229, 7,390,463, 7,381,471, 7,357,864, 7,351,592, 7,351,380, 7,338,637, 7,329,391, 7,323,140, 7,261,824, 7,258,837, 7,253,003, 7,238,324, 7,238,255, 7,233,865, 7,229,538, 7,201,881, 7,195,986, 7,189,581, 7,189,580, 7,189,368, 7,141,978, 7,138,062, 7,135,147, 7,125,711, 7,118,910, 7,118,661, 7,640,947, 7,666,361, 7,704,735; U.S. Patent Application Publication 20060035243; and International Patent Publication WO 2010/072410; each of which patents or applications are incorporated herein by reference in their entirety. Another example for use with methods disclosed herein is described in Chen et al., “Microfluidic isolation and transcriptome analysis of serum vesicles,” Lab on a Chip, Dec. 8, 2009 DOI 10.1039/b916199f.


Gene Expression Analysis by Massively Parallel Signature Sequencing (MPSS)


This method, described by Brenner et al. (2000) Nature Biotechnology 18:630-634, is a sequencing approach that combines non-gel-based signature sequencing with in vitro cloning of millions of templates on separate microbeads. First, a microbead library of DNA templates is constructed by in vitro cloning. This is followed by the assembly of a planar array of the template-containing microbeads in a flow cell at a high density. The free ends of the cloned templates on each microbead are analyzed simultaneously, using a fluorescence-based signature sequencing method that does not require DNA fragment separation. This method has been shown to simultaneously and accurately provide, in a single operation, hundreds of thousands of gene signature sequences from a cDNA library. MPSS data has many uses. The expression levels of nearly all transcripts can be quantitatively determined; the abundance of signatures is representative of the expression level of the gene in the analyzed tissue. Quantitative methods for the analysis of tag frequencies and detection of differences among libraries have been published and incorporated into public databases for SAGE™ data and are applicable to MPSS data. The availability of complete genome sequences permits the direct comparison of signatures to genomic sequences and further extends the utility of MPSS data. Because the targets for MPSS analysis are not pre-selected (like on a microarray), MPSS data can characterize the full complexity of transcriptomes. This is analogous to sequencing millions of ESTs at once, and genomic sequence data can be used so that the source of the MPSS signature can be readily identified by computational means.


Serial Analysis of Gene Expression (SAGE)


Serial analysis of gene expression (SAGE) is a method that allows the simultaneous and quantitative analysis of a large number of gene transcripts, without the need of providing an individual hybridization probe for each transcript. First, a short sequence tag (e.g., about 10-14 bp) is generated that contains sufficient information to uniquely identify a transcript, provided that the tag is obtained from a unique position within each transcript. Then, many transcripts are linked together to form long serial molecules, that can be sequenced, revealing the identity of the multiple tags simultaneously. The expression pattern of any population of transcripts can be quantitatively evaluated by determining the abundance of individual tags, and identifying the gene corresponding to each tag. See, e.g. Velculescu et al. (1995) Science 270:484-487; and Velculescu et al. (1997) Cell 88:243-51.


DNA Copy Number Profiling

Any method capable of determining a DNA copy number profile of a particular sample can be used for molecular profiling according to the invention as long as the resolution is sufficient to identify the biomarkers of the invention. The skilled artisan is aware of and capable of using a number of different platforms for assessing whole genome copy number changes at a resolution sufficient to identify the copy number of the one or more biomarkers of the invention. Some of the platforms and techniques are described in the embodiments below.


In some embodiments, the copy number profile analysis involves amplification of whole genome DNA by a whole genome amplification method. The whole genome amplification method can use a strand displacing polymerase and random primers.


In some aspects of these embodiments, the copy number profile analysis involves hybridization of whole genome amplified DNA with a high density array. In a more specific aspect, the high density array has 5,000 or more different probes. In another specific aspect, the high density array has 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1,000,000 or more different probes. In another specific aspect, each of the different probes on the array is an oligonucleotide having from about 15 to 200 bases in length. In another specific aspect, each of the different probes on the array is an oligonucleotide having from about 15 to 200, 15 to 150, 15 to 100, 15 to 75, 15 to 60, or 20 to 55 bases in length.


In some embodiments, a microarray is employed to aid in determining the copy number profile for a sample, e.g., cells from a tumor. Microarrays typically comprise a plurality of oligomers (e.g., DNA or RNA polynucleotides or oligonucleotides, or other polymers), synthesized or deposited on a substrate (e.g., glass support) in an array pattern. The support-bound oligomers are “probes”, which function to hybridize or bind with a sample material (e.g., nucleic acids prepared or obtained from the tumor samples), in hybridization experiments. The reverse situation can also be applied: the sample can be bound to the microarray substrate and the oligomer probes are in solution for the hybridization. In use, the array surface is contacted with one or more targets under conditions that promote specific, high-affinity binding of the target to one or more of the probes. In some configurations, the sample nucleic acid is labeled with a detectable label, such as a fluorescent tag, so that the hybridized sample and probes are detectable with scanning equipment. DNA array technology offers the potential of using a multitude (e.g., hundreds of thousands) of different oligonucleotides to analyze DNA copy number profiles. In some embodiments, the substrates used for arrays are surface-derivatized glass or silica, or polymer membrane surfaces (see e.g., in Z. Guo, et al., Nucleic Acids Res, 22, 5456-65 (1994); U. Maskos, E. M. Southern, Nucleic Acids Res, 20, 1679-84 (1992), and E. M. Southern, et al., Nucleic Acids Res, 22, 1368-73 (1994), each incorporated by reference herein). Modification of surfaces of array substrates can be accomplished by many techniques. For example, siliceous or metal oxide surfaces can be derivatized with bifunctional silanes, i.e., silanes having a first functional group enabling covalent binding to the surface (e.g., Si-halogen or Si-alkoxy group, as in —SiCl3 or —Si(OCH3)3, respectively) and a second functional group that can impart the desired chemical and/or physical modifications to the surface to covalently or non-covalently attach ligands and/or the polymers or monomers for the biological probe array. Silylated derivatizations and other surface derivatizations that are known in the art (see for example U.S. Pat. No. 5,624,711 to Sundberg, U.S. Pat. No. 5,266,222 to Willis, and U.S. Pat. No. 5,137,765 to Farnsworth, each incorporated by reference herein). Other processes for preparing arrays are described in U.S. Pat. No. 6,649,348, to Bass et. al., assigned to Agilent Corp., which disclose DNA arrays created by in situ synthesis methods.


Polymer array synthesis is also described extensively in the literature including in the following: WO 00/58516, U.S. Pat. Nos. 5,143,854, 5,242,974, 5,252,743, 5,324,633, 5,384,261, 5,405,783, 5,424,186, 5,451,683, 5,482,867, 5,491,074, 5,527,681, 5,550,215, 5,571,639, 5,578,832, 5,593,839, 5,599,695, 5,624,711, 5,631,734, 5,795,716, 5,831,070, 5,837,832, 5,856,101, 5,858,659, 5,936,324, 5,968,740, 5,974,164, 5,981,185, 5,981,956, 6,025,601, 6,033,860, 6,040,193, 6,090,555, 6,136,269, 6,269,846 and 6,428,752, 5,412,087, 6,147,205, 6,262,216, 6,310,189, 5,889,165, and 5,959,098 in PCT Applications Nos. PCT/US99/00730 (International Publication No. WO 99/36760) and PCT/US01/04285 (International Publication No. WO 01/58593), which are all incorporated herein by reference in their entirety for all purposes.


Nucleic acid arrays that are useful in the present invention include, but are not limited to, those that are commercially available from Affymetrix (Santa Clara, Calif.) under the brand name GeneChip™. Example arrays are shown on the website at affymetrix.com. Another microarray supplier is Illumina, Inc., of San Diego, Calif. with example arrays shown on their website at illumina.com.


In some embodiments, the inventive methods provide for sample preparation. Depending on the microarray and experiment to be performed, sample nucleic acid can be prepared in a number of ways by methods known to the skilled artisan. In some aspects of the invention, prior to or concurrent with genotyping (analysis of copy number profiles), the sample may be amplified any number of mechanisms. The most common amplification procedure used involves PCR. See, for example, PCR Technology: Principles and Applications for DNA Amplification (Ed. H. A. Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (Eds. Innis, et al., Academic Press, San Diego, Calif., 1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods and Applications 1, 17 (1991); PCR (Eds. McPherson et al., IRL Press, Oxford); and U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159 4,965,188, and 5,333,675, and each of which is incorporated herein by reference in their entireties for all purposes. In some embodiments, the sample may be amplified on the array (e.g., U.S. Pat. No. 6,300,070 which is incorporated herein by reference)


Other suitable amplification methods include the ligase chain reaction (LCR) (for example, Wu and Wallace, Genomics 4, 560 (1989), Landegren et al., Science 241, 1077 (1988) and Barringer et al. Gene 89:117 (1990)), transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173 (1989) and WO88/10315), self-sustained sequence replication (Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990) and WO90/06995), selective amplification of target polynucleotide sequences (U.S. Pat. No. 6,410,276), consensus sequence primed polymerase chain reaction (CP-PCR) (U.S. Pat. No. 4,437,975), arbitrarily primed polymerase chain reaction (AP-PCR) (U.S. Pat. Nos. 5,413,909, 5,861,245) and nucleic acid based sequence amplification (NABSA). (See, U.S. Pat. Nos. 5,409,818, 5,554,517, and 6,063,603, each of which is incorporated herein by reference). Other amplification methods that may be used are described in, U.S. Pat. Nos. 5,242,794, 5,494,810, 4,988,617 and in U.S. Ser. No. 09/854,317, each of which is incorporated herein by reference.


Additional methods of sample preparation and techniques for reducing the complexity of a nucleic sample are described in Dong et al., Genome Research 11, 1418 (2001), in U.S. Pat. Nos. 6,361,947, 6,391,592 and U.S. Ser. Nos. 09/916,135, 09/920,491 (U.S. Patent Application Publication 20030096235), Ser. No. 09/910,292 (U.S. Patent Application Publication 20030082543), and Ser. No. 10/013,598.


Methods for conducting polynucleotide hybridization assays are well developed in the art. Hybridization assay procedures and conditions used in the methods of the invention will vary depending on the application and are selected in accordance with the general binding methods known including those referred to in: Maniatis et al. Molecular Cloning: A Laboratory Manual (2.sup.nd Ed. Cold Spring Harbor, N.Y., 1989); Berger and Kimmel Methods in Enzymology, Vol. 152, Guide to Molecular Cloning Techniques (Academic Press, Inc., San Diego, Calif., 1987); Young and Davism, P.N.A.S, 80: 1194 (1983). Methods and apparatus for carrying out repeated and controlled hybridization reactions have been described in U.S. Pat. Nos. 5,871,928, 5,874,219, 6,045,996 and 6,386,749, 6,391,623 each of which are incorporated herein by reference.


The methods of the invention may also involve signal detection of hybridization between ligands in after (and/or during) hybridization. See U.S. Pat. Nos. 5,143,854, 5,578,832; 5,631,734; 5,834,758; 5,936,324; 5,981,956; 6,025,601; 6,141,096; 6,185,030; 6,201,639; 6,218,803; and 6,225,625, in U.S. Ser. No. 10/389,194 and in PCT Application PCT/US99/06097 (published as WO99/47964), each of which also is hereby incorporated by reference in its entirety for all purposes.


Methods and apparatus for signal detection and processing of intensity data are disclosed in, for example, U.S. Pat. Nos. 5,143,854, 5,547,839, 5,578,832, 5,631,734, 5,800,992, 5,834,758; 5,856,092, 5,902,723, 5,936,324, 5,981,956, 6,025,601, 6,090,555, 6,141,096, 6,185,030, 6,201,639; 6,218,803; and 6,225,625, in U.S. Ser. Nos. 10/389,194, 60/493,495 and in PCT Application PCT/US99/06097 (published as WO99/47964), each of which also is hereby incorporated by reference in its entirety for all purposes.


Immuno-Based Assays

Protein-based detection molecular profiling techniques include immunoaffinity assays based on antibodies selectively immunoreactive with mutant gene encoded protein according to the present invention. These techniques include without limitation immunoprecipitation, Western blot analysis, molecular binding assays, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunofiltration assay (ELIFA), fluorescence activated cell sorting (FACS) and the like. For example, an optional method of detecting the expression of a biomarker in a sample comprises contacting the sample with an antibody against the biomarker, or an immunoreactive fragment of the antibody thereof, or a recombinant protein containing an antigen binding region of an antibody against the biomarker; and then detecting the binding of the biomarker in the sample. Methods for producing such antibodies are known in the art. Antibodies can be used to immunoprecipitate specific proteins from solution samples or to immunoblot proteins separated by, e.g., polyacrylamide gels. Immunocytochemical methods can also be used in detecting specific protein polymorphisms in tissues or cells. Other well-known antibody-based techniques can also be used including, e.g., ELISA, radioimmunoassay (RIA), immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA), including sandwich assays using monoclonal or polyclonal antibodies. See, e.g., U.S. Pat. Nos. 4,376,110 and 4,486,530, both of which are incorporated herein by reference.


In alternative methods, the sample may be contacted with an antibody specific for a biomarker under conditions sufficient for an antibody-biomarker complex to form, and then detecting said complex. The presence of the biomarker may be detected in a number of ways, such as by Western blotting and ELISA procedures for assaying a wide variety of tissues and samples, including plasma or serum. A wide range of immunoassay techniques using such an assay format are available, see, e.g., U.S. Pat. Nos. 4,016,043, 4,424,279 and 4,018,653. These include both single-site and two-site or “sandwich” assays of the non-competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labelled antibody to a target biomarker.


A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabelled antibody is immobilized on a solid substrate, and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen complex, a second antibody specific to the antigen, labelled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labelled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal produced by the reporter molecule. The results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of biomarker.


Variations on the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent. In a typical forward sandwich assay, a first antibody having specificity for the biomarker is either covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g. 2-40 minutes or overnight if more convenient) and under suitable conditions (e.g. from room temperature to 40° C. such as between 25° C. and 32° C. inclusive) to allow binding of any subunit present in the antibody. Following the incubation period, the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of the biomarker. The second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the molecular marker.


An alternative method involves immobilizing the target biomarkers in the sample and then exposing the immobilized target to specific antibody which may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detectable by direct labelling with the antibody. Alternatively, a second labelled antibody, specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule. By “reporter molecule”, as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. The most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules.


In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, 3-galactosidase and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change. Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labelled antibody is added to the first antibody-molecular marker complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of biomarker which was present in the sample. Alternately, fluorescent compounds, such as fluorescein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic color visually detectable with a light microscope. As in the EIA, the fluorescent labelled antibody is allowed to bind to the first antibody-molecular marker complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength, the fluorescence observed indicates the presence of the molecular marker of interest. Immunofluorescence and EIA techniques are both very well established in the art. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.


Immunohistochemistry (IHC)


IHC is a process of localizing antigens (e.g., proteins) in cells of a tissue binding antibodies specifically to antigens in the tissues. The antigen-binding antibody can be conjugated or fused to a tag that allows its detection, e.g., via visualization. In some embodiments, the tag is an enzyme that can catalyze a color-producing reaction, such as alkaline phosphatase or horseradish peroxidase. The enzyme can be fused to the antibody or non-covalently bound, e.g., using a biotin-avadin system. Alternatively, the antibody can be tagged with a fluorophore, such as fluorescein, rhodamine, DyLight Fluor or Alexa Fluor. The antigen-binding antibody can be directly tagged or it can itself be recognized by a detection antibody that carries the tag. Using IHC, one or more proteins may be detected. The expression of a gene product can be related to its staining intensity compared to control levels. In some embodiments, the gene product is considered differentially expressed if its staining varies at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.5, 2.7, 3.0, 4, 5, 6, 7, 8, 9 or 10-fold in the sample versus the control.


IHC comprises the application of antigen-antibody interactions to histochemical techniques. In an illustrative example, a tissue section is mounted on a slide and is incubated with antibodies (polyclonal or monoclonal) specific to the antigen (primary reaction). The antigen-antibody signal is then amplified using a second antibody conjugated to a complex of peroxidase antiperoxidase (PAP), avidin-biotin-peroxidase (ABC) or avidin-biotin alkaline phosphatase. In the presence of substrate and chromogen, the enzyme forms a colored deposit at the sites of antibody-antigen binding. Immunofluorescence is an alternate approach to visualize antigens. In this technique, the primary antigen-antibody signal is amplified using a second antibody conjugated to a fluorochrome. On UV light absorption, the fluorochrome emits its own light at a longer wavelength (fluorescence), thus allowing localization of antibody-antigen complexes.


Epigenetic Status

Molecular profiling methods according to the invention also comprise measuring epigenetic change, i.e., modification in a gene caused by an epigenetic mechanism, such as a change in methylation status or histone acetylation. Frequently, the epigenetic change will result in an alteration in the levels of expression of the gene which may be detected (at the RNA or protein level as appropriate) as an indication of the epigenetic change. Often the epigenetic change results in silencing or down regulation of the gene, referred to as “epigenetic silencing.” The most frequently investigated epigenetic change in the methods of the invention involves determining the DNA methylation status of a gene, where an increased level of methylation is typically associated with the relevant cancer (since it may cause down regulation of gene expression). Aberrant methylation, which may be referred to as hypermethylation, of the gene or genes can be detected. Typically, the methylation status is determined in suitable CpG islands which are often found in the promoter region of the gene(s). The term “methylation,” “methylation state” or “methylation status” may refers to the presence or absence of 5-methylcytosine at one or a plurality of CpG dinucleotides within a DNA sequence. CpG dinucleotides are typically concentrated in the promoter regions and exons of human genes.


Diminished gene expression can be assessed in terms of DNA methylation status or in terms of expression levels as determined by the methylation status of the gene. One method to detect epigenetic silencing is to determine that a gene which is expressed in normal cells is less expressed or not expressed in tumor cells. Accordingly, the invention provides for a method of molecular profiling comprising detecting epigenetic silencing.


Various assay procedures to directly detect methylation are known in the art, and can be used in conjunction with the present invention. These assays rely onto two distinct approaches: bisulphite conversion based approaches and non-bisulphite based approaches. Non-bisulphite based methods for analysis of DNA methylation rely on the inability of methylation-sensitive enzymes to cleave methylation cytosines in their restriction. The bisulphite conversion relies on treatment of DNA samples with sodium bisulphite which converts unmethylated cytosine to uracil, while methylated cytosines are maintained (Furuichi Y, Wataya Y, Hayatsu H, Ukita T. Biochem Biophys Res Commun. 1970 December 9; 41(5): 1185-91). This conversion results in a change in the sequence of the original DNA. Methods to detect such changes include MS AP-PCR (Methylation-Sensitive Arbitrarily-Primed Polymerase Chain Reaction), a technology that allows for a global scan of the genome using CG-rich primers to focus on the regions most likely to contain CpG dinucleotides, and described by Gonzalgo et al., Cancer Research 57:594-599, 1997; MethyLight™, which refers to the art-recognized fluorescence-based real-time PCR technique described by Eads et al., Cancer Res. 59:2302-2306, 1999; the HeavyMethyl™assay, in the embodiment thereof implemented herein, is an assay, wherein methylation specific blocking probes (also referred to herein as blockers) covering CpG positions between, or covered by the amplification primers enable methylation-specific selective amplification of a nucleic acid sample; HeavyMethyl™MethyLight™ is a variation of the MethyLight™ assay wherein the MethyLight™ assay is combined with methylation specific blocking probes covering CpG positions between the amplification primers; Ms-SNuPE (Methylation-sensitive Single Nucleotide Primer Extension) is an assay described by Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531, 1997; MSP (Methylation-specific PCR) is a methylation assay described by Herman et al. Proc. Natl. Acad. Sci. USA 93:9821-9826, 1996, and by U.S. Pat. No. 5,786,146; COBRA (Combined Bisulfite Restriction Analysis) is a methylation assay described by Xiong & Laird, Nucleic Acids Res. 25:2532-2534, 1997; MCA (Methylated CpG Island Amplification) is a methylation assay described by Toyota et al., Cancer Res. 59:2307-12, 1999, and in WO 00/26401A1.


Other techniques for DNA methylation analysis include sequencing, methylation-specific PCR (MS-PCR), melting curve methylation-specific PCR (McMS-PCR), MLPA with or without bisulfite treatment, QAMA, MSRE-PCR, MethyLight, ConLight-MSP, bisulfite conversion-specific methylation-specific PCR (BS-MSP), COBRA (which relies upon use of restriction enzymes to reveal methylation dependent sequence differences in PCR products of sodium bisulfite-treated DNA), methylation-sensitive single-nucleotide primer extension conformation (MS-SNuPE), methylation-sensitive single-strand conformation analysis (MS-SSCA), Melting curve combined bisulfite restriction analysis (McCOBRA), PyroMethA, HeavyMethyl, MALDI-TOF, MassARRAY, Quantitative analysis of methylated alleles (QAMA), enzymatic regional methylation assay (ERMA), QBSUPT, MethylQuant, Quantitative PCR sequencing and oligonucleotide-based microarray systems, Pyrosequencing, Meth-DOP-PCR. A review of some useful techniques is provided in Nucleic acids research, 1998, Vol. 26, No. 10, 2255-2264; Nature Reviews, 2003, Vol. 3, 253-266; Oral Oncology, 2006, Vol. 42, 5-13, which references are incorporated herein in their entirety. Any of these techniques may be used in accordance with the present invention, as appropriate. Other techniques are described in U.S. Patent Publications 20100144836; and 20100184027, which applications are incorporated herein by reference in their entirety.


Through the activity of various acetylases and deacetylylases the DNA binding function of histone proteins is tightly regulated. Furthermore, histone acetylation and histone deactelyation have been linked with malignant progression. See Nature, 429: 457-63, 2004. Methods to analyze histone acetylation are described in U.S. Patent Publications 20100144543 and 20100151468, which applications are incorporated herein by reference in their entirety.


Sequence Analysis

Molecular profiling according to the present invention comprises methods for genotyping one or more biomarkers by determining whether an individual has one or more nucleotide variants (or amino acid variants) in one or more of the genes or gene products. Genotyping one or more genes according to the methods of the invention in some embodiments, can provide more evidence for selecting a treatment.


The biomarkers of the invention can be analyzed by any method useful for determining alterations in nucleic acids or the proteins they encode. According to one embodiment, the ordinary skilled artisan can analyze the one or more genes for mutations including deletion mutants, insertion mutants, frame shift mutants, nonsense mutants, missense mutant, and splice mutants.


Nucleic acid used for analysis of the one or more genes can be isolated from cells in the sample according to standard methodologies (Sambrook et al., 1989). The nucleic acid, for example, may be genomic DNA or fractionated or whole cell RNA, or miRNA acquired from exosomes or cell surfaces. Where RNA is used, it may be desired to convert the RNA to a complementary DNA. In one embodiment, the RNA is whole cell RNA; in another, it is poly-A RNA; in another, it is exosomal RNA. Normally, the nucleic acid is amplified. Depending on the format of the assay for analyzing the one or more genes, the specific nucleic acid of interest is identified in the sample directly using amplification or with a second, known nucleic acid following amplification. Next, the identified product is detected. In certain applications, the detection may be performed by visual means (e.g., ethidium bromide staining of a gel). Alternatively, the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (Affymax Technology; Bellus, 1994).


Various types of defects are known to occur in the biomarkers of the invention. Alterations include without limitation deletions, insertions, point mutations, and duplications. Point mutations can be silent or can result in stop codons, frame shift mutations or amino acid substitutions. Mutations in and outside the coding region of the one or more genes may occur and can be analyzed according to the methods of the invention. The target site of a nucleic acid of interest can include the region wherein the sequence varies. Examples include, but are not limited to, polymorphisms which exist in different forms such as single nucleotide variations, nucleotide repeats, multibase deletion (more than one nucleotide deleted from the consensus sequence), multibase insertion (more than one nucleotide inserted from the consensus sequence), microsatellite repeats (small numbers of nucleotide repeats with a typical 5-1000 repeat units), di-nucleotide repeats, tri-nucleotide repeats, sequence rearrangements (including translocation and duplication), chimeric sequence (two sequences from different gene origins are fused together), and the like. Among sequence polymorphisms, the most frequent polymorphisms in the human genome are single-base variations, also called single-nucleotide polymorphisms (SNPs). SNPs are abundant, stable and widely distributed across the genome.


Molecular profiling includes methods for haplotyping one or more genes. The haplotype is a set of genetic determinants located on a single chromosome and it typically contains a particular combination of alleles (all the alternative sequences of a gene) in a region of a chromosome. In other words, the haplotype is phased sequence information on individual chromosomes. Very often, phased SNPs on a chromosome define a haplotype. A combination ofhaplotypes on chromosomes can determine a genetic profile of a cell. It is the haplotype that determines a linkage between a specific genetic marker and a disease mutation. Haplotyping can be done by any methods known in the art. Common methods of scoring SNPs include hybridization microarray or direct gel sequencing, reviewed in Landgren et al., Genome Research, 8:769-776, 1998. For example, only one copy of one or more genes can be isolated from an individual and the nucleotide at each of the variant positions is determined. Alternatively, an allele specific PCR or a similar method can be used to amplify only one copy of the one or more genes in an individual, and the SNPs at the variant positions of the present invention are determined. The Clark method known in the art can also be employed for haplotyping. A high throughput molecular haplotyping method is also disclosed in Tost et al., Nucleic Acids Res., 30(19):e96 (2002), which is incorporated herein by reference.


Thus, additional variant(s) that are in linkage disequilibrium with the variants and/or haplotypes of the present invention can be identified by a haplotyping method known in the art, as will be apparent to a skilled artisan in the field of genetics and haplotyping. The additional variants that are in linkage disequilibrium with a variant or haplotype of the present invention can also be useful in the various applications as described below.


For purposes of genotyping and haplotyping, both genomic DNA and mRNA/cDNA can be used, and both are herein referred to generically as “gene.”


Numerous techniques for detecting nucleotide variants are known in the art and can all be used for the method of this invention. The techniques can be protein-based or nucleic acid-based. In either case, the techniques used must be sufficiently sensitive so as to accurately detect the small nucleotide or amino acid variations. Very often, a probe is used which is labeled with a detectable marker. Unless otherwise specified in a particular technique described below, any suitable marker known in the art can be used, including but not limited to, radioactive isotopes, fluorescent compounds, biotin which is detectable using streptavidin, enzymes (e.g., alkaline phosphatase), substrates of an enzyme, ligands and antibodies, etc. See Jablonski et al., Nucleic Acids Res., 14:6115-6128 (1986); Nguyen et al., Biotechniques, 13:116-123 (1992); Rigby et al., J. Mol. Biol., 113:237-251 (1977).


In a nucleic acid-based detection method, target DNA sample, i.e., a sample containing genomic DNA, cDNA, mRNA and/or miRNA, corresponding to the one or more genes must be obtained from the individual to be tested. Any tissue or cell sample containing the genomic DNA, miRNA, mRNA, and/or cDNA (or a portion thereof) corresponding to the one or more genes can be used. For this purpose, a tissue sample containing cell nucleus and thus genomic DNA can be obtained from the individual. Blood samples can also be useful except that only white blood cells and other lymphocytes have cell nucleus, while red blood cells are without a nucleus and contain only mRNA or miRNA. Nevertheless, miRNA and mRNA are also useful as either can be analyzed for the presence of nucleotide variants in its sequence or serve as template for cDNA synthesis. The tissue or cell samples can be analyzed directly without much processing. Alternatively, nucleic acids including the target sequence can be extracted, purified, and/or amplified before they are subject to the various detecting procedures discussed below. Other than tissue or cell samples, cDNAs or genomic DNAs from a cDNA or genomic DNA library constructed using a tissue or cell sample obtained from the individual to be tested are also useful.


To determine the presence or absence of a particular nucleotide variant, sequencing of the target genomic DNA or cDNA, particularly the region encompassing the nucleotide variant locus to be detected. Various sequencing techniques are generally known and widely used in the art including the Sanger method and Gilbert chemical method. The pyrosequencing method monitors DNA synthesis in real time using a luminometric detection system. Pyrosequencing has been shown to be effective in analyzing genetic polymorphisms such as single-nucleotide polymorphisms and can also be used in the present invention. See Nordstrom et al., Biotechnol. Appl. Biochem., 31(2):107-112 (2000); Ahmadian et al., Anal. Biochem., 280:103-110 (2000).


Nucleic acid variants can be detected by a suitable detection process. Non limiting examples of methods of detection, quantification, sequencing and the like are; mass detection of mass modified amplicons (e.g., matrix-assisted laser desorption ionization (MALDI) mass spectrometry and electrospray (ES) mass spectrometry), a primer extension method (e.g., iPLEX™; Sequenom, Inc.), microsequencing methods (e.g., a modification of primer extension methodology), ligase sequence determination methods (e.g., U.S. Pat. Nos. 5,679,524 and 5,952,174, and WO 01/27326), mismatch sequence determination methods (e.g., U.S. Pat. Nos. 5,851,770; 5,958,692; 6,110,684; and 6,183,958), direct DNA sequencing, fragment analysis (FA), restriction fragment length polymorphism (RFLP analysis), allele specific oligonucleotide (ASO) analysis, methylation-specific PCR (MSPCR), pyrosequencing analysis, acycloprime analysis, Reverse dot blot, GeneChip microarrays, Dynamic allele-specific hybridization (DASH), Peptide nucleic acid (PNA) and locked nucleic acids (LNA) probes, TaqMan, Molecular Beacons, Intercalating dye, FRET primers, AlphaScreen, SNPstream, genetic bit analysis (GBA), Multiplex minisequencing, SNaPshot, GOOD assay, Microarray miniseq, arrayed primer extension (APEX), Microarray primer extension (e.g., microarray sequence determination methods), Tag arrays, Coded microspheres, Template-directed incorporation (TDI), fluorescence polarization, Colorimetric oligonucleotide ligation assay (OLA), Sequence-coded OLA, Microarray ligation, Ligase chain reaction, Padlock probes, Invader assay, hybridization methods (e.g., hybridization using at least one probe, hybridization using at least one fluorescently labeled probe, and the like), conventional dot blot analyses, single strand conformational polymorphism analysis (SSCP, e.g., U.S. Pat. Nos. 5,891,625 and 6,013,499; Orita et al., Proc. Natl. Acad. Sci. U.S.A. 86: 27776-2770 (1989)), denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis, mismatch cleavage detection, and techniques described in Sheffield et al., Proc. Natl. Acad. Sci. USA 49: 699-706 (1991), White et al., Genomics 12: 301-306 (1992), Grompe et al., Proc. Natl. Acad. Sci. USA 86: 5855-5892 (1989), and Grompe, Nature Genetics 5: 111-117 (1993), cloning and sequencing, electrophoresis, the use of hybridization probes and quantitative real time polymerase chain reaction (QRT-PCR), digital PCR, nanopore sequencing, chips and combinations thereof. The detection and quantification of alleles or paralogs can be carried out using the “closed-tube” methods described in U.S. patent application Ser. No. 11/950,395, filed on Dec. 4, 2007. In some embodiments the amount of a nucleic acid species is determined by mass spectrometry, primer extension, sequencing (e.g., any suitable method, for example nanopore or pyrosequencing), Quantitative PCR (Q-PCR or QRT-PCR), digital PCR, combinations thereof, and the like.


The term “sequence analysis” as used herein refers to determining a nucleotide sequence, e.g., that of an amplification product. The entire sequence or a partial sequence of a polynucleotide, e.g., DNA or mRNA, can be determined, and the determined nucleotide sequence can be referred to as a “read” or “sequence read.” For example, linear amplification products may be analyzed directly without further amplification in some embodiments (e.g., by using single-molecule sequencing methodology). In certain embodiments, linear amplification products may be subject to further amplification and then analyzed (e.g., using sequencing by ligation or pyrosequencing methodology). Reads may be subject to different types of sequence analysis. Any suitable sequencing method can be used to detect, and determine the amount of, nucleotide sequence species, amplified nucleic acid species, or detectable products generated from the foregoing. Examples of certain sequencing methods are described hereafter.


A sequence analysis apparatus or sequence analysis component(s) includes an apparatus, and one or more components used in conjunction with such apparatus, that can be used by a person of ordinary skill to determine a nucleotide sequence resulting from processes described herein (e.g., linear and/or exponential amplification products). Examples of sequencing platforms include, without limitation, the 454 platform (Roche) (Margulies, M. et al. 2005 Nature 437, 376-380), Illumina Genomic Analyzer (or Solexa platform) or SOLID System (Applied Biosystems; see PCT patent application publications WO 06/084132 entitled “Reagents, Methods, and Libraries For Bead-Based Sequencing” and WO007/121,489 entitled “Reagents, Methods, and Libraries for Gel-Free Bead-Based Sequencing”), the Helicos True Single Molecule DNA sequencing technology (Harris T D et al. 2008 Science, 320, 106-109), the single molecule, real-time (SMRT™) technology of Pacific Biosciences, and nanopore sequencing (Soni G V and Meller A. 2007 Clin Chem 53: 1996-2001), Ion semiconductor sequencing (Ion Torrent Systems, Inc, San Francisco, Calif.), or DNA nanoball sequencing (Complete Genomics, Mountain View, Calif.), VisiGen Biotechnologies approach (Invitrogen) and polony sequencing. Such platforms allow sequencing of many nucleic acid molecules isolated from a specimen at high orders of multiplexing in a parallel manner (Dear Brief Funct Genomic Proteomic 2003; 1: 397-416; Haimovich, Methods, challenges, and promise of next-generation sequencing in cancer biology. Yale J Biol Med. 2011 December; 84(4):439-46). These non-Sanger-based sequencing technologies are sometimes referred to as NextGen sequencing, NGS, next-generation sequencing, next generation sequencing, and variations thereof. Typically they allow much higher throughput than the traditional Sanger approach. See Schuster, Next-generation sequencing transforms today's biology, Nature Methods 5:16-18 (2008); Metzker, Sequencing technologies—the next generation. Nat Rev Genet. 2010 January; 11(1):31-46. These platforms can allow sequencing of clonally expanded or non-amplified single molecules of nucleic acid fragments. Certain platforms involve, for example, sequencing by ligation of dye-modified probes (including cyclic ligation and cleavage), pyrosequencing, and single-molecule sequencing. Nucleotide sequence species, amplification nucleic acid species and detectable products generated there from can be analyzed by such sequence analysis platforms. Next-generation sequencing can be used in the methods of the invention, e.g., to determine mutations, copy number, or expression levels, as appropriate. The methods can be used to perform whole genome sequencing or sequencing of specific sequences of interest, such as a gene of interest or a fragment thereof.


Sequencing by ligation is a nucleic acid sequencing method that relies on the sensitivity of DNA ligase to base-pairing mismatch. DNA ligase joins together ends of DNA that are correctly base paired. Combining the ability of DNA ligase to join together only correctly base paired DNA ends, with mixed pools of fluorescently labeled oligonucleotides or primers, enables sequence determination by fluorescence detection. Longer sequence reads may be obtained by including primers containing cleavable linkages that can be cleaved after label identification. Cleavage at the linker removes the label and regenerates the 5′ phosphate on the end of the ligated primer, preparing the primer for another round of ligation. In some embodiments primers may be labeled with more than one fluorescent label, e.g., at least 1, 2, 3, 4, or 5 fluorescent labels.


Sequencing by ligation generally involves the following steps. Clonal bead populations can be prepared in emulsion microreactors containing target nucleic acid template sequences, amplification reaction components, beads and primers. After amplification, templates are denatured and bead enrichment is performed to separate beads with extended templates from undesired beads (e.g., beads with no extended templates). The template on the selected beads undergoes a 3′ modification to allow covalent bonding to the slide, and modified beads can be deposited onto a glass slide. Deposition chambers offer the ability to segment a slide into one, four or eight chambers during the bead loading process. For sequence analysis, primers hybridize to the adapter sequence. A set of four color dye-labeled probes competes for ligation to the sequencing primer. Specificity of probe ligation is achieved by interrogating every 4th and 5th base during the ligation series. Five to seven rounds of ligation, detection and cleavage record the color at every 5th position with the number of rounds determined by the type of library used. Following each round of ligation, a new complimentary primer offset by one base in the 5′ direction is laid down for another series of ligations. Primer reset and ligation rounds (5-7 ligation cycles per round) are repeated sequentially five times to generate 25-35 base pairs of sequence for a single tag. With mate-paired sequencing, this process is repeated for a second tag.


Pyrosequencing is a nucleic acid sequencing method based on sequencing by synthesis, which relies on detection of a pyrophosphate released on nucleotide incorporation. Generally, sequencing by synthesis involves synthesizing, one nucleotide at a time, a DNA strand complimentary to the strand whose sequence is being sought. Target nucleic acids may be immobilized to a solid support, hybridized with a sequencing primer, incubated with DNA polymerase, ATP sulfurylase, luciferase, apyrase, adenosine 5′ phosphosulfate and luciferin. Nucleotide solutions are sequentially added and removed. Correct incorporation of a nucleotide releases a pyrophosphate, which interacts with ATP sulfurylase and produces ATP in the presence of adenosine 5′ phosphosulfate, fueling the luciferin reaction, which produces a chemiluminescent signal allowing sequence determination. The amount of light generated is proportional to the number of bases added. Accordingly, the sequence downstream of the sequencing primer can be determined. An illustrative system for pyrosequencing involves the following steps: ligating an adaptor nucleic acid to a nucleic acid under investigation and hybridizing the resulting nucleic acid to a bead; amplifying a nucleotide sequence in an emulsion; sorting beads using a picoliter multiwell solid support; and sequencing amplified nucleotide sequences by pyrosequencing methodology (e.g., Nakano et al., “Single-molecule PCR using water-in-oil emulsion;” Journal of Biotechnology 102: 117-124 (2003)).


Certain single-molecule sequencing embodiments are based on the principal of sequencing by synthesis, and use single-pair Fluorescence Resonance Energy Transfer (single pair FRET) as a mechanism by which photons are emitted as a result of successful nucleotide incorporation. The emitted photons often are detected using intensified or high sensitivity cooled charge-couple-devices in conjunction with total internal reflection microscopy (TIRM). Photons are only emitted when the introduced reaction solution contains the correct nucleotide for incorporation into the growing nucleic acid chain that is synthesized as a result of the sequencing process. In FRET based single-molecule sequencing, energy is transferred between two fluorescent dyes, sometimes polymethine cyanine dyes Cy3 and Cy5, through long-range dipole interactions. The donor is excited at its specific excitation wavelength and the excited state energy is transferred, non-radiatively to the acceptor dye, which in turn becomes excited. The acceptor dye eventually returns to the ground state by radiative emission of a photon. The two dyes used in the energy transfer process represent the “single pair” in single pair FRET. Cy3 often is used as the donor fluorophore and often is incorporated as the first labeled nucleotide. Cy5 often is used as the acceptor fluorophore and is used as the nucleotide label for successive nucleotide additions after incorporation of a first Cy3 labeled nucleotide. The fluorophores generally are within 10 nanometers of each for energy transfer to occur successfully.


An example of a system that can be used based on single-molecule sequencing generally involves hybridizing a primer to a target nucleic acid sequence to generate a complex; associating the complex with a solid phase; iteratively extending the primer by a nucleotide tagged with a fluorescent molecule; and capturing an image of fluorescence resonance energy transfer signals after each iteration (e.g., U.S. Pat. No. 7,169,314; Braslavsky et al., PNAS 100(7): 3960-3964 (2003)). Such a system can be used to directly sequence amplification products (linearly or exponentially amplified products) generated by processes described herein. In some embodiments the amplification products can be hybridized to a primer that contains sequences complementary to immobilized capture sequences present on a solid support, a bead or glass slide for example. Hybridization of the primer-amplification product complexes with the immobilized capture sequences, immobilizes amplification products to solid supports for single pair FRET based sequencing by synthesis. The primer often is fluorescent, so that an initial reference image of the surface of the slide with immobilized nucleic acids can be generated. The initial reference image is useful for determining locations at which true nucleotide incorporation is occurring. Fluorescence signals detected in array locations not initially identified in the “primer only” reference image are discarded as non-specific fluorescence. Following immobilization of the primer-amplification product complexes, the bound nucleic acids often are sequenced in parallel by the iterative steps of, a) polymerase extension in the presence of one fluorescently labeled nucleotide, b) detection of fluorescence using appropriate microscopy, TIRM for example, c) removal of fluorescent nucleotide, and d) return to step a with a different fluorescently labeled nucleotide.


In some embodiments, nucleotide sequencing may be by solid phase single nucleotide sequencing methods and processes. Solid phase single nucleotide sequencing methods involve contacting target nucleic acid and solid support under conditions in which a single molecule of sample nucleic acid hybridizes to a single molecule of a solid support. Such conditions can include providing the solid support molecules and a single molecule of target nucleic acid in a “microreactor.” Such conditions also can include providing a mixture in which the target nucleic acid molecule can hybridize to solid phase nucleic acid on the solid support. Single nucleotide sequencing methods useful in the embodiments described herein are described in U.S. Provisional Patent Application Ser. No. 61/021,871 filed Jan. 17, 2008.


In certain embodiments, nanopore sequencing detection methods include (a) contacting a target nucleic acid for sequencing (“base nucleic acid,” e.g., linked probe molecule) with sequence-specific detectors, under conditions in which the detectors specifically hybridize to substantially complementary subsequences of the base nucleic acid; (b) detecting signals from the detectors and (c) determining the sequence of the base nucleic acid according to the signals detected. In certain embodiments, the detectors hybridized to the base nucleic acid are disassociated from the base nucleic acid (e.g., sequentially dissociated) when the detectors interfere with a nanopore structure as the base nucleic acid passes through a pore, and the detectors disassociated from the base sequence are detected. In some embodiments, a detector disassociated from a base nucleic acid emits a detectable signal, and the detector hybridized to the base nucleic acid emits a different detectable signal or no detectable signal. In certain embodiments, nucleotides in a nucleic acid (e.g., linked probe molecule) are substituted with specific nucleotide sequences corresponding to specific nucleotides (“nucleotide representatives”), thereby giving rise to an expanded nucleic acid (e.g., U.S. Pat. No. 6,723,513), and the detectors hybridize to the nucleotide representatives in the expanded nucleic acid, which serves as a base nucleic acid. In such embodiments, nucleotide representatives may be arranged in a binary or higher order arrangement (e.g., Soni and Meller, Clinical Chemistry 53(11): 1996-2001 (2007)). In some embodiments, a nucleic acid is not expanded, does not give rise to an expanded nucleic acid, and directly serves a base nucleic acid (e.g., a linked probe molecule serves as a non-expanded base nucleic acid), and detectors are directly contacted with the base nucleic acid. For example, a first detector may hybridize to a first subsequence and a second detector may hybridize to a second subsequence, where the first detector and second detector each have detectable labels that can be distinguished from one another, and where the signals from the first detector and second detector can be distinguished from one another when the detectors are disassociated from the base nucleic acid. In certain embodiments, detectors include a region that hybridizes to the base nucleic acid (e.g., two regions), which can be about 3 to about 100 nucleotides in length (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 nucleotides in length). A detector also may include one or more regions of nucleotides that do not hybridize to the base nucleic acid. In some embodiments, a detector is a molecular beacon. A detector often comprises one or more detectable labels independently selected from those described herein. Each detectable label can be detected by any convenient detection process capable of detecting a signal generated by each label (e.g., magnetic, electric, chemical, optical and the like). For example, a CD camera can be used to detect signals from one or more distinguishable quantum dots linked to a detector.


In certain sequence analysis embodiments, reads may be used to construct a larger nucleotide sequence, which can be facilitated by identifying overlapping sequences in different reads and by using identification sequences in the reads. Such sequence analysis methods and software for constructing larger sequences from reads are known to the person of ordinary skill (e.g., Venter et al., Science 291: 1304-1351 (2001)). Specific reads, partial nucleotide sequence constructs, and full nucleotide sequence constructs may be compared between nucleotide sequences within a sample nucleic acid (i.e., internal comparison) or may be compared with a reference sequence (i.e., reference comparison) in certain sequence analysis embodiments. Internal comparisons can be performed in situations where a sample nucleic acid is prepared from multiple samples or from a single sample source that contains sequence variations. Reference comparisons sometimes are performed when a reference nucleotide sequence is known and an objective is to determine whether a sample nucleic acid contains a nucleotide sequence that is substantially similar or the same, or different, than a reference nucleotide sequence. Sequence analysis can be facilitated by the use of sequence analysis apparatus and components described above.


Primer extension polymorphism detection methods, also referred to herein as “microsequencing” methods, typically are carried out by hybridizing a complementary oligonucleotide to a nucleic acid carrying the polymorphic site. In these methods, the oligonucleotide typically hybridizes adjacent to the polymorphic site. The term “adjacent” as used in reference to “microsequencing” methods, refers to the 3′ end of the extension oligonucleotide being sometimes 1 nucleotide from the 5′ end of the polymorphic site, often 2 or 3, and at times 4, 5, 6, 7, 8, 9, or 10 nucleotides from the 5′ end of the polymorphic site, in the nucleic acid when the extension oligonucleotide is hybridized to the nucleic acid. The extension oligonucleotide then is extended by one or more nucleotides, often 1, 2, or 3 nucleotides, and the number and/or type of nucleotides that are added to the extension oligonucleotide determine which polymorphic variant or variants are present. Oligonucleotide extension methods are disclosed, for example, in U.S. Pat. Nos. 4,656,127; 4,851,331; 5,679,524; 5,834,189; 5,876,934; 5,908,755; 5,912,118; 5,976,802; 5,981,186; 6,004,744; 6,013,431; 6,017,702; 6,046,005; 6,087,095; 6,210,891; and WO 01/20039. The extension products can be detected in any manner, such as by fluorescence methods (see, e.g., Chen & Kwok, Nucleic Acids Research 25: 347-353 (1997) and Chen et al., Proc. Natl. Acad. Sci. USA 94/20: 10756-10761 (1997)) or by mass spectrometric methods (e.g., MALDI-TOF mass spectrometry) and other methods described herein. Oligonucleotide extension methods using mass spectrometry are described, for example, in U.S. Pat. Nos. 5,547,835; 5,605,798; 5,691,141; 5,849,542; 5,869,242; 5,928,906; 6,043,031; 6,194,144; and 6,258,538.


Microsequencing detection methods often incorporate an amplification process that proceeds the extension step. The amplification process typically amplifies a region from a nucleic acid sample that comprises the polymorphic site. Amplification can be carried out using methods described above, or for example using a pair of oligonucleotide primers in a polymerase chain reaction (PCR), in which one oligonucleotide primer typically is complementary to a region 3′ of the polymorphism and the other typically is complementary to a region 5′ of the polymorphism. A PCR primer pair may be used in methods disclosed in U.S. Pat. Nos. 4,683,195; 4,683,202, 4,965,188; 5,656,493; 5,998,143; 6,140,054; WO 01/27327; and WO 01/27329 for example. PCR primer pairs may also be used in any commercially available machines that perform PCR, such as any of the GeneAmp™ Systems available from Applied Biosystems.


Other appropriate sequencing methods include multiplex polony sequencing (as described in Shendure et al., Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome, Sciencexpress, Aug. 4, 2005, pg 1 available at www.sciencexpress.org/4 Aug. 2005/Pagel/10.1126/science.1117389, incorporated herein by reference), which employs immobilized microbeads, and sequencing in microfabricated picoliter reactors (as described in Margulies et al., Genome Sequencing in Microfabricated High-Density Picolitre Reactors, Nature, August 2005, available at www.nature.com/nature (published online 31 Jul. 2005, doi:10.1038/nature03959, incorporated herein by reference).


Whole genome sequencing may also be used for discriminating alleles of RNA transcripts, in some embodiments. Examples of whole genome sequencing methods include, but are not limited to, nanopore-based sequencing methods, sequencing by synthesis and sequencing by ligation, as described above.


Nucleic acid variants can also be detected using standard electrophoretic techniques. Although the detection step can sometimes be preceded by an amplification step, amplification is not required in the embodiments described herein. Examples of methods for detection and quantification of a nucleic acid using electrophoretic techniques can be found in the art. A non-limiting example comprises running a sample (e.g., mixed nucleic acid sample isolated from maternal serum, or amplification nucleic acid species, for example) in an agarose or polyacrylamide gel. The gel may be labeled (e.g., stained) with ethidium bromide (see, Sambrook and Russell, Molecular Cloning: A Laboratory Manual 3d ed., 2001). The presence of a band of the same size as the standard control is an indication of the presence of a target nucleic acid sequence, the amount of which may then be compared to the control based on the intensity of the band, thus detecting and quantifying the target sequence of interest. In some embodiments, restriction enzymes capable of distinguishing between maternal and paternal alleles may be used to detect and quantify target nucleic acid species. In certain embodiments, oligonucleotide probes specific to a sequence of interest are used to detect the presence of the target sequence of interest. The oligonucleotides can also be used to indicate the amount of the target nucleic acid molecules in comparison to the standard control, based on the intensity of signal imparted by the probe.


Sequence-specific probe hybridization can be used to detect a particular nucleic acid in a mixture or mixed population comprising other species of nucleic acids. Under sufficiently stringent hybridization conditions, the probes hybridize specifically only to substantially complementary sequences. The stringency of the hybridization conditions can be relaxed to tolerate varying amounts of sequence mismatch. A number of hybridization formats are known in the art, which include but are not limited to, solution phase, solid phase, or mixed phase hybridization assays. The following articles provide an overview of the various hybridization assay formats: Singer et al., Biotechniques 4:230, 1986; Haase et al., Methods in Virology, pp. 189-226, 1984; Wilkinson, In situ Hybridization, Wilkinson ed., IRL Press, Oxford University Press, Oxford; and Hames and Higgins eds., Nucleic Acid Hybridization: A Practical Approach, IRL Press, 1987.


Hybridization complexes can be detected by techniques known in the art. Nucleic acid probes capable of specifically hybridizing to a target nucleic acid (e.g., mRNA or DNA) can be labeled by any suitable method, and the labeled probe used to detect the presence of hybridized nucleic acids. One commonly used method of detection is autoradiography, using probes labeled with 3H, 125I, 35S, 14C, 32P, 33P, or the like. The choice of radioactive isotope depends on research preferences due to ease of synthesis, stability, and half-lives of the selected isotopes. Other labels include compounds (e.g., biotin and digoxigenin), which bind to antiligands or antibodies labeled with fluorophores, chemiluminescent agents, and enzymes. In some embodiments, probes can be conjugated directly with labels such as fluorophores, chemiluminescent agents or enzymes. The choice of label depends on sensitivity required, ease of conjugation with the probe, stability requirements, and available instrumentation.


In embodiments, fragment analysis (referred to herein as “FA”) methods are used for molecular profiling. Fragment analysis (FA) includes techniques such as restriction fragment length polymorphism (RFLP) and/or (amplified fragment length polymorphism). If a nucleotide variant in the target DNA corresponding to the one or more genes results in the elimination or creation of a restriction enzyme recognition site, then digestion of the target DNA with that particular restriction enzyme will generate an altered restriction fragment length pattern. Thus, a detected RFLP or AFLP will indicate the presence of a particular nucleotide variant.


Terminal restriction fragment length polymorphism (TRFLP) works by PCR amplification of DNA using primer pairs that have been labeled with fluorescent tags. The PCR products are digested using RFLP enzymes and the resulting patterns are visualized using a DNA sequencer. The results are analyzed either by counting and comparing bands or peaks in the TRFLP profile, or by comparing bands from one or more TRFLP runs in a database.


The sequence changes directly involved with an RFLP can also be analyzed more quickly by PCR. Amplification can be directed across the altered restriction site, and the products digested with the restriction enzyme. This method has been called Cleaved Amplified Polymorphic Sequence (CAPS). Alternatively, the amplified segment can be analyzed by Allele specific oligonucleotide (ASO) probes, a process that is sometimes assessed using a Dot blot.


A variation on AFLP is cDNA-AFLP, which can be used to quantify differences in gene expression levels.


Another useful approach is the single-stranded conformation polymorphism assay (SSCA), which is based on the altered mobility of a single-stranded target DNA spanning the nucleotide variant of interest. A single nucleotide change in the target sequence can result in different intramolecular base pairing pattern, and thus different secondary structure of the single-stranded DNA, which can be detected in a non-denaturing gel. See Orita et al., Proc. Natl. Acad. Sci. USA, 86:2776-2770 (1989). Denaturing gel-based techniques such as clamped denaturing gel electrophoresis (CDGE) and denaturing gradient gel electrophoresis (DGGE) detect differences in migration rates of mutant sequences as compared to wild-type sequences in denaturing gel. See Miller et al., Biotechniques, 5:1016-24 (1999); Sheffield et al., Am. J. Hum, Genet., 49:699-706 (1991); Wartell et al., Nucleic Acids Res., 18:2699-2705 (1990); and Sheffield et al., Proc. Natl. Acad. Sci. USA, 86:232-236 (1989). In addition, the double-strand conformation analysis (DSCA) can also be useful in the present invention. See Arguello et al., Nat. Genet., 18:192-194 (1998).


The presence or absence of a nucleotide variant at a particular locus in the one or more genes of an individual can also be detected using the amplification refractory mutation system (ARMS) technique. See e.g., European Patent No. 0,332,435; Newton et al., Nucleic Acids Res., 17:2503-2515 (1989); Fox et al., Br. J. Cancer, 77:1267-1274 (1998); Robertson et al., Eur. Respir. J., 12:477-482 (1998). In the ARMS method, a primer is synthesized matching the nucleotide sequence immediately 5′ upstream from the locus being tested except that the 3′-end nucleotide which corresponds to the nucleotide at the locus is a predetermined nucleotide. For example, the 3′-end nucleotide can be the same as that in the mutated locus. The primer can be of any suitable length so long as it hybridizes to the target DNA under stringent conditions only when its 3′-end nucleotide matches the nucleotide at the locus being tested. Preferably the primer has at least 12 nucleotides, more preferably from about 18 to 50 nucleotides. If the individual tested has a mutation at the locus and the nucleotide therein matches the 3′-end nucleotide of the primer, then the primer can be further extended upon hybridizing to the target DNA template, and the primer can initiate a PCR amplification reaction in conjunction with another suitable PCR primer. In contrast, if the nucleotide at the locus is of wild type, then primer extension cannot be achieved. Various forms of ARMS techniques developed in the past few years can be used. See e.g., Gibson et al., Clin. Chem. 43:1336-1341(1997).


Similar to the ARMS technique is the mini sequencing or single nucleotide primer extension method, which is based on the incorporation of a single nucleotide. An oligonucleotide primer matching the nucleotide sequence immediately 5′ to the locus being tested is hybridized to the target DNA, mRNA or miRNA in the presence of labeled dideoxyribonucleotides. A labeled nucleotide is incorporated or linked to the primer only when the dideoxyribonucleotides matches the nucleotide at the variant locus being detected. Thus, the identity of the nucleotide at the variant locus can be revealed based on the detection label attached to the incorporated dideoxyribonucleotides. See Syvanen et al., Genomics, 8:684-692 (1990); Shumaker et al., Hum. Mutat., 7:346-354 (1996); Chen et al., Genome Res., 10:549-547 (2000).


Another set of techniques useful in the present invention is the so-called “oligonucleotide ligation assay” (OLA) in which differentiation between a wild-type locus and a mutation is based on the ability of two oligonucleotides to anneal adjacent to each other on the target DNA molecule allowing the two oligonucleotides joined together by a DNA ligase. See Landergren et al., Science, 241:1077-1080 (1988); Chen et al, Genome Res., 8:549-556 (1998); lannone et al., Cytometry, 39:131-140 (2000). Thus, for example, to detect a single-nucleotide mutation at a particular locus in the one or more genes, two oligonucleotides can be synthesized, one having the sequence just 5′ upstream from the locus with its 3′ end nucleotide being identical to the nucleotide in the variant locus of the particular gene, the other having a nucleotide sequence matching the sequence immediately 3′ downstream from the locus in the gene. The oligonucleotides can be labeled for the purpose of detection. Upon hybridizing to the target gene under a stringent condition, the two oligonucleotides are subject to ligation in the presence of a suitable ligase. The ligation of the two oligonucleotides would indicate that the target DNA has a nucleotide variant at the locus being detected.


Detection of small genetic variations can also be accomplished by a variety of hybridization-based approaches. Allele-specific oligonucleotides are most useful. See Conner et al., Proc. Natl. Acad. Sci. USA, 80:278-282 (1983); Saiki et al, Proc. Natl. Acad. Sci. USA, 86:6230-6234 (1989). Oligonucleotide probes (allele-specific) hybridizing specifically to a gene allele having a particular gene variant at a particular locus but not to other alleles can be designed by methods known in the art. The probes can have a length of, e.g., from 10 to about 50 nucleotide bases. The target DNA and the oligonucleotide probe can be contacted with each other under conditions sufficiently stringent such that the nucleotide variant can be distinguished from the wild-type gene based on the presence or absence of hybridization. The probe can be labeled to provide detection signals. Alternatively, the allele-specific oligonucleotide probe can be used as a PCR amplification primer in an “allele-specific PCR” and the presence or absence of a PCR product of the expected length would indicate the presence or absence of a particular nucleotide variant.


Other useful hybridization-based techniques allow two single-stranded nucleic acids annealed together even in the presence of mismatch due to nucleotide substitution, insertion or deletion. The mismatch can then be detected using various techniques. For example, the annealed duplexes can be subject to electrophoresis. The mismatched duplexes can be detected based on their electrophoretic mobility that is different from the perfectly matched duplexes. See Cariello, Human Genetics, 42:726 (1988). Alternatively, in an RNase protection assay, a RNA probe can be prepared spanning the nucleotide variant site to be detected and having a detection marker. See Giunta et al., Diagn. Mol. Path., 5:265-270 (1996); Finkelstein et al., Genomics, 7:167-172 (1990); Kinszler et al., Science 251:1366-1370 (1991). The RNA probe can be hybridized to the target DNA or mRNA forming a heteroduplex that is then subject to the ribonuclease RNase A digestion. RNase A digests the RNA probe in the heteroduplex only at the site of mismatch. The digestion can be determined on a denaturing electrophoresis gel based on size variations. In addition, mismatches can also be detected by chemical cleavage methods known in the art. See e.g., Roberts et al., Nucleic Acids Res., 25:3377-3378 (1997).


In the mutS assay, a probe can be prepared matching the gene sequence surrounding the locus at which the presence or absence of a mutation is to be detected, except that a predetermined nucleotide is used at the variant locus. Upon annealing the probe to the target DNA to form a duplex, the E. coli mutS protein is contacted with the duplex. Since the mutS protein binds only to heteroduplex sequences containing a nucleotide mismatch, the binding of the mutS protein will be indicative of the presence of a mutation. See Modrich et al., Ann. Rev. Genet., 25:229-253 (1991).


A great variety of improvements and variations have been developed in the art on the basis of the above-described basic techniques which can be useful in detecting mutations or nucleotide variants in the present invention. For example, the “sunrise probes” or “molecular beacons” use the fluorescence resonance energy transfer (FRET) property and give rise to high sensitivity. See Wolf et al., Proc. Nat. Acad. Sci. USA, 85:8790-8794 (1988). Typically, a probe spanning the nucleotide locus to be detected are designed into a hairpin-shaped structure and labeled with a quenching fluorophore at one end and a reporter fluorophore at the other end. In its natural state, the fluorescence from the reporter fluorophore is quenched by the quenching fluorophore due to the proximity of one fluorophore to the other. Upon hybridization of the probe to the target DNA, the 5′ end is separated apart from the 3′-end and thus fluorescence signal is regenerated. See Nazarenko et al., Nucleic Acids Res., 25:2516-2521 (1997); Rychlik et al., Nucleic Acids Res., 17:8543-8551 (1989); Sharkey et al., Bio/Technology 12:506-509 (1994); Tyagi et al., Nat. Biotechnol., 14:303-308 (1996); Tyagi et al., Nat. Biotechnol., 16:49-53 (1998). The homo-tag assisted non-dimer system (HANDS) can be used in combination with the molecular beacon methods to suppress primer-dimer accumulation. See Brownie et al., Nucleic Acids Res., 25:3235-3241 (1997).


Dye-labeled oligonucleotide ligation assay is a FRET-based method, which combines the OLA assay and PCR. See Chen et al., Genome Res. 8:549-556 (1998). TaqMan is another FRET-based method for detecting nucleotide variants. A TaqMan probe can be oligonucleotides designed to have the nucleotide sequence of the gene spanning the variant locus of interest and to differentially hybridize with different alleles. The two ends of the probe are labeled with a quenching fluorophore and a reporter fluorophore, respectively. The TaqMan probe is incorporated into a PCR reaction for the amplification of a target gene region containing the locus of interest using Taq polymerase. As Taq polymerase exhibits 5′-3′ exonuclease activity but has no 3′-5′ exonuclease activity, if the TaqMan probe is annealed to the target DNA template, the 5′-end of the TaqMan probe will be degraded by Taq polymerase during the PCR reaction thus separating the reporting fluorophore from the quenching fluorophore and releasing fluorescence signals. See Holland et al., Proc. Natl. Acad. Sci. USA, 88:7276-7280 (1991); Kalinina et al., Nucleic Acids Res., 25:1999-2004 (1997); Whitcombe et al., Clin. Chem., 44:918-923 (1998).


In addition, the detection in the present invention can also employ a chemiluminescence-based technique. For example, an oligonucleotide probe can be designed to hybridize to either the wild-type or a variant gene locus but not both. The probe is labeled with a highly chemiluminescent acridinium ester. Hydrolysis of the acridinium ester destroys chemiluminescence. The hybridization of the probe to the target DNA prevents the hydrolysis of the acridinium ester. Therefore, the presence or absence of a particular mutation in the target DNA is determined by measuring chemiluminescence changes. See Nelson et al., Nucleic Acids Res., 24:4998-5003 (1996).


The detection of genetic variation in the gene in accordance with the present invention can also be based on the “base excision sequence scanning” (BESS) technique. The BESS method is a PCR-based mutation scanning method. BESS T-Scan and BESS G-Tracker are generated which are analogous to T and G ladders of dideoxy sequencing. Mutations are detected by comparing the sequence of normal and mutant DNA. See, e.g., Hawkins et al., Electrophoresis, 20:1171-1176 (1999).


Mass spectrometry can be used for molecular profiling according to the invention. See Graber et al., Curr. Opin. Biotechnol., 9:14-18 (1998). For example, in the primer oligo base extension (PROBE™) method, a target nucleic acid is immobilized to a solid-phase support. A primer is annealed to the target immediately 5′ upstream from the locus to be analyzed. Primer extension is carried out in the presence of a selected mixture of deoxyribonucleotides and dideoxyribonucleotides. The resulting mixture of newly extended primers is then analyzed by MALDI-TOF. See e.g., Monforte et al., Nat. Med., 3:360-362 (1997).


In addition, the microchip or microarray technologies are also applicable to the detection method of the present invention. Essentially, in microchips, a large number of different oligonucleotide probes are immobilized in an array on a substrate or carrier, e.g., a silicon chip or glass slide. Target nucleic acid sequences to be analyzed can be contacted with the immobilized oligonucleotide probes on the microchip. See Lipshutz et al., Biotechniques, 19:442-447 (1995); Chee et al., Science, 274:610-614 (1996); Kozal et al., Nat. Med. 2:753-759 (1996); Hacia et al., Nat. Genet., 14:441-447 (1996); Saiki et al., Proc. Natl. Acad. Sci. USA, 86:6230-6234 (1989); Gingeras et al., Genome Res., 8:435-448 (1998). Alternatively, the multiple target nucleic acid sequences to be studied are fixed onto a substrate and an array of probes is contacted with the immobilized target sequences. See Drmanac et al., Nat. Biotechnol., 16:54-58 (1998). Numerous microchip technologies have been developed incorporating one or more of the above described techniques for detecting mutations. The microchip technologies combined with computerized analysis tools allow fast screening in a large scale. The adaptation of the microchip technologies to the present invention will be apparent to a person of skill in the art apprised of the present disclosure. See, e.g., U.S. Pat. No. 5,925,525 to Fodor et al; Wilgenbus et al., J. Mol. Med., 77:761-786 (1999); Graber et al., Curr. Opin. Biotechnol., 9:14-18 (1998); Hacia et al., Nat. Genet., 14:441-447 (1996); Shoemaker et al., Nat. Genet., 14:450-456 (1996); DeRisi et al., Nat. Genet., 14:457-460 (1996); Chee et al., Nat. Genet., 14:610-614 (1996); Lockhart et al., Nat. Genet., 14:675-680 (1996); Drobyshev et al., Gene, 188:45-52 (1997).


As is apparent from the above survey of the suitable detection techniques, it may or may not be necessary to amplify the target DNA, i.e., the gene, cDNA, mRNA, miRNA, or a portion thereof to increase the number of target DNA molecule, depending on the detection techniques used. For example, most PCR-based techniques combine the amplification of a portion of the target and the detection of the mutations. PCR amplification is well known in the art and is disclosed in U.S. Pat. Nos. 4,683,195 and 4,800,159, both which are incorporated herein by reference. For non-PCR-based detection techniques, if necessary, the amplification can be achieved by, e.g., in vivo plasmid multiplication, or by purifying the target DNA from a large amount of tissue or cell samples. See generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989. However, even with scarce samples, many sensitive techniques have been developed in which small genetic variations such as single-nucleotide substitutions can be detected without having to amplify the target DNA in the sample. For example, techniques have been developed that amplify the signal as opposed to the target DNA by, e.g., employing branched DNA or dendrimers that can hybridize to the target DNA. The branched or dendrimer DNAs provide multiple hybridization sites for hybridization probes to attach thereto thus amplifying the detection signals. See Detmer et al., J. Clin. Microbiol., 34:901-907 (1996); Collins et al., Nucleic Acids Res., 25:2979-2984 (1997); Horn et al., Nucleic Acids Res., 25:4835-4841 (1997); Horn et al., Nucleic Acids Res., 25:4842-4849 (1997); Nilsen et al., J. Theor. Biol., 187:273-284 (1997).


The Invader™ assay is another technique for detecting single nucleotide variations that can be used for molecular profiling according to the invention. The Invader™ assay uses a novel linear signal amplification technology that improves upon the long turnaround times required of the typical PCR DNA sequenced-based analysis. See Cooksey et al., Antimicrobial Agents and Chemotherapy 44:1296-1301 (2000). This assay is based on cleavage of a unique secondary structure formed between two overlapping oligonucleotides that hybridize to the target sequence of interest to form a “flap.” Each “flap” then generates thousands of signals per hour. Thus, the results of this technique can be easily read, and the methods do not require exponential amplification of the DNA target. The Invader™ system uses two short DNA probes, which are hybridized to a DNA target. The structure formed by the hybridization event is recognized by a special cleavase enzyme that cuts one of the probes to release a short DNA “flap.” Each released “flap” then binds to a fluorescently-labeled probe to form another cleavage structure. When the cleavase enzyme cuts the labeled probe, the probe emits a detectable fluorescence signal. See e.g. Lyamichev et al., Nat. Biotechnol., 17:292-296 (1999).


The rolling circle method is another method that avoids exponential amplification. Lizardi et al., Nature Genetics, 19:225-232 (1998) (which is incorporated herein by reference). For example, Sniper™, a commercial embodiment of this method, is a sensitive, high-throughput SNP scoring system designed for the accurate fluorescent detection of specific variants. For each nucleotide variant, two linear, allele-specific probes are designed. The two allele-specific probes are identical with the exception of the 3′-base, which is varied to complement the variant site. In the first stage of the assay, target DNA is denatured and then hybridized with a pair of single, allele-specific, open-circle oligonucleotide probes. When the 3′-base exactly complements the target DNA, ligation of the probe will preferentially occur. Subsequent detection of the circularized oligonucleotide probes is by rolling circle amplification, whereupon the amplified probe products are detected by fluorescence. See Clark and Pickering, Life Science News 6, 2000, Amersham Pharmacia Biotech (2000).


A number of other techniques that avoid amplification all together include, e.g., surface-enhanced resonance Raman scattering (SERRS), fluorescence correlation spectroscopy, and single-molecule electrophoresis. In SERRS, a chromophore-nucleic acid conjugate is absorbed onto colloidal silver and is irradiated with laser light at a resonant frequency of the chromophore. See Graham et al., Anal. Chem., 69:4703-4707 (1997). The fluorescence correlation spectroscopy is based on the spatio-temporal correlations among fluctuating light signals and trapping single molecules in an electric field. See Eigen et al., Proc. Natl. Acad. Sci. USA, 91:5740-5747 (1994). In single-molecule electrophoresis, the electrophoretic velocity of a fluorescently tagged nucleic acid is determined by measuring the time required for the molecule to travel a predetermined distance between two laser beams. See Castro et al., Anal. Chem., 67:3181-3186 (1995).


In addition, the allele-specific oligonucleotides (ASO) can also be used in in situ hybridization using tissues or cells as samples. The oligonucleotide probes which can hybridize differentially with the wild-type gene sequence or the gene sequence harboring a mutation may be labeled with radioactive isotopes, fluorescence, or other detectable markers. In situ hybridization techniques are well known in the art and their adaptation to the present invention for detecting the presence or absence of a nucleotide variant in the one or more gene of a particular individual should be apparent to a skilled artisan apprised of this disclosure.


Accordingly, the presence or absence of one or more genes nucleotide variant or amino acid variant in an individual can be determined using any of the detection methods described above.


Typically, once the presence or absence of one or more gene nucleotide variants or amino acid variants is determined, physicians or genetic counselors or patients or other researchers may be informed of the result. Specifically the result can be cast in a transmittable form that can be communicated or transmitted to other researchers or physicians or genetic counselors or patients. Such a form can vary and can be tangible or intangible. The result with regard to the presence or absence of a nucleotide variant of the present invention in the individual tested can be embodied in descriptive statements, diagrams, photographs, charts, images or any other visual forms. For example, images of gel electrophoresis of PCR products can be used in explaining the results. Diagrams showing where a variant occurs in an individual's gene are also useful in indicating the testing results. The statements and visual forms can be recorded on a tangible media such as papers, computer readable media such as floppy disks, compact disks, etc., or on an intangible media, e.g., an electronic media in the form of email or website on internet or intranet. In addition, the result with regard to the presence or absence of a nucleotide variant or amino acid variant in the individual tested can also be recorded in a sound form and transmitted through any suitable media, e.g., analog or digital cable lines, fiber optic cables, etc., via telephone, facsimile, wireless mobile phone, internet phone and the like.


Thus, the information and data on a test result can be produced anywhere in the world and transmitted to a different location. For example, when a genotyping assay is conducted offshore, the information and data on a test result may be generated and cast in a transmittable form as described above. The test result in a transmittable form thus can be imported into the U.S. Accordingly, the present invention also encompasses a method for producing a transmittable form of information on the genotype of the two or more suspected cancer samples from an individual. The method comprises the steps of (1) determining the genotype of the DNA from the samples according to methods of the present invention; and (2) embodying the result of the determining step in a transmittable form. The transmittable form is the product of the production method.


In Situ Hybridization

In situ hybridization assays are well known and are generally described in Angerer et al., Methods Enzymol. 152:649-660 (1987). In an in situ hybridization assay, cells, e.g., from a biopsy, are fixed to a solid support, typically a glass slide. If DNA is to be probed, the cells are denatured with heat or alkali. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of specific probes that are labeled. The probes are preferably labeled, e.g., with radioisotopes or fluorescent reporters, or enzymatically. FISH (fluorescence in situ hybridization) uses fluorescent probes that bind to only those parts of a sequence with which they show a high degree of sequence similarity. CISH (chromogenic in situ hybridization) uses conventional peroxidase or alkaline phosphatase reactions visualized under a standard bright-field microscope.


In situ hybridization can be used to detect specific gene sequences in tissue sections or cell preparations by hybridizing the complementary strand of a nucleotide probe to the sequence of interest. Fluorescent in situ hybridization (FISH) uses a fluorescent probe to increase the sensitivity of in situ hybridization.


FISH is a cytogenetic technique used to detect and localize specific polynucleotide sequences in cells. For example, FISH can be used to detect DNA sequences on chromosomes. FISH can also be used to detect and localize specific RNAs, e.g., mRNAs, within tissue samples. In FISH uses fluorescent probes that bind to specific nucleotide sequences to which they show a high degree of sequence similarity. Fluorescence microscopy can be used to find out whether and where the fluorescent probes are bound. In addition to detecting specific nucleotide sequences, e.g., translocations, fusion, breaks, duplications and other chromosomal abnormalities, FISH can help define the spatial-temporal patterns of specific gene copy number and/or gene expression within cells and tissues.


Various types of FISH probes can be used to detect chromosome translocations. Dual color, single fusion probes can be useful in detecting cells possessing a specific chromosomal translocation. The DNA probe hybridization targets are located on one side of each of the two genetic breakpoints. “Extra signal” probes can reduce the frequency of normal cells exhibiting an abnormal FISH pattern due to the random co-localization of probe signals in a normal nucleus. One large probe spans one breakpoint, while the other probe flanks the breakpoint on the other gene. Dual color, break apart probes are useful in cases where there may be multiple translocation partners associated with a known genetic breakpoint. This labeling scheme features two differently colored probes that hybridize to targets on opposite sides of a breakpoint in one gene. Dual color, dual fusion probes can reduce the number of normal nuclei exhibiting abnormal signal patterns. The probe offers advantages in detecting low levels of nuclei possessing a simple balanced translocation. Large probes span two breakpoints on different chromosomes. Such probes are available as Vysis probes from Abbott Laboratories, Abbott Park, Ill.


CISH, or chromogenic in situ hybridization, is a process in which a labeled complementary DNA or RNA strand is used to localize a specific DNA or RNA sequence in a tissue specimen. CISH methodology can be used to evaluate gene amplification, gene deletion, chromosome translocation, and chromosome number. CISH can use conventional enzymatic detection methodology, e.g., horseradish peroxidase or alkaline phosphatase reactions, visualized under a standard bright-field microscope. In a common embodiment, a probe that recognizes the sequence of interest is contacted with a sample. An antibody or other binding agent that recognizes the probe, e.g., via a label carried by the probe, can be used to target an enzymatic detection system to the site of the probe. In some systems, the antibody can recognize the label of a FISH probe, thereby allowing a sample to be analyzed using both FISH and CISH detection. CISH can be used to evaluate nucleic acids in multiple settings, e.g., formalin-fixed, paraffin-embedded (FFPE) tissue, blood or bone marrow smear, metaphase chromosome spread, and/or fixed cells. In an embodiment, CISH is performed following the methodology in the SPoT-Light® HER2 CISH Kit available from Life Technologies (Carlsbad, Calif.) or similar CISH products available from Life Technologies. The SPoT-Light® HER2 CISH Kit itself is FDA approved for in vitro diagnostics and can be used for molecular profiling of HER2. CISH can be used in similar applications as FISH. Thus, one of skill will appreciate that reference to molecular profiling using FISH herein can be performed using CISH, unless otherwise specified.


Silver-enhanced in situ hybridization (SISH) is similar to CISH, but with SISH the signal appears as a black coloration due to silver precipitation instead of the chromogen precipitates of CISH.


Modifications of the in situ hybridization techniques can be used for molecular profiling according to the invention. Such modifications comprise simultaneous detection of multiple targets, e.g., Dual ISH, Dual color CISH, bright field double in situ hybridization (BDISH). See e.g., the FDA approved INFORM HER2 Dual ISH DNA Probe Cocktail kit from Ventana Medical Systems, Inc. (Tucson, Ariz.); DuoCISH™, a dual color CISH kit developed by Dako Denmark A/S (Denmark).


Comparative Genomic Hybridization (CGH) comprises a molecular cytogenetic method of screening tumor samples for genetic changes showing characteristic patterns for copy number changes at chromosomal and subchromosomal levels. Alterations in patterns can be classified as DNA gains and losses. CGH employs the kinetics of in situ hybridization to compare the copy numbers of different DNA or RNA sequences from a sample, or the copy numbers of different DNA or RNA sequences in one sample to the copy numbers of the substantially identical sequences in another sample. In many useful applications of CGH, the DNA or RNA is isolated from a subject cell or cell population. The comparisons can be qualitative or quantitative. Procedures are described that permit determination of the absolute copy numbers of DNA sequences throughout the genome of a cell or cell population if the absolute copy number is known or determined for one or several sequences. The different sequences are discriminated from each other by the different locations of their binding sites when hybridized to a reference genome, usually metaphase chromosomes but in certain cases interphase nuclei. The copy number information originates from comparisons of the intensities of the hybridization signals among the different locations on the reference genome. The methods, techniques and applications of CGH are known, such as described in U.S. Pat. No. 6,335,167, and in U.S. App. Ser. No. 60/804,818, the relevant parts of which are herein incorporated by reference.


In an embodiment, CGH used to compare nucleic acids between diseased and healthy tissues. The method comprises isolating DNA from disease tissues (e.g., tumors) and reference tissues (e.g., healthy tissue) and labeling each with a different “color” or fluor. The two samples are mixed and hybridized to normal metaphase chromosomes. In the case of array or matrix CGH, the hybridization mixing is done on a slide with thousands of DNA probes. A variety of detection system can be used that basically determine the color ratio along the chromosomes to determine DNA regions that might be gained or lost in the diseased samples as compared to the reference.


Molecular Profiling for Treatment Selection

The methods of the invention provide a candidate treatment selection for a subject in need thereof. Molecular profiling can be used to identify one or more candidate therapeutic agents for an individual suffering from a condition in which one or more of the biomarkers disclosed herein are targets for treatment. For example, the method can identify one or more chemotherapy treatments for a cancer. In an aspect, the invention provides a method comprising: performing an immunohistochemistry (IHC) analysis on a sample from the subject to determine an IHC expression profile on at least five proteins; performing a microarray analysis on the sample to determine a microarray expression profile on at least ten genes; performing a fluorescent in-situ hybridization (FISH) analysis on the sample to determine a FISH mutation profile on at least one gene; performing DNA sequencing on the sample to determine a sequencing mutation profile on at least one gene; and comparing the IHC expression profile, microarray expression profile, FISH mutation profile and sequencing mutation profile against a rules database, wherein the rules database comprises a mapping of treatments whose biological activity is known against diseased cells that: i) overexpress or underexpress one or more proteins included in the IHC expression profile; ii) overexpress or underexpress one or more genes included in the microarray expression profile; iii) have zero or more mutations in one or more genes included in the FISH mutation profile; and/or iv) have zero or more mutations in one or more genes included in the sequencing mutation profile; and identifying the treatment if the comparison against the rules database indicates that the treatment should have biological activity against the diseased cells; and the comparison against the rules database does not contraindicate the treatment for treating the diseased cells. The disease can be a cancer. The molecular profiling steps can be performed in any order. In some embodiments, not all of the molecular profiling steps are performed. As a non-limiting example, microarray analysis is not performed if the sample quality does not meet a threshold value, as described herein. In another example, sequencing is performed only if FISH analysis meets a threshold value. Any relevant biomarker can be assessed using one or more of the molecular profiling techniques described herein or known in the art. The marker need only have some direct or indirect association with a treatment to be useful.


Molecular profiling comprises the profiling of at least one gene (or gene product) for each assay technique that is performed. Different numbers of genes can be assayed with different techniques. Any marker disclosed herein that is associated directly or indirectly with a target therapeutic can be assessed. For example, any “druggable target” comprising a target that can be modulated with a therapeutic agent such as a small molecule or binding agent such as an antibody, is a candidate for inclusion in the molecular profiling methods of the invention. The target can also be indirectly drug associated, such as a component of a biological pathway that is affected by the associated drug. The molecular profiling can be based on either the gene, e.g., DNA sequence, and/or gene product, e.g., mRNA or protein. Such nucleic acid and/or polypeptide can be profiled as applicable as to presence or absence, level or amount, activity, mutation, sequence, haplotype, rearrangement, copy number, or other measurable characteristic. In some embodiments, a single gene and/or one or more corresponding gene products is assayed by more than one molecular profiling technique. A gene or gene product (also referred to herein as “marker” or “biomarker”), e.g., an mRNA or protein, is assessed using applicable techniques (e.g., to assess DNA, RNA, protein), including without limitation FISH, microarray, IHC, sequencing or immunoassay. Therefore, any of the markers disclosed herein can be assayed by a single molecular profiling technique or by multiple methods disclosed herein (e.g., a single marker is profiled by one or more of IHC, FISH, sequencing, microarray, etc.). In some embodiments, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or at least about 100 genes or gene products are profiled by at least one technique, a plurality of techniques, or using a combination of FISH, microarray, IHC, and sequencing. In some embodiments, at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 28,000, 29,000, 30,000, 31,000, 32,000, 33,000, 34,000, 35,000, 36,000, 37,000, 38,000, 39,000, 40,000, 41,000, 42,000, 43,000, 44,000, 45,000, 46,000, 47,000, 48,000, 49,000, or at least 50,000 genes or gene products are profiled using various techniques. The number of markers assayed can depend on the technique used. For example, microarray and massively parallel sequencing lend themselves to high throughput analysis. Because molecular profiling queries molecular characteristics of the tumor itself, this approach provides information on therapies that might not otherwise be considered based on the lineage of the tumor.


In some embodiments, a sample from a subject in need thereof is profiled using methods which include but are not limited to IHC expression profiling, microarray expression profiling, FISH mutation profiling, and/or sequencing mutation profiling (such as by PCR, RT-PCR, pyrosequencing) for one or more of the following: ABCC1, ABCG2, ACE2, ADA, ADH1C, ADH4, AGT, AR, AREG, ASNS, BCL2, BCRP, BDCA1, beta III tubulin, BIRC5, B-RAF, BRCA1, BRCA2, CA2, caveolin, CD20, CD25, CD33, CD52, CDA, CDKN2A, CDKN1A, CDKN1B, CDK2, CDW52, CES2, CK 14, CK 17, CK 5/6, c-KIT, c-Met, c-Myc, COX-2, Cyclin D1, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, E-Cadherin, ECGF1, EGFR, EML4-ALK fusion, EPHA2, Epiregulin, ER, ERBR2, ERCC1, ERCC3, EREG, ESR1, FLT1, folate receptor, FOLR1, FOLR2, FSHB, FSHPRH1, FSHR, FYN, GART, GNA11, GNAQ, GNRH1, GNRHR1, GSTP1, HCK, HDAC1, hENT-1, Her2/Neu, HGF, HIF1A, HIG1, HSP90, HSP90AA1, HSPCA, IGF-1R, IGFRBP, IGFRBP3, IGFRBP4, IGFRBP5, IL13RA1, IL2RA, KDR, Ki67, KIT, K-RAS, LCK, LTB, Lymphotoxin Beta Receptor, LYN, MET, MGMT, MLH1, MMR, MRP1, MS4A1, MSH2, MSH5, Myc, NFKB1, NFKB2, NFKBIA, NRAS, ODC1, OGFR, p16, p21, p27, p53, p95, PARP-1, PDGFC, PDGFR, PDGFRA, PDGFRB, PGP, PGR, PI3K, POLA, POLA1, PPARG, PPARGC1, PR, PTEN, PTGS2, PTPN12, RAF1, RARA, ROS1, RRM1, RRM2, RRM2B, RXRB, RXRG, SIK2, SPARC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, Survivin, TK1, TLE3, TNF, TOP1, TOP2A, TOP2B, TS, TUBB3, TXN, TXNRD1, TYMS, VDR, VEGF, VEGFA, VEGFC, VHL, YES1, ZAP70.


Table 2 provides a listing of gene and corresponding protein symbols and names of many of the molecular profiling targets that are analyzed according to the methods of the invention. As understood by those of skill in the art, genes and proteins have developed a number of alternative names in the scientific literature. Thus, the listing in Table 2 comprises an illustrative but not exhaustive compilation. A further listing of gene aliases and descriptions can be found using a variety of online databases, including GeneCards® (www.genecards.org), HUGO Gene Nomenclature (www.genenames.org), Entrez Gene (www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene), UniProtKB/Swiss-Prot (www.uniprot.org), UniProtKB/TrEMBL (www.uniprot.org), OMIM (www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM), GeneLoc (genecards.weizmann.ac.il/geneloc/), and Ensembl (www.ensembl.org). Generally, gene symbols and names below correspond to those approved by HUGO, and protein names are those recommended by UniProtKB/Swiss-Prot. Common alternatives are provided as well. Where a protein name indicates a precursor, the mature protein is also implied. Throughout the application, gene and protein symbols may be used interchangeably and the meaning can be derived from context, e.g., FISH is used to analyze nucleic acids whereas IHC is used to analyze protein.









TABLE 2







Gene and Protein Names










Gene

Protein



Symbol
Gene Name
Symbol
Protein Name





ABCB1,
ATP-binding cassette, sub-family B
ABCB1,
Multidrug resistance protein 1; P-


PGP
(MDR/TAP), member 1
MDR1, PGP
glycoprotein


ABCC1,
ATP-binding cassette, sub-family C
MRP1,
Multidrug resistance-associated protein 1


MRP1
(CFTR/MRP), member 1
ABCC1


ABCG2,
ATP-binding cassette, sub-family G
ABCG2
ATP-binding cassette sub-family G


BCRP
(WHITE), member 2

member 2


ACE2
angiotensin I converting enzyme
ACE2
Angiotensin-converting enzyme 2



(peptidyl-dipeptidase A) 2

precursor


ADA
adenosine deaminase
ADA
Adenosine deaminase


ADH1C
alcohol dehydrogenase 1C (class I),
ADH1G
Alcohol dehydrogenase 1C



gamma polypeptide


ADH4
alcohol dehydrogenase 4 (class II), pi
ADH4
Alcohol dehydrogenase 4



polypeptide


AGT
angiotensinogen (serpin peptidase
ANGT, AGT
Angiotensinogen precursor



inhibitor, clade A, member 8)


ALK
anaplastic lymphoma receptor
ALK
ALK tyrosine kinase receptor precursor



tyrosine kinase


AR
androgen receptor
AR
Androgen receptor


AREG
amphiregulin
AREG
Amphiregulin precursor


ASNS
asparagine synthetase
ASNS
Asparagine synthetase [glutamine-





hydrolyzing]


BCL2
B-cell CLL/lymphoma 2
BCL2
Apoptosis regulator Bcl-2


BDCA1,
CD1c molecule
CD1C
T-cell surface glycoprotein CD1c


CD1C


precursor


BIRC5
baculoviral IAP repeat-containing 5
BIRC5,
Baculoviral IAP repeat-containing




Survivin
protein 5; Survivin


BRAF
v-raf murine sarcoma viral oncogene
B-RAF,
Serine/threonine-protein kinase B-raf



homolog B1
BRAF


BRCA1
breast cancer 1, early onset
BRCA1
Breast cancer type 1 susceptibility





protein


BRCA2
breast cancer 2, early onset
BRCA2
Breast cancer type 2 susceptibility





protein


CA2
carbonic anhydrase II
CA2
Carbonic anhydrase 2


CAV1
caveolin 1, caveolae protein, 22 kDa
CAV1
Caveolin-1


CCND1
cyclin D1
CCND1,
G1/S-specific cyclin-D1




Cyclin D1,




BCL-1


CD20,
membrane-spanning 4-domains,
CD20
B-lymphocyte antigen CD20


MS4A1
subfamily A, member 1


CD25,
interleukin 2 receptor, alpha
CD25
Interleukin-2 receptor subunit alpha


IL2RA


precursor


CD33
CD33 molecule
CD33
Myeloid cell surface antigen CD33





precursor


CD52,
CD52 molecule
CD52
CAMPATH-1 antigen precursor


CDW52


CDA
cytidine deaminase
CDA
Cytidine deaminase


CDH1,
cadherin 1, type 1, E-cadherin
E-Cad
Cadherin-1 precursor (E-cadherin)


ECAD
(epithelial)


CDK2
cyclin-dependent kinase 2
CDK2
Cell division protein kinase 2


CDKN1A,
cyclin-dependent kinase inhibitor 1A
CDKN1A,
Cyclin-dependent kinase inhibitor 1


P21
(p21, Cip1)
p21


CDKN1B
cyclin-dependent kinase inhibitor 1B
CDKN1B,
Cyclin-dependent kinase inhibitor 1B



(p27, Kip1)
p27


CDKN2A,
cyclin-dependent kinase inhibitor 2A
CD21A, p16
Cyclin-dependent kinase inhibitor 2A,


P16
(melanoma, p16, inhibits CDK4)

isoforms 1/2/3


CES2
carboxylesterase 2 (intestine, liver)
CES2, EST2
Carboxylesterase 2 precursor


CK 5/6
cytokeratin 5/cytokeratin 6
CK 5/6
Keratin, type II cytoskeletal 5; Keratin,





type II cytoskeletal 6


CK14,
keratin 14
CK14
Keratin, type I cytoskeletal 14


KRT14


CK17,
keratin 17
CK17
Keratin, type I cytoskeletal 17


KRT17


COX2,
prostaglandin-endoperoxide synthase
COX-2,
Prostaglandin G/H synthase 2 precursor


PTGS2
2 (prostaglandin G/H synthase and
PTGS2



cyclooxygenase)


DCK
deoxycytidine kinase
DCK
Deoxycytidine kinase


DHFR
dihydrofolate reductase
DHFR
Dihydrofolate reductase


DNMT1
DNA (cytosine-5-)-methyltransferase 1
DNMT1
DNA (cytosine-5)-methyltransferase 1


DNMT3A
DNA (cytosine-5-)-methyltransferase
DNMT3A
DNA (cytosine-5)-methyltransferase 3A



3 alpha


DNMT3B
DNA (cytosine-5-)-methyltransferase
DNMT3B
DNA (cytosine-5)-methyltransferase 3B



3 beta


ECGF1,
thymidine phosphorylase
TYMP, PD-
Thymidine phosphorylase precursor


TYMP

ECGF,




ECDF1


EGFR,
epidermal growth factor receptor
EGFR,
Epidermal growth factor receptor


ERBB1,
(erythroblastic leukemia viral (v-erb-
ERBB1,
precursor


HER1
b) oncogene homolog, avian)
HER1


EML4
echinoderm microtubule associated
EML4
Echinoderm microtubule-associated



protein like 4

protein-like 4


EPHA2
EPH receptor A2
EPHA2
Ephrin type-A receptor 2 precursor


ER, ESR1
estrogen receptor 1
ER, ESR1
Estrogen receptor


ERBB2,
v-erb-b2 erythroblastic leukemia
ERBB2,
Receptor tyrosine-protein kinase erbB-2


HER2/NEU
viral oncogene homolog 2,
HER2, HER-
precursor



neuro/glioblastoma derived oncogene
2/neu



homolog (avian)


ERCC1
excision repair cross-complementing
ERCC1
DNA excision repair protein ERCC-1



rodent repair deficiency,



complementation group 1 (includes



overlapping antisense sequence)


ERCC3
excision repair cross-complementing
ERCC3
TFIIH basal transcription factor complex



rodent repair deficiency,

helicase XPB subunit



complementation group 3 (xeroderma



pigmentosum group B



complementing)


EREG
Epiregulin
EREG
Proepiregulin precursor


FLT1
fms-related tyrosine kinase 1
FLT-1,
Vascular endothelial growth factor



(vascular endothelial growth
VEGFR1
receptor 1 precursor



factor/vascular permeability factor



receptor)


FOLR1
folate receptor 1 (adult)
FOLR1
Folate receptor alpha precursor


FOLR2
folate receptor 2 (fetal)
FOLR2
Folate receptor beta precursor


FSHB
follicle stimulating hormone, beta
FSHB
Follitropin subunit beta precursor



polypeptide


FSHPRH1,
centromere protein I
FSHPRH1,
Centromere protein I


CENP1

CENP1


FSHR
follicle stimulating hormone
FSHR
Follicle-stimulating hormone receptor



receptor

precursor


FYN
FYN oncogene related to SRC, FGR,
FYN
Tyrosine-protein kinase Fyn



YES


GART
phosphoribosylglycinamide
GART,
Trifunctional purine biosynthetic protein



formyltransferase,
PUR2
adenosine-3



phosphoribosylglycinamide



synthetase,



phosphoribosylaminoimidazole



synthetase


GNA11,
guanine nucleotide binding protein
GNA11, G
Guanine nucleotide-binding protein


GA11
(G protein), alpha 11 (Gq class)
alpha-11, G-
subunit alpha-11




protein




subunit




alpha-11


GNAQ,
guanine nucleotide binding protein
GNAQ
Guanine nucleotide-binding protein G(q)


GAQ
(G protein), q polypeptide

subunit alpha


GNRH1
gonadotropin-releasing hormone 1
GNRH1,
Progonadoliberin-1 precursor



(luteinizing-releasing hormone)
GON1


GNRHR1,
gonadotropin-releasing hormone
GNRHR1
Gonadotropin-releasing hormone


GNRHR
receptor

receptor


GSTP1
glutathione S-transferase pi 1
GSTP1
Glutathione S-transferase P


HCK
hemopoietic cell kinase
HCK
Tyrosine-protein kinase HCK


HDAC1
histone deacetylase 1
HDAC1
Histone deacetylase 1


HGF
hepatocyte growth factor
HGF
Hepatocyte growth factor precursor



(hepapoietin A; scatter factor)


HIF1A
hypoxia inducible factor 1, alpha
HIF1A
Hypoxia-inducible factor 1-alpha



subunit (basic helix-loop-helix



transcription factor)


HIG1,
HIG1 hypoxia inducible domain
HIG1,
HIG1 domain family member 1A


HIGD1A,
family, member 1A
HIGD1A,


HIG1A

HIG1A


HSP90AA1,
heat shock protein 90 kDa alpha
HSP90,
Heat shock protein HSP 90-alpha


HSP90,
(cytosolic), class A member 1
HSP90A


HSPCA


IGF1R
insulin-like growth factor 1 receptor
IGF-1R
Insulin-like growth factor 1 receptor





precursor


IGFBP3,
insulin-like growth factor binding
IGFBP-3,
Insulin-like growth factor-binding


IGFRBP3
protein 3
IBP-3
protein 3 precursor


IGFBP4,
insulin-like growth factor binding
IGFBP-4,
Insulin-like growth factor-binding


IGFRBP4
protein 4
IBP-4
protein 4 precursor


IGFBP5,
insulin-like growth factor binding
IGFBP-5,
Insulin-like growth factor-binding


IGFRBP5
protein 5
IBP-5
protein 5 precursor


IL13RA1
interleukin 13 receptor, alpha 1
IL-13RA1
Interleukin-13 receptor subunit alpha-1





precursor


KDR
kinase insert domain receptor (a type
KDR,
Vascular endothelial growth factor



III receptor tyrosine kinase)
VEGFR2
receptor 2 precursor


KIT, c-KIT
v-kit Hardy-Zuckerman 4 feline
KIT, c-KIT,
Mast/stem cell growth factor receptor



sarcoma viral oncogene homolog
CD117,
precursor




SCFR


KRAS
v-Ki-ras2 Kirsten rat sarcoma viral
K-RAS
GTPase KRas precursor



oncogene homolog


LCK
lymphocyte-specific protein tyrosine
LCK
Tyrosine-protein kinase Lck



kinase


LTB
lymphotoxin beta (TNF superfamily,
LTB, TNF3
Lymphotoxin-beta



member 3)


LTBR
lymphotoxin beta receptor (TNFR
LTBR,
Tumor necrosis factor receptor



superfamily, member 3)
LTBR3,
superfamily member 3 precursor




TNFR


LYN
v-yes-1 Yamaguchi sarcoma viral
LYN
Tyrosine-protein kinase Lyn



related oncogene homolog


MET, c-
met proto-oncogene (hepatocyte
MET, c-
Hepatocyte growth factor receptor


MET
growth factor receptor)
MET
precursor


MGMT
O-6-methylguanine-DNA
MGMT
Methylated-DNA--protein-cysteine



methyltransferase

methyltransferase


MKI67,
antigen identified by monoclonal
Ki67, Ki-67
Antigen KI-67


KI67
antibody Ki-67


MLH1
mutL homolog 1, colon cancer,
MLH1
DNA mismatch repair protein Mlh1



nonpolyposis type 2 (E. coli)


MMR
mismatch repair (refers to MLH1,



MSH2, MSH5)


MSH2
mutS homolog 2, colon cancer,
MSH2
DNA mismatch repair protein Msh2



nonpolyposis type 1 (E. coli)


MSH5
mutS homolog 5 (E. coli)
MSH5,
MutS protein homolog 5




hMSH5


MYC, c-
v-myc myelocytomatosis viral
MYC, c-
Myc proto-oncogene protein


MYC
oncogene homolog (avian)
MYC


NBN, P95
nibrin
NBN, p95
Nibrin


NDGR1
N-myc downstream regulated 1
NDGR1
Protein NDGR1


NFKB1
nuclear factor of kappa light
NFKB1
Nuclear factor NF-kappa-B p105



polypeptide gene enhancer in B-cells 1

subunit


NFKB2
nuclear factor of kappa light
NFKB2
Nuclear factor NF-kappa-B p100 subunit



polypeptide gene enhancer in B-cells



2 (p49/p100)


NFKBIA
nuclear factor of kappa light
NFKBIA
NF-kappa-B inhibitor alpha



polypeptide gene enhancer in B-cells



inhibitor, alpha


NRAS
neuroblastoma RAS viral (v-ras)
NRAS
GTPase NRas, Transforming protein N-



oncogene homolog

Ras


ODC1
ornithine decarboxylase 1
ODC
Ornithine decarboxylase


OGFR
opioid growth factor receptor
OGFR
Opioid growth factor receptor


PARP1
poly (ADP-ribose) polymerase 1
PARP-1
Poly [ADP-ribose] polymerase 1


PDGFC
platelet derived growth factor C
PDGF-C,
Platelet-derived growth factor C




VEGF-E
precursor


PDGFR
platelet-derived growth factor
PDGFR
Platelet-derived growth factor receptor



receptor


PDGFRA
platelet-derived growth factor
PDGFRA,
Alpha-type platelet-derived growth



receptor, alpha polypeptide
PDGFR2,
factor receptor precursor




CD140 A


PDGFRB
platelet-derived growth factor
PDGFRB,
Beta-type platelet-derived growth factor



receptor, beta polypeptide
PDGFR,
receptor precursor




PDGFR1,




CD140 B


PGR
progesterone receptor
PR
Progesterone receptor


PIK3CA
phosphoinositide-3-kinase, catalytic,
PI3K subunit
phosphoinositide-3-kinase, catalytic,



alpha polypeptide
p110α
alpha polypeptide


POLA1
polymerase (DNA directed), alpha 1,
POLA,
DNA polymerase alpha catalytic subunit



catalytic subunit; polymerase (DNA
POLA1,



directed), alpha, polymerase (DNA
p180



directed), alpha 1


PPARG,
peroxisome proliferator-activated
PPARG
Peroxisome proliferator-activated


PPARG1,
receptor gamma

receptor gamma


PPARG2,


PPAR-


gamma,


NR1C3


PPARGC1A,
peroxisome proliferator-activated
PGC-1-
Peroxisome proliferator-activated


LEM6,
receptor gamma, coactivator 1 alpha
alpha,
receptor gamma coactivator 1-alpha;


PGC1,

PPARGC-1-
PPAR-gamma coactivator 1-alpha


PGC1A,

alpha


PPARGC1


PSMD9,
proteasome (prosome, macropain)
p27
26S proteasome non-ATPase regulatory


P27
26S subunit, non-ATPase, 9

subunit 9


PTEN,
phosphatase and tensin homolog
PTEN
Phosphatidylinositol-3,4,5-trisphosphate


MMAC1,


3-phosphatase and dual-specificity


TEP1


protein phosphatase; Mutated in multiple





advanced cancers 1


PTPN12
protein tyrosine phosphatase, non-
PTPG1
Tyrosine-protein phosphatase non-



receptor type 12

receptor type 12; Protein-tyrosine





phosphatase G1


RAF1
v-raf-1 murine leukemia viral
RAF, RAF-
RAF proto-oncogene serine/threonine-



oncogene homolog 1
1, c-RAF
protein kinase


RARA
retinoic acid receptor, alpha
RAR, RAR-
Retinoic acid receptor alpha




alpha,




RARA


ROS1,
c-ros oncogene 1, receptor tyrosine
ROS1, ROS
Proto-oncogene tyrosine-protein kinase


ROS,
kinase

ROS


MCF3


RRM1
ribonucleotide reductase M1
RRM1, RR1
Ribonucleoside-diphosphate reductase





large subunit


RRM2
ribonucleotide reductase M2
RRM2,
Ribonucleoside-diphosphate reductase




RR2M, RR2
subunit M2


RRM2B
ribonucleotide reductase M2 B (TP53
RRM2B,
Ribonucleoside-diphosphate reductase



inducible)
P53R2
subunit M2 B


RXRB
retinoid X receptor, beta
RXRB
Retinoic acid receptor RXR-beta


RXRG
retinoid X receptor, gamma
RXRG,
Retinoic acid receptor RXR-gamma




RXRC


SIK2
salt-inducible kinase 2
SIK2,
Salt-inducible protein kinase 2;




Q9H0K1
Serine/threonine-protein kinase SIK2


SLC29A1
solute carrier family 29 (nucleoside
ENT-1
Equilibrative nucleoside transporter 1



transporters), member 1


SPARC
secreted protein, acidic, cysteine-rich
SPARC
SPARC precursor; Osteonectin



(osteonectin)


SRC
v-src sarcoma (Schmidt-Ruppin A-2)
SRC
Proto-oncogene tyrosine-protein kinase



viral oncogene homolog (avian)

Src


SSTR1
somatostatin receptor 1
SSTR1,
Somatostatin receptor type 1




SSR1, SS1R


SSTR2
somatostatin receptor 2
SSTR2,
Somatostatin receptor type 2




SSR2, SS2R


SSTR3
somatostatin receptor 3
SSTR3,
Somatostatin receptor type 3




SSR3, SS3R


SSTR4
somatostatin receptor 4
SSTR4,
Somatostatin receptor type 4




SSR4, SS4R


SSTR5
somatostatin receptor 5
SSTR5,
Somatostatin receptor type 5




SSR5, SS5R


TK1
thymidine kinase 1, soluble
TK1, KITH
Thymidine kinase, cytosolic


TLE3
transducin-like enhancer of split 3
TLE3
Transducin-like enhancer protein 3



(E(sp1) homolog, Drosophila)


TNF
tumor necrosis factor (TNF
TNF, TNF-
Tumor necrosis factor precursor



superfamily, member 2)
alpha, TNF-a


TOP1,
topoisomerase (DNA) I
TOP1,
DNA topoisomerase 1


TOPO1

TOPO1


TOP2A,
topoisomerase (DNA) II alpha
TOP2A,
DNA topoisomerase 2-alpha;


TOPO2A
170 kDa
TOP2,
Topoisomerase II alpha




TOPO2A


TOP2B,
topoisomerase (DNA) II beta
TOP2B,
DNA topoisomerase 2-beta;


TOPO2B
180 kDa
TOPO2B
Topoisomerase II beta


TP53
tumor protein p53
p53
Cellular tumor antigen p53


TUBB3
tubulin, beta 3
Beta III
Tubulin beta-3 chain




tubulin,




TUBB3,




TUBB4


TXN
thioredoxin
TXN, TRX,
Thioredoxin




TRX-1


TXNRD1
thioredoxin reductase 1
TXNRD1,
Thioredoxin reductase 1, cytoplasmic;




TXNR
Oxidoreductase


TYMS, TS
thymidylate synthetase
TYMS, TS
Thymidylate synthase


VDR
vitamin D (1,25-dihydroxyvitamin
VDR
Vitamin D3 receptor



D3) receptor


VEGFA,
vascular endothelial growth factor A
VEGF-A,
Vascular endothelial growth factor A


VEGF

VEGF
precursor


VEGFC
vascular endothelial growth factor C
VEGF-C
Vascular endothelial growth factor C





precursor


VHL
von Hippel-Lindau tumor suppressor
VHL
Von Hippel-Lindau disease tumor





suppressor


YES1
v-yes-1 Yamaguchi sarcoma viral
YES1, Yes,
Proto-oncogene tyrosine-protein kinase



oncogene homolog 1
p61-Yes
Yes


ZAP70
zeta-chain (TCR) associated protein
ZAP-70
Tyrosine-protein kinase ZAP-70



kinase 70 kDa









In some embodiments, additional molecular profiling methods are performed. These can include without limitation PCR, RT-PCR, Q-PCR, SAGE, MPSS, immunoassays and other techniques to assess biological systems described herein or known to those of skill in the art. The choice of genes and gene products to be assayed can be updated over time as new treatments and new drug targets are identified. Once the expression or mutation of a biomarker is correlated with a treatment option, it can be assessed by molecular profiling. One of skill will appreciate that such molecular profiling is not limited to those techniques disclosed herein but comprises any methodology conventional for assessing nucleic acid or protein levels, sequence information, or both. The methods of the invention can also take advantage of any improvements to current methods or new molecular profiling techniques developed in the future. In some embodiments, a gene or gene product is assessed by a single molecular profiling technique. In other embodiments, a gene and/or gene product is assessed by multiple molecular profiling techniques. In a non-limiting example, a gene sequence can be assayed by one or more of FISH and pyrosequencing analysis, the mRNA gene product can be assayed by one or more of RT-PCR and microarray, and the protein gene product can be assayed by one or more of IHC and immunoassay. One of skill will appreciate that any combination of biomarkers and molecular profiling techniques that will benefit disease treatment are contemplated by the invention.


Genes and gene products that are known to play a role in cancer and can be assayed by any of the molecular profiling techniques of the invention include without limitation 2AR, A DISINTEGRIN, ACTIVATOR OF THYROID AND RETINOIC ACID RECEPTOR (ACTR), ADAM 11, ADIPOGENESIS INHIBITORY FACTOR (ADIF), ALPHA 6 INTEGRIN SUBUNIT, ALPHA V INTEGRIN SUBUNIT, ALPHA-CATENIN, AMPLIFIED IN BREAST CANCER 1 (AIB1), AMPLIFIED IN BREAST CANCER 3 (AIB3), AMPLIFIED IN BREAST CANCER 4 (AIB4), AMYLOID PRECURSOR PROTEIN SECRETASE (APPS), AP-2 GAMMA, APPS, ATP-BINDING CASSETTE TRANSPORTER (ABCT), PLACENTA-SPECIFIC (ABCP), ATP-BINDING CASSETTE SUBFAMILY C MEMBER (ABCC1), BAG-1, BASIGIN (BSG), BCEI, B-CELL DIFFERENTIATION FACTOR (BCDF), B-CELL LEUKEMIA 2 (BCL-2), B-CELL STIMULATORY FACTOR-2 (BSF-2), BCL-1, BCL-2-ASSOCIATED X PROTEIN (BAX), BCRP, BETA 1 INTEGRIN SUBUNIT, BETA 3 INTEGRIN SUBUNIT, BETA 5 INTEGRIN SUBUNIT, BETA-2 INTERFERON, BETA-CATENIN, BETA-CATENIN, BONE SIALOPROTEIN (BSP), BREAST CANCER ESTROGEN-INDUCIBLE SEQUENCE (BCEI), BREAST CANCER RESISTANCE PROTEIN (BCRP), BREAST CANCER TYPE 1 (BRCA1), BREAST CANCER TYPE 2 (BRCA2), BREAST CARCINOMA AMPLIFIED SEQUENCE 2 (BCAS2), CADHERIN, EPITHELIAL CADHERIN-11, CADHERIN-ASSOCIATED PROTEIN, CALCITONIN RECEPTOR (CTR), CALCIUM PLACENTAL PROTEIN (CAPL), CALCYCLIN, CALLA, CAM5, CAPL, CARCINOEMBRYONIC ANTIGEN (CEA), CATENIN, ALPHA 1, CATHEPSIN B, CATHEPSIN D, CATHEPSIN K, CATHEPSIN L2, CATHEPSIN O, CATHEPSIN O1, CATHEPSIN V, CD10, CD146, CD147, CD24, CD29, CD44, CD51, CD54, CD61, CD66e, CD82, CD87, CD9, CEA, CELLULAR RETINOL-BINDING PROTEIN 1 (CRBP1), c-ERBB-2, CK7, CK8, CK18, CK19, CK20, CLAUDIN-7, c-MET, COLLAGENASE, FIBROBLAST, COLLAGENASE, INTERSTITIAL, COLLAGENASE-3, COMMON ACUTE LYMPHOCYTIC LEUKEMIA ANTIGEN (CALLA), CONNEXIN 26 (Cx26), CONNEXIN 43 (Cx43), CORTACTIN, COX-2, CTLA-8, CTR, CTSD, CYCLIN D1, CYCLOOXYGENASE-2, CYTOKERATIN 18, CYTOKERATIN 19, CYTOKERATIN 8, CYTOTOXIC T-LYMPHOCYTE-ASSOCIATED SERINE ESTERASE 8 (CTLA-8), DIFFERENTIATION-INHIBITING ACTIVITY (DIA), DNA AMPLIFIED IN MAMMARY CARCINOMA 1 (DAM1), DNA TOPOISOMERASE II ALPHA, DR-NM23, E-CADHERIN, EMMPRIN, EMS 1, ENDOTHELIAL CELL GROWTH FACTOR (ECGR), PLATELET-DERIVED (PD-ECGF), ENKEPHALINASE, EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR), EPISIALIN, EPITHELIAL MEMBRANE ANTIGEN (EMA), ER-ALPHA, ERBB2, ERBB4, ER-BETA, ERF-1, ERYTHROID-POTENTIATING ACTIVITY (EPA), ESR1, ESTROGEN RECEPTOR-ALPHA, ESTROGEN RECEPTOR-BETA, ETS-1, EXTRACELLULAR MATRIX METALLOPROTEINASE INDUCER (EMMPRIN), FIBRONECTIN RECEPTOR, BETA POLYPEPTIDE (FNRB), FIBRONECTIN RECEPTOR BETA SUBUNIT (FNRB), FLK-1, GA15.3, GA733.2, GALECTIN-3, GAMMA-CATENIN, GAP JUNCTION PROTEIN (26 kDa), GAP JUNCTION PROTEIN (43 kDa), GAP JUNCTION PROTEIN ALPHA-1 (GJA1), GAP JUNCTION PROTEIN BETA-2 (GJB2), GCP1, GELATINASE A, GELATINASE B, GELATINASE (72 kDa), GELATINASE (92 kDa), GLIOSTATIN, GLUCOCORTICOID RECEPTOR INTERACTING PROTEIN 1 (GRIPI), GLUTATHIONE S-TRANSFERASE p, GM-CSF, GRANULOCYTE CHEMOTACTIC PROTEIN 1 (GCP1), GRANULOCYTE-MACROPHAGE-COLONY STIMULATING FACTOR, GROWTH FACTOR RECEPTOR BOUND-7 (GRB-7), GSTp, HAP, HEAT-SHOCK COGNATE PROTEIN 70 (HSC70), HEAT-STABLE ANTIGEN, HEPATOCYTE GROWTH FACTOR (HGF), HEPATOCYTE GROWTH FACTOR RECEPTOR (HGFR), HEPATOCYTE-STIMULATING FACTOR III (HSF III), HER-2, HER2/NEU, HERMES ANTIGEN, HET, HHM, HUMORAL HYPERCALCEMIA OF MALIGNANCY (HHM), ICERE-1, INT-1, INTERCELLULAR ADHESION MOLECULE-1 (ICAM-1), INTERFERON-GAMMA-INDUCING FACTOR (IGIF), INTERLEUKIN-1 ALPHA (IL-1A), INTERLEUKIN-1 BETA (IL-1B), INTERLEUKIN-11 (IL-11), INTERLEUKIN-17 (IL-17), INTERLEUKIN-18 (IL-18), INTERLEUKIN-6 (IL-6), INTERLEUKIN-8 (IL-8), INVERSELY CORRELATED WITH ESTROGEN RECEPTOR EXPRESSION-1 (ICERE-1), KAI1, KDR, KERATIN 8, KERATIN 18, KERATIN 19, KISS-1, LEUKEMIA INHIBITORY FACTOR (LIF), LIF, LOST IN INFLAMMATORY BREAST CANCER (LIBC), LOT (“LOST ON TRANSFORMATION”), LYMPHOCYTE HOMING RECEPTOR, MACROPHAGE-COLONY STIMULATING FACTOR, MAGE-3, MAMMAGLOBIN, MASPIN, MC56, M-CSF, MDC, MDNCF, MDR, MELANOMA CELL ADHESION MOLECULE (MCAM), MEMBRANE METALLOENDOPEPTIDASE (MME), MEMBRANE-ASSOCIATED NEUTRAL ENDOPEPTIDASE (NEP), CYSTEINE-RICH PROTEIN (MDC), METASTASIN (MTS-1), MLN64, MMP1, MMP2, MMP3, MMP7, MMP9, MMP11, MMP13, MMP14, MMP15, MMP16, MMP17, MOESIN, MONOCYTE ARGININE-SERPIN, MONOCYTE-DERIVED NEUTROPHIL CHEMOTACTIC FACTOR, MONOCYTE-DERIVED PLASMINOGEN ACTIVATOR INHIBITOR, MTS-1, MUC-1, MUC18, MUCIN LIKE CANCER ASSOCIATED ANTIGEN (MCA), MUCIN, MUC-1, MULTIDRUG RESISTANCE PROTEIN 1 (MDR, MDR1), MULTIDRUG RESISTANCE RELATED PROTEIN-1 (MRP, MRP-1), N-CADHERIN, NEP, NEU, NEUTRAL ENDOPEPTIDASE, NEUTROPHIL-ACTIVATING PEPTIDE 1 (NAP1), NM23-H1, NM23-H2, NME1, NME2, NUCLEAR RECEPTOR COACTIVATOR-1 (NCoA-1), NUCLEAR RECEPTOR COACTIVATOR-2 (NCoA-2), NUCLEAR RECEPTOR COACTIVATOR-3 (NCoA-3), NUCLEOSIDE DIPHOSPHATE KINASE A (NDPKA), NUCLEOSIDE DIPHOSPHATE KINASE B (NDPKB), ONCOSTATIN M (OSM), ORNITHINE DECARBOXYLASE (ODC), OSTEOCLAST DIFFERENTIATION FACTOR (ODF), OSTEOCLAST DIFFERENTIATION FACTOR RECEPTOR (ODFR), OSTEONECTIN (OSN, ON), OSTEOPONTIN (OPN), OXYTOCIN RECEPTOR (OXTR), p27/kip1, p300/CBP COINTEGRATOR ASSOCIATE PROTEIN (p/CIP), p53, p9Ka, PAI-1, PAI-2, PARATHYROID ADENOMATOSIS 1 (PRAD 1), PARATHYROID HORMONE-LIKE HORMONE (PTHLH), PARATHYROID HORMONE-RELATED PEPTIDE (PTHrP), P-CADHERIN, PD-ECGF, PDGF, PEANUT-REACTIVE URINARY MUCIN (PUM), P-GLYCOPROTEIN (P-GP), PGP-1, PHGS-2, PHS-2, PIP, PLAKOGLOBIN, PLASMINOGEN ACTIVATOR INHIBITOR (TYPE 1), PLASMINOGEN ACTIVATOR INHIBITOR (TYPE 2), PLASMINOGEN ACTIVATOR (TISSUE-TYPE), PLASMINOGEN ACTIVATOR (UROKINASE-TYPE), PLATELET GLYCOPROTEIN IIIa (GP3A), PLAU, PLEOMORPHIC ADENOMA GENE-LIKE 1 (PLAGL1), POLYMORPHIC EPITHELIAL MUCIN (PEM), PRADI, PROGESTERONE RECEPTOR (PgR), PROGESTERONE RESISTANCE, PROSTAGLANDIN ENDOPEROXIDE SYNTHASE-2, PROSTAGLANDIN G/H SYNTHASE-2, PROSTAGLANDIN H SYNTHASE-2, pS2, PS6K, PSORIASIN, PTHLH, PTHrP, RAD51, RAD52, RAD54, RAP46, RECEPTOR-ASSOCIATED COACTIVATOR 3 (RAC3), REPRESSOR OF ESTROGEN RECEPTOR ACTIVITY (REA), S100A4, S100A6, S100A7, S6K, SART-1, SCAFFOLD ATTACHMENT FACTOR B (SAF-B), SCATTER FACTOR (SF), SECRETED PHOSPHOPROTEIN-1 (SPP-1), SECRETED PROTEIN, ACIDIC AND RICH IN CYSTEINE (SPARC), STANNICALCIN, STEROID RECEPTOR COACTIVATOR-1 (SRC-1), STEROID RECEPTOR COACTIVATOR-2 (SRC-2), STEROID RECEPTOR COACTIVATOR-3 (SRC-3), STEROID RECEPTOR RNA ACTIVATOR (SRA), STROMELYSIN-1, STROMELYSIN-3, TENASCIN-C(TN-C), TESTES-SPECIFIC PROTEASE 50, THROMBOSPONDIN I, THROMBOSPONDIN II, THYMIDINE PHOSPHORYLASE (TP), THYROID HORMONE RECEPTOR ACTIVATOR MOLECULE 1 (TRAM-1), TIGHT JUNCTION PROTEIN 1 (TJPI), TIMP1, TIMP2, TIMP3, TIMP4, TISSUE-TYPE PLASMINOGEN ACTIVATOR, TN-C, TP53, tPA, TRANSCRIPTIONAL INTERMEDIARY FACTOR 2 (TIF2), TREFOIL FACTOR 1 (TFF1), TSG101, TSP-1, TSP1, TSP-2, TSP2, TSP50, TUMOR CELL COLLAGENASE STIMULATING FACTOR (TCSF), TUMOR-ASSOCIATED EPITHELIAL MUCIN, uPA, uPAR, UROKINASE, UROKINASE-TYPE PLASMINOGEN ACTIVATOR, UROKINASE-TYPE PLASMINOGEN ACTIVATOR RECEPTOR (uPAR), UVOMORULIN, VASCULAR ENDOTHELIAL GROWTH FACTOR, VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR-2 (VEGFR2), VASCULAR ENDOTHELIAL GROWTH FACTOR-A, VASCULAR PERMEABILITY FACTOR, VEGFR2, VERY LATE T-CELL ANTIGEN BETA (VLA-BETA), VIMENTIN, VITRONECTIN RECEPTOR ALPHA POLYPEPTIDE (VNRA), VITRONECTIN RECEPTOR, VON WILLEBRAND FACTOR, VPF, VWF, WNT-1, ZAC, ZO-1, and ZONULA OCCLUDENS-1.


The gene products used for IHC expression profiling include without limitation one or more of AR, BCRP, BCRPI, BRCA1, CAV-1, CK 5/6, CK14, CK17, c-Kit, cMET, cMYC, COX2, Cyclin D1, ECAD, EGFR, ER, ERCC1, Her2/Neu, IGF1R, IGFRBP1, IGFRBP2, IGFRBP3, IGFRBP4, IGFRBP5, IGFRBP6, IGFRBP7, Ki67, MGMT, MRP1, P53, P95, PDGFR, PDGFRA, PGP (MDR1), PR, PTEN, RRM1, SPARC, TLE3, TOP1, TOP2, TOP2A, TS, and TUBB3. In an embodiment, the IHC is performed on AR, BCRP, CAV-1, CK 5/6, CK14, CK17, c-Kit, COX2, Cyclin D1, ECAD, EGFR, ER, ERCC1, Her2/Neu, IGF1R, Ki67, MGMT, MRP1, P53, P95, PDGFRa, PGP (MDR1), PR, PTEN, RRM1, SPARC, TLE3, TOP1, TOP2A, TS, and TUBB3. In some embodiments, IHC analysis includes one or more of c-Met, EML4-ALK fusion, hENT-1, IGF-1R, MMR, p16, p21, p27, PARP-1, PI3K, and TLE3. IHC profiling of EGFR can also be performed. IHC is also used to detect or test for various gene products, including without limitation one or more of the following: EGFR, SPARC, C-kit, ER, PR, Androgen receptor, PGP, RRM1, TOPO1, BRCP1, MRP1, MGMT, PDGFR, DCK, ERCC1, Thymidylate synthase, Her2/neu, or TOPO2A. In some embodiments, IHC is used to detect on or more of the following proteins, including without limitation: ADA, AR, ASNA, BCL2, BRCA2, c-Met, CD33, CDW52, CES2, DNMT1, EGFR, EML4-ALK fusion, ERBB2, ERCC3, ESR1, FOLR2, GART, GSTP1, HDAC1, hENT-1, HIF1A, HSPCA, IGF-1R, IL2RA, KIT, MLH1, MMR, MS4A1, MASH2, NFKB2, NFKBIA, OGFR, p16, p21, p27, PARP-1, PI3K, PDGFC, PDGFRA, PDGFRB, PGR, POLA, PTEN, PTGS2, RAF1, RARA, RXRB, SPARC, SSTR1, TK1, TLE3, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGF, VHL, or ZAP70. The proteins can be detected by IHC using monoclonal or polyclonal antibodies. In some embodiments, both are used. As an illustrative example, SPARC can be detected by anti-SPARC monoclonal (SPARC mono, SPARC m) and/or anti-SPARC polyclonal (SPARC poly, SPARC p) antibodies.


In some embodiments, IHC analysis according to the methods of the invention includes one or more of AR, c-Kit, COX2, CAV-1, CK 5/6, CK14, CK17, ECAD, ER, Her2/Neu, Ki67, MRP1, P53, PDGFR, PGP, PR, PTEN, SPARC, TLE3 and TS. All of these genes can be examined. As indicated by initial results of IHC or other molecular profiling methods as described herein, additional IHC assayscan be performed. In one embodiment, the additional IHC comprises that of p95, or p95, Cyclin D1 and EGFR. IHC can also be performed on IGFRBP3, IGFRBP4, IGFRBP5, or other forms of IGFRBP (e.g., IGFRBP1, IGFRBP2, IGFRBP6, IGFRBP7). In another embodiment, the additional IHC comprises that of one or more of BCRP, ERCC1, MGMT, P95, RRM1, TOP2A, and TOP1. In still another embodiment, the additional IHC comprises that of one or more of BCRP, Cyclin D1, EGFR, ERCC1, MGMT, P95, RRM1, TOP2A, and TOP1. Any useful subset or all of these genes can be examined. The additional IHC can be selected on the basis of molecular characteristics of the tumor so that IHC is only performed where it is likely to indicate a candidate therapy for treating the cancer. As described herein, the molecular characteristics of the tumor determined can be determined by IHC combined with one or more of FISH, DNA microarray and mutation analysis. The genes and/or gene products used for IHC analysis can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or all of the genes and/or gene products listed in Table 2.


Microarray expression profiling can be used to simultaneously measure the expression of one or more genes or gene products, including without limitation ABCC1, ABCG2, ADA, AR, ASNS, BCL2, BIRC5, BRCA1, BRCA2, CD33, CD52, CDA, CES2, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2, ERBB2, ERCC1, ERCC3, ESR1, FLT1, FOLR2, FYN, GART, GNRH1, GSTP1, HCK, HDAC1, HIF1A, HSP90AA1, IGFBP3, IGFBP4, IGFBP5, IL2RA, KDR, KIT, LCK, LYN, MET, MGMT, MLH1, MS4A1, MSH2, NFKB1, NFKB2, NFKBIA, OGFR, PARP1, PDGFC, PDGFRA, PDGFRB, PGP, PGR, POLA1, PTEN, PTGS2, RAF1, RARA, RRM1, RRM2, RRM2B, RXRB, RXRG, SIK2, SPARC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGFA, VHL, YES1, and ZAP70. In some embodiments, the genes used for the microarray expression profiling comprise one or more of: EGFR, SPARC, C-kit, ER, PR, Androgen receptor, PGP, RRM1, TOPO1, BRCP1, MRP1, MGMT, PDGFR, DCK, ERCC1, Thymidylate synthase, Her2/neu, TOPO2A, ADA, AR, ASNA, BCL2, BRCA2, CD33, CDW52, CES2, DNMT1, EGFR, ERBB2, ERCC3, ESR1, FOLR2, GART, GSTP1, HDAC1, HIF1A, HSPCA, IL2RA, KIT, MLH1, MS4A1, MASH2, NFKB2, NFKBIA, OGFR, PDGFC, PDGFRA, PDGFRB, PGR, POLA, PTEN, PTGS2, RAF1, RARA, RXRB, SPARC, SSTR1, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGF, VHL, and ZAP70. One or more of the following genes can also be assessed by microarray expression profiling: ALK, EML4, hENT-1, IGF-1R, HSP90AA1, MMR, p16, p21, p27, PARP-1, PI3K and TLE3. The microarray expression profiling can be performed using a low density microarray, an expression microarray, a comparative genomic hybridization (CGH) microarray, a single nucleotide polymorphism (SNP) microarray, a proteomic array an antibody array, or other array as disclosed herein or known to those of skill in the art. In some embodiments, high throughput expression arrays are used. Such systems include without limitation commercially available systems from Affymetrix, Agilent or Illumina, as described in more detail herein.


Microarray expression profiling can be used to simultaneously measure the expression of one or more genes or gene products, including without limitation ABCC1, ABCG2, ADA, AR, ASNS, BCL2, BIRC5, BRCA1, BRCA2, CD33, CD52, CDA, CES2, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2, ERBB2, ERCC1, ERCC3, ESR1, FLT1, FOLR2, FYN, GART, GNRH1, GSTP1, HCK, HDAC1, HIF1A, HSP90AA1, IGFBP3, IGFBP4, IGFBP5, IL2RA, KDR, KIT, LCK, LYN, MET, MGMT, MLH1, MS4A1, MSH2, NFKB1, NFKB2, NFKBIA, OGFR, PARP1, PDGFC, PDGFRA, PDGFRB, PGP, PGR, POLA1, PTEN, PTGS2, PTPN12, RAF1, RARA, RRM1, RRM2, RRM2B, RXRB, RXRG, SIK2, SPARC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGFA, VHL, YES1, and ZAP70. The genes and/or gene products used for RT-PCR profiling analysis can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or all of the genes and/or gene products listed in Table 2.


Expression profiling can be performed using PCR, e.g., real-time PCR (qPCR or RT-PCR). RT-PCR can be used to measure the expression of one or more genes or gene products, including without limitation ABCC1, ABCG2, ACE2, ADA, ADH1C, ADH4, AGT, AR, AREG, ASNS, BCL2, BCRP, BDCA1, beta III tubulin, BIRC5, B-RAF, BRCA1, BRCA2, CA2, caveolin, CD20, CD25, CD33, CD52, CDA, CDKN2A, CDKN1A, CDKN1B, CDK2, CDW52, CES2, CK 14, CK 17, CK 5/6, c-KIT, c-Met, c-Myc, COX-2, Cyclin D1, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, E-Cadherin, ECGF1, EGFR, EML4-ALK fusion, EPHA2, Epiregulin, ER, ERBR2, ERCC1, ERCC3, EREG, ESR1, FLT1, folate receptor, FOLR1, FOLR2, FSHB, FSHPRH1, FSHR, FYN, GART, GNA11, GNAQ, GNRH1, GNRHR1, GSTP1, HCK, HDAC1, hENT-1, Her2/Neu, HGF, HIF1A, HIG1, HSP90, HSP90AA1, HSPCA, IGF-1R, IGFRBP, IGFRBP3, IGFRBP4, IGFRBP5, IL13RA1, IL2RA, KDR, Ki67, KIT, K-RAS, LCK, LTB, Lymphotoxin Beta Receptor, LYN, MET, MGMT, MLH1, MMR, MRP1, MS4A1, MSH2, MSH5, Myc, NFKB1, NFKB2, NFKBIA, NRAS, ODC1, OGFR, p16, p21, p27, p53, p95, PARP-1, PDGFC, PDGFR, PDGFRA, PDGFRB, PGP, PGR, PI3K, POLA, POLA1, PPARG, PPARGC1, PR, PTEN, PTGS2, PTPN12, RAF1, RARA, ROS1, RRM1, RRM2, RRM2B, RXRB, RXRG, SIK2, SPARC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, Survivin, TK1, TLE3, TNF, TOP1, TOP2A, TOP2B, TS, TUBB3, TXN, TXNRD1, TYMS, VDR, VEGF, VEGFA, VEGFC, VHL, YES1, ZAP70. For example, the genes assessed by RT-PCR can include AREG, BRCA1, EGFR, ERBB3, ERCC1, EREG, PGP (MDR-1), RRM1, TOPO1, TOPO2A, TS, TUBB3 and VEGFR2. The genes and/or gene products used for real-time PCR analysis can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or all of the genes and/or gene products listed in Table 2. The PCR can be performed in a high throughput fashion, e.g., using multiplex amplification, microfluidics, and/or using a low density microarray.


FISH analysis can be used to profile one or more of HER2, CMET, PIK3CA, EGFR, TOP2A, CMYC and EML4-ALK fusion. In some embodiments, FISH is used to detect or test for one or more of the following genes, including without limitation: EGFR, SPARC, C-kit, ER, PR, AR, PGP, RRM1, TOPO1, BRCP1, MRP1, MGMT, PDGFR, DCK, ERCC1, TS, HER2, or TOPO2A. In some embodiments, FISH is used to detect or test for one or more of EML4-ALK fusion and IGF-1R. In some embodiments, FISH is used to detect or test various biomarkers, including without limitation one or more of the following: ADA, AR, ASNA, BCL2, BRCA2, c-Met, CD33, CDW52, CES2, DNMT1, EGFR, EML4-ALK fusion, ERBB2, ERCC3, ESR1, FOLR2, GART, GSTP1, HDAC1, hENT-1, HIF1A, HSPCA, IGF-1R, IL2RA, KIT, MLH1, MMR, MS4A1, MASH2, NFKB2, NFKBIA, OGFR, p16, p21, p27, PARP-1, PI3K, PDGFC, PDGFRA, PDGFRB, PGR, POLA, PTEN, PTGS2, RAF1, RARA, RXRB, SPARC, SSTR1, TK1, TLE3, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGF, VHL, or ZAP70.


In some embodiments, FISH is used to detect or test for HER2, and depending on the results of the HER2 analysis and other molecular profiling techniques, additional FISH testing may be performed. The additional FISH testing can comprise that of CMYC and/or TOP2A. For example, FISH testing may indicate that a cancer is HER2+. The cancer may be a breast cancer. HER2+ cancers may then be followed up by FISH testing for CMYC and TOP2A, whereas HER2− cancers are followed up with FISH testing for CMYC. For some cancers, e.g., triple negative breast cancer (i.e., ER−/PR−/HER2−), additional FISH testing may not be performed. The decision whether to perform additional FISH testing can be guided by whether the additional FISH testing is likely to reveal information about candidate therapies for the cancer. The additional FISH can be selected on the basis of molecular characteristics of the tumor so that FISH is only performed where it is likely to indicate a candidate therapy for treating the cancer. As described herein, the molecular characteristics of the tumor determined can be determined by one or more of IHC, FISH, DNA microarray and sequence analysis. The genes and/or gene products used for FISH analysis can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or all of the genes and/or gene products listed in Table 2.


In some embodiments, the genes used for the mutation profiling comprise one or more of PIK3CA, EGFR, cKIT, KRAS, NRAS and BRAF. Mutation profiling can be determined by sequencing, including Sanger sequencing, array sequencing, pyrosequencing, NextGen sequencing, etc. Sequence analysis may reveal that genes harbor activating mutations so that drugs that inhibit activity are indicated for treatment. Alternately, sequence analysis may reveal that genes harbor mutations that inhibit or eliminate activity, thereby indicating treatment for compensating therapies. In embodiments, sequence analysis comprises that of exon 9 and 11 of c-KIT. Sequencing may also be performed on EGFR-kinase domain exons 18, 19, 20, and 21. Mutations, amplifications or misregulations of EGFR or its family members are implicated in about 30% of all epithelial cancers. Sequencing can also be performed on PI3K, encoded by the PIK3CA gene. This gene is a found mutated in many cancers. Sequencing analysis can also comprise assessing mutations in one or more ABCC1, ABCG2, ADA, AR, ASNS, BCL2, BIRC5, BRCA1, BRCA2, CD33, CD52, CDA, CES2, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2, ERBB2, ERCC1, ERCC3, ESR1, FLT1, FOLR2, FYN, GART, GNRH1, GSTP1, HCK, HDAC1, HIF1A, HSP90AA1, IGFBP3, IGFBP4, IGFBP5, IL2RA, KDR, KIT, LCK, LYN, MET, MGMT, MLH1, MS4A1, MSH2, NFKB1, NFKB2, NFKBIA, NRAS, OGFR, PARP1, PDGFC, PDGFRA, PDGFRB, PGP, PGR, POLA1, PTEN, PTGS2, PTPN12, RAF1, RARA, RRM1, RRM2, RRM2B, RXRB, RXRG, SIK2, SPARC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGFA, VHL, YES1, and ZAP70. One or more of the following genes can also be assessed by sequence analysis: ALK, EML4, hENT-1, IGF-1R, HSP90AA1, MMR, p16, p21, p27, PARP-1, PI3K and TLE3. The genes and/or gene products used for mutation or sequence analysis can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or all of the genes and/or gene products listed in Table 2, Table 6 or Table 25.


In some embodiments, mutational analysis is performed on PIK3CA. The decision whether to perform mutational analysis on PIK3CA can be guided by whether this testing is likely to reveal information about candidate therapies for the cancer. The PIK3CA mutational analysis can be selected on the basis of molecular characteristics of the tumor so that the analysis is only performed where it is likely to indicate a candidate therapy for treating the cancer. As described herein, the molecular characteristics of the tumor determined can be determined by one or more of IHC, FISH, DNA microarray and sequence analysis. In one embodiment, PIK3CA is analyzed for a HER2+ cancer. The cancer can be a breast cancer.


In a related aspect, the invention provides a method of identifying a candidate treatment for a subject in need thereof by using molecular profiling of sets of known biomarkers. For example, the method can identify a chemotherapeutic agent for an individual with a cancer. The method comprises: obtaining a sample from the subject; performing an immunohistochemistry (IHC) analysis on the sample to determine an IHC expression profile on one or more, e.g. 2, 3, 4, 5, 6,7, 8, 9, 10 or more, of: SPARC, PGP, Her2/neu, ER, PR, c-kit, AR, CD52, PDGFR, TOP2A, TS, ERCC1, RRM1, BCRP, TOPO1, PTEN, MGMT, MRP1, c-Met, EML4-ALK fusion, hENT-1, IGF-1R, MMR, p16, p21, p27, PARP-1, PI3K, COX2 and TLE3; performing a microarray analysis on the sample to determine a microarray expression profile on one or more, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, of: ABCC1, ABCG2, ADA, AR, ASNS, BCL2, BIRC5, BRCA1, BRCA2, CD33, CD52, CDA, CES2, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2, ERBB2, ERCC1, ERCC3, ESR1, FLT1, FOLR2, FYN, GART, GNRH1, GSTP1, HCK, HDAC1, HIF1A, HSP90AA1, IGFBP3, IGFBP4, IGFBP5, IL2RA, KDR, KIT, LCK, LYN, MET, MGMT, MLH1, MS4A1, MSH2, NFKB1, NFKB2, NFKBIA, OGFR, PARP1, PDGFC, PDGFRA, PDGFRB, PGP, PGR, POLA1, PTEN, PTGS2, PTPN12, RAF1, RARA, RRM1, RRM2, RRM2B, RXRB, RXRG, SIK2, SPARC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGFA, VHL, YES1, and ZAP70; performing a fluorescent in-situ hybridization (FISH) analysis on the sample to determine a FISH mutation profile on at least one of EGFR, HER2, EML4-ALK fusion and IGF-1R; performing DNA sequencing on the sample to determine a sequencing mutation profile on at least one of KRAS, BRAF, c-KIT, PI3K (PIK3CA), NRAS and EGFR; and comparing the IHC expression profile, microarray expression profile, FISH mutation profile and sequencing mutation profile against a rules database, wherein the rules database comprises a mapping of treatments whose biological activity is known against diseased cells that: i) overexpress or underexpress one or more proteins included in the IHC expression profile; ii) overexpress or underexpress one or more genes included in the microarray expression profile; iii) have zero or more mutations in one or more genes included in the FISH mutation profile; and/or iv) have zero or more mutations in one or more genes included in the sequencing mutation profile; and identifying the treatment if the comparison against the rules database indicates that the treatment should have biological activity against the disease; and the comparison against the rules database does not contraindicate the treatment for treating the disease. The disease can be a cancer. The molecular profiling steps can be performed in any order. In some embodiments, not all of the molecular profiling steps are performed. As a non-limiting example, microarray analysis is not performed if the sample quality does not meet a threshold value, as described herein. In some embodiments, the IHC expression profiling is performed on at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the gene products above. In some embodiments, the microarray expression profiling is performed on at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the genes listed above. In some embodiments, the IHC expression profiling is performed on all of the gene products above. In some embodiments, the microarray profiling is performed on all of the genes listed above. In some embodiments, the FISH profiling is performed on all of the gene products above. In some embodiments, the sequence profiling is performed on all of the genes listed above.


In a related aspect, the invention provides a method of identifying a candidate treatment for a subject in need thereof by using molecular profiling of defined sets of known biomarkers. For example, the method can identify a chemotherapeutic agent for an individual with a cancer. The method comprises: obtaining a sample from the subject, wherein the sample comprises formalin-fixed paraffin-embedded (FFPE) tissue or fresh frozen tissue, and wherein the sample comprises cancer cells; performing an immunohistochemistry (IHC) analysis on the sample to determine an IHC expression profile on at least: SPARC, PGP, Her2/neu, ER, PR, c-kit, AR, CD52, PDGFR, TOP2A, TS, ERCC1, RRM1, BCRP, TOPO1, PTEN, MGMT, MRP1, c-Met, EML4-ALK fusion, hENT-1, IGF-1R, MMR, p16, p21, p27, PARP-1, PI3K, and TLE3; performing a microarray analysis on the sample to determine a microarray expression profile on at least: ABCC1, ABCG2, ADA, AR, ASNS, BCL2, BIRC5, BRCA1, BRCA2, CD33, CD52, CDA, CES2, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2, ERBB2, ERCC1, ERCC3, ESR1, FLT1, FOLR2, FYN, GART, GNRH1, GSTP1, HCK, HDAC1, HIF1A, HSP90AA1, IGFBP3, IGFBP4, IGFBP5, IL2RA, KDR, KIT, LCK, LYN, MET, MGMT, MLH1, MS4A1, MSH2, NFKB1, NFKB2, NFKBIA, OGFR, PARP, PDGFC, PDGFRA, PDGFRB, PGP, PGR, POLA1, PTEN, PTGS2, PTPN12, RAF1, RARA, RRM1, RRM2, RRM2B, RXRB, RXRG, SIK2, SPARC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGFA, VHL, YES1, and ZAP70; performing a fluorescent in-situ hybridization (FISH) analysis on the sample to determine a FISH mutation profile on at least one of EGFR, HER2, EML4-ALK fusion and IGF-1R; performing DNA sequencing on the sample to determine a sequencing mutation profile on at least KRAS, BRAF, c-KIT, PI3K (PIK3CA), NRAS and EGFR. The IHC expression profile, microarray expression profile, FISH mutation profile and sequencing mutation profile are compared against a rules database, wherein the rules database comprises a mapping of treatments whose biological activity is known against diseased cells that: i) overexpress or underexpress one or more proteins included in the IHC expression profile; ii) overexpress or underexpress one or more genes included in the microarray expression profile; iii) have zero or more mutations in one or more genes included in the FISH mutation profile; or iv) have zero or more mutations in one or more genes included in the sequencing mutation profile; and identifying the treatment if the comparison against the rules database indicates that the treatment should have biological activity against the disease; and the comparison against the rules database does not contraindicate the treatment for treating the disease. The disease can be a cancer. The molecular profiling steps can be performed in any order. In some embodiments, not all of the molecular profiling steps are performed. As a non-limiting example, microarray analysis is not performed if the sample quality does not meet a threshold value, as described herein. In some embodiments, the biological material is mRNA and the quality control test comprises a A260/A280 ratio and/or a Ct value of RT-PCR using a housekeeping gene, e.g., RPL13a. In embodiments, the mRNA does not pass the quality control test if the A260/A280 ratio <1.5 or the RPL13a Ct value is >30. In that case, microarray analysis may not be performed. Alternately, microarray results may be attenuated, e.g., given a lower priority as compared to the results of other molecular profiling techniques.


In some embodiments, molecular profiling is always performed on certain genes or gene products, whereas the profiling of other genes or gene products is optional. For example, IHC expression profiling may be performed on at least SPARC, TOP2A and/or PTEN. Similarly, microarray expression profiling may be performed on at least CD52. In other embodiments, genes in addition to those listed above are used to identify a treatment. For example, the group of genes used for the IHC expression profiling can further comprise DCK, EGFR, BRCA1, CK 14, CK 17, CK 5/6, E-Cadherin, p95, PARP-1, SPARC and TLE3. In some embodiments, the group of genes used for the IHC expression profiling further comprises Cox-2 and/or Ki-67. In some embodiments, HSPCA is assayed by microarray analysis. In some embodiments, FISH mutation is performed on c-Myc and TOP2A. In some embodiments, sequencing is performed on PI3K.


The methods of the invention can be used in any setting wherein differential expression or mutation analysis have been linked to efficacy of various treatments. In some embodiments, the methods are used to identify candidate treatments for a subject having a cancer. Under these conditions, the sample used for molecular profiling preferably comprises cancer cells. The percentage of cancer in a sample can be determined by methods known to those of skill in the art, e.g., using pathology techniques. Cancer cells can also be enriched from a sample, e.g., using microdissection techniques or the like. A sample may be required to have a certain threshold of cancer cells before it is used for molecular profiling. The threshold can be at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 95% cancer cells. The threshold can depend on the analysis method. For example, a technique that reveals expression in individual cells may require a lower threshold that a technique that used a sample extracted from a mixture of different cells. In some embodiments, the diseased sample is compared to a normal sample taken from the same patient, e.g., adjacent but non-cancer tissue.


In embodiments, the methods of the invention are used detect gene fusions, such as those listed in U.S. patent application Ser. No. 12/658,770, filed Feb. 12, 2010; International PCT Patent Application PCT/US2010/000407, filed Feb. 11, 2010; and International PCT Patent Application PCT/US2010/54366, filed Oct. 27, 2010; all of which applications are incorporated by reference herein in their entirety. A fusion gene is a hybrid gene created by the juxtaposition of two previously separate genes. This can occur by chromosomal translocation or inversion, deletion or via trans-splicing. The resulting fusion gene can cause abnormal temporal and spatial expression of genes, leading to abnormal expression of cell growth factors, angiogenesis factors, tumor promoters or other factors contributing to the neoplastic transformation of the cell and the creation of a tumor. For example, such fusion genes can be oncogenic due to the juxtaposition of: 1) a strong promoter region of one gene next to the coding region of a cell growth factor, tumor promoter or other gene promoting oncogenesis leading to elevated gene expression, or 2) due to the fusion of coding regions of two different genes, giving rise to a chimeric gene and thus a chimeric protein with abnormal activity. Fusion genes are characteristic of many cancers. Once a therapeutic intervention is associated with a fusion, the presence of that fusion in any type of cancer identifies the therapeutic intervention as a candidate therapy for treating the cancer.


The presence of fusion genes, e.g., those described in U.S. patent application Ser. No. 12/658,770, filed Feb. 12, 2010; International PCT Patent Application PCT/US2010/000407, filed Feb. 11, 2010; and International PCT Patent Application PCT/US2010/54366, filed Oct. 27, 2010 or elsewhere herein, can be used to guide therapeutic selection. For example, the BCR-ABL gene fusion is a characteristic molecular aberration in ˜90% of chronic myelogenous leukemia (CML) and in a subset of acute leukemias (Kurzrock et al., Annals of Internal Medicine 2003; 138:819-830). The BCR-ABL results from a translocation between chromosomes 9 and 22, commonly referred to as the Philadelphia chromosome or Philadelphia translocation. The translocation brings together the 5′ region of the BCR gene and the 3′ region of ABL1, generating a chimeric BCR-ABL1 gene, which encodes a protein with constitutively active tyrosine kinase activity (Mittleman et al., Nature Reviews Cancer 2007; 7:233-245). The aberrant tyrosine kinase activity leads to de-regulated cell signaling, cell growth and cell survival, apoptosis resistance and growth factor independence, all of which contribute to the pathophysiology of leukemia (Kurzrock et al., Annals of Internal Medicine 2003; 138:819-830). Patients with the Philadelphia chromosome are treated with imatinib and other targeted therapies. Imatinib binds to the site of the constitutive tyrosine kinase activity of the fusion protein and prevents its activity. Imatinib treatment has led to molecular responses (disappearance of BCR-ABL+blood cells) and improved progression-free survival in BCR-ABL+CML patients (Kantarjian et al., Clinical Cancer Research 2007; 13:1089-1097).


Another fusion gene, IGH-MYC, is a defining feature of ˜80% of Burkitt's lymphoma (Ferry et al. Oncologist 2006; 11:375-83). The causal event for this is a translocation between chromosomes 8 and 14, bringing the c-Myc oncogene adjacent to the strong promoter of the immunoglobulin heavy chain gene, causing c-myc overexpression (Mittleman et al., Nature Reviews Cancer 2007; 7:233-245). The c-myc rearrangement is a pivotal event in lymphomagenesis as it results in a perpetually proliferative state. It has wide ranging effects on progression through the cell cycle, cellular differentiation, apoptosis, and cell adhesion (Ferry et al. Oncologist 2006; 11:375-83).


A number of recurrent fusion genes have been catalogued in the Mittleman database (cgap.nci.nih.gov/Chromosomes/Mitelman). The gene fusions can be used to characterize neoplasms and cancers and guide therapy using the subject methods described herein. For example, TMPRSS2-ERG, TMPRSS2-ETV and SLC45A3-ELK4 fusions can be detected to characterize prostate cancer; and ETV6-NTRK3 and ODZ4-NRG1 can be used to characterize breast cancer. The EML4-ALK, RLF-MYCL1, TGF-ALK, or CD74-ROS1 fusions can be used to characterize a lung cancer. The ACSL3-ETV1, C150RF21-ETV1, FLJ35294-ETV1, HERV-ETV1, TMPRSS2-ERG, TMPRSS2-ETV1/4/5, TMPRSS2-ETV4/5, SLC5A3-ERG, SLC5A3-ETV1, SLC5A3-ETV5 or KLK2-ETV4 fusions can be used to characterize a prostate cancer. The GOPC-ROS1 fusion can be used to characterize a brain cancer. The CHCHD7-PLAG1, CTNNB1-PLAG1, FHIT-HMGA2, HMGA2-NFIB, LIFR-PLAG1, or TCEA1-PLAG1 fusions can be used to characterize a head and neck cancer. The ALPHA-TFEB, NONO-TFE3, PRCC-TFE3, SFPQ-TFE3, CLTC-TFE3, or MALAT1-TFEB fusions can be used to characterize a renal cell carcinoma (RCC). The AKAP9-BRAF, CCDC6-RET, ERC1-RET, GOLGA5-RET, HOOK3-RET, HRH4-RET, KTN1-RET, NCOA4-RET, PCM1-RET, PRKARA1A-RET, RFG-RET, RFG9-RET, Ria-RET, TGF-NTRK1, TPM3-NTRK1, TPM3-TPR, TPR-MET, TPR-NTRK1, TRIM24-RET, TRIM27-RET or TRIM33-RET fusions can be used to characterize a thyroid cancer and/or papillary thyroid carcinoma; and the PAX8-PPARy fusion can be analyzed to characterize a follicular thyroid cancer. Fusions that are associated with hematological malignancies include without limitation TTL-ETV6, CDK6-MLL, CDK6-TLX3, ETV6-FLT3, ETV6-RUNX1, ETV6-TTL, MLL-AFF1, MLL-AFF3, MLL-AFF4, MLL-GAS7, TCBA1-ETV6, TCF3-PBX1 or TCF3-TFPT, which are characteristic of acute lymphocytic leukemia (ALL); BCL 11B-TLX3, IL2-TNFRFS17, NUP214-ABL1, NUP98-CCDC28A, TAL1-STIL, or ETV6-ABL2, which are characteristic of T-cell acute lymphocytic leukemia (T-ALL); ATIC-ALK, KIAA1618-ALK, MSN-ALK, MYH9-ALK, NPM1-ALK, TGF-ALK or TPM3-ALK, which are characteristic of anaplastic large cell lymphoma (ALCL); BCR-ABL1, BCR-JAK2, ETV6-EVI1, ETV6-MN1 or ETV6-TCBA1, characteristic of chronic myelogenous leukemia (CML); CBFB-MYH11, CHIC2-ETV6, ETV6-ABL1, ETV6-ABL2, ETV6-ARNT, ETV6-CDX2, ETV6-HLXB9, ETV6-PER1, MEF2D-DAZAP, AML-AFF1, MLL-ARHGAP26, MLL-ARHGEF12, MLL-CASC5, MLL-CBL,MLL-CREBBP, MLL-DAB21P, MLL-ELL, MLL-EP300, MLL-EPS 15, MLL-FNBP 1, MLL-FOXO3A, MLL-GMPS, MLL-GPHN, MLL-MLLT1, MLL-MLLT11, MLL-MLLT3, MLL-MLLT6, MLL-MYO1F, MLL-PICALM, MLL-SEPT2, MLL-SEPT6, MLL-SORBS2, MYST3-SORBS2, MYST-CREBBP, NPM1-MLF1, NUP98-HOXA13, PRDM16-EVI1, RABEP 1-PDGFRB, RUNX1-EVI1, RUNX1-MDS1, RUNX1-RPL22, RUNX1-RUNX1TI, RUNX1-SH3D19, RUNX1-USP42, RUNX1-YTHDF2, RUNX1-ZNF687, or TAF15-ZNF-384, which are characteristic of acute myeloid leukemia (AML); CCND1-FSTL3, which is characteristic of chronic lymphocytic leukemia (CLL); BCL3-MYC, MYC-BTG1, BCL7A-MYC, BRWD3-ARHGAP20 or BTG1-MYC, which are characteristic of B-cell chronic lymphocytic leukemia (B-CLL); CITTA-BCL6, CLTC-ALK, IL21R-BCL6, PIM1-BCL6, TFCR-BCL6, IKZF1-BCL6 or SEC31A-ALK, which are characteristic of diffuse large B-cell lymphomas (DLBCL); FLIP1-PDGFRA, FLT3-ETV6, KIAA1509-PDGFRA, PDE4DIP-PDGFRB, NIN-PDGFRB, TP53BP1-PDGFRB, or TPM3-PDGFRB, which are characteristic of hyper eosinophilia/chronic eosinophilia; and IGH-MYC or LCP1-BCL6, which are characteristic of Burkitt's lymphoma. One of skill will understand that additional fusions, including those yet to be identified to date, can be used to guide treatment once their presence is associated with a therapeutic intervention.


The fusion genes and gene products can be detected using one or more techniques described herein. In some embodiments, the sequence of the gene or corresponding mRNA is determined, e.g., using Sanger sequencing, NextGen sequencing, pyrosequencing, DNA microarrays, etc. Chromosomal abnormalities can be assessed using FISH or PCR techniques, among others. For example, a break apart probe can be used for FISH detection of ALK fusions such as EML4-ALK, KIF5B-ALK and/or TFG-ALK. As an alternate, PCR can be used to amplify the fusion product, wherein amplification or lack thereof indicates the presence or absence of the fusion, respectively. In some embodiments, the fusion protein fusion is detected. Appropriate methods for protein analysis include without limitation mass spectroscopy, electrophoresis (e.g., 2D gel electrophoresis or SDS-PAGE) or antibody related techniques, including immunoassay, protein array or immunohistochemistry. The techniques can be combined. As a non-limiting example, indication of an ALK fusion by FISH can be confirmed for ALK expression using IHC, or vice versa.


Treatment Selection

The systems and methods allow identification of one or more therapeutic targets whose projected efficacy can be linked to therapeutic efficacy, ultimately based on the molecular profiling. Illustrative schemes for using molecular profiling to identify a treatment regime are shown in FIGS. 2, 49A-B and 50, each of which is described in further detail herein. The invention comprises use of molecular profiling results to suggest associations with treatment responses. In an embodiment, the appropriate biomarkers for molecular profiling are selected on the basis of the subject's tumor type. These suggested biomarkers can be used to modify a default list of biomarkers. In other embodiments, the molecular profiling is independent of the source material. In some embodiments, rules are used to provide the suggested chemotherapy treatments based on the molecular profiling test results. In an embodiment, the rules are generated from abstracts of the peer reviewed clinical oncology literature. Expert opinion rules can be used but are optional. In an embodiment, clinical citations are assessed for their relevance to the methods of the invention using a hierarchy derived from the evidence grading system used by the United States Preventive Services Taskforce. The “best evidence” can be used as the basis for a rule. The simplest rules are constructed in the format of “if biomarker positive then treatment option one, else treatment option two.” Treatment options comprise no treatment with a specific drug, treatment with a specific drug or treatment with a combination of drugs. In some embodiments, more complex rules are constructed that involve the interaction of two or more biomarkers. In such cases, the more complex interactions are typically supported by clinical studies that analyze the interaction between the biomarkers included in the rule. Finally, a report can be generated that describes the association of the chemotherapy response and the biomarker and a summary statement of the best evidence supporting the treatments selected. Ultimately, the treating physician will decide on the best course of treatment.


As a non-limiting example, molecular profiling might reveal that the EGFR gene is amplified or overexpressed, thus indicating selection of a treatment that can block EGFR activity, such as the monoclonal antibody inhibitors cetuximab and panitumumab, or small molecule kinase inhibitors effective in patients with activating mutations in EGFR such as gefitinib, erlotinib, and lapatinib. Other anti-EGFR monoclonal antibodies in clinical development include zalutumumab, nimotuzumab, and matuzumab. The candidate treatment selected can depend on the setting revealed by molecular profiling. For example, kinase inhibitors are often prescribed with EGFR is found to have activating mutations. Continuing with the illustrative embodiment, molecular profiling may also reveal that some or all of these treatments are likely to be less effective. For example, patients taking gefitinib or erlotinib eventually develop drug resistance mutations in EGFR. Accordingly, the presence of a drug resistance mutation would contraindicate selection of the small molecule kinase inhibitors. One of skill will appreciate that this example can be expanded to guide the selection of other candidate treatments that act against genes or gene products whose differential expression is revealed by molecular profiling. Similarly, candidate agents known to be effective against diseased cells carrying certain nucleic acid variants can be selected if molecular profiling reveals such variants.


As another example, consider the drug imatinib, currently marketed by Novartis as Gleevec in the US in the form of imatinib mesylate. Imatinib is a 2-phenylaminopyrimidine derivative that functions as a specific inhibitor of a number of tyrosine kinase enzymes. It occupies the tyrosine kinase active site, leading to a decrease in kinase activity. Imatinib has been shown to block the activity of Abelson cytoplasmic tyrosine kinase (ABL), c-Kit and the platelet-derived growth factor receptor (PDGFR). Thus, imatinib can be indicated as a candidate therapeutic for a cancer determined by molecular profiling to overexpress ABL, c-KIT or PDGFR. Imatinib can be indicated as a candidate therapeutic for a cancer determined by molecular profiling to have mutations in ABL, c-KIT or PDGFR that alter their activity, e.g., constitutive kinase activity of ABLs caused by the BCR-ABL mutation. As an inhibitor of PDGFR, imatinib mesylate appears to have utility in the treatment of a variety of dermatological diseases.


Cancer therapies that can be identified as candidate treatments by the methods of the invention include without limitation: 13-cis-Retinoic Acid, 2-CdA, 2-Chlorodeoxyadenosine, 5-Azacitidine, 5-Fluorouracil, 5-FU, 6-Mercaptopurine, 6-MP, 6-TG, 6-Thioguanine, Abraxane, Accutane®, Actinomycin-D, Adriamycin®, Adrucil®, Afinitor®, Agrylin®, Ala-Cort®, Aldesleukin, Alemtuzumab, ALIMTA, Alitretinoin, Alkaban-AQ®, Alkeran®, All-transretinoic Acid, Alpha Interferon, Altretamine, Amethopterin, Amifostine, Aminoglutethimide, Anagrelide, Anandron®, Anastrozole, Arabinosylcytosine, Ara-C, Aranesp®, Aredia®, Arimidex®, Aromasin®, Arranon®, Arsenic Trioxide, Asparaginase, ATRA, Avastin®, Azacitidine, BCG, BCNU, Bendamustine, Bevacizumab, Bexarotene, BEXXAR®, Bicalutamide, BiCNU, Blenoxane®, Bleomycin, Bortezomib, Busulfan, Busulfex®, C225, Calcium Leucovorin, Campath®, Camptosar®, Camptothecin-11, Capecitabine, Carac™, Carboplatin, Carmustine, Carmustine Wafer, Casodex®, CC-5013, CCI-779, CCNU, CDDP, CeeNU, Cerubidine®, Cetuximab, Chlorambucil, Cisplatin, Citrovorum Factor, Cladribine, Cortisone, Cosmegen®, CPT-11, Cyclophosphamide, Cytadren®, Cytarabine, Cytarabine Liposomal, Cytosar-U®, Cytoxan®, Dacarbazine, Dacogen, Dactinomycin, Darbepoetin Alfa, Dasatinib, Daunomycin Daunorubicin, Daunorubicin Hydrochloride, Daunorubicin Liposomal, DaunoXome®, Decadron, Decitabine, Delta-Cortef®, Deltasone®, Denileukin, Diftitox, DepoCyt™, Dexamethasone, Dexamethasone Acetate Dexamethasone Sodium Phosphate, Dexasone, Dexrazoxane, DHAD, DIC, Diodex Docetaxel, Doxil®, Doxorubicin, Doxorubicin Liposomal, Droxia™, DTIC, DTIC-Dome®, Duralone®, Efudex®, Eligard™, Ellence™, Eloxatin™, Elspar®, Emcyt®, Epirubicin, Epoetin Alfa, Erbitux, Erlotinib, Erwinia Lasparaginase, Estramustine, Ethyol Etopophos®, Etoposide, Etoposide Phosphate, Eulexin®, Everolimus, Evista®, Exemestane, Fareston®, Faslodex®, Femara®, Filgrastim, Floxuridine, Fludara®, Fludarabine, Fluoroplex®, Fluorouracil, Fluorouracil (cream), Fluoxymesterone, Flutamide, Folinic Acid, FUDR®, Fulvestrant, G-CSF, Gefitinib, Gemcitabine, Gemtuzumab ozogamicin, Gemzar, Gleevec™, Gliadel® Wafer, GM-CSF, Goserelin, Granulocyte-Colony Stimulating Factor, Granulocyte Macrophage Colony Stimulating Factor, Halotestin®, Herceptin®, Hexadrol, Hexalen®, Hexamethylmelamine, HMM, Hycamtin®, Hydrea®, Hydrocort Acetate®, Hydrocortisone, Hydrocortisone Sodium Phosphate, Hydrocortisone Sodium Succinate, Hydrocortone Phosphate, Hydroxyurea, Ibritumomab, Ibritumomab, Tiuxetan, Idamycin®, Idarubicin, Ifex®, IFN-alpha, Ifosfamide, IL-11, IL-2, Imatinib mesylate, Imidazole Carboxamide, Interferon alfa, Interferon Alfa-2b (PEG Conjugate), Interleukin-2, Interleukin-11, Intron A® (interferon alfa-2b), Iressa®, Irinotecan, Isotretinoin, Ixabepilone, Ixempra™, Kidrolase (t), Lanacort®, Lapatinib, L-asparaginase, LCR, Lenalidomide, Letrozole, Leucovorin, Leukeran, Leukine™, Leuprolide, Leurocristine, Leustatin™, Liposomal Ara-C Liquid Pred®, Lomustine, L-PAM, L-Sarcolysin, Lupron®, Lupron Depot®, Matulane®, Maxidex, Mechlorethamine, Mechlorethamine Hydrochloride, Medralone®, Medrol®, Megace®, Megestrol, Megestrol Acetate, Melphalan, Mercaptopurine, Mesna, Mesnex™, Methotrexate, Methotrexate Sodium, Methylprednisolone, Meticorten®, Mitomycin, Mitomycin-C, Mitoxantrone, M-Prednisol®, MTC, MTX, Mustargen®, Mustine, Mutamycin®, Myleran®, Mylocel™, Mylotarg®, Navelbine®, Nelarabine, Neosar®, Neulasta™, Neumega®, Neupogen®, Nexavar®, Nilandron®, Nilutamide, Nipent®, Nitrogen Mustard, Novaldex®, Novantrone®, Octreotide, Octreotide acetate, Oncospar®, Oncovin®, Ontak®, Onxal™, Oprevelkin, Orapred®, Orasone®, Oxaliplatin, Paclitaxel, Paclitaxel Protein-bound, Pamidronate, Panitumumab, Panretin®, Paraplatin®, Pediapred®, PEG Interferon, Pegaspargase, Pegfilgrastim, PEG-INTRON™, PEG-L-asparaginase, PEMETREXED, Pentostatin, Phenylalanine Mustard, Platinol®, Platinol-AQ®, Prednisolone, Prednisone, Prelone®, Procarbazine, PROCRIT®, Proleukin®, Prolifeprospan 20 with Carmustine Implant, Purinethol®, Raloxifene, Revlimid®, Rheumatrex®, Rituxan®, Rituximab, Roferon-A® (Interferon Alfa-2a), Rubex®, Rubidomycin hydrochloride, Sandostatin®, Sandostatin LAR®, Sargramostim, Solu-Cortef®, Solu-Medrol®, Sorafenib, SPRYCEL™, STI-571, Streptozocin, SU11248, Sunitinib, Sutent®, Tamoxifen, Tarceva®, Targretin®, Taxol®, Taxotere®, Temodar®, Temozolomide, Temsirolimus, Teniposide, TESPA, Thalidomide, Thalomid®, TheraCys®, Thioguanine, Thioguanine Tabloid®, Thiophosphoamide, Thioplex®, Thiotepa, TICE®, Toposar®, Topotecan, Toremifene, Torisel®, Tositumomab, Trastuzumab, Treanda®, Tretinoin, Trexall™, Trisenox®, TSPA, TYKERB®, VCR, Vectibix™, Velban®, Velcade®, VePesid®, Vesanoid®, Viadur™, Vidaza®, Vinblastine, Vinblastine Sulfate, Vincasar Pfs®, Vincristine, Vinorelbine, Vinorelbine tartrate, VLB, VM-26, Vorinostat, VP-16, Vumon®, Xeloda®, Zanosar®, Zevalin™, Zinecard®, Zoladex®, Zoledronic acid, Zolinza, Zometa®, and any appropriate combinations thereof.


The candidate treatments identified according to the subject methods can be chosen from the class of therapeutic agents identified as Anthracyclines and related substances, Anti-androgens, Anti-estrogens, Antigrowth hormones (e.g., Somatostatin analogs), Combination therapy (e.g., vincristine, bcnu, melphalan, cyclophosphamide, prednisone (VBMCP)), DNA methyltransferase inhibitors, Endocrine therapy—Enzyme inhibitor, Endocrine therapy—other hormone antagonists and related agents, Folic acid analogs (e.g., methotrexate), Folic acid analogs (e.g., pemetrexed), Gonadotropin releasing hormone analogs, Gonadotropin-releasing hormones, Monoclonal antibodies (EGFR-Targeted—e.g., panitumumab, cetuximab), Monoclonal antibodies (Her2-Targeted—e.g., trastuzumab), Monoclonal antibodies (Multi-Targeted—e.g., alemtuzumab), Other alkylating agents, Other antineoplastic agents (e.g., asparaginase), Other antineoplastic agents (e.g., ATRA), Other antineoplastic agents (e.g., bexarotene), Other antineoplastic agents (e.g., celecoxib), Other antineoplastic agents (e.g., gemcitabine), Other antineoplastic agents (e.g., hydroxyurea), Other antineoplastic agents (e.g., irinotecan, topotecan), Other antineoplastic agents (e.g., pentostatin), Other cytotoxic antibiotics, Platinum compounds, Podophyllotoxin derivatives (e.g., etoposide), Progestogens, Protein kinase inhibitors (EGFR-Targeted), Protein kinase inhibitors (Her2 targeted therapy—e.g., lapatinib), Pyrimidine analogs (e.g., cytarabine), Pyrimidine analogs (e.g., fluoropyrimidines), Salicylic acid and derivatives (e.g., aspirin), Src-family protein tyrosine kinase inhibitors (e.g., dasatinib), Taxanes, Taxanes (e.g., nab-paclitaxel), Vinca Alkaloids and analogs, Vitamin D and analogs, Monoclonal antibodies (Multi-Targeted—e.g., bevacizumab), Protein kinase inhibitors (e.g., imatinib, sorafenib, sunitinib), Tyrosine Kinase inhibitors (TKI) (e.g., vemurafenib, sorafenib, imatinib, sunitinib, erlotinib, gefitinib, crizotinib, lapatinib).


In some embodiments, the candidate treatments identified according to the subject methods are chosen from at least the groups of treatments consisting of 5-fluorouracil, abarelix, alemtuzumab, aminoglutethimide, anastrozole, asparaginase, aspirin, ATRA, azacitidine, bevacizumab, bexarotene, bicalutamide, calcitriol, capecitabine, carboplatin, celecoxib, cetuximab, chemotherapy, cholecalciferol, cisplatin, cytarabine, dasatinib, daunorubicin, decitabine, doxorubicin, epirubicin, erlotinib, etoposide, exemestane, flutamide, fulvestrant, gefitinib, gemcitabine, gonadorelin, goserelin, hydroxyurea, imatinib, irinotecan, lapatinib, letrozole, leuprolide, liposomal-doxorubicin, medroxyprogesterone, megestrol, megestrol acetate, methotrexate, mitomycin, nab-paclitaxel, octreotide, oxaliplatin, paclitaxel, panitumumab, pegaspargase, pemetrexed, pentostatin, sorafenib, sunitinib, tamoxifen, Taxanes, temozolomide, toremifene, trastuzumab, VBMCP, and vincristine. The candidate treatments can be any of those in Tables 3-5, 7-22, 28, 29, 33, 36 or 37 herein.


Rules Engine

In some embodiments, a database is created that maps treatments and molecular profiling results. The treatment information can include the projected efficacy of a therapeutic agent against cells having certain attributes that can be measured by molecular profiling. The molecular profiling can include differential expression or mutations in certain genes, proteins, or other biological molecules of interest. Through the mapping, the results of the molecular profiling can be compared against the database to select treatments. The database can include both positive and negative mappings between treatments and molecular profiling results. In some embodiments, the mapping is created by reviewing the literature for links between biological agents and therapeutic agents. For example, a journal article, patent publication or patent application publication, scientific presentation, etc can be reviewed for potential mappings. The mapping can include results of in vivo, e.g., animal studies or clinical trials, or in vitro experiments, e.g., cell culture. Any mappings that are found can be entered into the database, e.g., cytotoxic effects of a therapeutic agent against cells expressing a gene or protein. In this manner, the database can be continuously updated. It will be appreciated that the methods of the invention are updated as well.


The rules can be generated by evidence-based literature review. Biomarker research continues to provide a better understanding of the clinical behavior and biology of cancer. This body of literature can be maintained in an up-to-date data repository incorporating recent clinical studies relevant to treatment options and potential clinical outcomes. The studies can be ranked so that only those with the strongest or most reliable evidence are selected for rules generation. For example, the rules generation can employ the grading system from the current methods of the U.S. Preventive Services Task Force. The literature evidence can be reviewed and evaluated based on the strength of clinical evidence supporting associations between biomarkers and treatments in the literature study. This process can be performed by a staff of scientists, physicians and other skilled reviewers. The process can also be automated in whole or in part by using language search and heuristics to identify relevant literature. The rules can be generated by a review of a plurality of literature references, e.g., tens, hundreds, thousands or more literature articles.


In another aspect, the invention provides a method of generating a set of evidence-based associations, comprising: (a) searching one or more literature database by a computer using an evidence-based medicine search filter to identify articles comprising a gene or gene product thereof, a disease, and one or more therapeutic agent; (b) filtering the articles identified in (a) to compile evidence-based associations comprising the expected benefit and/or the expected lack of benefit of the one or more therapeutic agent for treating the disease given the status of the gene or gene product; (c) adding the evidence-based associations compiled in (b) to the set of evidence-based associations; and (d) repeating steps (a)-(c) for an additional gene or gene product thereof. The status of the gene can include one or more assessments as described herein which relate to a biological state, e.g., one or more of an expression level, a copy number, and a mutation. The genes or gene products thereof can be one or more genes or gene products thereof selected from Table 2, Table 6 or Table 25. For example, the method can be repeated for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 or more of the genes or gene products thereof in Table 2, Table 6 or Table 25. The disease can be a disease described here, e.g., in embodiment the disease comprises a cancer. The one or more literature database can be selected from the group consisting of the National Library of Medicine's (NLM's) MEDLINE™ database of citations, a patent literature database, and a combination thereof.


Evidence-based medicine (EBM) or evidence-based practice (EBP) aims to apply the best available evidence gained from the scientific method to clinical decision making. This approach assesses the strength of evidence of the risks and benefits of treatments (including lack of treatment) and diagnostic tests. Evidence quality can be assessed based on the source type (from meta-analyses and systematic reviews of double-blind, placebo-controlled clinical trials at the top end, down to conventional wisdom at the bottom), as well as other factors including statistical validity, clinical relevance, currency, and peer-review acceptance. Evidence-based medicine filters are searches that have been developed to facilitate searches in specific areas of clinical medicine related to evidence-based medicine (diagnosis, etiology, meta-analysis, prognosis and therapy). They are designed to retrieve high quality evidence from published studies appropriate to decision-making. The evidence-based medicine filter used in the invention can be selected from the group consisting of a generic evidence-based medicine filter, a McMaster University optimal search strategy evidence-based medicine filter, a University of York statistically developed search evidence-based medicine filter, and a University of California San Francisco systemic review evidence-based medicine filter. See e.g., US Patent Publication 20080215570; Shojania and Bero. Taking advantage of the explosion of systematic reviews: an efficient MEDLINE search strategy. Eff Clin Pract. 2001 July-August; 4(4): 157-62; Ingui and Rogers. Searching for clinical prediction rules in MEDLINE. J Am Med Inform Assoc. 2001 July-August; 8(4):391-7; Haynes et al., Optimal search strategies for retrieving scientifically strong studies of treatment from Medline: analytical survey. BMJ. 2005 May 21; 330(7501):1179; Wilczynski and Haynes. Consistency and accuracy of indexing systematic review articles and meta-analyses in medline. Health Info Libr J. 2009 September; 26(3):203-10; which references are incorporated by reference herein in their entirety. A generic filter can be a customized filter based on an algorithm to identify the desired references from the one or more literature database. For example, the method can use one or more approach as described in U.S. Pat. No. 5,168,533 to Kato et al., U.S. Pat. No. 6,886,010 to Kostoff, or US Patent Application Publication No. 20040064438 to Kostoff; which references are incorporated by reference herein in their entirety.


The further filtering of articles identified by the evidence-based medicine filter can be performed using a computer, by one or more expert user, or combination thereof. The one or more expert can be a trained scientist or physician. In embodiments, the set of evidence-based associations comprise one or more of the rules in any of Tables 3-4, 7-25 or 27. For example, the set of evidence-based associations can include at least 5, 10, 25, 50 or 100 rules in Tables 3-4, 7-25 or 27. In some embodiments, the set of evidence-based associations comprises or consists of all of the rules in any of Tables 3-4, 7-25 or 27. In an aspect, the invention provides a computer readable medium comprising the set of evidence-based associations generated by the subject methods. The invention further provides a computer readable medium comprising one or more rules in any of Tables 3-4, 7-25 or 27 herein. In an embodiment, the computer readable medium comprises at least 5, 10, 25, 50 or 100 rules in any of Tables 3-4, 7-25 or 27. For example, the computer readable medium can comprise all rules in any of Tables 3-4, 7-25 or 27., e.g., all rules in Tables 3-4, 7-25 or 27.


The rules for the mappings can contain a variety of supplemental information. In some embodiments, the database contains prioritization criteria. For example, a treatment with more projected efficacy in a given setting can be preferred over a treatment projected to have lesser efficacy. A mapping derived from a certain setting, e.g., a clinical trial, may be prioritized over a mapping derived from another setting, e.g., cell culture experiments. A treatment with strong literature support may be prioritized over a treatment supported by more preliminary results. A treatment generally applied to the type of disease in question, e.g., cancer of a certain tissue origin, may be prioritized over a treatment that is not indicated for that particular disease. Mappings can include both positive and negative correlations between a treatment and a molecular profiling result. In a non-limiting example, one mapping might suggest use of a kinase inhibitor like erlotinib against a tumor having an activating mutation in EGFR, whereas another mapping might suggest against that treatment if the EGFR also has a drug resistance mutation. Similarly, a treatment might be indicated as effective in cells that overexpress a certain gene or protein but indicated as not effective if the gene or protein is underexpressed.


The selection of a candidate treatment for an individual can be based on molecular profiling results from any one or more of the methods described. Alternatively, selection of a candidate treatment for an individual can be based on molecular profiling results from more than one of the methods described. For example, selection of treatment for an individual can be based on molecular profiling results from FISH alone, IHC alone, or microarray analysis alone. In other embodiments, selection of treatment for an individual can be based on molecular profiling results from IHC, FISH, and microarray analysis; IHC and FISH; IHC and microarray analysis, or FISH and microarray analysis. Selection of treatment for an individual can also be based on molecular profiling results from sequencing or other methods of mutation detection. Molecular profiling results may include mutation analysis along with one or more methods, such as IHC, immunoassay, and/or microarray analysis. Different combinations and sequential results can be used. For example, treatment can be prioritized according the results obtained by molecular profiling. In an embodiment, the prioritization is based on the following algorithm: 1) IHC/FISH and microarray indicates same target as a first priority; 2) IHC positive result alone next priority; or 3) microarray positive result alone as last priority. Sequencing can also be used to guide selection. In some embodiments, sequencing reveals a drug resistance mutation so that the effected drug is not selected even if techniques including IHC, microarray and/or FISH indicate differential expression of the target molecule. Any such contraindication, e.g., differential expression or mutation of another gene or gene product may override selection of a treatment.


An illustrative listing of microarray expression results versus predicted treatments is presented in Table 3. As disclosed herein, molecular profiling is performed to determine whether a gene or gene product is differentially expressed in a sample as compared to a control. The expression status of the gene or gene product is used to select agents that are predicted to be efficacious or not. For example, Table 3 shows that overexpression of the ADA gene or protein points to pentostatin as a possible treatment. On the other hand, underexpression of the ADA gene or protein implicates resistance to cytarabine, suggesting that cytarabine is not an optimal treatment.









TABLE 3







Molecular Profiling Results and Predicted Treatments










Gene Name
Expression Status
Candidate Agent(s)
Possible Resistance





ADA
Overexpressed
pentostatin



ADA
Underexpressed

cytarabine


AR
Overexpressed
abarelix, bicalutamide,





flutamide, gonadorelin,





goserelin, leuprolide



ASNS
Underexpressed
asparaginase,





pegaspargase



BCRP (ABCG2)
Overexpressed

cisplatin, carboplatin,





irinotecan, topotecan


BRCA1
Underexpressed
mitomycin



BRCA2
Underexpressed
mitomycin



CD52
Overexpressed
alemtuzumab



CDA
Overexpressed

cytarabine


CES2
Overexpressed
irinotecan



c-kit
Overexpressed
sorafenib, sunitinib,





imatinib



COX-2
Overexpressed
celecoxib



DCK
Overexpressed
gemcitabine
cytarabine


DHFR
Underexpressed
methotrexate,





pemetrexed



DHFR
Overexpressed

methotrexate


DNMT 1
Overexpressed
azacitidine, decitabine



DNMT3A
Overexpressed
azacitidine, decitabine



DNMT3B
Overexpressed
azacitidine, decitabine



EGFR
Overexpressed
erlotinib, gefitinib,





cetuximab, panitumumab



EML4-ALK
Overexpressed
crizotinib




(present)




EPHA2
Overexpressed
dasatinib



ER
Overexpressed
anastrazole, exemestane,





fulvestrant, letrozole,





megestrol, tamoxifen,





medroxyprogesterone,





toremifene,





aminoglutethimide



ERCC1
Overexpressed

carboplatin, cisplatin


GART
Underexpressed
pemetrexed



HER-2 (ERBB2)
Overexpressed
trastuzumab, lapatinib



HIF-1α
Overexpressed
sorafenib, sunitinib,





bevacizumab



IκB-α
Overexpressed
bortezomib



MGMT
Underexpressed
temozolomide



MGMT
Overexpressed

temozolomide


MRP1 (ABCC1)
Overexpressed

etoposide, paclitaxel,





docetaxel,





vinblastine,





vinorelbine,





topotecan, teniposide


P-gp (ABCB1)
Overexpressed

doxorubicin,





etoposide, epirubicin,





paclitaxel, docetaxel,





vinblastine,





vinorelbine,





topotecan, teniposide,





liposomal





doxorubicin


PDGFR-α
Overexpressed
sorafenib, sunitinib,





imatinib



PDGFR-β
Overexpressed
sorafenib, sunitinib,





imatinib



PR
Overexpressed
exemestane, fulvestrant,





gonadorelin, goserelin,





medroxyprogesterone,





megestrol, tamoxifen,





toremifene



RARA
Overexpressed
ATRA



RRM1
Underexpressed
gemcitabine,





hydroxyurea



RRM2
Underexpressed
gemcitabine,





hydroxyurea



RRM2B
Underexpressed
gemcitabine,





hydroxyurea



RXR-α
Overexpressed
bexarotene



RXR-β
Overexpressed
bexarotene



SPARC
Overexpressed
nab-paclitaxel



SRC
Overexpressed
dasatinib



SSTR2
Overexpressed
octreotide



SSTR5
Overexpressed
octreotide



TOPO I
Overexpressed
irinotecan, topotecan



TOPO IIα
Overexpressed
doxorubicin, epirubicin,





liposomal- doxorubicin



TOPO IIβ
Overexpressed
doxorubicin, epirubicin,





liposomal- doxorubicin



TS
Underexpressed
capecitabine, 5-





fluorouracil, pemetrexed



TS
Overexpressed

capecitabine, 5-





fluorouracil


VDR
Overexpressed
calcitriol, cholecalciferol



VEGFR1 (Flt1)
Overexpressed
sorafenib, sunitinib,





bevacizumab



VEGFR2
Overexpressed
sorafenib, sunitinib,





bevacizumab



VHL
Underexpressed
sorafenib, sunitinib









Table 4 presents a selection of illustrative rules for treatment selection. The table is ordered by groups of related therapeutic agents. Each row describes a rule that maps the information derived from molecular profiling with an indication of benefit or lack of benefit for the therapeutic agent. Thus, the database contains a mapping of treatments whose biological activity is known against cancer cells that have alterations in certain genes or gene products, including gene copy alterations, chromosomal abnormalities, overexpression of or underexpression of one or more genes or gene products, or have various mutations. For each agent, a Lineage is presented as applicable which corresponds to a type of cancer associated with use of the agent. In this example, the agents can be used for all cancers. Agents with Benefit are listed along with a Benefit Summary Statement that describes molecular profiling information that relates to the predicted beneficial agent. Similarly, agents with Lack of Benefit are listed along with a Lack of Benefit Summary Statement that describes molecular profiling information that relates to the lack of benefit associated with the agent. Finally, the molecular profiling Criteria are shown. In the criteria, results from analysis using DNA microarray (DMA), IHC, FISH, and mutation analysis (MA) for one or more biomarkers is listed. For microarray analysis, expression can be reported as over (overexpressed) or under (underexpressed). When these criteria are met according to the application of the molecular profiling techniques to a sample, then the therapeutic agent or agents are predicted to have a benefit or lack of benefit as indicated in the corresponding row.


Further drug associations and rules that can be used in embodiments of the invention are found in U.S. Patent Application Publication 20100304989, filed Feb. 12, 2010; International PCT Patent Application WO/2010/093465, filed Feb. 11, 2010; and International PCT Patent Application WO/2011/056688, filed Oct. 27, 2010; all of which applications are incorporated by reference herein in their entirety. See e.g., “Table 4: Rules Summary for Treatment Selection” of WO/2011/056688.









TABLE 4







Exemplary Rules Summary for Treatment Selection

















Agents
Lack of





Agents
Benefit
with
Benefit



Therapeutic

with
Summary
Lack of
Summary



Agent
Lineage
Benefit
Statement
Benefit
Statement
Criteria





Protein kinase
None
sunitinib,
Presence of c-


DMA: VEGFR1


inhibitors

sorafenib
Kit mutation in


overexpressed.


(imatinib,


exon 9 has


DMA: HIF1A


sorafenib,


been


overexpressed.


sunitinib)


associated with


DMA: VEGFR2





benefit from


overexpressed.





sunitinib. In


DMA: KIT





addition, over


overexpressed.





expression of


DMA: PDGFRA





HIF1A,


overexpressed.





VEGFR1,


DMA: PDGFRB





VEGFR2, c-


overexpressed.





Kit, PDGFRA


DMA: VHL





and PDGFRB,


underexpressed.





and under


MA: c-kit mutated -





expression of


Exon 9





VHL have








been








associated with








benefit from








sunitinib and








sorafenib.





Protein kinase
None
sunitinib,
Presence of c-


DMA: VEGFR1


inhibitors

sorafenib
Kit mutation in


overexpressed.


(imatinib,


exon 9 has


DMA: HIF1A


sorafenib,


been


overexpressed.


sunitinib)


associated with


DMA: VEGFR2





benefit from


overexpressed.





sunitinib. In


DMA: KIT





addition, over


overexpressed.





expression of


DMA: PDGFRA





HIF1A,


overexpressed.





VEGFR1,


DMA: PDGFRB





VEGFR2, c-


overexpressed.





Kit, PDGFRA


DMA: VHL. MA:





and PDGFRB


c-kit mutated -





have been


Exon 9





associated with








benefit from








sunitinib and








sorafenib.





Protein kinase
None
sunitinib,
Presence of c-


DMA: VEGFR1


inhibitors

sorafenib
Kit mutation in


overexpressed.


(imatinib,


exon 9 has


DMA: HIF1A


sorafenib,


been


overexpressed.


sunitinib)


associated with


DMA: VEGFR2.





benefit from


DMA: KIT





sunitinib. In


overexpressed.





addition, over


DMA: PDGFRA





expression of


overexpressed.





HIF1A,


DMA: PDGFRB





VEGFR1, c-


overexpressed.





Kit, PDGFRA


DMA: VHL





and PDGFRB,


underexpressed.





and under


MA: c-kit mutated -





expression of


Exon 9





VHL have








been








associated with








benefit from








sunitinib and








sorafenib.





Protein kinase
None
sunitinib,
Presence of c-


DMA: VEGFR1


inhibitors

sorafenib
Kit mutation in


overexpressed.


(imatinib,


exon 9 has


DMA: HIF1A


sorafenib,


been


overexpressed.


sunitinib)


associated with


DMA: VEGFR2.





benefit from


DMA: KIT





sunitinib. In


overexpressed.





addition, over


DMA: PDGFRA





expression of


overexpressed.





HIF1A,


DMA: PDGFRB





VEGFR1, c-


overexpressed.





Kit, PDGFRA


DMA: VHL.





and PDGFRB


MA: c-kit mutated -





have been


Exon 9,





associated with








benefit from








sunitinib and








sorafenib.





Protein kinase
None
sunitinib,
Presence of c-


DMA: VEGFR1.


inhibitors

sorafenib
Kit mutation in


DMA: HIF1A


(imatinib,


exon 9 has


overexpressed.


sorafenib,


been


DMA: VEGFR2


sunitinib)


associated with


overexpressed.





benefit from


DMA: KIT





sunitinib. In


overexpressed.





addition, over


DMA: PDGFRA





expression of


overexpressed.





HIF1A,


DMA: PDGFRB





VEGFR2, c-


overexpressed.





Kit, PDGFRA


DMA: VHL





and PDGFRB,


underexpressed.





and under


MA: c-kit mutated -





expression of


Exon 9





VHL have








been








associated with








benefit from








sunitinib and








sorafenib.





Protein kinase
None
sunitinib,
Presence of c-


DMA: VEGFR1.


inhibitors

sorafenib
Kit mutation in


DMA: HIF1A


(imatinib,


exon 9 has


overexpressed.


sorafenib,


been


DMA: VEGFR2


sunitinib)


associated with


overexpressed.





benefit from


DMA: KIT





sunitinib. In


overexpressed.





addition, over


DMA: PDGFRA





expression of


overexpressed.





HIF1A,


DMA: PDGFRB





VEGFR2, c-


overexpressed.





Kit, PDGFRA


DMA: VHL.





and PDGFRB


MA: c-kit mutated -





have been


Exon 9





associated with








benefit from








sunitinib and








sorafenib.





Protein kinase
None
sunitinib,
Presence of c-


DMA: VEGFR1.


inhibitors

sorafenib
Kit mutation in


DMA: HIF1A


(imatinib,


exon 9 has


overexpressed.


sorafenib,


been


DMA: VEGFR2.


sunitinib)


associated with


DMA: KIT





benefit from


overexpressed.





sunitinib. In


DMA: PDGFRA





addition, over


overexpressed.





expression of


DMA: PDGFRB





HIF1A, c-Kit,


overexpressed.





PDGFRA and


DMA: VHL





PDGFRB, and


underexpressed.





under


MA: c-kit mutated -





expression of


Exon 9





VHL have








been








associated with








benefit from








sunitinib and








sorafenib.





Protein kinase
None
sunitinib,
Presence of c-


DMA: VEGFR1.


inhibitors

sorafenib
Kit mutation in


DMA: HIF1A


(imatinib,


exon 9 has


overexpressed.


sorafenib,


been


DMA: VEGFR2.


sunitinib)


associated with


DMA: KIT





benefit from


overexpressed.





sunitinib. In


DMA: PDGFRA





addition, over


overexpressed.





expression of


DMA: PDGFRB





HIF1A, c-Kit,


overexpressed.





PDGFRA and


DMA: VHL.





PDGFRB have


MA: c-kit mutated -





been


Exon 9





associated with








benefit from








sunitinib and








sorafenib.





Protein kinase
None
sunitinib,
Presence of c-


DMA: VEGFR1


inhibitors

sorafenib
Kit mutation in


overexpressed.


(imatinib,


exon 9 has


DMA: HIF1A


sorafenib,


been


overexpressed.


sunitinib)


associated with


DMA: VEGFR2





benefit from


overexpressed.





sunitinib. In


DMA: KIT





addition, over


overexpressed.





expression of


DMA: PDGFRA





HIF1A,


overexpressed.





VEGFR1,


DMA: PDGFRB.





VEGFR2, c-


DMA: VHL





Kit and


underexpressed.





PDGFRA, and


MA: c-kit mutated -





under


Exon 9





expression of








VHL have








been








associated with








benefit from








sunitinib and








sorafenib.









The efficacy of various therapeutic agents given particular assay results, such as those in Table 4 above, is derived from reviewing, analyzing and rendering conclusions on empirical evidence, such as that is available the medical literature or other medical knowledge base. The results are used to guide the selection of certain therapeutic agents in a prioritized list for use in treatment of an individual. When molecular profiling results are obtained, e.g., differential expression or mutation of a gene or gene product, the results can be compared against the database to guide treatment selection. The set of rules in the database can be updated as new treatments and new treatment data become available. In some embodiments, the rules database is updated continuously. In some embodiments, the rules database is updated on a periodic basis. Any relevant correlative or comparative approach can be used to compare the molecular profiling results to the rules database. In one embodiment, a gene or gene product is identified as differentially expressed by molecular profiling. The rules database is queried to select entries for that gene or gene product. Treatment selection information selected from the rules database is extracted and used to select a treatment. The information, e.g., to recommend or not recommend a particular treatment, can be dependent on whether the gene or gene product is over or underexpressed, or has other abnormalities at the genetic or protein levels as compared to a reference. In some cases, multiple rules and treatments may be pulled from a database comprising the comprehensive rules set depending on the results of the molecular profiling. In some embodiments, the treatment options are presented in a prioritized list. In some embodiments, the treatment options are presented without prioritization information. In either case, an individual, e.g., the treating physician or similar caregiver may choose from the available options.


The methods described herein are used to prolong survival of a subject by providing personalized treatment. In some embodiments, the subject has been previously treated with one or more therapeutic agents to treat the disease, e.g., a cancer. The cancer may be refractory to one of these agents, e.g., by acquiring drug resistance mutations. In some embodiments, the cancer is metastatic. In some embodiments, the subject has not previously been treated with one or more therapeutic agents identified by the method. Using molecular profiling, candidate treatments can be selected regardless of the stage, anatomical location, or anatomical origin of the cancer cells.


Progression-free survival (PFS) denotes the chances of staying free of disease progression for an individual or a group of individuals suffering from a disease, e.g., a cancer, after initiating a course of treatment. It can refer to the percentage of individuals in a group whose disease is likely to remain stable (e.g., not show signs of progression) after a specified duration of time. Progression-free survival rates are an indication of the effectiveness of a particular treatment. Similarly, disease-free survival (DFS) denotes the chances of staying free of disease after initiating a particular treatment for an individual or a group of individuals suffering from a cancer. It can refer to the percentage of individuals in a group who are likely to be free of disease after a specified duration of time. Disease-free survival rates are an indication of the effectiveness of a particular treatment. Treatment strategies can be compared on the basis of the PFS or DFS that is achieved in similar groups of patients. Disease-free survival is often used with the term overall survival when cancer survival is described.


The candidate treatment selected by molecular profiling according to the invention can be compared to a non-molecular profiling selected treatment by comparing the progression free survival (PFS) using therapy selected by molecular profiling (period B) with PFS for the most recent therapy on which the patient has just progressed (period A). See FIG. 40. In one setting, a PFS(B)/PFS(A) ratio≧1.3 was used to indicate that the molecular profiling selected therapy provides benefit for patient (Robert Temple, Clinical measurement in drug evaluation. Edited by Wu Ningano and G. T. Thicker John Wiley and Sons Ltd. 1995; Von Hoff D. D. Clin Can Res. 4: 1079, 1999: Dhani et al. Clin Cancer Res. 15: 118-123, 2009). Other methods of comparing the treatment selected by molecular profiling to a non-molecular profiling selected treatment include determining response rate (RECIST) and percent of patients without progression or death at 4 months. The term “about” as used in the context of a numerical value for PFS means a variation of +/−ten percent (10%) relative to the numerical value. The PFS from a treatment selected by molecular profiling can be extended by at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90% as compared to a non-molecular profiling selected treatment. In some embodiments, the PFS from a treatment selected by molecular profiling can be extended by at least 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or at least about 1000% as compared to a non-molecular profiling selected treatment. In yet other embodiments, the PFS ratio (PFS on molecular profiling selected therapy or new treatment/PFS on prior therapy or treatment) is at least about 1.3. In yet other embodiments, the PFS ratio is at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0. In yet other embodiments, the PFS ratio is at least about 3, 4, 5, 6, 7, 8, 9 or 10.


Similarly, the DFS can be compared in patients whose treatment is selected with or without molecular profiling. In embodiments, DFS from a treatment selected by molecular profiling is extended by at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90% as compared to a non-molecular profiling selected treatment. In some embodiments, the DFS from a treatment selected by molecular profiling can be extended by at least 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or at least about 1000% as compared to a non-molecular profiling selected treatment. In yet other embodiments, the DFS ratio (DFS on molecular profiling selected therapy or new treatment/DFS on prior therapy or treatment) is at least about 1.3. In yet other embodiments, the DFS ratio is at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0. In yet other embodiments, the DFS ratio is at least about 3, 4, 5, 6, 7, 8, 9 or 10.


In some embodiments, the candidate treatment of the invention will not increase the PFS ratio or the DFS ratio in the patient, nevertheless molecular profiling provides invaluable patient benefit. For example, in some instances no preferable treatment has been identified for the patient. In such cases, molecular profiling provides a method to identify a candidate treatment where none is currently identified. The molecular profiling may extend PFS, DFS or lifespan by at least 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months or 2 years. The molecular profiling may extend PFS, DFS or lifespan by at least 2½ years, 3 years, 4 years, 5 years, or more. In some embodiments, the methods of the invention improve outcome so that patient is in remission.


The effectiveness of a treatment can be monitored by other measures. A complete response (CR) comprises a complete disappearance of the disease: no disease is evident on examination, scans or other tests. A partial response (PR) refers to some disease remaining in the body, but there has been a decrease in size or number of the lesions by 30% or more. Stable disease (SD) refers to a disease that has remained relatively unchanged in size and number of lesions. Generally, less than a 50% decrease or a slight increase in size would be described as stable disease. Progressive disease (PD) means that the disease has increased in size or number on treatment. In some embodiments, molecular profiling according to the invention results in a complete response or partial response. In some embodiments, the methods of the invention result in stable disease. In some embodiments, the invention is able to achieve stable disease where non-molecular profiling results in progressive disease.


Computer Systems

The practice of the present invention may also employ conventional biology methods, software and systems. Computer software products of the invention typically include computer readable medium having computer-executable instructions for performing the logic steps of the method of the invention. Suitable computer readable medium include floppy disk, CD-ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM, magnetic tapes and etc. The computer executable instructions may be written in a suitable computer language or combination of several languages. Basic computational biology methods are described in, for example Setubal and Meidanis et al., Introduction to Computational Biology Methods (PWS Publishing Company, Boston, 1997); Salzberg, Searles, Kasif, (Ed.), Computational Methods in Molecular Biology, (Elsevier, Amsterdam, 1998); Rashidi and Buehler, Bioinformatics Basics: Application in Biological Science and Medicine (CRC Press, London, 2000) and Ouelette and Bzevanis Bioinformatics: A Practical Guide for Analysis of Gene and Proteins (Wiley & Sons, Inc., 2.sup.nd ed., 2001). See U.S. Pat. No. 6,420,108.


The present invention may also make use of various computer program products and software for a variety of purposes, such as probe design, management of data, analysis, and instrument operation. See, U.S. Pat. Nos. 5,593,839, 5,795,716, 5,733,729, 5,974,164, 6,066,454, 6,090,555, 6,185,561, 6,188,783, 6,223,127, 6,229,911 and 6,308,170.


Additionally, the present invention relates to embodiments that include methods for providing genetic information over networks such as the Internet as shown in U.S. Ser. Nos. 10/197,621, 10/063,559 (U.S. Publication Number 20020183936), Ser. Nos. 10/065,856, 10/065,868, 10/328,818, 10/328,872, 10/423,403, and 60/482,389. For example, one or more molecular profiling techniques can be performed in one location, e.g., a city, state, country or continent, and the results can be transmitted to a different city, state, country or continent. Treatment selection can then be made in whole or in part in the second location. The methods of the invention comprise transmittal of information between different locations.


Conventional data networking, application development and other functional aspects of the systems (and components of the individual operating components of the systems) may not be described in detail herein but are part of the invention. Furthermore, the connecting lines shown in the various figures contained herein are intended to represent illustrative functional relationships and/or physical couplings between the various elements. It should be noted that many alternative or additional functional relationships or physical connections may be present in a practical system.


The various system components discussed herein may include one or more of the following: a host server or other computing systems including a processor for processing digital data; a memory coupled to the processor for storing digital data; an input digitizer coupled to the processor for inputting digital data; an application program stored in the memory and accessible by the processor for directing processing of digital data by the processor; a display device coupled to the processor and memory for displaying information derived from digital data processed by the processor; and a plurality of databases. Various databases used herein may include: patient data such as family history, demography and environmental data, biological sample data, prior treatment and protocol data, patient clinical data, molecular profiling data of biological samples, data on therapeutic drug agents and/or investigative drugs, a gene library, a disease library, a drug library, patient tracking data, file management data, financial management data, billing data and/or like data useful in the operation of the system. As those skilled in the art will appreciate, user computer may include an operating system (e.g., Windows NT, 95/98/2000, OS2, UNIX, Linux, Solaris, MacOS, etc.) as well as various conventional support software and drivers typically associated with computers. The computer may include any suitable personal computer, network computer, workstation, minicomputer, mainframe or the like. User computer can be in a home or medical/business environment with access to a network. In an illustrative embodiment, access is through a network or the Internet through a commercially-available web-browser software package.


As used herein, the term “network” shall include any electronic communications means which incorporates both hardware and software components of such. Communication among the parties may be accomplished through any suitable communication channels, such as, for example, a telephone network, an extranet, an intranet, Internet, point of interaction device, personal digital assistant (e.g., Palm Pilot®, Blackberry®), cellular phone, kiosk, etc.), online communications, satellite communications, off-line communications, wireless communications, transponder communications, local area network (LAN), wide area network (WAN), networked or linked devices, keyboard, mouse and/or any suitable communication or data input modality. Moreover, although the system is frequently described herein as being implemented with TCP/IP communications protocols, the system may also be implemented using IPX, Appletalk, IP-6, NetBIOS, OSI or any number of existing or future protocols. If the network is in the nature of a public network, such as the Internet, it may be advantageous to presume the network to be insecure and open to eavesdroppers. Specific information related to the protocols, standards, and application software used in connection with the Internet is generally known to those skilled in the art and, as such, need not be detailed herein. See, for example, DILIP NAIK, INTERNET STANDARDS AND PROTOCOLS (1998); JAVA 2 COMPLETE, various authors, (Sybex 1999); DEBORAH RAY AND ERIC RAY, MASTERING HTML 4.0 (1997); and LOSHIN, TCP/IP CLEARLY EXPLAINED (1997) and DAVID GOURLEY AND BRIAN TOTTY, HTTP, THE DEFINITIVE GUIDE (2002), the contents of which are hereby incorporated by reference.


The various system components may be independently, separately or collectively suitably coupled to the network via data links which includes, for example, a connection to an Internet Service Provider (ISP) over the local loop as is typically used in connection with standard modem communication, cable modem, Dish networks, ISDN, Digital Subscriber Line (DSL), or various wireless communication methods, see, e.g., GILBERT HELD, UNDERSTANDING DATA COMMUNICATIONS (1996), which is hereby incorporated by reference. It is noted that the network may be implemented as other types of networks, such as an interactive television (ITV) network. Moreover, the system contemplates the use, sale or distribution of any goods, services or information over any network having similar functionality described herein.


As used herein, “transmit” may include sending electronic data from one system component to another over a network connection. Additionally, as used herein, “data” may include encompassing information such as commands, queries, files, data for storage, and the like in digital or any other form.


The system contemplates uses in association with web services, utility computing, pervasive and individualized computing, security and identity solutions, autonomic computing, commodity computing, mobility and wireless solutions, open source, biometrics, grid computing and/or mesh computing.


Any databases discussed herein may include relational, hierarchical, graphical, or object-oriented structure and/or any other database configurations. Common database products that may be used to implement the databases include DB2 by IBM (White Plains, N.Y.), various database products available from Oracle Corporation (Redwood Shores, Calif.), Microsoft Access or Microsoft SQL Server by Microsoft Corporation (Redmond, Wash.), or any other suitable database product. Moreover, the databases may be organized in any suitable manner, for example, as data tables or lookup tables. Each record may be a single file, a series of files, a linked series of data fields or any other data structure. Association of certain data may be accomplished through any desired data association technique such as those known or practiced in the art. For example, the association may be accomplished either manually or automatically. Automatic association techniques may include, for example, a database search, a database merge, GREP, AGREP, SQL, using a key field in the tables to speed searches, sequential searches through all the tables and files, sorting records in the file according to a known order to simplify lookup, and/or the like. The association step may be accomplished by a database merge function, for example, using a “key field” in pre-selected databases or data sectors.


More particularly, a “key field” partitions the database according to the high-level class of objects defined by the key field. For example, certain types of data may be designated as a key field in a plurality of related data tables and the data tables may then be linked on the basis of the type of data in the key field. The data corresponding to the key field in each of the linked data tables is preferably the same or of the same type. However, data tables having similar, though not identical, data in the key fields may also be linked by using AGREP, for example. In accordance with one embodiment, any suitable data storage technique may be used to store data without a standard format. Data sets may be stored using any suitable technique, including, for example, storing individual files using an ISO/IEC 7816-4 file structure; implementing a domain whereby a dedicated file is selected that exposes one or more elementary files containing one or more data sets; using data sets stored in individual files using a hierarchical filing system; data sets stored as records in a single file (including compression, SQL accessible, hashed vione or more keys, numeric, alphabetical by first tuple, etc.); Binary Large Object (BLOB); stored as ungrouped data elements encoded using ISO/IEC 7816-6 data elements; stored as ungrouped data elements encoded using ISO/IEC Abstract Syntax Notation (ASN. 1) as in ISO/IEC 8824 and 8825; and/or other proprietary techniques that may include fractal compression methods, image compression methods, etc.


In one illustrative embodiment, the ability to store a wide variety of information in different formats is facilitated by storing the information as a BLOB. Thus, any binary information can be stored in a storage space associated with a data set. The BLOB method may store data sets as ungrouped data elements formatted as a block of binary via a fixed memory offset using either fixed storage allocation, circular queue techniques, or best practices with respect to memory management (e.g., paged memory, least recently used, etc.). By using BLOB methods, the ability to store various data sets that have different formats facilitates the storage of data by multiple and unrelated owners of the data sets. For example, a first data set which may be stored may be provided by a first party, a second data set which may be stored may be provided by an unrelated second party, and yet a third data set which may be stored, may be provided by a third party unrelated to the first and second party. Each of these three illustrative data sets may contain different information that is stored using different data storage formats and/or techniques. Further, each data set may contain subsets of data that also may be distinct from other subsets.


As stated above, in various embodiments, the data can be stored without regard to a common format. However, in one illustrative embodiment, the data set (e.g., BLOB) may be annotated in a standard manner when provided for manipulating the data. The annotation may comprise a short header, trailer, or other appropriate indicator related to each data set that is configured to convey information useful in managing the various data sets. For example, the annotation may be called a “condition header”, “header”, “trailer”, or “status”, herein, and may comprise an indication of the status of the data set or may include an identifier correlated to a specific issuer or owner of the data. Subsequent bytes of data may be used to indicate for example, the identity of the issuer or owner of the data, user, transaction/membership account identifier or the like. Each of these condition annotations are further discussed herein.


The data set annotation may also be used for other types of status information as well as various other purposes. For example, the data set annotation may include security information establishing access levels. The access levels may, for example, be configured to permit only certain individuals, levels of employees, companies, or other entities to access data sets, or to permit access to specific data sets based on the transaction, issuer or owner of data, user or the like. Furthermore, the security information may restrict/permit only certain actions such as accessing, modifying, and/or deleting data sets. In one example, the data set annotation indicates that only the data set owner or the user are permitted to delete a data set, various identified users may be permitted to access the data set for reading, and others are altogether excluded from accessing the data set. However, other access restriction parameters may also be used allowing various entities to access a data set with various permission levels as appropriate. The data, including the header or trailer may be received by a standalone interaction device configured to add, delete, modify, or augment the data in accordance with the header or trailer.


One skilled in the art will also appreciate that, for security reasons, any databases, systems, devices, servers or other components of the system may consist of any combination thereof at a single location or at multiple locations, wherein each database or system includes any of various suitable security features, such as firewalls, access codes, encryption, decryption, compression, decompression, and/or the like.


The computing unit of the web client may be further equipped with an Internet browser connected to the Internet or an intranet using standard dial-up, cable, DSL or any other Internet protocol known in the art. Transactions originating at a web client may pass through a firewall in order to prevent unauthorized access from users of other networks. Further, additional firewalls may be deployed between the varying components of CMS to further enhance security.


Firewall may include any hardware and/or software suitably configured to protect CMS components and/or enterprise computing resources from users of other networks. Further, a firewall may be configured to limit or restrict access to various systems and components behind the firewall for web clients connecting through a web server. Firewall may reside in varying configurations including Stateful Inspection, Proxy based and Packet Filtering among others. Firewall may be integrated within an web server or any other CMS components or may further reside as a separate entity.


The computers discussed herein may provide a suitable website or other Internet-based graphical user interface which is accessible by users. In one embodiment, the Microsoft Internet Information Server (IIS), Microsoft Transaction Server (MTS), and Microsoft SQL Server, are used in conjunction with the Microsoft operating system, Microsoft NT web server software, a Microsoft SQL Server database system, and a Microsoft Commerce Server. Additionally, components such as Access or Microsoft SQL Server, Oracle, Sybase, Informix MySQL, Interbase, etc., may be used to provide an Active Data Object (ADO) compliant database management system.


Any of the communications, inputs, storage, databases or displays discussed herein may be facilitated through a website having web pages. The term “web page” as it is used herein is not meant to limit the type of documents and applications that might be used to interact with the user. For example, a typical website might include, in addition to standard HTML documents, various forms, Java applets, JavaScript, active server pages (ASP), common gateway interface scripts (CGI), extensible markup language (XML), dynamic HTML, cascading style sheets (CSS), helper applications, plug-ins, and the like. A server may include a web service that receives a request from a web server, the request including a URL (http://yahoo.com/stockquotes/ge) and an IP address (123.56.789.234). The web server retrieves the appropriate web pages and sends the data or applications for the web pages to the IP address. Web services are applications that are capable of interacting with other applications over a communications means, such as the internet. Web services are typically based on standards or protocols such as XML, XSLT, SOAP, WSDL and UDDI. Web services methods are well known in the art, and are covered in many standard texts. See, e.g., ALEX NGHIEM, IT WEB SERVICES: A ROADMAP FOR THE ENTERPRISE (2003), hereby incorporated by reference.


The web-based clinical database for the system and method of the present invention preferably has the ability to upload and store clinical data files in native formats and is searchable on any clinical parameter. The database is also scalable and may use an EAV data model (metadata) to enter clinical annotations from any study for easy integration with other studies. In addition, the web-based clinical database is flexible and may be XML and XSLT enabled to be able to add user customized questions dynamically. Further, the database includes exportability to CDISC ODM.


Practitioners will also appreciate that there are a number of methods for displaying data within a browser-based document. Data may be represented as standard text or within a fixed list, scrollable list, drop-down list, editable text field, fixed text field, pop-up window, and the like. Likewise, there are a number of methods available for modifying data in a web page such as, for example, free text entry using a keyboard, selection of menu items, check boxes, option boxes, and the like.


The system and method may be described herein in terms of functional block components, screen shots, optional selections and various processing steps. It should be appreciated that such functional blocks may be realized by any number of hardware and/or software components configured to perform the specified functions. For example, the system may employ various integrated circuit components, e.g., memory elements, processing elements, logic elements, look-up tables, and the like, which may carry out a variety of functions under the control of one or more microprocessors or other control devices. Similarly, the software elements of the system may be implemented with any programming or scripting language such as C, C++, Macromedia Cold Fusion, Microsoft Active Server Pages, Java, COBOL, assembler, PERL, Visual Basic, SQL Stored Procedures, extensible markup language (XML), with the various algorithms being implemented with any combination of data structures, objects, processes, routines or other programming elements. Further, it should be noted that the system may employ any number of conventional techniques for data transmission, signaling, data processing, network control, and the like. Still further, the system could be used to detect or prevent security issues with a client-side scripting language, such as JavaScript, VBScript or the like. For a basic introduction of cryptography and network security, see any of the following references: (1) “Applied Cryptography: Protocols, Algorithms, And Source Code In C,” by Bruce Schneier, published by John Wiley & Sons (second edition, 1995); (2) '7 Java Cryptography” by Jonathan Knudson, published by O'Reilly & Associates (1998); (3) “Cryptography & Network Security: Principles & Practice” by William Stallings, published by Prentice Hall; all of which are hereby incorporated by reference.


As used herein, the term “end user”, “consumer”, “customer”, “client”, “treating physician”, “hospital”, or “business” may be used interchangeably with each other, and each shall mean any person, entity, machine, hardware, software or business. Each participant is equipped with a computing device in order to interact with the system and facilitate online data access and data input. The customer has a computing unit in the form of a personal computer, although other types of computing units may be used including laptops, notebooks, hand held computers, set-top boxes, cellular telephones, touch-tone telephones and the like. The owner/operator of the system and method of the present invention has a computing unit implemented in the form of a computer-server, although other implementations are contemplated by the system including a computing center shown as a main frame computer, a mini-computer, a PC server, a network of computers located in the same of different geographic locations, or the like. Moreover, the system contemplates the use, sale or distribution of any goods, services or information over any network having similar functionality described herein.


In one illustrative embodiment, each client customer may be issued an “account” or “account number”. As used herein, the account or account number may include any device, code, number, letter, symbol, digital certificate, smart chip, digital signal, analog signal, biometric or other identifier/indicia suitably configured to allow the consumer to access, interact with or communicate with the system (e.g., one or more of an authorization/access code, personal identification number (PIN), Internet code, other identification code, and/or the like). The account number may optionally be located on or associated with a charge card, credit card, debit card, prepaid card, embossed card, smart card, magnetic stripe card, bar code card, transponder, radio frequency card or an associated account. The system may include or interface with any of the foregoing cards or devices, or a fob having a transponder and RFID reader in RF communication with the fob. Although the system may include a fob embodiment, the invention is not to be so limited. Indeed, system may include any device having a transponder which is configured to communicate with RFID reader via RF communication. Typical devices may include, for example, a key ring, tag, card, cell phone, wristwatch or any such form capable of being presented for interrogation. Moreover, the system, computing unit or device discussed herein may include a “pervasive computing device,” which may include a traditionally non-computerized device that is embedded with a computing unit. The account number may be distributed and stored in any form of plastic, electronic, magnetic, radio frequency, wireless, audio and/or optical device capable of transmitting or downloading data from itself to a second device.


As will be appreciated by one of ordinary skill in the art, the system may be embodied as a customization of an existing system, an add-on product, upgraded software, a standalone system, a distributed system, a method, a data processing system, a device for data processing, and/or a computer program product. Accordingly, the system may take the form of an entirely software embodiment, an entirely hardware embodiment, or an embodiment combining aspects of both software and hardware. Furthermore, the system may take the form of a computer program product on a computer-readable storage medium having computer-readable program code means embodied in the storage medium. Any suitable computer-readable storage medium may be used, including hard disks, CD-ROM, optical storage devices, magnetic storage devices, and/or the like.


The system and method is described herein with reference to screen shots, block diagrams and flowchart illustrations of methods, apparatus (e.g., systems), and computer program products according to various embodiments. It will be understood that each functional block of the block diagrams and the flowchart illustrations, and combinations of functional blocks in the block diagrams and flowchart illustrations, respectively, can be implemented by computer program instructions.


These computer program instructions may be loaded onto a general purpose computer, special purpose computer, or other programmable data processing apparatus to produce a machine, such that the instructions that execute on the computer or other programmable data processing apparatus create means for implementing the functions specified in the flowchart block or blocks. These computer program instructions may also be stored in a computer-readable memory that can direct a computer or other programmable data processing apparatus to function in a particular manner, such that the instructions stored in the computer-readable memory produce an article of manufacture including instruction means which implement the function specified in the flowchart block or blocks. The computer program instructions may also be loaded onto a computer or other programmable data processing apparatus to cause a series of operational steps to be performed on the computer or other programmable apparatus to produce a computer-implemented process such that the instructions which execute on the computer or other programmable apparatus provide steps for implementing the functions specified in the flowchart block or blocks.


Accordingly, functional blocks of the block diagrams and flowchart illustrations support combinations of means for performing the specified functions, combinations of steps for performing the specified functions, and program instruction means for performing the specified functions. It will also be understood that each functional block of the block diagrams and flowchart illustrations, and combinations of functional blocks in the block diagrams and flowchart illustrations, can be implemented by either special purpose hardware-based computer systems which perform the specified functions or steps, or suitable combinations of special purpose hardware and computer instructions. Further, illustrations of the process flows and the descriptions thereof may make reference to user windows, web pages, websites, web forms, prompts, etc. Practitioners will appreciate that the illustrated steps described herein may comprise in any number of configurations including the use of windows, web pages, web forms, popup windows, prompts and the like. It should be further appreciated that the multiple steps as illustrated and described may be combined into single web pages and/or windows but have been expanded for the sake of simplicity. In other cases, steps illustrated and described as single process steps may be separated into multiple web pages and/or windows but have been combined for simplicity.


Molecular Profiling Methods


FIG. 1 illustrates a block diagram of an illustrative embodiment of a system 10 for determining individualized medical intervention for a particular disease state that uses molecular profiling of a patient's biological specimen. System 10 includes a user interface 12, a host server 14 including a processor 16 for processing data, a memory 18 coupled to the processor, an application program 20 stored in the memory 18 and accessible by the processor 16 for directing processing of the data by the processor 16, a plurality of internal databases 22 and external databases 24, and an interface with a wired or wireless communications network 26 (such as the Internet, for example). System 10 may also include an input digitizer 28 coupled to the processor 16 for inputting digital data from data that is received from user interface 12.


User interface 12 includes an input device 30 and a display 32 for inputting data into system 10 and for displaying information derived from the data processed by processor 16. User interface 12 may also include a printer 34 for printing the information derived from the data processed by the processor 16 such as patient reports that may include test results for targets and proposed drug therapies based on the test results.


Internal databases 22 may include, but are not limited to, patient biological sample/specimen information and tracking, clinical data, patient data, patient tracking, file management, study protocols, patient test results from molecular profiling, and billing information and tracking. External databases 24 nay include, but are not limited to, drug libraries, gene libraries, disease libraries, and public and private databases such as UniGene, OMIM, GO, TIGR, GenBank, KEGG and Biocarta.


Various methods may be used in accordance with system 10. FIG. 2 shows a flowchart of an illustrative embodiment of a method 50 for determining individualized medical intervention for a particular disease state that uses molecular profiling of a patient's biological specimen that is non disease specific. In order to determine a medical intervention for a particular disease state using molecular profiling that is independent of disease lineage diagnosis (i.e. not single disease restricted), at least one test is performed for at least one target from a biological sample of a diseased patient in step 52. A target is defined as any molecular finding that may be obtained from molecular testing. For example, a target may include one or more genes, one or more gene expressed proteins, one or more molecular mechanisms, and/or combinations of such. For example, the expression level of a target can be determined by the analysis of mRNA levels or the target or gene, or protein levels of the gene. Tests for finding such targets may include, but are not limited, fluorescent in-situ hybridization (FISH), in-situ hybridization (ISH), and other molecular tests known to those skilled in the art. PCR-based methods, such as real-time PCR or quantitative PCR can be used. Furthermore, microarray analysis, such as a comparative genomic hybridization (CGH) micro array, a single nucleotide polymorphism (SNP) microarray, a proteomic array, or antibody array analysis can also be used in the methods disclosed herein. In some embodiments, microarray analysis comprises identifying whether a gene is up-regulated or down-regulated relative to a reference with a significance of p<0.001. Tests or analyses of targets can also comprise immunohistochemical (IHC) analysis. In some embodiments, IHC analysis comprises determining whether 30% or more of a sample is stained, if the staining intensity is +2 or greater, or both.


Furthermore, the methods disclosed herein also including profiling more than one target. For example, the expression of a plurality of genes can be identified. Furthermore, identification of a plurality of targets in a sample can be by one method or by various means. For example, the expression of a first gene can be determined by one method and the expression level of a second gene determined by a different method. Alternatively, the same method can be used to detect the expression level of the first and second gene. For example, the first method can be IHC and the second by microarray analysis, such as detecting the gene expression of a gene.


In some embodiments, molecular profiling can also including identifying a genetic variant, such as a mutation, polymorphism (such as a SNP), deletion, or insertion of a target. For example, identifying a SNP in a gene can be determined by microarray analysis, real-time PCR, or sequencing. Other methods disclosed herein can also be used to identify variants of one or more targets.


Accordingly, one or more of the following may be performed: an IHC analysis in step 54, a microanalysis in step 56, and other molecular tests know to those skilled in the art in step 58.


Biological samples are obtained from diseased patients by taking a biopsy of a tumor, conducting minimally invasive surgery if no recent tumor is available, obtaining a sample of the patient's blood, or a sample of any other biological fluid including, but not limited to, cell extracts, nuclear extracts, cell lysates or biological products or substances of biological origin such as excretions, blood, sera, plasma, urine, sputum, tears, feces, saliva, membrane extracts, and the like.


In step 60, a determination is made as to whether one or more of the targets that were tested for in step 52 exhibit a change in expression compared to a normal reference for that particular target. In one illustrative method of the invention, an IHC analysis may be performed in step 54 and a determination as to whether any targets from the IHC analysis exhibit a change in expression is made in step 64 by determining whether 30% or more of the biological sample cells were +2 or greater staining for the particular target. It will be understood by those skilled in the art that there will be instances where +1 or greater staining will indicate a change in expression in that staining results may vary depending on the technician performing the test and type of target being tested. In another illustrative embodiment of the invention, a micro array analysis may be performed in step 56 and a determination as to whether any targets from the micro array analysis exhibit a change in expression is made in step 66 by identifying which targets are up-regulated or down-regulated by determining whether the fold change in expression for a particular target relative to a normal tissue of origin reference is significant at p<0.001. A change in expression may also be evidenced by an absence of one or more genes, gene expressed proteins, molecular mechanisms, or other molecular findings.


After determining which targets exhibit a change in expression in step 60, at least one non-disease specific agent is identified that interacts with each target having a changed expression in step 70. An agent may be any drug or compound having a therapeutic effect. A non-disease specific agent is a therapeutic drug or compound not previously associated with treating the patient's diagnosed disease that is capable of interacting with the target from the patient's biological sample that has exhibited a change in expression. Some of the non-disease specific agents that have been found to interact with specific targets found in different cancer patients are shown in Table 5 below.









TABLE 5







Illustrative target-drug associations









Patients
Target(s) Found
Treatment(s)





Advanced Pancreatic Cancer
HER 2/neu
Trastuzumab


Advanced Pancreatic Cancer
EGFR, HIF 1α
Cetuximab, Sirolimus


Advanced Ovarian Cancer
ERCC3
Irofulven


Advanced Adenoid Cystic
Vitamin D receptors,
Calcitriol, Flutamide


Carcinoma
Androgen receptors









Finally, in step 80, a patient profile report may be provided which includes the patient's test results for various targets and any proposed therapies based on those results. An illustrative patient profile report 100 is shown in FIGS. 3A-3D. Patient profile report 100 shown in FIG. 3A identifies the targets tested 102, those targets tested that exhibited significant changes in expression 104, and proposed non-disease specific agents for interacting with the targets 106. Patient profile report 100 shown in FIG. 3B identifies the results 108 of immunohistochemical analysis for certain gene expressed proteins 110 and whether a gene expressed protein is a molecular target 112 by determining whether 30% or more of the tumor cells were +2 or greater staining. Report 100 also identifies immunohistochemical tests that were not performed 114. Patient profile report 100 shown in FIG. 3C identifies the genes analyzed 116 with a micro array analysis and whether the genes were under expressed or over expressed 118 compared to a reference. Finally, patient profile report 100 shown in FIG. 3D identifies the clinical history 120 of the patient and the specimens that were submitted 122 from the patient. Molecular profiling techniques can be performed anywhere, e.g., a foreign country, and the results sent by network to an appropriate party, e.g., the patient, a physician, lab or other party located remotely.



FIG. 4 shows a flowchart of an illustrative embodiment of a method 200 for identifying a drug therapy/agent capable of interacting with a target. In step 202, a molecular target is identified which exhibits a change in expression in a number of diseased individuals. Next, in step 204, a drug therapy/agent is administered to the diseased individuals. After drug therapy/agent administration, any changes in the molecular target identified in step 202 are identified in step 206 in order to determine if the drug therapy/agent administered in step 204 interacts with the molecular targets identified in step 202. If it is determined that the drug therapy/agent administered in step 204 interacts with a molecular target identified in step 202, the drug therapy/agent may be approved for treating patients exhibiting a change in expression of the identified molecular target instead of approving the drug therapy/agent for a particular disease.



FIGS. 5-14 are flowcharts and diagrams illustrating various parts of an information-based personalized medicine drug discovery system and method in accordance with the present invention. FIG. 5 is a diagram showing an illustrative clinical decision support system of the information-based personalized medicine drug discovery system and method of the present invention. Data obtained through clinical research and clinical care such as clinical trial data, biomedical/molecular imaging data, genomics/proteomics/chemical library/literature/expert curation, biospecimen tracking/LIMS, family history/environmental records, and clinical data are collected and stored as databases and datamarts within a data warehouse. FIG. 6 is a diagram showing the flow of information through the clinical decision support system of the information-based personalized medicine drug discovery system and method of the present invention using web services. A user interacts with the system by entering data into the system via form-based entry/upload of data sets, formulating queries and executing data analysis jobs, and acquiring and evaluating representations of output data. The data warehouse in the web based system is where data is extracted, transformed, and loaded from various database systems. The data warehouse is also where common formats, mapping and transformation occurs. The web based system also includes datamarts which are created based on data views of interest.


A flow chart of an illustrative clinical decision support system of the information-based personalized medicine drug discovery system and method of the present invention is shown in FIG. 7. The clinical information management system includes the laboratory information management system and the medical information contained in the data warehouses and databases includes medical information libraries, such as drug libraries, gene libraries, and disease libraries, in addition to literature text mining. Both the information management systems relating to particular patients and the medical information databases and data warehouses come together at a data junction center where diagnostic information and therapeutic options can be obtained. A financial management system may also be incorporated in the clinical decision support system of the information-based personalized medicine drug discovery system and method of the present invention.



FIG. 8 is a diagram showing an illustrative biospecimen tracking and management system which may be used as part of the information-based personalized medicine drug discovery system and method of the present invention. FIG. 8 shows two host medical centers which forward specimens to a tissue/blood bank. The specimens may go through laboratory analysis prior to shipment. Research may also be conducted on the samples via micro array, genotyping, and proteomic analysis. This information can be redistributed to the tissue/blood bank. FIG. 9 depicts a flow chart of an illustrative biospecimen tracking and management system which may be used with the information-based personalized medicine drug discovery system and method of the present invention. The host medical center obtains samples from patients and then ships the patient samples to a molecular profiling laboratory which may also perform RNA and DNA isolation and analysis.


A diagram showing a method for maintaining a clinical standardized vocabulary for use with the information-based personalized medicine drug discovery system and method of the present invention is shown in FIG. 10. FIG. 10 illustrates how physician observations and patient information associated with one physician's patient may be made accessible to another physician to enable the other physician to use the data in making diagnostic and therapeutic decisions for their patients.



FIG. 11 shows a schematic of an illustrative microarray gene expression database which may be used as part of the information-based personalized medicine drug discovery system and method of the present invention. The micro array gene expression database includes both external databases and internal databases which can be accessed via the web based system. External databases may include, but are not limited to, UniGene, GO, TIGR, GenBank, KEGG. The internal databases may include, but are not limited to, tissue tracking, LIMS, clinical data, and patient tracking. FIG. 12 shows a diagram of an illustrative micro array gene expression database data warehouse which may be used as part of the information-based personalized medicine drug discovery system and method of the present invention. Laboratory data, clinical data, and patient data may all be housed in the micro array gene expression database data warehouse and the data may in turn be accessed by public/private release and used by data analysis tools.


Another schematic showing the flow of information through an information-based personalized medicine drug discovery system and method of the present invention is shown in FIG. 13. Like FIG. 7, the schematic includes clinical information management, medical and literature information management, and financial management of the information-based personalized medicine drug discovery system and method of the present invention. FIG. 14 is a schematic showing an illustrative network of the information-based personalized medicine drug discovery system and method of the present invention. Patients, medical practitioners, host medical centers, and labs all share and exchange a variety of information in order to provide a patient with a proposed therapy or agent based on various identified targets.



FIGS. 15-25 are computer screen print outs associated with various parts of the information-based personalized medicine drug discovery system and method shown in FIGS. 5-14. FIGS. 15 and 16 show computer screens where physician information and insurance company information is entered on behalf of a client. FIGS. 17-19 show computer screens in which information can be entered for ordering analysis and tests on patient samples.



FIG. 20 is a computer screen showing micro array analysis results of specific genes tested with patient samples. This information and computer screen is similar to the information detailed in the patient profile report shown in FIG. 3C. FIG. 22 is a computer screen that shows immunohistochemistry test results for a particular patient for various genes. This information is similar to the information contained in the patient profile report shown in FIG. 3B.



FIG. 21 is a computer screen showing selection options for finding particular patients, ordering tests and/or results, issuing patient reports, and tracking current cases/patients.



FIG. 23 is a computer screen which outlines some of the steps for creating a patient profile report as shown in FIGS. 3A through 3D. FIG. 24 shows a computer screen for ordering an immunohistochemistry test on a patient sample and FIG. 25 shows a computer screen for entering information regarding a primary tumor site for micro array analysis. It will be understood by those skilled in the art that any number and variety of computer screens may be used to enter the information necessary for using the information-based personalized medicine drug discovery system and method of the present invention and to obtain information resulting from using the information-based personalized medicine drug discovery system and method of the present invention.



FIGS. 26-31 represent tables that show the frequency of a significant change in expression of certain genes and/or gene expressed proteins by tumor type, i.e. the number of times that a gene and/or gene expressed protein was flagged as a target by tumor type as being significantly overexpressed or underexpressed. The tables show the total number of times a gene and/or gene expressed protein was overexpressed or underexpressed in a particular tumor type and whether the change in expression was determined by immunohistochemistry analysis (FIG. 26, FIG. 28) or gene expression analysis (FIGS. 27, 30). The tables also identify the total number of times an overexpression of any gene expressed protein occurred in a particular tumor type using immunohistochemistry and the total number of times an overexpression or underexpression of any gene occurred in a particular tumor type using gene microarray analysis.


The systems of the invention can be used to automate the steps of identifying a molecular profile to assess a cancer. In an aspect, the invention provides a method of generating a report comprising a molecular profile. The method comprises: performing a search on an electronic medium to obtain a data set, wherein the data set comprises a plurality of scientific publications corresponding to plurality of cancer biomarkers; and analyzing the data set to identify a rule set linking a characteristic of each of the plurality of cancer biomarkers with an expected benefit of a plurality of treatment options, thereby identifying the cancer biomarkers included within a molecular profile. The method can further comprise performing molecular profiling on a sample from a subject to assess the characteristic of each of the plurality of cancer biomarkers, and compiling a report comprising the assessed characteristics into a list, thereby generating a report that identifies a molecular profile for the sample. The report can further comprise a list describing the expected benefit of the plurality of treatment options based on the assessed characteristics, thereby identifying candidate treatment options for the subject. The sample from the subject may comprise cancer cells. The cancer can be any cancer disclosed herein or known in the art.


The characteristic of each of the plurality of cancer biomarkers can be any useful characteristic for molecular profiling as disclosed herein or known in the art. Such characteristics include without limitation mutations (point mutations, insertions, deletions, rearrangements, etc), epigenetic modifications, copy number, nucleic acid or protein expression levels, post-translational modifications, and the like.


In an embodiment, the method further comprises identifying a priority list as amongst said plurality of cancer biomarkers. The priority list can be sorted according to any appropriate priority criteria. In an embodiment, the priority list is sorted according to strength of evidence in the plurality of scientific publications linking the cancer biomarkers to the expected benefit. In another embodiment, the priority list is sorted according to strength of the expected benefit. In still another embodiment, the priority list is sorted according to strength of the expected benefit. One of skill will appreciate that the priority list can be sorted according to a combination of these or other appropriate priority criteria. The candidate treatment options can be sorted according to the priority list, thereby identifying a ranked list of treatment options for the subject.


The candidate treatment options can be categorized by expected benefit to the subject. For example, the candidate treatment options can categorized as those that are expected to provide benefit, those that are not expected to provide benefit, or those whose expected benefit cannot be determined.


The candidate treatment options can include regulatory approved and/or on-compendium treatments for the cancer. The candidate treatment options can include regulatory approved but off-label treatments for the cancer, such as a treatment that has been approved for a cancer of another lineage. The candidate treatment options can include treatments that are under development, such as in ongoing clinical trials. The report may identify treatments as approved, on- or off-compendium, in clinical trials, and the like.


In some embodiments, the method further comprises analyzing the data set to select a laboratory technique to assess the characteristics of the biomarkers, thereby designating a technique that can be used to assess the characteristic for each of the plurality of biomarkers. In other embodiments, the laboratory technique is chosen based on its applicability to assess the characteristic of each of the biomarkers. The laboratory techniques can be those disclosed herein, including without limitation FISH for gene copy number or mutation analysis, IHC for protein expression levels, RT-PCR for mutation or expression analysis, sequencing or fragment analysis for mutation analysis. Sequencing includes any useful sequencing method disclosed herein or known in the art, including without limitation Sanger sequencing, pyrosequencing, or next generation sequencing methods.


In a related aspect, the invention provides a method comprising: performing a search on an electronic medium to obtain a data set comprising a plurality of scientific publications corresponding to plurality of cancer biomarkers; analyzing the data set to select a method to assess a characteristic of each of the cancer biomarkers, thereby designating a method for characterizing each of the biomarkers; further analyzing the data set to select a rule set that identifies a priority list as amongst the biomarkers; performing tumor profiling on a tumor sample from a subject comprising the selected methods to determine the status of the characteristic of each of the biomarkers; and compiling the status in a report according to said priority list; thereby generating a report that identifies a tumor profile.


Molecular Profiling Targets

The present invention provides methods and systems for analyzing diseased tissue using molecular profiling as previously described above. Because the methods rely on analysis of the characteristics of the tumor under analysis, the methods can be applied in for any tumor or any stage of disease, such an advanced stage of disease or a metastatic tumor of unknown origin. As described herein, a tumor or cancer sample is analyzed for molecular characteristics in order to predict or identify a candidate therapeutic treatment. The molecular characteristics can include the expression of genes or gene products, assessment of gene copy number, or mutational analysis. Any relevant determinable characteristic that can assist in prediction or identification of a candidate therapeutic can be included within the methods of the invention.


The biomarker patterns or biomarker signature sets can be determined for tumor types, diseased tissue types, or diseased cells including without limitation adipose, adrenal cortex, adrenal gland, adrenal gland—medulla, appendix, bladder, blood vessel, bone, bone cartilage, brain, breast, cartilage, cervix, colon, colon sigmoid, dendritic cells, skeletal muscle, endometrium, esophagus, fallopian tube, fibroblast, gallbladder, kidney, larynx, liver, lung, lymph node, melanocytes, mesothelial lining, myoepithelial cells, osteoblasts, ovary, pancreas, parotid, prostate, salivary gland, sinus tissue, skeletal muscle, skin, small intestine, smooth muscle, stomach, synovium, joint lining tissue, tendon, testis, thymus, thyroid, uterus, and uterus corpus.


The methods of the present invention can be used for selecting a treatment of any cancer or tumor type, including but not limited to breast cancer (including HER2+ breast cancer, HER2− breast cancer, ER/PR+, HER2− breast cancer, or triple negative breast cancer), pancreatic cancer, cancer of the colon and/or rectum, leukemia, skin cancer, bone cancer, prostate cancer, liver cancer, lung cancer, brain cancer, cancer of the larynx, gallbladder, parathyroid, thyroid, adrenal, neural tissue, head and neck, stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma of both ulcerating and papillary type, metastatic skin carcinoma, osteo sarcoma, Ewing's sarcoma, veticulum cell sarcoma, myeloma, giant cell tumor, small-cell lung tumor, islet cell carcinoma, primary brain tumor, acute and chronic lymphocytic and granulocytic tumors, hairy-cell tumor, adenoma, hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuroma, intestinal ganglioneuroma, hyperplastic corneal nerve tumor, marfanoid habitus tumor, Wilm's tumor, seminoma, ovarian tumor, leiomyoma, cervical dysplasia and in situ carcinoma, neuroblastoma, retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skin lesion, mycosis fungoides, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic and other sarcoma, malignant hypercalcemia, renal cell tumor, polycythermia vera, adenocarcinoma, glioblastoma multiforma, leukemias, lymphomas, malignant melanomas, and epidermoid carcinomas. The cancer or tumor can comprise, without limitation, a carcinoma, a sarcoma, a lymphoma or leukemia, a germ cell tumor, a blastoma, or other cancers. Carcinomas that can be assessed using the subject methods include without limitation epithelial neoplasms, squamous cell neoplasms, squamous cell carcinoma, basal cell neoplasms basal cell carcinoma, transitional cell papillomas and carcinomas, adenomas and adenocarcinomas (glands), adenoma, adenocarcinoma, linitis plastica insulinoma, glucagonoma, gastrinoma, vipoma, cholangiocarcinoma, hepatocellular carcinoma, adenoid cystic carcinoma, carcinoid tumor of appendix, prolactinoma, oncocytoma, hurthle cell adenoma, renal cell carcinoma, grawitz tumor, multiple endocrine adenomas, endometrioid adenoma, adnexal and skin appendage neoplasms, mucoepidermoid neoplasms, cystic, mucinous and serous neoplasms, cystadenoma, pseudomyxoma peritonei, ductal, lobular and medullary neoplasms, acinar cell neoplasms, complex epithelial neoplasms, warthin's tumor, thymoma, specialized gonadal neoplasms, sex cord stromal tumor, thecoma, granulosa cell tumor, arrhenoblastoma, sertoli leydig cell tumor, glomus tumors, paraganglioma, pheochromocytoma, glomus tumor, nevi and melanomas, melanocytic nevus, malignant melanoma, melanoma, nodular melanoma, dysplastic nevus, lentigo maligna melanoma, superficial spreading melanoma, and malignant acral lentiginous melanoma. Sarcoma that can be assessed using the subject methods include without limitation Askin's tumor, botryodies, chondrosarcoma, Ewing's sarcoma, malignant hemangio endothelioma, malignant schwannoma, osteosarcoma, soft tissue sarcomas including: alveolar soft part sarcoma, angiosarcoma, cystosarcoma phyllodes, dermatofibrosarcoma, desmoid tumor, desmoplastic small round cell tumor, epithelioid sarcoma, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, hemangiopericytoma, hemangiosarcoma, kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma, and synovialsarcoma. Lymphoma and leukemia that can be assessed using the subject methods include without limitation chronic lymphocytic leukemia/small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma (such as waldenström macroglobulinemia), splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, monoclonal immunoglobulin deposition diseases, heavy chain diseases, extranodal marginal zone B cell lymphoma, also called malt lymphoma, nodal marginal zone B cell lymphoma (nmzl), follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, burkitt lymphoma/leukemia, T cell prolymphocytic leukemia, T cell large granular lymphocytic leukemia, aggressive NK cell leukemia, adult T cell leukemia/lymphoma, extranodal NK/T cell lymphoma, nasal type, enteropathy-type T cell lymphoma, hepatosplenic T cell lymphoma, blastic NK cell lymphoma, mycosis fungoides/sezary syndrome, primary cutaneous CD30-positive T cell lymphoproliferative disorders, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T cell lymphoma, peripheral T cell lymphoma, unspecified, anaplastic large cell lymphoma, classical Hodgkin lymphomas (nodular sclerosis, mixed cellularity, lymphocyte-rich, lymphocyte depleted or not depleted), and nodular lymphocyte-predominant Hodgkin lymphoma. Germ cell tumors that can be assessed using the subject methods include without limitation germinoma, dysgerminoma, seminoma, nongerminomatous germ cell tumor, embryonal carcinoma, endodermal sinus turmor, choriocarcinoma, teratoma, polyembryoma, and gonadoblastoma. Blastoma includes without limitation nephroblastoma, medulloblastoma, and retinoblastoma. Other cancers include without limitation labial carcinoma, larynx carcinoma, hypopharynx carcinoma, tongue carcinoma, salivary gland carcinoma, gastric carcinoma, adenocarcinoma, thyroid cancer (medullary and papillary thyroid carcinoma), renal carcinoma, kidney parenchyma carcinoma, cervix carcinoma, uterine corpus carcinoma, endometrium carcinoma, chorion carcinoma, testis carcinoma, urinary carcinoma, melanoma, brain tumors such as glioblastoma, astrocytoma, meningioma, medulloblastoma and peripheral neuroectodermal tumors, gall bladder carcinoma, bronchial carcinoma, multiple myeloma, basalioma, teratoma, retinoblastoma, choroidea melanoma, seminoma, rhabdomyosarcoma, craniopharyngeoma, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma, Ewing sarcoma, and plasmocytoma.


In an embodiment, the cancer may be a acute myeloid leukemia (AML), breast carcinoma, cholangiocarcinoma, colorectal adenocarcinoma, extrahepatic bile duct adenocarcinoma, female genital tract malignancy, gastric adenocarcinoma, gastroesophageal adenocarcinoma, gastrointestinal stromal tumors (GIST), glioblastoma, head and neck squamous carcinoma, leukemia, liver hepatocellular carcinoma, low grade glioma, lung bronchioloalveolar carcinoma (BAC), lung non-small cell lung cancer (NSCLC), lung small cell cancer (SCLC), lymphoma, male genital tract malignancy, malignant solitary fibrous tumor of the pleura (MSFT), melanoma, multiple myeloma, neuroendocrine tumor, nodal diffuse large B-cell lymphoma, non epithelial ovarian cancer (non-EOC), ovarian surface epithelial carcinoma, pancreatic adenocarcinoma, pituitary carcinomas, oligodendroglioma, prostatic adenocarcinoma, retroperitoneal or peritoneal carcinoma, retroperitoneal or peritoneal sarcoma, small intestinal malignancy, soft tissue tumor, thymic carcinoma, thyroid carcinoma, or uveal melanoma.


In a further embodiment, the cancer may be a lung cancer including non-small cell lung cancer and small cell lung cancer (including small cell carcinoma (oat cell cancer), mixed small cell/large cell carcinoma, and combined small cell carcinoma), colon cancer, breast cancer, prostate cancer, liver cancer, pancreas cancer, brain cancer, kidney cancer, ovarian cancer, stomach cancer, skin cancer, bone cancer, gastric cancer, breast cancer, pancreatic cancer, glioma, glioblastoma, hepatocellular carcinoma, papillary renal carcinoma, head and neck squamous cell carcinoma, leukemia, lymphoma, myeloma, or a solid tumor.


In embodiments, the cancer comprises an acute lymphoblastic leukemia; acute myeloid leukemia; adrenocortical carcinoma; AIDS-related cancers; AIDS-related lymphoma; anal cancer; appendix cancer; astrocytomas; atypical teratoid/rhabdoid tumor; basal cell carcinoma; bladder cancer; brain stem glioma; brain tumor (including brain stem glioma, central nervous system atypical teratoid/rhabdoid tumor, central nervous system embryonal tumors, astrocytomas, craniopharyngioma, ependymoblastoma, ependymoma, medulloblastoma, medulloepithelioma, pineal parenchymal tumors of intermediate differentiation, supratentorial primitive neuroectodermal tumors and pineoblastoma); breast cancer; bronchial tumors; Burkitt lymphoma; cancer of unknown primary site; carcinoid tumor; carcinoma of unknown primary site; central nervous system atypical teratoid/rhabdoid tumor; central nervous system embryonal tumors; cervical cancer; childhood cancers; chordoma; chronic lymphocytic leukemia; chronic myelogenous leukemia; chronic myeloproliferative disorders; colon cancer; colorectal cancer; craniopharyngioma; cutaneous T-cell lymphoma; endocrine pancreas islet cell tumors; endometrial cancer; ependymoblastoma; ependymoma; esophageal cancer; esthesioneuroblastoma; Ewing sarcoma; extracranial germ cell tumor; extragonadal germ cell tumor; extrahepatic bile duct cancer; gallbladder cancer; gastric (stomach) cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal cell tumor; gastrointestinal stromal tumor (GIST); gestational trophoblastic tumor; glioma; hairy cell leukemia; head and neck cancer; heart cancer; Hodgkin lymphoma; hypopharyngeal cancer; intraocular melanoma; islet cell tumors; Kaposi sarcoma; kidney cancer; Langerhans cell histiocytosis; laryngeal cancer; lip cancer; liver cancer; malignant fibrous histiocytoma bone cancer; medulloblastoma; medulloepithelioma; melanoma; Merkel cell carcinoma; Merkel cell skin carcinoma; mesothelioma; metastatic squamous neck cancer with occult primary; mouth cancer; multiple endocrine neoplasia syndromes; multiple myeloma; multiple myeloma/plasma cell neoplasm; mycosis fungoides; myelodysplastic syndromes; myeloproliferative neoplasms; nasal cavity cancer; nasopharyngeal cancer; neuroblastoma; Non-Hodgkin lymphoma; nonmelanoma skin cancer; non-small cell lung cancer; oral cancer; oral cavity cancer; oropharyngeal cancer; osteosarcoma; other brain and spinal cord tumors; ovarian cancer; ovarian epithelial cancer; ovarian germ cell tumor; ovarian low malignant potential tumor; pancreatic cancer; papillomatosis; paranasal sinus cancer; parathyroid cancer; pelvic cancer; penile cancer; pharyngeal cancer; pineal parenchymal tumors of intermediate differentiation; pineoblastoma; pituitary tumor; plasma cell neoplasm/multiple myeloma; pleuropulmonary blastoma; primary central nervous system (CNS) lymphoma; primary hepatocellular liver cancer; prostate cancer; rectal cancer; renal cancer; renal cell (kidney) cancer; renal cell cancer; respiratory tract cancer; retinoblastoma; rhabdomyosarcoma; salivary gland cancer; Sdzary syndrome; small cell lung cancer; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma; squamous neck cancer; stomach (gastric) cancer; supratentorial primitive neuroectodermal tumors; T-cell lymphoma; testicular cancer; throat cancer; thymic carcinoma; thymoma; thyroid cancer; transitional cell cancer; transitional cell cancer of the renal pelvis and ureter; trophoblastic tumor; ureter cancer; urethral cancer; uterine cancer; uterine sarcoma; vaginal cancer; vulvar cancer; Waldenstrim macroglobulinemia; or Wilm's tumor.


The methods of the invention can be used to determine biomarker patterns or biomarker signature sets in a number of tumor types, diseased tissue types, or diseased cells including accessory, sinuses, middle and inner ear, adrenal glands, appendix, hematopoietic system, bones and joints, spinal cord, breast, cerebellum, cervix uteri, connective and soft tissue, corpus uteri, esophagus, eye, nose, eyeball, fallopian tube, extrahepatic bile ducts, other mouth, intrahepatic bile ducts, kidney, appendix-colon, larynx, lip, liver, lung and bronchus, lymph nodes, cerebral, spinal, nasal cartilage, excl. retina, eye, nos, oropharynx, other endocrine glands, other female genital, ovary, pancreas, penis and scrotum, pituitary gland, pleura, prostate gland, rectum renal pelvis, ureter, peritonem, salivary gland, skin, small intestine, stomach, testis, thymus, thyroid gland, tongue, unknown, urinary bladder, uterus, nos, vagina & labia, and vulva, nos.


In some embodiments, the molecular profiling methods are used to identify a treatment for a cancer of unknown primary (CUP). Approximately 40,000 CUP cases are reported annually in the US. Most of these are metastatic and/or poorly differentiated tumors. Because molecular profiling can identify a candidate treatment depending only upon the diseased sample, the methods of the invention can be used in the CUP setting. Moreover, molecular profiling can be used to create signatures of known tumors, which can then be used to classify a CUP and identify its origin. In an aspect, the invention provides a method of identifying the origin of a CUP, the method comprising performing molecular profiling on a panel of diseased samples to determine a panel of molecular profiles that correlate with the origin of each diseased sample, performing molecular profiling on a CUP sample, and correlating the molecular profile of the CUP sample with the molecular profiling of the panel of diseased samples, thereby identifying the origin of the CUP sample. The identification of the origin of the CUP sample can be made by matching the molecular profile of the CUP sample with the molecular profiles that correlate most closely from the panel of disease samples. The molecular profiling can use any of the techniques described herein, e.g., IHC, FISH, microarray and sequencing. The diseased samples and CUP samples can be derived from a patient sample, e.g., a biopsy sample, including a fine needle biopsy. In one embodiment, DNA microarray and IHC profiling are performed on the panel of diseased samples, DNA microarray is performed on the CUP samples, and then IHC is performed on the CUP sample for a subset of the most informative genes as indicated by the DNA microarray analysis. This approach can identify the origin of the CUP sample while avoiding the expense of performing unnecessary IHC testing. The IHC can be used to confirm the microarray findings.


The biomarker patterns or biomarker signature sets of the cancer or tumor can be used to determine a therapeutic agent or therapeutic protocol that is capable of interacting with the biomarker pattern or signature set. For example, with advanced breast cancer, immunohistochemistry analysis can be used to determine one or more gene expressed proteins that are overexpressed. Accordingly, a biomarker pattern or biomarker signature set can be identified for advanced stage breast cancer and a therapeutic agent or therapeutic protocol can be identified which is capable of interacting with the biomarker pattern or signature set.


These examples of biomarker patterns or biomarker signature sets for advanced stage breast cancer are just one example of the extensive number of biomarker patterns or biomarker signature sets for a number of advanced stage diseases or cancers that can be identified from the tables depicted in FIGS. 26-31. In addition, a number of non disease specific therapies or therapeutic protocols may be identified for treating patients with these biomarker patterns or biomarker signature sets by using method steps of the present invention described above such as depicted in FIGS. 1-2 and FIGS. 5-14.


The biomarker patterns and/or biomarker signature sets disclosed in the table depicted in FIGS. 26 and 28, and the tables depicted in FIGS. 27 and 30 may be used for a number of purposes including, but not limited to, specific cancer/disease detection, specific cancer/disease treatment, and identification of new drug therapies or protocols for specific cancers/diseases. The biomarker patterns and/or biomarker signature sets disclosed in the table depicted in FIGS. 26 and 28, and the tables depicted in FIGS. 27 and 30 can also represent drug resistant expression profiles for the specific tumor type or cancer type. The biomarker patterns and/or biomarker signature sets disclosed in the table depicted in FIGS. 26 and 28, and the tables depicted in FIGS. 27 and 30 represent advanced stage drug resistant profiles.


The biomarker patterns and/or biomarker signature sets can comprise at least one biomarker. In yet other embodiments, the biomarker patterns or signature sets can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 biomarkers. In some embodiments, the biomarker signature sets or biomarker patterns can comprise at least 15, 20, 30, 40, 50, or 60 biomarkers. In some embodiments, the biomarker signature sets or biomarker patterns can comprise at least 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000 or 50,000 biomarkers. Analysis of the one or more biomarkers can be by one or more methods. For example, analysis of 2 biomarkers can be performed using microarrays. Alternatively, one biomarker may be analyzed by IHC and another by microarray. Any such combinations of methods and biomarkers are contemplated herein.


The one or more biomarkers can be selected from the group consisting of, but not limited to: Her2/Neu, ER, PR, c-kit, EGFR, MLH1, MSH2, CD20, p53, Cyclin D1, bcl2, COX-2, Androgen receptor, CD52, PDGFR, AR, CD25, VEGF, HSP90, PTEN, RRM1, SPARC, Survivin, TOP2A, BCL2, HIF1A, AR, ESR1, PDGFRA, KIT, PDGFRB, CDW52, ZAP70, PGR, SPARC, GART, GSTP1, NFKBIA, MSH2, TXNRD1, HDAC1, PDGFC, PTEN, CD33, TYMS, RXRB, ADA, TNF, ERCC3, RAF1, VEGF, TOP1, TOP2A, BRCA2, TK1, FOLR2, TOP2B, MLH1, IL2RA, DNMT1, HSPCA, ERBR2, ERBB2, SSTR1, VHL, VDR, PTGS2, POLA, CES2, EGFR, OGFR, ASNS, NFKB2, RARA, MS4A1, DCK, DNMT3A, EREG, Epiregulin, FOLR1, GNRH1, GNRHR1, FSHB, FSHR, FSHPRH1, folate receptor, HGF, HIG1, IL13RA1, LTB, ODC1, PPARG, PPARGC1, Lymphotoxin Beta Receptor, Myc, Topoisomerase II, TOPO2B, TXN, VEGFC, ACE2, ADH1C, ADH4, AGT, AREG, CA2, CDK2, caveolin, NFKB1, ASNS, BDCA1, CD52, DHFR, DNMT3B, EPHA2, FLT1, HSP90AA1, KDR, LCK, MGMT, RRM1, RRM2, RRM2B, RXRG, SRC, SSTR2, SSTR3, SSTR4, SSTR5, VEGFA, or YES1.


For example, a biological sample from an individual can be analyzed to determine a biomarker pattern or biomarker signature set that comprises a biomarker such as HSP90, Survivin, RRM1, SSTRS3, DNMT3B, VEGFA, SSTR4, RRM2, SRC, RRM2B, HSP90AA1, STR2, FLT1, SSTR5, YES1, BRCA1, RRM1, DHFR, KDR, EPHA2, RXRG, or LCK. In other embodiments, the biomarker SPARC, HSP90, TOP2A, PTEN, Survivin, or RRM1 forms part of the biomarker pattern or biomarker signature set. In yet other embodiments, the biomarker MGMT, SSTRS3, DNMT3B, VEGFA, SSTR4, RRM2, SRC, RRM2B, HSP90AA1, STR2, FLT1, SSTR5, YES1, BRCA1, RRM1, DHFR, KDR, EPHA2, RXRG, CD52, or LCK is included in a biomarker pattern or biomarker signature set. In still other embodiments, the biomarker hENT1, cMet, P21, PARP-1, TLE3 or IGF1R is included in a biomarker pattern or biomarker signature set.


The expression level of HSP90, Survivin, RRM1, SSTRS3, DNMT3B, VEGFA, SSTR4, RRM2, SRC, RRM2B, HSP90AA1, STR2, FLT1, SSTR5, YES1, BRCA1, RRM1, DHFR, KDR, EPHA2, RXRG, or LCK can be determined and used to identify a therapeutic for an individual. The expression level of the biomarker can be used to form a biomarker pattern or biomarker signature set. Determining the expression level can be by analyzing the levels of mRNA or protein, such as by microarray analysis or IHC. In some embodiments, the expression level of a biomarker is performed by IHC, such as for SPARC, TOP2A, or PTEN, and used to identify a therapeutic for an individual. The results of the IHC can be used to form a biomarker pattern or biomarker signature set. In yet other embodiments, a biological sample from an individual or subject is analyzed for the expression level of CD52, such as by determining the mRNA expression level by methods including, but not limited to, microarray analysis. The expression level of CD52 can be used to identify a therapeutic for the individual. The expression level of CD52 can be used to form a biomarker pattern or biomarker signature set. In still other embodiments, the biomarkers hENT1, cMet, P21, PARP-1, TLE3 and/or IGF1R are assessed to identify a therapeutic for the individual.


As described herein, the molecular profiling of one or more targets can be used to determine or identify a therapeutic for an individual. For example, the expression level of one or more biomarkers can be used to determine or identify a therapeutic for an individual. The one or more biomarkers, such as those disclosed herein, can be used to form a biomarker pattern or biomarker signature set, which is used to identify a therapeutic for an individual. In some embodiments, the therapeutic identified is one that the individual has not previously been treated with. For example, a reference biomarker pattern has been established for a particular therapeutic, such that individuals with the reference biomarker pattern will be responsive to that therapeutic. An individual with a biomarker pattern that differs from the reference, for example the expression of a gene in the biomarker pattern is changed or different from that of the reference, would not be administered that therapeutic. In another example, an individual exhibiting a biomarker pattern that is the same or substantially the same as the reference is advised to be treated with that therapeutic. In some embodiments, the individual has not previously been treated with that therapeutic and thus a new therapeutic has been identified for the individual.


Molecular profiling according to the invention can take on a biomarker-centric or a therapeutic-centric point of view. Although the approaches are not mutually exclusive, the biomarker-centric approach focuses on sets of biomarkers that are expected to be informative for a tumor of a given tumor lineage, whereas the therapeutic-centric point approach identifies candidate therapeutics using biomarker panels that are lineage independent. In a biomarker-centric view, panels of specific biomarkers are run on different tumor types. See FIG. 32A. This approach provides a method of identifying a candidate therapeutic by collecting a sample from a subject with a cancer of known origin, and performing molecular profiling on the cancer for specific biomarkers depending on the origin of the cancer. The molecular profiling can be performed using any of the various techniques disclosed herein. As an example, FIG. 32A shows biomarker panels for breast cancer, ovarian cancer, colorectal cancer, lung cancer, and a “complete” profile to run on any cancer. In the figure, markers shown in italics are assessed using mutational analysis (e.g., sequencing approaches), marker shown underlined are analyzed by FISH, and the remainder are analyzed using IHC. DNA microarray profiling can be performed on any sample. The candidate therapeutic is selected based on the molecular profiling results according to the subject methods. An advantage to the bio-marker centric approach is only performing assays that are most likely to yield informative results. Another advantage is that this approach can focus on identifying therapeutics conventionally used to treat cancers of the specific lineage. In a therapeutic-centric approach, the biomarkers assessed are not dependent on the origin of the tumor. See FIG. 32B. This approach provides a method of identifying a candidate therapeutic by collecting a sample from a subject with a cancer, and performing molecular profiling on the cancer for a panel of biomarkers without regards to the origin of the cancer. The molecular profiling can be performed using any of the various techniques disclosed herein. As an example, in FIG. 32B, markers shown in italics are assessed using mutational analysis (e.g., sequencing approaches), marker shown underlined are analyzed by FISH, and the remainder are analyzed using IHC. DNA microarray profiling can be performed on any sample. The candidate therapeutic is selected based on the molecular profiling results according to the subject methods. An advantage to the therapeutic-marker centric approach is that the most promising therapeutics are identified only taking into account the molecular characteristics of the tumor itself. Another advantage is that the method can be preferred for a cancer of unidentified primary origin (CUP). In some embodiments, a hybrid of biomarker-centric and therapeutic-centric points of view is used to identify a candidate therapeutic. This method comprises identifying a candidate therapeutic by collecting a sample from a subject with a cancer of known origin, and performing molecular profiling on the cancer for a comprehensive panel of biomarkers, wherein a portion of the markers assessed depend on the origin of the cancer. For example, consider a breast cancer. A comprehensive biomarker panel is run on the breast cancer, e.g., the complete panel as shown in FIG. 32B, but additional sequencing analysis is performed on one or more additional markers, e.g., BRCA1 or any other marker with mutations informative for theranosis or prognosis of the breast cancer. Theranosis can be used to refer to the likely efficacy of a therapeutic treatment. Prognosis refers to the likely outcome of an illness. One of skill will apprecitate that the hybrid approach can be used to identify a candidate therapeutic for any cancer having additional biomarkers that provide theranostic or prognostic information, including the cancers disclosed herein.


Methods for providing a theranosis of disease include selecting candidate therapeutics for various cancers by assessing a sample from a subject in need thereof (i.e., suffering from a particular cancer). The sample is assessed by performing an immunohistochemistry (IHC) to determine of the presence or level of: AR, BCRP, c-KIT, ER, ERCC1, HER2, IGF1R, MET (also referred to herein as cMet), MGMT, MRP1, PDGFR, PGP, PR, PTEN, RRM1, SPARC, TOPO1, TOP2A, TS, COX-2, CK5/6, CK14, CK17, Ki67, p53, CAV-1, CYCLIN D1, EGFR, E-cadherin, p95, TLE3 or a combination thereof; performing a microarray analysis on the sample to determine a microarray expression profile on one or more (such as at least five, 10, 15, 20, 25, 30, 40, 50, 60, 70 or all) of: ABCC1, ABCG2, ADA, AR, ASNS, BCL2, BIRC5, BRCA1, BRCA2, CD33, CD52, CDA, CES2, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2, ERBB2, ERCC1, ERCC3, ESR1, FLT1, FOLR2, FYN, GART, GNRH1, GSTP1, HCK, HDAC1, HIF1A, HSP90AA1, IGFBP3, IGFBP4, IGFBP5, IL2RA, KDR, KIT, LCK, LYN, MET, MGMT, MLH1, MS4A1, MSH2, NFKB1, NFKB2, NFKBIA, OGFR, PARP1, PDGFC, PDGFRA, PDGFRB, PGP, PGR, POLA1, PTEN, PTGS2, PTPN12, RAF1, RARA, RRM1, RRM2, RRM2B, RXRB, RXRG, SIK2, SPARC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, TK1, TNF, TOP, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGFA, VHL, YES1, and ZAP70; comparing the results obtained from the IHC and microarray analysis against a rules database, wherein the rules database comprises a mapping of candidate treatments whose biological activity is known against a cancer cell that expresses one or more proteins included in the IHC expression profile and/or expresses one or more genes included in the microarray expression profile; and determining a candidate treatment if the comparison indicates that the candidate treatment has biological activity against the cancer.


Assessment can further comprise determining a fluorescent in-situ hybridization (FISH) profile of EGFR, HER2, cMYC, TOP2A, MET, or a combination thereof, comparing the FISH profile against a rules database comprising a mapping of candidate treatments predetermined as effective against a cancer cell having a mutation profile for EGFR, HER2, cMYC, TOP2A, MET, or a combination thereof, and determining a candidate treatment if the comparison of the FISH profile against the rules database indicates that the candidate treatment has biological activity against the cancer.


As explained further herein, the FISH analysis can be performed based on the origin of the sample. This can avoid unnecessary laboratory procedures and concomitant expenses by targeting analysis of genes that are known to play a role in a particular disorder, e.g., a particular type of cancer. In an embodiment, EGFR, HER2, cMYC, and TOP2A are assessed for breast cancer. In another embodiment, EGFR and MET are assessed for lung cancer. Alternately, FISH analysis of all of EGFR, HER2, cMYC, TOP2A, MET can be performed on a sample. The complete panel may be assessed, e.g., when a sample is of unknown or mixed origin, to provide a comprehensive view of an unusual sample, or when economies of scale dictate that it is more efficient to perform FISH on the entire panel than to make individual assessments.


In an additional embodiment, the sample is assessed by performing nucleic acid sequencing on the sample to determine a presence of a mutation of KRAS, BRAF, NRAS, PIK3CA (also referred to as PI3K), c-Kit, EGFR, or a combination thereof, comparing the results obtained from the sequencing against a rules database comprising a mapping of candidate treatments predetermined as effective against a cancer cell having a mutation profile for KRAS, BRAF, NRAS, PIK3CA, c-Kit, EGFR, or a combination thereof; and determining a candidate treatment if the comparison of the sequencing to the mutation profile indicates that the candidate treatment has biological activity against the cancer.


As explained further herein, the nucleic acid sequencing can be performed based on the origin of the sample. This can avoid unnecessary laboratory procedures and concomitant expenses by targeting analysis of genes that are known to play a role in a particular disorder, e.g., a particular type of cancer. In an embodiment, the sequences of PIK3CA and c-KIT are assessed for breast cancer. In another embodiment, the sequences of KRAS and BRAF are assessed for GI cancers such as colorectal cancer. In still another embodiment, the sequences of KRAS, BRAF and EGFR are assessed for lung cancer. Alternately, sequencing of all of KRAS, BRAF, NRAS, PIK3CA, c-Kit, EGFR can be performed on a sample. The complete panel may be sequenced, e.g., when a sample is of unknown or mixed origin, to provide a comprehensive view of an unusual sample, or when economies of scale dictate that it is more efficient to sequence the entire panel than to make individual assessments.


The genes and gene products used for molecular profiling, e.g., by microarray, IHC, FISH, sequencing, and/or PCR (e.g., qPCR), can be selected from those listed in Table 2, Table 6 or Table 25. In an embodiment, IHC is performed for one or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more, of: AR, BCRP, CAV-1, CD20, CD52, CK 5/6, CK14, CK17, c-kit, CMET, COX-2, Cyclin D1, E-Cad, EGFR, ER, ERCC1, HER-2, IGF1R, Ki67, MGMT, MRP1, P53, p95, PDGFR, PGP, PR, PTEN, RRM1, SPARC, TLE3, TOPO1, TOPO2A, TS, TUBB3; expression analysis (e.g., microarray or RT-PCR) is performed on one or more, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 or more, of: ABCC1, ABCG2, ADA, AR, ASNS, BCL2, BIRC5, BRCA1, BRCA2, CD33, CD52, CDA, CES2, cKit, c-MYC, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2, ERCC1, ERCC3, ESR1, FLT1, FOLR2, FYN, GART, GNRH1, GSTP1, HCK, HDAC1, HER2/ERBB2, HIF1A, HSP90, IGFBP3, IGFBP4, IGFBP5, IL2RA, KDR, LCK, LYN, MET, MGMT, MLH1, MS4A1, MSH2, NFKB1, NFKB2, NFKBIA, OGFR, PARP1, PDGFC, PDGFRa, PDGFRA, PDGFRB, PGP, PGR, POLA1, PTEN, PTGS2, RAF1, RARA, ROS1, RRM1, RRM2, RRM2B, RXRB, RXRG, SIK2, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, SPARC, TK1, TNF, TOP2B, TOP2A, TOPO1, TXNRD1, TYMS, VDR, VEGFA, VHL, YES1, and ZAP70; fluorescent in-situ hybridization (FISH) is performed on 1, 2, 3, 4, 5, 6 or 7 of ALK, cMET, c-MYC, EGFR, HER-2, PIK3CA, and TOPO2A; and DNA sequencing or PCR are performed on 1, 2, 3, 4, 5 or 6 of BRAF, c-kit, EGFR, KRAS, NRAS, and PIK3CA. In an embodiment, all of these genes and/or the gene products thereof are assessed.


Assessing one or more biomarkers disclosed herein can be used for characterizing any of the cancers disclosed herein. Characterizing includes the diagnosis of a disease or condition, the prognosis of a disease or condition, the determination of a disease stage or a condition stage, a drug efficacy, a physiological condition, organ distress or organ rejection, disease or condition progression, therapy-related association to a disease or condition, or a specific physiological or biological state.


A cancer in a subject can be characterized by obtaining a biological sample from a subject and analyzing one or more biomarkers from the sample. For example, characterizing a cancer for a subject or individual may include detecting a disease or condition (including pre-symptomatic early stage detecting), determining the prognosis, diagnosis, or theranosis of a disease or condition, or determining the stage or progression of a disease or condition. Characterizing a cancer can also include identifying appropriate treatments or treatment efficacy for specific diseases, conditions, disease stages and condition stages, predictions and likelihood analysis of disease progression, particularly disease recurrence, metastatic spread or disease relapse. Characterizing can also be identifying a distinct type or subtype of a cancer. The products and processes described herein allow assessment of a subject on an individual basis, which can provide benefits of more efficient and economical decisions in treatment.


In an aspect, characterizing a cancer includes predicting whether a subject is likely to respond to a treatment for the cancer. As used herein, a “responder” responds to or is predicted to respond to a treatment and a “non-responder” does not respond or is predicted to not respond to the treatment. Biomarkers can be analyzed in the subject and compared to biomarker profiles of previous subjects that were known to respond or not to a treatment. If the biomarker profile in a subject more closely aligns with that of previous subjects that were known to respond to the treatment, the subject can be characterized, or predicted, as a responder to the treatment. Similarly, if the biomarker profile in the subject more closely aligns with that of previous subjects that did not respond to the treatment, the subject can be characterized, or predicted as a non-responder to the treatment.


The sample used for characterizing a cancer can be any disclosed herein, including without limitation a tissue sample, tumor sample, or a bodily fluid. Bodily fluids that can be used included without limitation peripheral blood, sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen (including prostatic fluid), Cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, malignant effusion, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates or other lavage fluids. In an embodiment, the sample comprises vesicles. The biomarkers can be associated with the vesicles. In some embodiments, vesicles are isolated from the sample and the biomarkers associated with the vesicles are assessed.


Comprehensive and Standard-of-Care Molecular Profiling

Molecular profiling according to the invention can be used to guide treatment selection for cancers at any stage of disease or prior treatment. Molecular profiling comprises assessment of DNA mutations, gene rearrangements, gene copy number variation, RNA expression, protein expression, as well as assessment of other biological entities and phenomena that can inform clinical decision making. In some embodiments, the methods herein are used to guide selection of candidate treatments using the standard of care treatments for a particular type or lineage of cancer. Profiling of biomarkers that implicate standard-of-care treatments may be used to assist in treatment selection for a newly diagnosed cancer having multiple treatment options. Such profiling may be referred to herein as “select” profiling. Standard-of-care treatments may comprise NCCN on-compendium treatments or other standard treatments used for a cancer of a given lineage. One of skill will appreciate that such profiles can be updated as the standard of care and/or availability of experimental agents for a given disease lineage change. In other embodiments, molecular profiling is performed for additional biomarkers to identify treatments as beneficial or not beyond that go beyond the standard-of-care for a particular lineage or stage of the cancer. Such “comprehensive” profiling can be performed to assess a wide panel of druggable or drug-associated biomarker targets for any biological sample or specimen of interest. One of skill will appreciate that the select profiles generally comprise subsets of the comprehensive profile. The comprehensive profile can also be used to guide selection of candidate treatments for any cancer at any point of care. The comprehensive profile may also be preferable when standard-of-care treatments not expected to provide further benefit, such as in the salvage treatment setting for recurrent cancer or wherein all standard treatments have been exhausted. For example, the comprehensive profile may be used to assist in treatment selection when standard therapies are not an option for any reason including, without limitation, when standard treatments have been exhausted for the patient. The comprehensive profile may be used to assist in treatment selection for highly aggressive or rare tumors with uncertain treatment regimens. For example, a comprehensive profile can be used to identify a candidate treatment for a newly diagnosed case or when the patient has exhausted standard of care therapies or has an aggressive disease. In practice, molecular profiling according to the invention has indeed identified beneficial therapies for a cancer patient when all standard-of-care treatments were exhausted the treating physician was unsure of what treatment to select next. See the Examples herein. One of skill in the art will appreciate that by its very nature a comprehensive molecular profiling can be used to select a therapy for any appropriate indication independent of the nature of the indication (e.g., source, stage, prior treatment, etc). However, in some embodiments, a comprehensive molecular profile is tailored for a particular indication. For example, biomarkers associated with treatments that are known to be ineffective for a cancer from a particular lineage or anatomical origin may not be assessed as part of a comprehensive molecular profile for that particular cancer. Similarly, biomarkers associated with treatments that have been previously used and failed for a particular patient may not be assessed as part of a comprehensive molecular profile for that particular patient. In yet another non-limiting example, biomarkers associated with treatments that are only known to be effective for a cancer from a particular anatomical origin may only be assessed as part of a comprehensive molecular profile for that particular cancer. One of skill will further appreciate that the comprehensive molecular profile can be updated to reflect advancements, e.g., new treatments, new biomarker-drug associations, and the like, as available.


Molecular Intelligence Profiles (5.0)

The invention provides molecular intelligence (MI) molecular profiles using a variety of techniques to assess panels of biomarkers in order to select or not select a candidate therapeutic for treating a cancer. Such techniques comprise IHC for expression profiling, CISH/FISH for DNA copy number, and Sanger, Pyrosequencing, PCR, RFLP, fragment analysis and Next Generation sequencing for mutational analysis. Such profiles are described in FIGS. 33A-33Q. The profiling is performed using the rules for the biomarker-drug associations for the various cancer lineages as described for FIGS. 33A-33Q and Tables 7-24. MI profiles for all solid tumors or that have additional analyses based on tumor lineage include NextGen analysis of a panel of biomarkers linked to known therapies and clinical trials. The MI profiles can further be expanded to “MI PLUS” profiles that include sequencing of set of genes that are known to be involved in cancer and have alternative clinical utilities including predictive, prognostic or diagnostic uses.


The biomarkers which comprise the molecular intelligence molecular profiles can include genes or gene products that are known to be associated directly with a particular drug or class of drugs. The biomarkers can also be genes or gene products that interact with such drug associated targets, e.g., as members of a common pathway. The biomarkers can be selected from Table 2. In some embodiments, the genes and/or gene products included in the molecular intelligence (MI) molecular profiles are selected from Table 6.









TABLE 6







Exemplary Genes and Gene Products and Related Therapies








Biomarker
Description





ALK
ALK rearrangements may indicate the fusion of ALK (anaplastic lymphoma



kinase) gene with fusion partners, such as EML4. EML4-ALK fusion results in the



pathologic expression of a fusion protein with constitutively active ALK kinase,



resulting in aberrant activation of downstream signaling pathways including RAS-



ERK, JAK3-STAT3 and PI3K-AKT. Patients with ALK rearrangements such as



EML4-ALK are likely to respond to the ALK-targeted agent crizotinib.


AR
The androgen receptor (AR) is a member of the nuclear hormone receptor



superfamily. Prostate tumor dependency on androgens/AR signaling is the basis



for hormone withdrawal, or androgen ablation therapy, to treat men with prostate



cancer. Androgen receptor antagonists as well as agents which block androgen



production are indicated for the treatment of AR expressing prostate cancers.


AREG
AREG, also known as amphiregulin, is a ligand of the epidermal growth factor



receptor. Overexpression of AREG in primary colorectal cancer patients has been



associated with increased clinical benefit from cetuximab in KRAS wildtype



patients.


BRAF
BRAF encodes a protein belonging to the raf/mil family of serine/threonine protein



kinases. This protein plays a role in regulating the MAP kinase/ERK signaling



pathway initiated by EGFR activation, which affects cell division, differentiation,



and secretion. Patients with mutated BRAF genes have a reduced likelihood of



response to EGFR targeted monoclonal antibodies, such as cetuximab in colorectal



cancer. A BRAF enzyme inhibitor, vemurafenib, was approved by FDA to treat



unresectable or metastatic melanoma patients harboring BRAF V600E mutations.


BRCA1
BRCA1, breast cancer type 1 susceptibility gene, is a gene involved in cell growth,



cell division, and DNA-damage repair. Low expression of the BRCA1 gene has



been associated with clinical benefit from cisplatin and carboplatin in cancers of



the lung and ovary.


c-kit
c-Kit is a cytokine receptor expressed on the surface of hematopoietic stem cells as



well as other cell types. This receptor binds to stem cell factor (SCF, a cell growth



factor). As c-Kit is a receptor tyrosine kinase, ligand binding causes receptor



dimerization and initiates a phosphorylation cascade resulting in changes in gene



expression. These changes affect cell proliferation, apoptosis, chemotaxis and



adhesion. c-Kit is inhibited by multi-targeted agents including imatinib, sunitinib



and sorafenib.


cMET
C-Met is a tyrosine kinase receptor for hepatocyte growth factor (HGF) or scatter



factor (SF) and is overexpressed and amplified in a wide range of tumors. cMET



overexpression has been associated with a more aggressive biology and a worse



prognosis in many human malignancies. Amplification or overexpression of cMET



has been implicated in the development of acquired resistance to erlotinib and



gefitinib in NSCLC.


EGFR
EGFR (epidermal growth factor receptor) is a receptor tyrosine kinase and its



abnormalities contribute to the growth and proliferation of many human cancers.



Sensitizing mutations are commonly detected in NSCLC and patients harboring



such mutations may respond to EGFR-targeted tyrosine kinase inhibitors including



erlotinib and gefitinib. Lung cancer patients overexpressing EGFR protein are



known to respond to the EGFR monoclonal antibody, cetuximab. Increased gene



expression of EGFR is associated with response to irinotecan containing regimen



in colorectal cancer patients.


ER
The estrogen receptor (ER) is a member of the nuclear hormone family of



intracellular receptors which is activated by the hormone estrogen. It functions as a



DNA binding transcription factor to regulate estrogen-mediated gene expression.



Estrogen receptors overexpressing breast cancers are referred to as “ER positive.”



Estrogen binding to ER on cancer cells leads to cancer cell proliferation. Breast



tumors over-expressing ER are indicated for treatment with hormone-based anti-



estrogen therapy.


ERBB3
ERBB3 encodes for HER3, a member of the epidermal growth factor receptor



(EGFR) family. This protein forms heterodimers with other EGF receptor family



members which do have kinase activity. Amplification and/or overexpression of



ERBB3 have been reported in numerous cancers, including breast cancer. ERBB3



is a target for drug development.


ERCC1
Nucleotide excision repair (NER) is a DNA repair mechanism necessary for the



repair of DNA damage from a vast variety of sources including chemicals and



ultraviolet (UV) light from the sun. ERCC1 (excision repair cross-



complementation group 1) is an important enzyme in the NER pathway. Platinum-



based drugs induce DNA cross-links that interfere with DNA replication. Tumors



with low ERCC1 expression and, hence, less DNA repair capacity, are more likely



to benefit from platinum-based DNA damaging agents.


EREG
EREG, also known as epiregulin, is a ligand of the epidermal growth factor



receptor. Overexpression of EREG in primary colorectal cancer patients has been



shown to significantly predict clinical outcome in KRAS wildtype patients treated



with cetuximab indicating ligand driven autocrine oncogenic EGFR signaling.


GNA11
G proteins are a family of heterotrimeric proteins coupling seven-transmembrane



domain receptor. These heterotrimeric proteins are composed of three subunits:



Galpha, Gbeta, and Ggamma The GNA11 gene encodes the alpha-11 subunit



(Galpha11). Recent data suggests that over half of uveal melanoma patients lacking



a mutation in GNAQ exhibit mutations in GNA11. Clinical trials are underway



with HDAC inhibitors and MEK inhibitors in patients harboring GNA11



mutations.


GNAQ
G proteins are a family of heterotrimeric proteins coupling seven-transmembrane



domain receptors. G proteins are potential drivers of MAPK activation. In uveal



melanomas 46-53% of patients exhibit a GNAQ mutation which encodes the q



class of G-protein alpha subunit. Clinical trials are underway with HDAC



inhibitors and MEK inhibitors in patients harboring GNAQ mutations.


Her2/Neu
ErbB2/Her2 encodes a member of the epidermal growth factor (EGF) receptor



family of receptor tyrosine kinases. Her2 has no ligand-binding domain of its own



and, therefore, cannot bind growth factors. It does, however, bind tightly to other



ligand-bound EGF receptor family members to form a heterodimer and enhances



kinase-mediated activation of downstream signaling pathways leading to cell



proliferation. Her2 is overexpressed in 15-30% of newly diagnosed breast cancers



and is also expressed in various other cancers. Her2 is a target for the monoclonal



antibodies trastuzumab and pertuzumab which bind to the receptor extracellularly;



the kinase inhibitor lapatinib binds and blocks the receptor intracellularly.


IDH2
IDH2 encodes for the mitochondrial form of isocitrate dehydrogenase, a key



enzyme in the citric acid cycle, which is essential for cell respiration. Mutation in



IDH2 may results in impaired catalytic function of the enzyme, and cause the



overproduction of an onco-metabolite, 2-hydroxy-glutarate, which can extensively



alter the methylation profile in cancer. IDH2 mutation is mutually exclusive of



IDH1 mutation, and has been found in 2% of gliomas and 10% of AML, as well as



in cartilaginous tumors and cholangiocarcinoma. In gliomas, IDH2 mutations are



associated with lower grade astrocytomas, oligodendrogliomas (grade II/III), as



well as secondary glioblastoma (transformed from a lower grade glioma), and are



associated with a better prognosis. In secondary glioblastoma, preliminary



evidence suggests that IDH2 mutation may associate with a better response to



alkylating agent temozolomide. IDH mutations have also been suggested to



associate with a benefit from using hypomethylating agents in cancers including



AML. Various clinical trials investigating agents which target this gene and/or its



downstream or upstream effectors may be available, which include the following:



NCT01534845, NCT01537744. Germline IDH2 mutation has been indicated to



associate with a rare inherited neurometabolic disorder D-2-hydroxyglutaric



aciduria.


KRAS
Proto-oncogene of the Kirsten murine sarcoma virus (KRAS) is a signaling



intermediate involved in many signaling cascades including the EGFR pathway.



Mutations at activating hotspots are associated with resistance to EGFR tyrosine



kinase inhibitors (erlotinib, gefitinib) and monoclonal antibodies (cetuximab,



panitumumab).


MGMT
O-6-methylguanine-DNA methyltransferase (MGMT) encodes a DNA repair



enzyme. Loss of MGMT expression leads to compromised DNA repair in cells and



may play a significant role in cancer formation. Low MGMT expression has been



correlated with response to alkylating agents like temozolomide and dacarbazine.



MGMT expression can be downregulated by promoter hyper methylation.


NRAS
NRAS is an oncogene and a member of the (GTPase) ras family, which includes



KRAS and HRAS. This biomarker has been detected in multiple cancers including



melanoma, colorectal cancer, AML and bladder cancer. Evidence suggests that an



acquired mutation in NRAS may be associated with resistance to vemurafenib in



melanoma patients. In other cancers, e.g., colorectal cancer, NRAS mutation is



associated with resistance to EGFR-targeted monoclonal antibodies.


PGP
P-glycoprotein (MDR1, ABCB1) is an ATP-dependent, transmembrane drug efflux



pump with broad substrate specificity, which pumps antitumor drugs out of cells.



Its expression is often induced by chemotherapy drugs and is thought to be a major



mechanism of chemotherapy resistance. Overexpression of PGP is associated with



resistance to anthracylines (doxorubicin, epirubicin). PGP remains the most



important and dominant representative of Multi-Drug Resistance phenotype and is



correlated with disease state and resistant phenotype.


PIK3CA
The hot spot missense mutations in the gene PIK3CA are present in various



malignancies, e.g., breast, colon and NSCLC, resulting in activation of the PI3



kinase pathway. This pathway is an active target for drug development. PIK3CA



mutations have been associated with benefit from mTOR inhibitors (everolimus,



temsirolimus). Evidence suggests that breast cancer patients with activation of the



PI3K pathway due to PTEN loss or PIK3CA mutation/amplification have a



significantly shorter survival following trastuzumab treatment. PIK3CA mutated



(exon 20) colorectal cancer patients are less likely to respond to EGFR targeted



monoclonal antibody therapy.


PR
The progesterone receptor (PR or PGR) is an intracellular steroid receptor that



specifically binds progesterone, an important hormone that fuels breast cancer



growth. PR positivity in a tumor indicates that the tumor is more likely to be



responsive to hormone therapy by anti-estrogens, aromatase inhibitors and



progestogens.


PTEN
PTEN (phosphatase and tensin homolog) is a tumor suppressor gene that prevents



cells from proliferating. Loss of PTEN protein is one of the most common



occurrences in multiple advanced human cancers. PTEN is an important mediator



in signaling downstream of EGFR, and its loss is associated with reduced benefit to



trastuzumab and EGFR-targeted therapies. Intra-tumoral PTEN loss has been



associated with benefit from mTOR inhibitors (everolimus, temsirolimus).


RET
The RET proto-oncogene is a member of the cadherin superfamily and encodes a



receptor tyrosine kinase cell-surface molecule involved in numerous cellular



mechanisms including cell proliferation, neuronal navigation, cell migration, and



cell differentiation upon binding with glial cell derived neurotrophic factor family



ligands.. Gain of function mutations in RET are associated with the development of



various types of human cancers. Vandetanib is a tyrosine kinase inhibitor that can



inhibit several receptors, including VEGFR, EGFR, and RET.


ROS1
ROS1 (c-ros oncogene 1, receptor tyrosine kinase) is a tyrosine kinase that plays a



role in epithelial cell differentiation and regionalization of the proximal epididymal



epithelium. ROS1 may activate several downstream signaling pathways related to



cell differentiation, proliferation, growth and survival including the PI3 kinase-



mTOR signaling pathway. TKI inhibitors such as crizotinib or other ROS1



inhibitor compounds can have benefit when mutations or rearrangements in ROS1



are identified.


RRM1
Ribonucleotide reductase subunit M1 (RRM1) is a component of the



ribonucleotide reductase holoenzyme consisting of M1 and M2 subunits. The



ribonucleotide reductase is a rate-limiting enzyme involved in the production of



nucleotides required for DNA synthesis. Gemcitabine is a deoxycitidine analogue



which inhibits ribonucleotide reductase activity. High RRM1 level is associated



with resistance to gemcitabine.


SPARC
SPARC (secreted protein acidic and rich in cysteine) is a calcium-binding



matricellular glycoprotein secreted by many types of cells. Studies indicate SPARC



over-expression improves the response to the anticancer drug, nab-paclitaxel. The



improved response is thought to be related to SPARC's role in accumulating



albumin and albumin-targeted agents within tumor tissue.


TLE3
TLE3 is a member of the transducin-like enhancer of split (TLE) family of proteins



that have been implicated in tumorigenesis. It acts downstream of APC and beta-



catenin to repress transcription of a number of oncogenes, which influence growth



and microtubule stability. Studies indicate that TLE3 expression is associated with



response to taxane therapy in various cancers, e.g., breast, ovarian and lung



cancers.


TOP2A
TOPOIIA is an enzyme that alters the supercoiling of double-stranded DNA and



allows chromosomal segregation into daughter cells. Due to its essential role in



DNA synthesis and repair, and frequent overexpression in tumors, TOPOIIA is an



ideal target for antineoplastic agents. In breast cancer, co-amplification of



TOPOIIA and HER2 has been associated with benefit from anthracycline-based



therapy. In HER2 negative breast cancers, patients with low gene expression of



TOPOIIA may derive benefit from anthracycline-based therapy.


TOPO1
Topoisomerase I is an enzyme that alters the supercoiling of double-stranded DNA.



TOPOI acts by transiently cutting one strand of the DNA to relax the coil and



extend the DNA molecule. Higher expression of TOPOI has been associated with



response to TOPOI inhibitors including irinotecan and topotecan.


TS
Thymidylate synthase (TS) is an enzyme involved in DNA synthesis that generates



thymidine monophosphate (dTMP), which is subsequently phosphorylated to



thymidine triphosphate for use in DNA synthesis and repair. Low levels of TS are



predictive of response to fluoropyrimidines and other folate analogues.


TUBB3
Class III β-Tubulin (TUBB3) is part of a class of proteins that provide the



framework for microtubules, major structural components of the cytoskeleton. Due



to their importance in maintaining structural integrity of the cell, microtubules are



ideal targets for anti-cancer agents. Low expression of TUBB3 is associated with



potential clinical benefit to taxanes and vinca alkaloids in certain tumor types.


VEGFR2
VEGFR2, vascular endothelial growth factor 2, is one of three main subtypes of



VEGFR. This protein is an important signaling protein in angiogenesis. Evidence



suggests that increased levels of VEGFR2 may be predictive of response to anti-



angiogenic drugs.









Tables 7, 9, 11, 13, 15, 17 and 21 present views of the information that can be gathered and reported for the MI and MI Plus molecular profiles. Profiles for various lineages are indicated by the table headers. Modifications made dependent on cancer lineage are indicated as appropriate. The columns headed “Agent/Biomarker Status Reported” provide either candidate agents (e.g., drugs) or biomarker status to be included in the report. Where agents are indicated, the association of the agent with the indicated biomarker is included in the report. Where a status is indicated (e.g., mutational status, protein expression status, gene copy number status), the biomarker status is indicated in the report instead of drug associations. The candidate agents may comprise those undergoing clinical trials, as indicated. Platform abbreviations are as used throughout the application, e.g., IHC: immunohistochemistry; FISH: fluorescent in situ hybridication; CISH: colorimetric in situ hybridization; NGS: next generation sequencing; PCR: polymerase chain reaction.


In an embodiment, the invention provides molecular intelligence (MI) profiles for an ovarian cancer comprising assessment of one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 or 58, of: ABL1, AKT1, ALK, APC, AR, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ER, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HER2, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MGMT, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PGP, PIK3CA, PR, PTEN, PTPN11, RB1, RET, RRM1, SMAD4, SMARCB1, SMO, SPARC, STK11, TLE3, TOP2A, TOPO1, TP53, TS, TUBB3, VHL. The invention further provides a method of selecting a candidate treatment for an ovarian cancer comprising assessment of one or more members of the ovarian cancer molecular profile using one or more molecular profiling technique presented herein, e.g., ISH (e.g., FISH, CISH), IHC, RT-PCR, expression array, mutation analysis (e.g., NextGen sequencing, Sanger sequencing, pyrosequencing, Fragment analysis (FA, e.g., RFLP), PCR), etc. In one embodiment, ISH is used to assess one or more, e.g., 1 or 2, of: cMET, HER2. Any useful ISH technique can be used. For example, FISH can be used to assess cMET and/or HER2; or CISH can be used to assess cMET and/or HER2. In an embodiment, protein analysis such as IHC is used to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 of: AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3. “m” and “p” as in SPARC (m/p) refer to IHC performed with monoclonal (“m”) or polyclonal (“p”) primary antibodies. In some embodiments, sequence analysis is used to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 of: ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, STK11, TP53, VHL. For example, the sequence analysis can be performed on one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 of ABL1, APC, BRAF, EGFR, FLT3, GNAQ, IDH1, JAK2, cKIT, KRAS, MPL, NRAS, PDGFRA, VHL. The sequence analysis can also be performed on one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 of ABL1, APC, BRAF, EGFR, FLT3, GNAQ, IDH1, JAK2, cKIT, KRAS, MPL, NPM1, NRAS, PDGFRA, VHL. The sequencing may be performed using Next Generation sequencing technology or other technologies as described herein. The molecular profile can be based on assessing the biomarkers as illustrated in FIGS. 33C-D or Table 7 below.


In an embodiment, the invention provides a molecular intelligence (MI) profile for an ovarian cancer comprising analysis of the biomarkers in FIG. 33C, which may be assessed as indicated in the paragraph above and/or as in FIG. 33C or Table 7 below. For example, the MI profile for ovarian cancer may comprise: 1) ISH to assess one or more, e.g., 1 or 2, of: cMET, HER2; 2) IHC to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 of: AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3; and/or 3) sequence analysis to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 of: ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL. In another embodiment, the invention provides a molecular intelligence (MI) PLUS profile for an ovarian cancer comprising analysis of the biomarkers in the molecular intelligence (MI) profile and the additional biomarker in FIG. 33D, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1, PTPN11, RB1, SMAD4, SMARCB1 and STK11, which may be assessed as indicated this paragraph and/or as in FIG. 33D or Table 7 below. The invention further provides a report comprising results of the ovarian cancer molecular profiling and corresponding candidate treatments that are identified as likely beneficial or likely not beneficial, as further described herein.


Table 7 presents a view of the information that is reported for the ovarian cancer molecular intelligence molecular profiles. The columns headed “Agent/Biomarker Expression Reported” provide either candidate agents (e.g., drugs) or biomarker status to be included in the report. Where agents are indicated, the association of the agent with the indicated biomarker is included in the report. Where a status is indicated (e.g., mutational status, protein expression status, gene copy number status), the biomarker status is indicated in the report instead of drug associations. The candidate agents may comprise those undergoing clinical trials, as indicated. The ovarian cancer profiles provide standard of care therapies for ovarian cancer according to the NCCN guidelines as well as additional non-standard candidate therapies for treating the cancer. As will be evident to one of skill, the same biomarkers in Table 7 can be assessed using the indicated methodology for both MI and MI Plus molecular profiling.









TABLE 7







Molecular Profile and Report Parameters: Ovarian Cancer









Agent(s)/Biomarker Status Reported
Biomarker
Platform





docetaxel, paclitaxel, nab-paclitaxel
TUBB3
IHC



Pgp
IHC



SPARCm
IHC



SPARCp
IHC


irinotecan, topotecan
TOPO1
IHC


gemcitabine
RRM1
IHC


doxorubicin, liposomal-doxorubicin,
HER2
FISH/CISH


epirubicin
TOP2A
IHC



Pgp
IHC


fulvestrant, tamoxifen, letrozole,
ER
IHC


anastrozole




megestrol acetate, leuprolide
ER
IHC



PR
IHC


pemetrexed, capecitabine, fluorouracil
TS
IHC


trastuzumab, pertuzumab, T-DM1,
HER2
IHC,


clinical trials

FISH/CISH


everolimus, temsirolimus, clinical trials
PIK3CA
NGS


protein expression status
AR
IHC



TLE3
IHC


imatinib
cKIT
NGS



PDGFRA
NGS


temozolomide, dacarbazine
MGMT
IHC


vandetanib
RET
NGS


clinical trials
PTEN
IHC


clinical trials
cMET
IHC,




FISH/CISH


clinical trials
VHL
NGS


clinical trials
PTEN
NGS


clinical trials
KRAS
NGS


clinical trials
IDH1
NGS


clinical trials
BRAF
NGS


clinical trials
NRAS
NGS


clinical trials
ABL1
NGS


clinical trials
AKT1
NGS


clinical trials
ALK
NGS


clinical trials
APC
NGS


clinical trials
ATM
NGS


clinical trials
CSF1R
NGS


clinical trials
CTNNB1
NGS


clinical trials
EGFR
NGS


clinical trials
ERBB2
NGS



(HER2)



clinical trials
FGFR1
NGS


clinical trials
FGFR2
NGS


clinical trials
FLT3
NGS


clinical trials
GNAQ
NGS


clinical trials
GNA11
NGS


clinical trials
GNAS
NGS


clinical trials
HRAS
NGS


clinical trials
JAK2
NGS


clinical trials
KDR
NGS



(VEGFR2)



clinical trials
cMET
NGS


clinical trials
MLH1
NGS


clinical trials
MPL
NGS


clinical trials
NOTCH1
NGS


clinical trials
SMO
NGS


clinical trials
TP53
NGS









The invention further provides a set of biomarker-treatment association rules for an ovarian cancer, wherein the rules comprise a predicted likelihood of benefit or lack of benefit of a certain treatment for the cancer given an assessment of one or more biomarker. The associations/rules for an ovarian cancer may comprise those presented in Table 8. Tables 10, 12, 14, 16, 18, 19, 20 and 22 are interpreted similarly. In the tables, the class of drug and illustrative drugs of the indicated class are indicated in the columns “Class of Drugs” and “Drugs,” respectively. The columns headed “Biomarker Result” illustrate illustrative methods of profiling the indicated biomarkers, wherein the results are generally true (“T”) or false (“F”), “Any,” or “No Data.” The data can also be labeled “Equivocal,” “Equivocal Low,” or “Equivocal High,” e.g., for IHC where the observed expression level is near or at the threshold set to determine whether a protein is under-expressed, over-expressed, or expressed at normal levels. For mutations, in some cases a particular mutation (e.g., BRAF V600E or V600K) or region/mutational hotspot is called out (e.g., c-KIT exon11 or exon13). In some cases, a particular mutation is called out from others in the “Biomarker Result.” For example, in the case of cKIT, the V654A mutation or mutations in exon 14, exon 17, or exon 18 are called out in the rules for the tyrosine kinase inhibitor (“TKI”) imatinib. Similarly, in the case of PDGFRA mutations, the PDGFRA D842V mutation may be called out in the tables apart from other PDGFRA mutations. In the case of the taxanes paclitaxel, docetaxel, nab-paclitaxel, certain biomarker results only implicate the likely benefit or not of nab-paclitaxel while others implicate the likely benefit or not of all of paclitaxel, docetaxel, and nab-paclitaxel. Such determinations can be based on the available evidence. One of skill will appreciate that alternative methods can be used to analyze the biomarkers as appropriate. For example, sequencing analysis performed by Next Generation methodology could also be performed by Sanger sequencing or other forms of sequence analysis method such as those described herein or known in the art that yield similar biological information (e.g., an expression or mutation status). The biomarker results combine to predict a benefit or lack of benefit from treatment with the indicated candidate drugs. As an example in Table 8, consider that PIK3CA exon20 is mutated as determined by sequencing (PIK3CA Mutated exon20=T), then the mTOR inhibitor agents everolimus and/or temsirolimus are predicted to have treatment benefit (Overall Benefit=T). However, if PIK3CA exon20 mutation is determined to be false (“F”) or is not determined (“No Data”), then the overall benefit of the mTOR inhibitors is indeterminate. As another example in Table 8, consider that the sample is determined to be ER positive by IHC. In such case, overall benefit from the hormonal agents leuprolide and/or megestrol acetate is expected to be likely (i.e., true or “T”). These results are independent of the status of PR as also determined by IHC. If ER is determined to not be overexpressed (i.e., false “F”) or no data is available, and PR is determined to be positive by IHC, then overall benefit from the hormonal agents leuprolide and/or megestrol acetate is also expected to be likely (i.e., true or “T”). If neither ER nor PR are expressed (i.e., ER Positive=false (“F”) and PR Positive=false (“F”)), then overall benefit from the hormonal agents leuprolide and/or megestrol acetate is expected to be not likely (i.e., false or “F”). The expected overall benefit from the hormonal agents leuprolide and/or megestrol acetate is indeterminate (i.e., “Indet.”) in either of the following situations: 1) ER is not expressed or data is unavailable (i.e., ER Positive=“No Data”) and data is unavailable for PR (i.e., PR Positive=“No Data”); or 2) data is unavailable for ER (i.e., ER Positive=“No Data”) and PR is not expressed (i.e., PR Positive=“F”).


Abbreviations used in Tables 8, 10, 12, 14, 16, 18, 19, 20 and 22 include: tyrosine kinase inhibitor (“TKI”); Sequencing (“Seq.”); Indeterminate (“Indet.”); True (“T”); False (“F”).









TABLE 8







Rules for Ovarian Cancer Biomarker - Drug Associations















Biomarker
Biomarker
Biomarker
Biomarker
Overall


Class of Drugs
Drugs
Result
Result
Result
Result
benefit





Topo1
irinotecan,
TOPO1



Overall


inhibitors
topotecan
Positive



benefit




(IHC)








T



T




F



F




No Data



Indet.


Antimetabolites
gemcitabine
RRM1



Overall




Negative



benefit




(IHC)








T



T




F



F




No Data



Indet.


Hormonal
tamoxifen,
ER



Overall


Agents
fulvestrant,
Positive



Benefit



letrozole,
(IHC)







anastrozole









T



T




F



F




No Data



Indet.


Hormonal
leuprolide,
ER
PR


Overall


Agents
megestrol
Positive
Positive


Benefit



acetate
(IHC)
(IHC)







T
Any


T




F or No
T


T




Data








F
F


F




F or No
No Data


Indet.




Data








No Data
F


Indet.


Antimetabolites
fluorouracil,
TS



Overall



capecitabine,
Negative



benefit



pemetrexed
(IHC)








T



T




F



F




No Data



Indet.


Alkylating
temozolomide,
MGMT



Overall


agents
dacarbazine
Negative



benefit




(IHC)








T



T




F



F




No Data



Indet.


Monoclonal
trastuzumab,
HER2
HER2


Overall


antibodies
pertuzumab,
Positive
Amplified


Benefit


(Her2-
ado-
(IHC)
(ISH)





Targeted)
trastuzumab








emtansine (T-








DM1)









T
Any


T




F,
T or


T




Equivocal
Equivocal







or No Data
High







F or
F or


F




Equivocal
Equivocal








Low







F or
No Data


Indet.




Equivocal








No Data
F,


Indet.





Equivocal








Low or No








Data





mTOR
everolimus,
PIK3CA



Overall


inhibitors
temsirolimus
exon 20



Benefit




(Seq.)








T



T




F or No



Indet.




Data






TKI
imatinib
c-KIT
PDGFRA


Overall




exon 11 |
exon 12 |


Benefit




exon 13
exon 14 |







(Seq.)
exon 18








(Seq.)







Any
D842V


F




V654A
Any


F




T
Any other


T




F, exon 14,
T


T




exon 17,








exon 18 or








No Data








F, exon 14,
F or No


Indet.




exon 17,
Data







exon 18 or








No Data






TKI
crizotinib
ALK
ROS1


Overall




Positive
Positive


Benefit




(ISH)
(ISH)







T
Any


T




F or No
T


T




Data








F
F or No


F





Data







No Data
F or No


Indet.





Data





Anthracyclines
doxorubicin,
TOP2A
Her2
TOP2A
PGP
Overall


and related
liposomal-
Amplified
Amplified
Positive
Positive
Benefit


substances
doxorubicin,
(ISH)
(ISH)
(IHC)
(IHC)




epirubicin









T
Any
Any
Any
T




F or No
T or
Any
Any
T




Data
Equivocal








High







F or No
F,
T
Any
T




Data
Equivocal








Low or No








Data







F
F,
F or No
Any
F





Equivocal
Data







Low or No








Data







No Data
F or
F or No
Any
F





Equivocal
Data







Low







No Data
No Data
F
Any
F




No Data
No Data
No Data
T
F




No Data
No Data
No Data
F
T




No Data
No Data
No Data
No Data
Indet.


TKI (RET-
vandetanib
RET



Overall


targeted)

Mutated



benefit




(Seq.)








T



T




F or No



Indet.




Data






Taxanes
paclitaxel,
SPARC
SPARC
TUBB3
PGP
Overall



docetaxel, nab-
Positive
Positive
Positive
Positive
Benefit



paclitaxel
(Mono
(Poly IHC)
(IHC)
(IHC)





IHC)







nab-paclitaxel
T
Any
T or No
Any
T






Data





paclitaxel,
T
Any
F
Any
T



docetaxel, nab-








paclitaxel








nab-paclitaxel
F or No
T
T or No
Any
T




Data

Data





paclitaxel,
F or No
T
F
Any
T



docetaxel, nab-
Data







paclitaxel








paclitaxel,
F or No
F or No
T
Any
F



docetaxel, nab-
Data
Data






paclitaxel








paclitaxel,
F or No
F or No
F
Any
T



docetaxel, nab-
Data
Data






paclitaxel








nab-paclitaxel
F
F or No
No Data
Any
F





Data






nab-paclitaxel
No Data
F
No Data
Any
F



paclitaxel,
No Data
No Data
No Data
Any
Indet.



docetaxel, nab-








paclitaxel









In an aspect, the invention provides molecular intelligence (MI) profiles for breast cancer comprising assessment of one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 or 58, of: ABL1, AKT1, ALK, APC, AR, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ER, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HER2, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MGMT, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PGP, PIK3CA, PR, PTEN, PTPN11, RB1, RET, RRM1, SMAD4, SMARCB1, SMO, SPARC, STK11, TLE3, TOP2A, TOPO1, TP53, TS, TUBB3, VHL. The invention further provides a method of selecting a candidate treatment for a breast cancer comprising assessment of one or more members of the breast cancer molecular profile using one or more molecular profiling technique presented herein, e.g., ISH (e.g., FISH, CISH), IHC, RT-PCR, expression array, mutation analysis (e.g., NextGen sequencing, Sanger sequencing, pyrosequencing, Fragment analysis (FA, e.g., RFLP), PCR), etc. In one embodiment, ISH is used to assess one or more, e.g., 1, 2 or 3, of: cMET, HER2, TOP2A. Any useful ISH technique can be used. For example, FISH can be used to assess TOP2A and CISH can be used to assess HER2 and cMET. CISH can also be used to assess TOP2A. As desired, FISH can be used to assess HER2 and/or cMET. In an embodiment, protein analysis such as IHC is used to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 of: AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOPO1, TS, TUBB3. “m” and “p” as in SPARC (m/p) refer to IHC performed with monoclonal (“m”) or polyclonal (“p”) primary antibodies. In some embodiments, sequence analysis is used to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 of: ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, STK11, TP53, VHL. For example, the sequence analysis can be performed on one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 of ABL1, APC, BRAF, EGFR, FLT3, GNAQ, IDH1, JAK2, cKIT, KRAS, MPL, NRAS, PDGFRA, VHL. The sequence analysis can also be performed on one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 of ABL1, APC, BRAF, EGFR, FLT3, GNAQ, IDH1, JAK2, cKIT, KRAS, MPL, NPM1, NRAS, PDGFRA, VHL. The sequencing may be performed using Next Generation sequencing technology or other technologies as described herein. The molecular profile can be based on assessing the biomarkers as illustrated in FIGS. 33K-L or Table 9 below.


In an embodiment, the invention provides a molecular intelligence (MI) profile for a breast cancer comprising analysis of the biomarkers in FIG. 33K or Table 9 below. For example, the MI profile for breast cancer may comprise: 1) ISH to assess one or more, e.g., 1, 2 or 3, of: cMET, HER2, TOP2A; 2) IHC to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 of: AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOPO1, TS, TUBB3; and/or 3) sequence analysis to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 of: ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL. In another embodiment, the invention provides a molecular intelligence (MI) PLUS profile for a breast cancer comprising analysis of the biomarkers in the molecular intelligence (MI) profile and the additional biomarker in FIGS. 33L, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1, PTPN11, RB1, SMAD4, SMARCB1 and STK11, which may be assessed as indicated this paragraph and/or as in FIG. 33L or Table 9 below. The invention further provides a report comprising results of the breast cancer molecular profiling and corresponding candidate treatments that are identified as likely beneficial or likely not beneficial, as further described herein.


Table 9 presents a view of the information that is reported for the breast cancer molecular intelligence molecular profiles, which can be interpreted as described for Table 7 above. The biomarker-treatment associations for the molecular profile for breast cancer may comprise those associations in Table 10, which can be interpreted as described for Table 8 above.









TABLE 9







Molecular Profile and Report Parameters: Breast Cancer









Agent(s)/Biomarker Status Reported
Biomarker
Platform





fulvestrant, tamoxifen, toremifene;
ER
IHC


anastrozole, exemestane, letrozole;
PR
IHC


leuprolide, goserelin, megestrol acetate




trastuzumab
HER2
IHC;




FISH/CISH



PTEN
IHC



PIK3CA
NGS


lapatinib, pertuzumab, T-DM1, clinical
HER2
IHC;


trials

FISH/CISH


doxorubicin, liposomal-doxorubicin,
TOP2A
FISH/CISH


epirubicin
HER2
FISH/CISH


fluorouracil, capecitabine, pemetrexed
TS
IHC


docetaxel, paclitaxel, nab-paclitaxel
TLE3
IHC



Pgp
IHC



SPARCm
IHC



SPARCp
IHC


gemcitabine
RRM1
IHC


irinotecan
TOPO1
IHC


everolimus, temsirolimus, clinical trials
ER
IHC



HER2
IHC,




FISH/CISH



PIK3CA
NGS


protein expression status
TUBB3
IHC


imatinib
cKIT
NGS


vandetanib
RET
NGS


clinical trials
AR
IHC


clinical trials
cMET
IHC,




FISH/CISH


clinical trials
BRAF
NGS



KRAS
NGS



NRAS
NGS


clinical trials
IDH1
NGS


clinical trials
VHL
NGS


clinical trials
PTEN
NGS


Clinical Trials
ABL1
NGS


Clinical Trials
AKT1
NGS


Clinical Trials
ALK
NGS


Clinical Trials
APC
NGS


Clinical Trials
ATM
NGS


Clinical Trials
CSF1R
NGS


Clinical Trials
CTNNB1
NGS


Clinical Trials
EGFR
NGS


Clinical Trials
ERBB2
NGS



(HER2)



Clinical Trials
FGFR1
NGS


Clinical Trials
FGFR2
NGS


Clinical Trials
FLT3
NGS


Clinical Trials
GNAQ
NGS


Clinical Trials
GNA11
NGS


Clinical Trials
GNAS
NGS


Clinical Trials
HRAS
NGS


Clinical Trials
JAK2
NGS


Clinical Trials
KDR
NGS



(VEGFR2)



Clinical Trials
cMET
NGS


Clinical Trials
MLH1
NGS


Clinical Trials
MPL
NGS


Clinical Trials
NOTCH1
NGS


Clinical Trials
SMO
NGS


Clinical Trials
TP53
NGS
















TABLE 10







Rules for Breast Cancer Biomarker - Drug Associations















Biomarker
Biomarker
Biomarker
Biomarker
Overall


Drug Class
Drugs
Result
Result
Result
Result
benefit





Antimetabolites
gemcitabine
RRM1



Overall




Negative



benefit




(IHC)








T



T




F



F




No Data



Indet.


Antimetabolites
fluorouracil,
TS



Overall



capecitabine,
Negative



benefit



pemetrexed
(IHC)








T



T




F



F




No Data



Indet.


Topo1
irinotecan
TOPO1



Overall


inhibitors

Positive



benefit




(IHC)








T



T




F



F




No Data



Indet.


Hormonal
tamoxifen,
ER
PR


Overall


Agents
toremifene,
Positive
Positive


Benefit



fulvestrant,
(IHC)
(IHC)






letrozole,








anastrozole,








exemestane,








megestrol








acetate,








leuprolide,








goserelin









T
Any


T




F or No
T


T




Data








F
F


F




F
No Data


Indet.




No Data
F or No


Indet.





Data





Her2-targeted
lapatinib,
HER2
HER2


Overall


Agents
pertuzumab,
Positive
Amplified


Benefit



ado-
(IHC)
(ISH)






trastuzumab








emtansine (T-








DM1)









T
Any


T




F,
T or


T




Equivocal
Equiviocal







or No Data
High







F or
F or


F




Equivocal
Equivocal








Low







F or
No Data


Indet.




Equivocal








No Data
F,


Indet.





Equivocal








Low or No








Data





Anthracyclines
doxorubicin,
TOP2A
HER2


Overall


and related
liposomal-
Amplified
Amplified


Benefit


substances
doxorubicin,
(ISH)
(ISH)






epirubicin









T
Any


T




F or No
T or


T




Data
Equivocal








High







F
F, No Data


F





or








Equivocal








Low







No Data
F or


F





Equivocal








Low







No Data
No Data


Indet.


TKI
crizotinib
ALK
ROS1


Overall




Positive
Positive


Benefit




(ISH)
(ISH)







T
Any


T




F or No
T


T




Data








F
F or No


F





Data







No Data
F or No


Indet.





Data





Monoclonal
trastuzumab
HER2
HER2
PTEN
PIK3CA
Overall


antibodies

Positive
Amplified
Negative
Mutated |
Benefit


(Her2-

(IHC)
(ISH)
(IHC)
exon 20



Targeted -




(Seq.)



trastuzumab)

T
Any
Any
Any
T




F,
T or
Any
Any
T




Equivocal
Equivocal







or No Data
High







F or
F or
Any
Any
F




Equivocal
Equivocal








Low







F or
No Data
Any
Any
Indet.




Equivocal








No Data
F,
Any
Any
Indet.





Equivocal








Low or No








Data





Alkylating
temozolomide,
MGMT



Overall


agents
dacarbazine
Negative



benefit




(IHC)








T



T




F



F




No Data



Indet.


mTOR
everolimus,
ER
Her2
Her2
PIK3CA
Overall


inhibitors
temsirolimus
Positive
Positive
Amplified
exon 20
Benefit




T
T
Any
Any
F




T
F,
T or
Any
F





Equivocal
Equivocal







or No Data
High






T
F,
F,
Any
T





Equivocal
Equivocal







or No Data
Low or No








Data






F
Any
Any
Any
F




No Data
T
Any
Any
F




No Data
F,
T or
Any
F





Equivocal
Equivocal







or No Data
High






No Data
F,
F,
Any
Indet.





Equivocal
Equivocal







or No Data
Low or No








Data




TKI (RET-
vandetanib
RET



Overall


targeted)

Mutated



benefit




(Seq.)








T



T




F or No



Indet.




Data






Taxanes
paclitaxel,
SPARC
SPARC
TLE3
PGP
Overall



docetaxel, nab-
Positive
Positive
Positive
Positive
Benefit



paclitaxel
(Mono
(Poly IHC)
(IHC)
(IHC)





IHC)







paclitaxel,
Any
Any
T
Any
T



docetaxel, nab-








paclitaxel








nab-paclitaxel
T or F
Any
F or No
Any
T






Data





paclitaxel,
F or No
F
F
Any
F



docetaxel, nab-
Data







paclitaxel








nab-paclitaxel
F
F or No
No Data
Any
F





Data






paclitaxel,
F
No Data
F
Any
F



docetaxel, nab-








paclitaxel








nab-paclitaxel
No Data
T
F or No
Any
T






Data





nab-paclitaxel
No Data
F
No Data
Any
F



paclitaxel,
No Data
No Data
F
Any
F



docetaxel, nab-








paclitaxel








paclitaxel,
No Data
No Data
No Data
Any
Indet.



docetaxel, nab-








paclitaxel







TKI
imatinib
c-KIT
PDGFRA


Overall




exon 11 |
exon 12 |


Benefit




exon 13
exon 14 |







(Seq.)
exon 18








(Seq.)







Any
D842V


F




V654A
Any


F




T
Any other


T




F, exon 14,
T


T




exon 17,








exon 18 or








No Data








F, exon 14,
F or No


Indet.




exon 17,
Data







exon 18 or








No Data









In an aspect, the invention provides molecular intelligence (MI) profiles for melanoma comprising assessment of one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 or 58, of: ABL1, AKT1, ALK, APC, AR, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ER, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HER2, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MGMT, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PGP, PIK3CA, PR, PTEN, PTPN11, RB1, RET, RRM1, SMAD4, SMARCB1, SMO, SPARC, STK11, TLE3, TOP2A, TOPO1, TP53, TS, TUBB3, VHL. The invention further provides a method of selecting a candidate treatment for a melanoma comprising assessment of one or more members of the melanoma molecular profile using one or more molecular profiling technique presented herein, e.g., ISH (e.g., FISH, CISH), IHC, RT-PCR, expression array, mutation analysis (e.g., NextGen sequencing, Sanger sequencing, pyrosequencing, Fragment analysis (FA, e.g., RFLP), PCR), etc. In one embodiment, ISH is used to assess one or more, e.g., 1 or 2, of: cMET, HER2. Any useful ISH technique can be used. For example, FISH can be used to assess cMET and/or HER2; or CISH can be used to assess cMET and/or HER2. PCR, e.g., the Cobas V600E test, can be used to assess BRAF. In an embodiment, protein analysis such as IHC is used to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, of: AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3. “m” and “p” as in SPARC (m/p) refer to IHC performed with monoclonal (“m”) or polyclonal (“p”) primary antibodies. In some embodiments, sequence analysis is used to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 of: ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, STK11, TP53, VHL. For example, the sequence analysis can be performed on one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 of ABL1, APC, BRAF, EGFR, FLT3, GNAQ, IDH1, JAK2, cKIT, KRAS, MPL, NRAS, PDGFRA, VHL. The sequence analysis can also be performed on one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 of ABL1, APC, BRAF, EGFR, FLT3, GNAQ, IDH1, JAK2, cKIT, KRAS, MPL, NPM1, NRAS, PDGFRA, VHL. The sequencing may be performed using Next Generation sequencing technology or other technologies as described herein. The molecular profile can be based on assessing the biomarkers as illustrated in FIGS. 33E-F or Table 11 below.


In an embodiment, the invention provides a molecular intelligence (MI) profile for a melanoma comprising analysis of the biomarkers FIG. 33E or Table 11 below. For example, the MI profile for melanoma may comprise: 1) ISH to assess one or more, e.g., 1 or 2, of: cMET, HER2; 2) IHC to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 of: AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3; 3) PCR to assess BRAF; and/or 4) sequence analysis to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 of: ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL. In another embodiment, the invention provides a molecular intelligence (MI) PLUS profile for a melanoma comprising analysis of the biomarkers in the molecular intelligence (MI) profile and the additional biomarker in FIG. 33F, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1, PTPN11, RB1, SMAD4, SMARCB1 and STK11, which may be assessed as indicated this paragraph and/or as in FIG. 33F or Table 11 below. The invention further provides a report comprising results of the molecular profiling and corresponding candidate treatments that are identified as likely beneficial or likely not beneficial, as further described herein.


Table 11 presents a view of the information that is reported for the melanoma molecular intelligence molecular profiles, which can be interpreted as described for Table 7 above. The biomarker-treatment associations for the molecular intelligence molecular profiles for melanoma may comprise those associations in Table 12, which can be interpreted as described for Table 8 above.









TABLE 11







Molecular Profile and Report Parameters: Melanoma









Agent(s)/Biomarker Status




Reported
Biomarker
Platform





vemurafenib, dabrafenib, trametinib*
BRAF
cobas PCR




NGS


temozolomide, dacarbazine
MGMT
IHC


imatinib
cKIT
NGS



PDGFRA
NGS


everolimus, temsirolimus, clinical
PIK3CA
NGS


trials




protein expression status
AR
IHC



ER
IHC



PR
IHC


paclitaxel, docetaxel, nab-paclitaxel
TLE3
IHC



TUBB3
IHC



Pgp
IHC



SPARCm
IHC



SPARCp
IHC


doxorubicin, liposomal-doxorubicin,
HER2
FISH/CISH


epirubicin
TOP2A
IHC



Pgp
IHC


trastuzumab, lapatinib, pertuzumab,
HER2
IHC, FISH/CISH


T-DM1




gemcitabine
RRM1
IHC


irinotecan
TOPO1
IHC


fluorouracil, capecitabine, pemetrexed
TS
IHC


vandetanib
RET
NGS


clinical trials
PTEN
IHC


clinical trials
cMET
IHC, FISH/CISH


clinical trials
BRAF
cobas PCR


clinical trials

NGS


clinical trials
IDH1
NGS


clinical trials
KRAS
NGS


clinical trials
NRAS
NGS


clinical trials
VHL
NGS


clinical trials
PTEN
NGS


clinical trials
ABL1
NGS


clinical trials
AKT1
NGS


clinical trials
ALK
NGS


clinical trials
APC
NGS


clinical trials
ATM
NGS


clinical trials
CSF1R
NGS


clinical trials
CTNNB1
NGS


clinical trials
EGFR
NGS


clinical trials
ERBB2
NGS



(HER2)



clinical trials
FGFR1
NGS


clinical trials
FGFR2
NGS


clinical trials
FLT3
NGS


clinical trials
GNAQ
NGS


clinical trials
GNA11
NGS


clinical trials
GNAS
NGS


clinical trials
HRAS
NGS


clinical trials
JAK2
NGS


clinical trials
KDR
NGS



(VEGFR2)



clinical trials
cMET
NGS


clinical trials
MLH1
NGS


clinical trials
MPL
NGS


clinical trials
NOTCH1
NGS


clinical trials
SMO
NGS


clinical trials
TP53
NGS





*trametinib association will include BRAF by NGS testing for V600K mutations.













TABLE 12







Rules for Melanoma Biomarker - Drug Associations
















Biomarker
Biomarker
Biomarker
Biomarker
Biomarker
Overall


Class of Drugs
Drugs
Result
Result
Result
Result
Result
benefit





Antimetabolites
gemcitabine
RRM1




Overall




Negative




benefit




(IHC)









T




T




F




F




No Data




Indet.


Antimetabolites
fluorouracil,
TS




Overall



capecitabine,
Negative




benefit



pemetrexed
(IHC)









T




T




F




F




No Data




Indet.


Topo1
irinotecan
TOPO1




Overall


inhibitors

Positive




benefit




(IHC)









T




T




F




F




No Data




Indet.


Alkylating
temozolomide,
MGMT




Overall


agents
dacarbazine
Negative




benefit




(IHC)









T




T




F




F




No Data




Indet.


TKI
vemurafenib,
BRAF
BRAF



Overall



dabrafenib,
V600E
mutated |



Benefit



trametinib
(PCR)
V600E |









V600K









(Seq.)








T
Any



T




F
Any



F




No Data
Any



Indet.


mTOR
everolimus,
PIK3CA




Overall


inhibitors
temsirolimus
exon 20




Benefit




(Seq.)









T




T




F or No




Indet.




Data







TKI
imatinib
c-KIT
PDGFRA



Overall




exon 11 |
exon 12 |



Benefit




exon 13
exon 14 |








(Seq.)
exon 18









(Seq.)








Any
D842V



F




V654A
Any



F




T
Any other



T




F, exon 14,
T



T




exon 17,









exon 18 or









No Data









F, exon 14,
F or No



Indet.




exon 17,
Data








exon 18 or









No Data







TKI
lapatinib
HER2
HER2



Overall




Positive
Amplified



Benefit




(IHC)
(ISH)








T
Any



T




F,
T or



T




Equivocal
Equivocal








or No Data
High








F or
F or



F




Equivocal
Equivocal









Low








F or
No Data



Indet.




Equivocal









No Data
F,



Indet.





Equivocal









Low or No









Data






Monoclonal
trastuzumab,
HER2
HER2



Overall


antibodies
pertuzumab,
Positive
Amplified



Benefit


(Her2-
ado-
(IHC)
(ISH)






Targeted)
trastuzumab









emtansine (T-









DM1)










T
Any



T




F,
T or



T




Equivocal
Equivocal








or No Data
High








F or
F or








Equivocal
Equivocal









Low



F




F or
No Data



Indet.




Equivocal









No Data
F,



Indet.





Equivocal









Low or No









Data






Anthracyclines
doxorubicin,
TOP2A
Her2
TOP2A
PGP

Overall


and related
liposomal-
Amplified
Amplified
Positive
Positive

Benefit


substances
doxorubicin,
(ISH)
(ISH)
(IHC)
(IHC)





epirubicin










T
Any
Any
Any

T




F or No
T or
Any
Any

T




Data
Equivocal









High








F or No
F,
T
Any

T




Data
Equivocal









Low or No









Data








F
F,
F or No
Any

F





Equivocal
Data








Low or No









Data








No Data
F,
F
Any

F





Equivocal









Low or No









Data








No Data
F or
No Data
Any

F





Equivocal









Low








No Data
No Data
No Data
T

F




No Data
No Data
No Data
F

T




No Data
No Data
No Data
No Data

Indet.


TKI
crizotinib
ALK
ROS1



Overall




Positive
Positive



Benefit




(ISH)
(ISH)








T
Any



T




F or No
T



T




Data









F
F or No



F





Data








No Data
F or No



Indet.





Data






TKI (RET-
vandetanib
RET




Overall


targeted)

Mutated




benefit




(Seq.)









T




T




F or No




Indet.




Data







Taxanes
paclitaxel,
SPARC
SPARC
TLE3
TUBB3
PGP
Overall



docetaxel,
Positive
Positive
Positive
Positive
Positive
Benefit



nab-paclitaxel
(Mono
(Poly IHC)
(IHC)
(IHC)
(IHC)





IHC)








paclitaxel,
Any
Any
T
Any
Any
T



docetaxel, nab-









paclitaxel









nab-paclitaxel
T
Any
F or No
T or No
Any
T






Data
Data





paclitaxel,
T
Any
F or No
F
Any
T



docetaxel, nab-


Data






paclitaxel









nab-paclitaxel
F or No
T
F or No
T or No
Any
T




Data

Data
Data





paclitaxel,
F or No
T
F or No
F
Any
T



docetaxel, nab-
Data

Data






paclitaxel









paclitaxel,
F or No
F
F
T or No
Any
F



docetaxel, nab-
Data


Data





paclitaxel









paclitaxel,
F or No
F
F or No
F
Any
T



docetaxel, nab-
Data

Data






paclitaxel









paclitaxel,
F or No
F
No Data
T
Any
F



docetaxel, nab-
Data








paclitaxel









nab-paclitaxel
F
F or No
No Data
No Data
Any
F





Data







paclitaxel,
F
No Data
F
T or No
Any
F



docetaxel, nab-



Data





paclitaxel









paclitaxel,
F or No
No Data
F or No
F
Any
T



docetaxel, nab-
Data

Data






paclitaxel









paclitaxel,
F or No
No Data
No Data
T
Any
F



docetaxel, nab-
Data








paclitaxel









nab-paclitaxel
No Data
F
No Data
No Data
Any
F



paclitaxel,
No Data
No Data
F
T or No
Any
F



docetaxel, nab-



Data





paclitaxel









paclitaxel,
No Data
No Data
No Data
No Data
Any
Indet.



docetaxel, nab-









paclitaxel









In an aspect, the invention provides molecular intelligence (MI) profiles for uveal melanoma comprising assessment of one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 or 58, of: ABL1, AKT1, ALK, APC, AR, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ER, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HER2, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MGMT, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PGP, PIK3CA, PR, PTEN, PTPN11, RB1, RET, RRM1, SMAD4, SMARCB1, SMO, SPARC, STK11, TLE3, TOP2A, TOPO1, TP53, TS, TUBB3, VHL. The invention further provides a method of selecting a candidate treatment for a uveal melanoma comprising assessment of one or more members of the uveal melanoma molecular profile using one or more molecular profiling technique presented herein, e.g., ISH (e.g., FISH, CISH), IHC, RT-PCR, expression array, mutation analysis (e.g., NextGen sequencing, Sanger sequencing, pyrosequencing, Fragment analysis (FA, e.g., RFLP), PCR), etc. In one embodiment, ISH is used to assess one or more, e.g., 1 or 2, of: cMET, HER2. Any useful ISH technique can be used. For example, FISH can be used to assess cMET and/or HER2; or CISH can be used to assess cMET and/or HER2. PCR, e.g., the Cobas V600E test, can be used to assess BRAF. In an embodiment, protein analysis such as IHC is used to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, of: AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3. “m” and “p” as in SPARC (m/p) refer to IHC performed with monoclonal (“m”) or polyclonal (“p”) primary antibodies. In some embodiments, sequence analysis is used to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 of: ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, STK11, TP53, VHL. For example, the sequence analysis can be performed on one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 of ABL1, APC, BRAF, EGFR, FLT3, GNAQ, IDH1, JAK2, cKIT, KRAS, MPL, NRAS, PDGFRA, VHL. The sequence analysis can also be performed on one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 of ABL1, APC, BRAF, EGFR, FLT3, GNAQ, IDH1, JAK2, cKIT, KRAS, MPL, NPM1, NRAS, PDGFRA, VHL. The sequencing may be performed using Next Generation sequencing technology or other technologies as described herein. The molecular profile can be based on assessing the biomarkers as illustrated in FIGS. 33G-H or Table 11.


In an embodiment, the invention provides a molecular intelligence (MI) profile for a uveal melanoma comprising analysis of the biomarkers in FIG. 33G, which may be assessed as indicated in the paragraph above and/or as in FIG. 33G or Table 11. For example, the MI profile for uveal melanoma may comprise: 1) ISH to assess one or more, e.g., 1 or 2, of: cMET, HER2; 2) IHC to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 of: AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3; 3) PCR to assess BRAF; and/or 4) sequence analysis to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 of: ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL. In another embodiment, the invention provides a molecular intelligence (MI) PLUS profile for a uveal melanoma comprising analysis of the biomarkers in the molecular intelligence (MI) profile and the additional biomarker in FIG. 33H, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1, PTPN11, RB1, SMAD4, SMARCB1 and STK11, which may be assessed as indicated this paragraph and/or as in FIG. 33H or Table 11 below. The invention further provides a report comprising results of the molecular profiling and corresponding candidate treatments that are identified as likely beneficial or likely not beneficial, as further described herein.


Table 13 presents a view of the information that is reported for the uveal melanoma molecular intelligence molecular profiles, which can be interpreted as described for Table 7 above. The biomarker-treatment associations for the molecular intelligence molecular profiles for uveal melanoma may comprise those associations in Table 14, which can be interpreted as described for Table 8 above.









TABLE 13







Molecular Profile and Report Parameters: Uveal Melanoma









Agent(s)/Biomarker Status Reported
Biomarker
Platform





vemurafenib
BRAF
cobas PCR


temozolomide, dacarbazine
MGMT
IHC


imatinib
cKIT
NGS



PDGFRA
NGS


everolimus, temsirolimus, clinical trials
PIK3CA
NGS


protein expression status
AR
IHC



ER
IHC



PR
IHC


paclitaxel, docetaxel, nab-paclitaxel
TLE3
IHC



TUBB3
IHC



Pgp
IHC



SPARCm
IHC



SPARCp
IHC


doxorubicin, liposomal-doxorubicin ,
HER2
FISH/CISH


epirubicin
TOP2A
IHC



Pgp
IHC


trastuzumab, lapatinib, pertuzumab,
HER2
IHC,


T-DM1

FISH/CISH


gemcitabine
RRM1
IHC


irinotecan
TOPO1
IHC


fluorouracil, capecitabine, pemetrexed
TS
IHC


vandetanib
RET
NGS


clinical trials
cMET
IHC,




FISH/CISH


clinical trials
PTEN
IHC


clinical trials
IDH1
NGS


clinical trials
BRAF
NGS


clinical trials
KRAS
NGS


clinical trials
NRAS
NGS


clinical trials
GNA11
NGS


clinical trials
VHL
NGS


clinical trials
PTEN
NGS


clinical trials
ABL1
NGS


clinical trials
AKT1
NGS


clinical trials
ALK
NGS


clinical trials
APC
NGS


clinical trials
ATM
NGS


clinical trials
CSF1R
NGS


clinical trials
CTNNB1
NGS


clinical trials
EGFR
NGS


clinical trials
ERBB2
NGS



(HER2)



clinical trials
FGFR1
NGS


clinical trials
FGFR2
NGS


clinical trials
FLT3
NGS


clinical trials
GNAQ
NGS


clinical trials
GNAS
NGS


clinical trials
HRAS
NGS


clinical trials
JAK2
NGS


clinical trials
KDR
NGS



(VEGFR2)



clinical trials
cMET
NGS


clinical trials
MLH1
NGS


clinical trials
MPL
NGS


clinical trials
NOTCH1
NGS


clinical trials
SMO
NGS


clinical trials
TP53
NGS
















TABLE 14







Rules for Uveal Melanoma Biomarker - Drug Associations
















Biomarker
Biomarker
Biomarker
Biomarker
Biomarker
Overall


Class of Drugs
Drugs
Result
Result
Result
Result
Result
benefit





Antimetabolites
gemcitabine
RRM1




Overall




Negative




benefit




(IHC)









T




T




F




F




No Data




Indet.


Antimetabolites
fluorouracil,
TS Negative




Overall



capecitabine,
(IHC)




benefit



pemetrexed










T




T




F




F




No Data




Indet.


Topo1 inhibitors
irinotecan
TOPO1




Overall




Positive




benefit




(IHC)









T




T




F




F




No Data




Indet.


Alkylating
temozolomide,
MGMT




Overall


agents
dacarbazine
Negative




benefit




(IHC)









T




T




F




F




No Data




Indet.


TKI
vemurafenib
BRAF
BRAF



Overall




V600E
mutated |



Benefit




(PCR)
V600E |









V600K |









(Seq.)








T
Any



T




F
Any



F




No Data
Any



Indet.


mTOR
everolimus,
PIK3CA




Overall


inhibitors
temsirolimus
exon 20




Benefit




(Seq.)









T




T




F or No Data




Indet.


TKI
imatinib
c-KIT
PDGFRA



Overall




exon 11 |
exon 12 |



Benefit




exon 13
exon 14 |








(Seq.)
exon 18









(Seq.)








Any
D842V



F




V654A
Any



F




T
Any other



T




F, exon 14,
T



T




exon 17,









exon 18 or









No Data









F, exon 14,
F or No



Indet.




exon 17,
Data








exon 18 or









No Data







TKI
lapatinib
HER2
HER2



Overall




Positive
Amplified



Benefit




(IHC)
(ISH)








T
Any



T




F, Equivocal
T or



T




or No Data
Equivocal









High








F or
F or



F




Equivocal
Equivocal









Low








F or
No Data



Indet.




Equivocal









No Data
F, Equivocal



Indet.





Low or No









Data






Monoclonal
trastuzumab,
HER2
HER2



Overall


antibodies
pertuzumab,
Positive
Amplified



Benefit


(Her2-Targeted)
ado-trastuzumab
(IHC)
(ISH)







emtansine (T-









DM1)










T
Any



T




F, Equivocal
T or



T




or No Data
Equivocal









High








F or
F or



F




Equivocal
Equivocal









Low








F or
No Data



Indet.




Equivocal









No Data
F, Equivocal



Indet.





Low or No









Data






TKI
crizotinib
ALK
ROS1



Overall




Positive
Positive



Benefit




(ISH)
(ISH)








T
Any



T




F or No Data
T



T




F
F or No









Data



F




No Data
F or No



Indet.





Data






Anthracyclines
doxorubicin,
TOP2A
Her2
TOP2A
PGP

Overall


and related
liposomal-
Amplified
Amplified
Positive
Positive

Benefit


substances
doxorubicin,
(ISH)
(ISH)
(IHC)
(IHC)





epirubicin










T
Any
Any
Any

T




F or No Data
T or
Any
Any

T





Equivocal









High








F or No Data
F, Equivocal
T
Any

T





Low or No









Data








F
F, Equivocal
F or No
Any

F





Low or No
Data








Data








No Data
F, Equivocal
F
Any

F





Low or No









Data








No Data
F or
No Data
Any

F





Equivocal









Low








No Data
No Data
No Data
T

F




No Data
No Data
No Data
F

T




No Data
No Data
No Data
No Data

Indet.


TKI (RET-
vandetanib
RET




Overall


targeted)

Mutated




benefit




(Seq.)









T




T




F or No Data




Indet.


Taxanes
paclitaxel,
SPARC
SPARC
TLE3
TUBB3
PGP
Overall



docetaxel, nab-
Positive
Positive
Positive
Positive
Positive
Benefit



paclitaxel
(Mono IHC)
(Poly IHC)
(IHC)
(IHC)
(IHC)




paclitaxel,
Any
Any
T
Any
Any
T



docetaxel, nab-









paclitaxel









nab-paclitaxel
T
Any
F or No
T or No
Any
T






Data
Data





paclitaxel,
T
Any
F or No
F
Any
T



docetaxel, nab-


Data






paclitaxel









nab-paclitaxel
F or No Data
T
F or No
T or No
Any
T






Data
Data





paclitaxel,
F or No Data
T
F or No
F
Any
T



docetaxel, nab-


Data






paclitaxel









paclitaxel,
F or No Data
F
F
T or No
Any
F



docetaxel, nab-



Data





paclitaxel









paclitaxel,
F or No Data
F
F or No
F
Any
T



docetaxel, nab-


Data






paclitaxel









paclitaxel,
F or No Data
F
No Data
T
Any
F



docetaxel, nab-









paclitaxel









nab-paclitaxel
F
F or No
No Data
No Data
Any
F





Data







paclitaxel,
F
No Data
F
T or No
Any
F



docetaxel, nab-



Data





paclitaxel









paclitaxel,
F or No Data
No Data
F or No
F
Any
T



docetaxel, nab-


Data






paclitaxel









paclitaxel,
F or No Data
No Data
No Data
T
Any
F



docetaxel, nab-









paclitaxel









nab-paclitaxel
No Data
No Data
No Data
Any
F
F



paclitaxel,
No Data
No Data
F
T or No
Any
F



docetaxel, nab-



Data





paclitaxel
No Data
No Data
No Data
No Data
Any
Indet.



paclitaxel,









docetaxel, nab-









paclitaxel









In an aspect, the invention provides molecular intelligence (MI) profiles for colorectal cancer (CRC) comprising assessment of one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 or 58, of: ABL1, AKT1, ALK, APC, AR, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ER, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HER2, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MGMT, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PGP, PIK3CA, PR, PTEN, PTPN11, RB1, RET, RRM1, SMAD4, SMARCB1, SMO, SPARC, STK11, TLE3, TOP2A, TOPO1, TP53, TS, TUBB3, VHL. The invention further provides a method of selecting a candidate treatment for a CRC comprising assessment of one or more members of the CRC molecular profile using one or more molecular profiling technique presented herein, e.g., ISH (e.g., FISH, CISH), IHC, RT-PCR, expression array, mutation analysis (e.g., NextGen sequencing, Sanger sequencing, pyrosequencing, Fragment analysis (FA, e.g., RFLP), PCR), etc. In one embodiment, ISH is used to assess one or more, e.g., 1 or 2, of: cMET, HER2. Any useful ISH technique can be used. For example, FISH can be used to assess cMET and/or HER2; or CISH can be used to assess cMET and/or HER2. In an embodiment, protein analysis such as IHC is used to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, of: AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3. “m” and “p” as in SPARC (m/p) refer to IHC performed with monoclonal (“m”) or polyclonal (“p”) primary antibodies. In some embodiments, sequence analysis is used to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 of: ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, STK11, TP53, VHL. For example, the sequence analysis can be performed on one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 of ABL1, APC, BRAF, EGFR, FLT3, GNAQ, IDH1, JAK2, cKIT, KRAS, MPL, NRAS, PDGFRA, VHL. The sequence analysis can also be performed on one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 of ABL1, APC, BRAF, EGFR, FLT3, GNAQ, IDH1, JAK2, cKIT, KRAS, MPL, NPM1, NRAS, PDGFRA, VHL. The sequencing may be performed using Next Generation sequencing technology or other technologies as described herein. The molecular profile can be based on assessing the biomarkers as illustrated in FIGS. 33M-N or Table 15 below.


In an embodiment, the invention provides a molecular intelligence (MI) profile for a CRC comprising analysis of the biomarkers in FIG. 33M, which may be assessed as indicated in FIG. 33M or Table 15 below. For example, the MI profile for colorectal cancer may comprise: 1) ISH to assess one or more, e.g., 1 or 2, of: cMET, HER2; 2) IHC to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 of: AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3; and/or 3) sequence analysis to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 of: ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL. In another embodiment, the invention provides a molecular intelligence (MI) PLUS profile for a CRC comprising analysis of the biomarkers in the molecular intelligence (MI) profile and the additional biomarker in FIG. 33N, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1, PTPN11, RB1, SMAD4, SMARCB1 and STK11, which may be assessed as indicated this paragraph and/or as in FIG. 33N or Table 15 below. The invention further provides a report comprising results of the molecular profiling and corresponding candidate treatments that are identified as likely beneficial or likely not beneficial, as further described herein.


Table 15 presents a view of the information that is reported for the colorectal cancer molecular intelligence molecular profiles, which can be interpreted as described for Table 7 above. The biomarker-treatment associations for the molecular intelligence molecular profiles for colorectal cancer may comprise those associations in Table 16, which can be interpreted as described for Table 8 above.









TABLE15







Molecular Profile and Report Parameters: Colorectal Cancer (CRC)









Agent(s)/Biomarker Status Reported
Biomarker
Platform





cetuximab, panitumumab
KRAS
NGS



BRAF
NGS



NRAS
NGS



PIK3CA
NGS



PTEN
IHC


fluorouracil, capecitabine, pemetrexed
TS
IHC


irinotecan
TOPO1
IHC


protein expression status
AR
IHC



ER
IHC



PR
IHC


imatinib
cKIT
NGS



PDGFRA
NGS


doxorubicin, liposomal-doxorubicin,
HER2
FISH/CISH


epirubicin
TOP2A
IHC



Pgp
IHC


trastuzumab, lapatinib, pertuzumab,
HEA
IHC,


T-DM1

FISH/CISH


gemcitabine
RRM1
IHC


temozolomide, dacarbazine
MGMT
IHC


docetaxel, paclitaxel, nab-paclitaxel
TLE3
IHC



TUBB3
IHC



Pgp
IHC



SPARCm
IHC



SPARCp
IHC


vandetanib
RET
NGS


clinical trials
cMET
IHC,




FISH/CISH


clinical trials
VHL
NGS


clinical trials
PTEN
NGS


clinical trials
IDH1
NGS


clinical trials
ABL1
NGS


clinical trials
AKT1
NGS


clinical trials
ALK
NGS


clinical trials
APC
NGS


clinical trials
ATM
NGS


clinical trials
CSF1R
NGS


clinical trials
CTNNB1
NGS


clinical trials
EGFR
NGS


clinical trials
ERBB2
NGS



(HER2)



clinical trials
FGFR1
NGS


clinical trials
FGFR2
NGS


clinical trials
FLT3
NGS


clinical trials
GNAQ
NGS


clinical trials
GNA11
NGS


clinical trials
GNAS
NGS


clinical trials
HRAS
NGS


clinical trials
JAK2
NGS


clinical trials
KDR
NGS



(VEGFR2)



clinical trials
cMET
NGS


clinical trials
MLH1
NGS


clinical trials
MPL
NGS


clinical trials
NOTCH1
NGS


clinical trials
SMO
NGS


clinical trials
TP53
NGS
















TABLE 16







Rules for Colorectal Cancer Biomarker - Drug Associations
















Biomarker
Biomarker
Biomarker
Biomarker
Biomarker
Overall


Drug Class
Drugs
Result
Result
Result
Result
Result
Benefit





Monoclonal
cetuximab,
KRAS
BRAF
NRAS
PIK3CA
PTEN
Overall


antibodies
panitumumab
Mutated
V600E
Mutated
Mutated |
Negative
Benefit


(EGFR-

(Seq.)
(Seq.)
(Seq.)
exon 20
(IHC)



targeted)




(Seq.)






T
Any
Any
Any
Any
F




F or G13D
Any
Any
Any
Any
T




No Data
Any
Any
Any
Any
Indet.


Antimetabolites
gemcitabine
RRM1




Overall




Negative




benefit




(IHC)









T




T




F




F




No Data




Indet.


Antimetabolites
fluorouracil,
TS




Overall



capecitabine,
Negative




benefit



pemetrexed
(IHC)









T




T




F




F




No Data




Indet.


Topo1
irinotecan
TOPO1




Overall


inhibitors

Positive




benefit




(IHC)









T




T




F




F




No Data




Indet.


Alkylating
temozolomide,
MGMT




Overall


agents
dacarbazine
Negative




benefit




(IHC)









T




T




F




F




No Data




Indet.


TKI
lapatinib
HER2
HER2



Overall




Positive
Amplified



Benefit




(IHC)
(ISH)








T
Any



T




F,
T or



T




Equivocal
Equivocal








or No Data
High








F or
F or



F




Equivocal
Equivocal









Low








F or
No Data



Indet.




Equivocal









No Data
F,



Indet.





Equivocal









Low or No









Data






Monoclonal
trastuzumab,
HER2
HER2



Overall


antibodies
pertuzumab, ado-
Positive
Amplified



Benefit


(Her2-
trastuzumab
(IHC)
(ISH)






Targeted)
emtansine (T-









DM1)










T
Any



T




F,
T or








Equivocal
Equivocal



T




or No Data
High








F or
F or



F




Equivocal
Equivocal









Low








F or
No Data



Indet.




Equivocal









No Data
F,



Indet.





Equivocal









Low or No









Data






TKI
crizotinib
ALK
ROS1



Overall




Positive
Positive



Benefit




(ISH)
(ISH)








T
Any



T




F or No
T



T




Data









F
F or No



F





Data








No Data
F or No



Indet.





Data






Anthracyclines
doxorubicin,
TOP2A
Her2
TOP2A
PGP

Overall


and related
liposomal-
Amplified
Amplified
Positive
Positive

Benefit


substances
doxorubicin,
(ISH)
(ISH)
(IHC)
(IHC)





epirubicin










T
Any
Any
Any

T




F or No
T or
Any
Any






Data
Equivocal









High



T




F or No
F,
T
Any

T




Data
Equivocal









Low or No









Data








F
F,
F or No
Any

F





Equivocal
Data








Low or No









Data








No Data
F,
F
Any

F





Equivocal









Low or No









Data








No Data
F or
No Data
Any

F





Equivocal









Low








No Data
No Data
No Data
T

F




No Data
No Data
No Data
F

T




No Data
No Data
No Data
No Data

Indet.


TKI
imatinib
c-KIT
PDGFRA



Overall




exon 11 |
exon 12 |



Benefit




exon 13
exon 14 |








(Seq.)
exon 18









(Seq.)








Any
D842V



F




V654A
Any



F




T
Any other



T




F, exon 14,
T



T




exon 17,









exon 18 or









No Data









F, exon 14,
F or No



Indet.




exon 17,
Data








exon 18 or









No Data







TKI (RET-
vandetanib
RET




Overall


targeted)

Mutated




benefit




(Seq.)









T




T




F or No




Indet.




Data







Taxanes
paclitaxel,
SPARC
SPARC
TLE3
TUBB3
PGP
Overall



docetaxel, nab-
Positive
Positive
Positive
Positive
Positive
BenefiT



paclitaxel
(Mono
(Poly IHC)
(IHC)
(IHC)
(IHC)





IHC)








paclitaxel,
Any
Any
T
Any
Any
T



docetaxel, nab-









paclitaxel









nab-paclitaxel
T
Any
F or No
T or No
Any
T






Data
Data





paclitaxel,
T
Any
F or No
F
Any
T



docetaxel, nab-


Data






paclitaxel









nab-paclitaxel
F or No
T
F or No
T or No
Any
T




Data

Data
Data





paclitaxel,
F or No
T
F or No
F
Any
T



docetaxel, nab-
Data

Data






paclitaxel









paclitaxel,
F or No
F
F
T or No
Any
F



docetaxel, nab-
Data


Data





paclitaxel









paclitaxel,
F or No
F
F or No
F
Any
T



docetaxel, nab-
Data

Data






paclitaxel









paclitaxel,
F or No
F
No Data
T
Any
F



docetaxel, nab-
Data








paclitaxel









nab-paclitaxel
F
F or No
No Data
No Data
Any
F





Data







paclitaxel,
F
No Data
F
T or No
Any
F



docetaxel, nab-



Data





paclitaxel









paclitaxel,
F or No
No Data
F or No
F
Any
T



docetaxel, nab-
Data

Data






paclitaxel









paclitaxel,
F or No
No Data
No Data
T
Any
F



docetaxel, nab-
Data




F



paclitaxel









nab-paclitaxel
No Data
F
No Data
No Data
Any
F



paclitaxel,
No Data
No Data
F
T or No
Any
F



docetaxel, nab-



Data





paclitaxel









paclitaxel,
No Data
No Data
No Data
No Data
Any
Indet.



docetaxel, nab-









paclitaxel









In an aspect, the invention provides molecular intelligence (MI) profiles for a lung cancer, including without limitation a non-small cell lung cancer (NSCLC) or bronchioloalveolar cancer (BAC or LBAC), comprising assessment of one or more, e.g., e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 or 59 of: ABL1, AKT1, ALK, APC, AR, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ER, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HER2, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MGMT, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PGP, PIK3CA, PR, PTEN, PTPN11, RB1, RET, ROS1, RRM1, SMAD4, SMARCB1, SMO, SPARC, STK11, TLE3, TOP2A, TOPO1, TP53, TS, TUBB3, VHL. In one embodiment, ISH is used to assess one or more, e.g., 1, 2, 3, or 4, of: ALK, cMET, HER2, ROS1. Any useful ISH technique can be used. For example, FISH can be used to assess one or two of: ALK and ROS1; and CISH can be used to assess HER2 and cMET. CISH can also be used to assess ALK and/or ROS1. As desired, FISH can be used to assess HER2 and/or cMET. In an embodiment, protein analysis such as IHC is used to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 of: AR, cMET, EGFR (H-score), ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3. “m” and “p” as in SPARC (m/p) refer to IHC performed with monoclonal (“m”) or polyclonal (“p”) primary antibodies. EGFR can be assessed using an H-score, as described herein. In some embodiments, sequence analysis is used to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 of: ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, STK11, TP53, VHL. For example, the sequence analysis can be performed on one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 of ABL1, APC, BRAF, EGFR, FLT3, GNAQ, IDH1, JAK2, cKIT, KRAS, MPL, NRAS, PDGFRA, VHL. The sequence analysis can also be performed on one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 of ABL1, APC, BRAF, EGFR, FLT3, GNAQ, IDH1, JAK2, cKIT, KRAS, MPL, NPM1, NRAS, PDGFRA, VHL. The sequencing may be performed using Next Generation sequencing technology or other technologies as described herein. The molecular profile can be based on assessing the biomarkers as illustrated in FIGS. 33I-J or Table 17 below.


In an embodiment, the invention provides a molecular intelligence (MI) profile for a lung cancer comprising analysis of the biomarkers in FIG. 33I, which may be assessed as indicated in the paragraph above and/or as in FIG. 33I or Table 17 below. For example, the MI profile for lung cancer may comprise: 1) ISH to assess one or more, e.g., 1, 2, 3 or 4, of: ALK, cMET, HER2, ROS1; 2) IHC to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 of: AR, cMET, EGFR (H-score), ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3; and/or 3) sequence analysis to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 of: ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL. In another embodiment, the invention provides a molecular intelligence (MI) PLUS profile for a lung cancer comprising analysis of the biomarkers in the molecular intelligence (MI) profile and the additional biomarker in FIG. 33J, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1, PTPN11, RB1, SMAD4, SMARCB1 and STK11, which may be assessed as indicated this paragraph and/or as in FIG. 33J or Table 17 below. The invention further provides a report comprising results of the molecular profiling and corresponding candidate treatments that are identified as likely beneficial or likely not beneficial, as further described herein.


Table 17 presents a view of the information that is reported for the lung cancer molecular intelligence molecular intelligence molecular profiles, which can be interpreted as described for Table 7 above. The biomarker—treatment associations for the molecular intelligence molecular profiles for lung cancer may comprise those associations in Table 18, which can be interpreted as described for Table 8 above.









TABLE 17







Molecular Profile and Report Parameters: Lung Cancer, e.g., NSCLC or BAC








Agent(s)/Biomarker Status










Reported
Biomarker
Platform





erlotinib, gefitinib
EGFR
NGS



KRAS
NGS



cMET
FISH/CISH



PIK3CA
NGS



PTEN
IHC


afatinib
EGFR
NGS


crizotinib
ALK
FISH



ROS1
FISH


pemetrexed, fluorouracil, capecitabine
TS
IHC


gemcitabine
RRM1
IHC


docetaxel, paclitaxel, nab-paclitaxel
TLE3
IHC



TUBB3
IHC



Pgp
IHC



SPARCm
IHC



SPARCp
IHC


cetuximab
EGFR
IHC (H-Score)


everolimus, temsirolimus, clinical
PIK3CA
NGS


trials




protein expression status
AR
IHC



ER
IHC



PR
IHC


imatinib
cKIT
NGS



PDGFRA
NGS


doxorubicin, liposomal-doxorubicin,
HER2
FISH/CISH


epirubicin
TOP2A
IHC



Pgp
IHC


irinotecan
TOPO1
IHC


temozolomide, dacarbazine
MGMT
IHC


vandetanib
RET
NGS


clinical trials
cMET
IHC


trastuzumab, lapatinib, pertuzumab,
HER2
IHC,


T-DM1, clinical trials

FISH/CISH


clinical trials
BRAF
NGS



KRAS
NGS



NRAS
NGS


clinical trials
IDH1
NGS


clinical trials
VHL
NGS


clinical trials
PTEN
NGS


clinical trials
ABL1
NGS


clinical trials
AKT1
NGS


clinical trials
ALK
NGS


clinical trials
APC
NGS


clinical trials
ATM
NGS


clinical trials
CSF1R
NGS


clinical trials
CTNNB1
NGS


clinical trials
EGFR
NGS


clinical trials
ERBB2
NGS



(HER2)



clinical trials
FGFR1
NGS


clinical trials
FGFR2
NGS


clinical trials
FLT3
NGS


clinical trials
GNAQ
NGS


clinical trials
GNA11
NGS


clinical trials
GNAS
NGS


clinical trials
HRAS
NGS


clinical trials
JAK2
NGS


clinical trials
KDR
NGS



(VEGFR2)



clinical trials
cMET
NGS


clinical trials
MLH1
NGS


clinical trials
MPL
NGS


clinical trials
NOTCH1
NGS


clinical trials
SMO
NGS


clinical trials
TP53
NGS
















TABLE 18







Rules for Lung Cancer Biomarker - Drug Associations


















Biomarker
Biomarker
Biomarker
Biomarker
Biomarker
Biomarker
Biomarker
Overall


Class of Drugs
Drugs
Result
Result
Result
Result
Result
Result
Result
benefit





TKI
erlotinib,
EGFR
EGFR
KRAS
EGFR
cMET
PIK3CA
PTEN
Overall



gefitinib
Exon 20
Activating
Mutated |
T790M
Amplified
Mutated |
Negative
Benefit




insert
Mutation |
G13D
Present
(ISH)
exon 20
(IHC)





Present
Exon 21
(Seq.)
(Seq.)

(Seq.)






(Seq.)
L858R |











Exon 19











del (Seq.)










T
T
F or No
Any
Any
Any
Any
T






Data









Any
T
T
Any
Any
Any
Any
Indet.




Any
F
Any
Any
Any
Any
Any
F




F or No
T
F or No
Any
Any
Any
Any
T




Data

Data









F
No Data
F
Any
Any
Any
Any
Indet.




F
No Data
T or No
Any
Any
Any
Any
F






Data









No Data
No Data
F or No
Any
Any
Any
Any
Indet.






Data









No Data
No Data
T
Any
Any
Any
Any
F


Antimetabolite
gemcitabine
RRM1






Overall




Negative






benefit




(IHC)











T






T




F






F




No Data






Indet.


Antimetabolite
fluorouracil,
TS






Overall



capecitabine,
Negative






benefit



pemetrexed
(IHC)











T






T




F






F




No Data






Indet.


mTOR
everolimus,
PIK3CA






Overall


inhibitors
temsirolimus
exon 20






Benefit




(Seq.)











T






T




F or No






Indet.




Data









Monoclonal
cetuximab
EGFR






Benefit


Antibodies

Positive






Overall


(EGFR

(IHC H-









Targeted-

Overall









cetuximab)

Score)











T






T




F






F




No Data






Indet.


TKI
crizotinib
ALK
ROS1





Benefit




Positive
Positive





Overall




(FISH)
(FISH)










T
Any





T




F or No
T





T




Data











F
F or No





F





Data





Indet.




No Data
F or No











Data








Topo1
irinotecan
TOPO1






Overall


inhibitors

Positive






benefit




(IHC)











T






T




F






F




No Data






Indet.


Alkylating
temozolomide,
MGMT






Overall


Agents
dacarbazine
Negative






benefit




(IHC)











T






T




F






F




No Data






Indet.


TKI
lapatinib
HER2
HER2





Overall




Positive
Amplified





Benefit




(IHC)
(FISH)










T
Any





T




F,
T or





T




Equivocal
Equivocal










or No Data
High










F or
F or





F




Equivocal
Equivocal











Low










F or
No Data





Indet.




Equivocal











No Data
F,





Indet.





Equivocal











Low or No











Data








Monoclonal
trastuzumab,
HER2
HER2





Overall


antibodies
pertuzumab,
Positive
Amplified





Benefit


(Her2-
ado-
(IHC)
(FISH)








Targeted)
trastuzumab











emtansine (T-











DM1)












T
Any





T




F,
T or





T




Equivocal
Equivocal










or No Data
High










F or
F or





F




Equivocal
Equivocal











Low










F or
No Data





Indet.




Equivocal











No Data
F,





Indet.





Equivocal











Low or No











Data








Anthracyclines
doxorubicin,
TOP2A
Her2
TOP2A
PGP



Overall


and related
liposomal-
Amplified
Amplified
Positive
Positive



Benefit


substances
doxorubicin,
(FISH)
(FISH)
(IHC)
(IHC)







epirubicin












T
Any
Any
Any



T




F or No
T or
Any
Any



T




Data
Equivocal











High










F or No
F,
T
Any



T




Data
Equivocal











Low or No











Data










F
F,
F or No
Any



F





Equivocal
Data










Low or No











Data










No Data
F,
F
Any



F





Equivocal











Low or No











Data










No Data
F or
No Data
Any



F





Equivocal











Low










No Data
No Data
No Data
T



F




No Data
No Data
No Data
F



T




No Data
No Data
No Data
No Data



Indet.


TKI
imatinib
c-KIT
PDGFRA





Overall




exon 11 |
exon 12 |





Benefit




exon 13
exon 14 |










(Seq.)
exon 18











(Seq.)










Any
D842V





F




V654A
Any





F




T
Any other





T




F, exon 14,
T





T




exon 17,











exon 18 or











No Data











F, exon 14,
F or No





Indet.




exon 17,
Data










exon 18 or











No Data









TKI (RET-
vandetanib
RET






Overall


targeted)

mutated






benefit




(Seq.)











T






T




F or No






Indet.




Data









Taxanes
paclitaxel,
SPARC
SPARC
TLE3
TUBB3
PGP


Overall



docetaxel,
Positive
Positive
Positive
Positive
Positive


Benefit



nab-paclitaxel
(Mono
(Poly
(IHC)
(IHC)
(IHC)







IHC)
IHC)









paclitaxel,
Any
Any
T
Any
Any


T



docetaxel, nab-











paclitaxel











nab-paclitaxel
T
Any
F or No
T or No
Any


T






Data
Data







paclitaxel,
T
Any
F or No
F
Any


T



docetaxel, nab-


Data








paclitaxel







T



nab-paclitaxel
F or No
T
F or No
T or No
Any


T




Data

Data
Data







paclitaxel,
F or No
T
F or No
F
Any


F



docetaxel, nab-
Data

Data








paclitaxel











paclitaxel,
F or No
F
F
T or No
Any


F



docetaxel, nab-
Data


Data







paclitaxel











paclitaxel,
F or No
F
F or No
F
Any


T



docetaxel, nab-
Data

Data








paclitaxel











paclitaxel,
F or No
F
No Data
T
Any


F



docetaxel, nab-
Data










paclitaxel











nab-paclitaxel
F
F or No
No Data
No Data
Any


F





Data









paclitaxel,
F
No Data
F
T or No
Any


F



docetaxel, nab-



Data







paclitaxel











paclitaxel,
F or No
No Data
F or No
F
Any


T



docetaxel, nab-
Data

Data








paclitaxel











paclitaxel,
F or No
No Data
No Data
T
Any


F



docetaxel, nab-
Data










paclitaxel











nab-paclitaxel
No Data
F
No Data
No Data
Any


F



paclitaxel,
No Data
No Data
F
T or No
Any


F



docetaxel, nab-



Data







paclitaxel











paclitaxel,
No Data
No Data
No Data
No Data
Any


Indet.



docetaxel, nab-











paclitaxel










TKI (EGFR-
afatinib
EGFR
EGFR
EGFR




Overall


targeted)

activating
T790M
Exon 20




benefit




mutation
Present
insert









(Seq.)
(Seq.)
Present











(Seq.)









T, F,
Any
Any




Indet.




exon 20 ins











or No Data











Exon 21
Any
Any




T




L858R or











Exon 19











del











F
F or No
F or No




F





Data
Data









When assessing lung cancer, the T790M mutation in EGFR may further implicate treatment decisions as follows. First, the following information can be reported when EGFR T790M is detected concomitantly with an exon19 deletion or L858R EGFR mutation: The presence of T790M mutation in EGFR has been associated with higher likelihood of prolonged efficacy (PFS/OS) with afatinib than gefitinib or erlotinib. See, e.g., Metro, G., L. Crino, (2011) “The LUX-Lung clinical trial program of afatinib for non-small-cell lung cancer.” Expert Rev Anticancer Ther. 11(5):673-82; which reference is incorporated herein in its entirety. Recent data including AMP, CAP and NCCN guidelines support the continued use of EGFR TKIs in lung adenocarcinoma patients with EGFR activating mutations after the acquisition of a secondary mutation in EGFR-T790M that renders the kinase resistant to erlotinib or gefitinib. To overcome resistance, EGFR remains a drug target and discontinuation ofEGFR TKIs may lead to further progression of the disease. See, e.g., Lindeman, N. I., M. Ladanyi, et al. (2013) “Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology.” Arch Pathol Lab Med, 137(6):828-60; which reference is incorporated herein in its entirety. Second, the following information can be reported when T790M is detected concomitantly with an activating EGFR mutation other than an exon 19 deletion or L858R: Recent data including AMP, CAP and NCCN guidelines support the continued use of EGFR TKIs in lung adenocarcinoma patients with EGFR activating mutations after the acquisition of a secondary mutation in EGFR-T790M that renders the kinase resistant to erlotinib or gefitinib. To overcome resistance, EGFR remains a drug target and discontinuation of EGFR TKIs may lead to further progression of the disease. See e.g., Lindeman, et al. 2013.


In an aspect, the invention provides molecular intelligence (MI) profiles for a glioma comprising assessment of one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or 61, of: ABL1, AKT1, ALK, APC, AR, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, EGFRvIII, ER, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HER2, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR (VEGFR2), KRAS, MGMT-Me, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PGP, PIK3CA, PR, PTEN, PTPN11, RB1, RET, RRM1, SMAD4, SMARCB1, SMO, SPARCm, SPARCp, STK11, TLE3, TOP2A, TOPO1, TP53, TS, TUBB3, VHL. The invention further provides a method of selecting a candidate treatment for a glioma comprising assessment of one or more members of the glioma molecular profile using one or more molecular profiling technique presented herein, e.g., ISH (e.g., FISH, CISH), IHC, RT-PCR, expression array, mutation analysis (e.g., NextGen sequencing, Sanger sequencing, pyrosequencing, Fragment analysis (FA, e.g., RFLP), PCR), etc. In one embodiment, ISH is used to assess one or more, e.g., 1 or 2, of: cMET, HER2. Any useful ISH technique can be used. For example, FISH can be used to assess cMET and/or HER2; or CISH can be used to assess cMET and/or HER2. In an embodiment, protein analysis such as IHC is used to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, of: AR, cMET, ER, HER2, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3. “m” and “p” as in SPARC (m/p) refer to IHC performed with monoclonal (“m”) or polyclonal (“p”) primary antibodies. In some embodiments, sequence analysis is used to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 of: ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, STK11, TP53, VHL. For example, the sequence analysis can be performed on one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 of ABL1, APC, BRAF, EGFR, FLT3, GNAQ, IDH1, JAK2, cKIT, KRAS, MPL, NRAS, PDGFRA, VHL. The sequence analysis can also be performed on one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 of ABL1, APC, BRAF, EGFR, FLT3, GNAQ, IDH1, JAK2, cKIT, KRAS, MPL, NPM1, NRAS, PDGFRA, VHL. The sequencing may be performed using Next Generation sequencing technology or other technologies as described herein. Sequence analysis can also be performed for one or more of MGMT, IDH2 and EGFRvIII. For example, methylation of the MGMT promoter region can be assessed using pyrosequencing, mutation of IDH2 can be assess by Sanger sequencing, and/or the presence of EGFRvIII can be detected using fragment analysis. The molecular profile can be based on assessing the biomarkers as illustrated in FIGS. 33O-P or Table 21 below.


In an embodiment, the invention provides a molecular intelligence (MI) profile for a glioma comprising analysis of the biomarkers in FIG. 33O, which may be assessed as indicated in the paragraph above and/or as in FIG. 33O or Table 21 below. For example, the MI profile for a glioma may comprise: 1) ISH to assess one or more, e.g., 1 or 2, of: cMET, HER2; 2) IHC to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, of: AR, cMET, ER, HER2, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3; 3) sequence analysis to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 of: ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL; 4) sequence analysis, e.g., pyrosequencing, to assess promoter methylation of MGMT; 5) sequence analysis, e.g., Sanger sequencing, or IDH2; and/or 6) detection of the EGFRvIII variant, e.g., as assessed by fragment analysis.


In another embodiment, the invention provides a molecular intelligence (MI) PLUS profile for a glioma comprising analysis of the biomarkers in the molecular intelligence (MI) profile and the additional biomarker in FIG. 33P, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1, PTPN11, RB1, SMAD4, SMARCB1 and STK11, which may be assessed as indicated this paragraph and/or as in FIG. 33P or Table 21 below. The invention further provides a report comprising results of the molecular profiling and corresponding candidate treatments that are identified as likely beneficial or likely not beneficial, as further described herein.


Table 21 below presents a view of the information that is reported for the glioblastoma molecular intelligence molecular profile, which can be interpreted as described for Table 7 above. The biomarker-treatment associations for the molecular profile for glioblastoma may comprise those associations in Table 19, which can be interpreted as described for Table 8 above.









TABLE 19







Rules for Glioma Biomarker - Drug Associations
















Biomarker
Biomarker
Biomarker
Biomarker
Biomarker
Overall


Class of Drugs
Drugs
Result
Result
Result
Result
Result
benefit





Antimetabolites
gemcitabine
RRM1




Overall


(gemcitabine)

Negative




benefit




(IHC)









T




T




F




F




No Data




Indet.


Antimetabolites
fluorouracil,
TS




Overall



capecitabine,
Negative




benefit



pemetrexed
(IHC)









T




T




F




F




No Data




Indet.


Topo1
irinotecan,
TOPO1




Overall


inhibitors
topotecan
Positive




benefit




(IHC)









T




T




F




F




No Data




Indet.


Alkylating
temozolomide,
MGMT
MGMT



Overall


agents
dacarbazine
Negative
Methylated



benefit




(IHC)
(Pyro.)








Any
T



T




Any
F



F




T
Equivocal or



T





No Data








F
Equivocal or



F





No Data








No Data
Equivocal or



Indet.





No Data






mTOR
everolimus,
PIK3CA




Overall


inhibitors
temsirolimus
exon 20




Benefit




(Seq.)









T




T




F or No




Indet.




Data







Anti-androgens
bicalutamide,
AR Positive




Overall



flutamide,
(IHC)




Benefit



abiraterone










T




T




F




F




No Data




Indet.


Hormonal
tamoxifen,
ER Positive
PR Positive



Benefit


Agents
toremifene,
(IHC)
(IHC)



Overall



fulvestrant,









letrozole,









anastrozole,









exemestane,









megestrol









acetate,









leuprolide,









goserelin










T
Any



T




F or No
T



T




Data









F
F



F




F
No Data



Indet.




No Data
F or No



Indet.





Data






TKI (lapatinib)
lapatinib
HER2
HER2



Overall




Positive
Amplified



Benefit




(IHC)
(ISH)








T
Any



T




F,
T or



T




Equivocal
Equivocal








or No Data
High








F or
F or



F




Equivocal
Equivocal









Low








F or
No Data



Indet.




Equivocal









No Data
F, Equivocal



Indet.





Low or No









Data






Monoclonal
trastuzumab,
HER2
HER2



Overall


antibodies
pertuzumab, ado-
Positive
Amplified



Benefit


(Her2-
trastuzumab
(IHC)
(ISH)






Targeted)
emtansine (T-









DM1)










T
Any



T




F,
T or



T




Equivocal
Equivocal








or No Data
High








F or
F or



F




Equivocal
Equivocal









Low








F or
No Data



Indet.




Equivocal









No Data
F, Equivocal



Indet.





Low or No









Data






Anthracyclines
doxorubicin,
TOP2A
Her2
TOP2A
PGP

Overall


and related
liposomal-
Amplified
Amplified
Positive
Positive

Benefit


substances
doxorubicin,
(ISH)
(ISH)
(IHC)
(IHC)





epirubicin










T
Any
Any
Any

T




F or No
T or
Any
Any

T




Data
Equivocal









High








F or No
F, Equivocal
T
Any

T




Data
Low or No









Data








F
F, Equivocal
F or No
Any

F





Low or No
Data








Data








No Data
F, Equivocal
F
Any

F





Low or No









Data








No Data
F or
No Data
Any

F





Equivocal









Low








No Data
No Data
No Data
T

F




No Data
No Data
No Data
F

T




No Data
No Data
No Data
No Data

Indet.


TKI
imatinib
c-KIT
PDGFRA



Overall




exon 11 |
exon 12 |



Benefit




exon 13
exon 14 |








(Seq.)
exon 18









(Seq.)








Any
D842V



F




V654A
Any



F




T
Any other



T




F, exon 14,
T



T




exon 17,









exon 18 or









No Data









F, exon 14,
F or No



Indet.




exon 17,
Data








exon 18 or









No Data







TKI
crizotinib
ALK
ROS1



Overall




Positive
Positive



Benefit




(ISH)
(ISH)








T
Any



T




F or No
T



T




Data









F
F or No



F





Data








No Data
F or No



Indet.





Data






TKI (RET-
vandetanib
RET




Overall


targeted)

mutated




benefit




(Seq.)









T




T




F or No




Indet.




Data







Taxanes
paclitaxel,
SPARC
SPARC
TLE3
TUBB3
PGP
Overall



docetaxel, nab-
(Mono
(Poly IHC)
Positive
Positive
Positive
Benefit



paclitaxel
IHC)

(IHC)
(IHC)
(IHC)




paclitaxel,
Any
Any
T
Any
Any
T



docetaxel, nab-









paclitaxel









nab-paclitaxel
T
Any
F or No
T or No
Any
T






Data
Data





paclitaxel,
T
Any
F or No
F
Any
T



docetaxel, nab-


Data






paclitaxel









nab-paclitaxel
F or No
T
F or No
T or No
Any
T




Data

Data
Data





paclitaxel,
F or No
T
F or No
F
Any
T



docetaxel, nab-
Data

Data






paclitaxel









paclitaxel,
F or No
F
F
T or No
Any
F



docetaxel, nab-
Data


Data





paclitaxel









paclitaxel,
F or No
F
F or No
F
Any
T



docetaxel, nab-
Data

Data






paclitaxel









paclitaxel,
F or No
F
No Data
T
Any
F



docetaxel, nab-
Data








paclitaxel









nab-paclitaxel
F
F or No
No Data
No Data
Any
F





Data







paclitaxel,
F
No Data
F
T or No
Any
F



docetaxel, nab-



Data





paclitaxel









paclitaxel,
F or No
No Data
F or No
F
Any
T



docetaxel, nab-
Data

Data






paclitaxel









paclitaxel,
F or No
No Data
No Data
T
Any
F



docetaxel, nab-
Data








paclitaxel









nab-paclitaxel
No Data
F
No Data
No Data
Any
F



paclitaxel,
No Data
No Data
F
T or No
Any
F



docetaxel, nab-



Data





paclitaxel









paclitaxel,
No Data
No Data
No Data
No Data
Any
Indet.



docetaxel, nab-









paclitaxel









In an aspect, the invention provides molecular intelligence (MI) profiles for a gastrointestinal stromal tumor (GIST) comprising assessment of one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 or 58, of: ABL1, AKT1, ALK, APC, AR, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ER, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HER2, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MGMT, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PGP, PIK3CA, PR, PTEN, PTPN11, RB1, RET, RRM1, SMAD4, SMARCB1, SMO, SPARC, STK11, TLE3, TOP2A, TOPO1, TP53, TS, TUBB3, VHL. The invention further provides a method of selecting a candidate treatment for GIST comprising assessment of one or more members of the GIST cancer molecular profile using one or more molecular profiling technique presented herein, e.g., ISH (e.g., FISH, CISH), IHC, RT-PCR, expression array, mutation analysis (e.g., NextGen sequencing, Sanger sequencing, pyrosequencing, Fragment analysis (FA, e.g., RFLP), PCR), etc. In one embodiment, ISH is used to assess one or more, e.g., 1 or 2, of: cMET, HER2. Any useful ISH technique can be used. For example, FISH can be used to assess cMET and/or HER2; or CISH can be used to assess cMET and/or HER2. In an embodiment, protein analysis such as IHC is used to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 of: AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3. “m” and “p” as in SPARC (m/p) refer to IHC performed with monoclonal (“m”) or polyclonal (“p”) primary antibodies. In some embodiments, sequence analysis is used to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 of: ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, STK11, TP53, VHL. For example, the sequence analysis can be performed on one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 of ABL1, APC, BRAF, EGFR, FLT3, GNAQ, IDH1, JAK2, cKIT, KRAS, MPL, NRAS, PDGFRA, VHL. The sequence analysis can also be performed on one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 of ABL1, APC, BRAF, EGFR, FLT3, GNAQ, IDH1, JAK2, cKIT, KRAS, MPL, NPM1, NRAS, PDGFRA, VHL. The sequencing may be performed using Next Generation sequencing technology or other technologies as described herein. The molecular profile can be based on assessing the biomarkers as illustrated in Table 21 below, which table presents a molecular profile for any cancer, including without limitation a solid tumor.


In an embodiment, the invention provides a molecular intelligence (MI) profile for a GIST comprising analysis of the biomarkers in the molecular profile for a GIST, which may be assessed as indicated in the paragraph above and/or as in Table 21 below. For example, the MI profile for GIST may comprise: 1) ISH to assess one or more, e.g., 1 or 2, of: cMET, HER2; 2) IHC to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 of: AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3; and/or 3) sequence analysis to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 of: ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL. In another embodiment, the invention provides a molecular intelligence (MI) PLUS profile for GIST comprising analysis of the biomarkers in the molecular intelligence (MI) profile 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1, PTPN11, RB1, SMAD4, SMARCB1 and STK11, which may be assessed as indicated this paragraph and/or as in Table 21 below. The invention further provides a report comprising results of the molecular profiling and corresponding candidate treatments that are identified as likely beneficial or likely not beneficial, as further described herein.


Table 21 below presents a view of the information that is reported for GIST molecular intelligence molecular profile, which can be interpreted as described for Table 7 above. The biomarker-treatment associations for the molecular profile for GIST may comprise those associations in Table 20, which can be interpreted as described for Table 8 above.









TABLE 20







Rules for GIST Biomarker - Drug Associations
















Biomarker
Biomarker
Biomarker
Biomarker
Biomarker
Overall


Class of Drugs
Drugs
Result
Result
Result
Result
Result
benefit







RRM1









Negative




Overall


Antimetabolites
gemcitabine
(IHC)




benefit




T




T




F




F




No Data




Indeterminate



fluorouracil,
TS



capecitabine,
Negative




Overall


Antimetabolites
pemetrexed
(IHC)




benefit




T




T




F




F




No Data




Indeterminate




TOPO1


Topo1
irinotecan,
Positive




Overall


inhibitors
topotecan
(IHC)




benefit




T




T




F




F




No Data




Indeterminate




MGMT


Alkylating
temozolomide,
Negative




Overall


agents
dacarbazine
(IHC)




benefit




T




T




F




F




No Data




Indeterminate




PIK3CA


mTOR
everolimus,
exon20




Overall


inhibitors
temsirolimus
(Seq.)




Benefit




T




T




F or No




indeterminate




Data



bicalutamide,
AR



flutamide,
Positive




Overall


Anti-androgens
abiraterone
(IHC)




Benefit




T




T




F




F




No Data




Indeterminate



tamoxifen,



toremifene,



fulvestrant,



letrozole,



anastrozole,



exemestane,



megestrol



acetate,
ER
PR


Hormonal
leuprolide,
Positive
Positive



Overall


Agents
goserelin
(IHC)
(IHC)



Benefit




T
Any



T




F or No
T



T




Data




F
F



F




F
No Data



Indet.




No Data
F or No



Indet.





Data




HER2




Positive
HER2



Overall


TKI
lapatinib
(IHC)
Amplified



Benefit




T
Any



T




F,
T or



T




Equivocal
Equivocal




or No Data
High




F or
F or



F




Equivocal
Equivocal





Low




F or
No Data



Indet.




Equivocal




No Data
F,



Indet.





Equivocal





Low or No





Data



trastuzumab,



pertuzumab,


Monoclonal
ado-


antibodies
trastuzumab
HER2
HER2


(Her2-
emtansine (T-
Positive
Amplified



Overall


Targeted)
DM1)
(IHC)
(ISH)



Benefit




T
Any



T




F,
T or



T




Equivocal
Equivocal




or No Data
High




F or
F or



F




Equivocal
Equivocal





Low




F or
No Data



Indet.




Equivocal




No Data
F,



Indet.





Equivocal





Low or No





Data




c-KIT




exon9|




V654A|




exon 14




Overall


TKI
sunitinib
(Seq.)




Benefit




T or F




T




Exon 11,




F




Exon 13,




Exon 17 or




Exon 18




No Data




Indeterminate




c-KIT
PDGFRA




exon9|
exon 12|




exon11|
exon 14|




exon13
exon 18



Overall


TKI
imatinib
(Seq.)
(Seq.)



Benefit




Any
D842V



F




V654A
Any



F




T
Any other



T




F, exon 14,
T



T




exon 17,




exon 18 or




No Data




F, exon 14,
F or No



Indet.




exon 17,
Data




exon 18 or




No Data




ALK
ROS1




Positive
Positive



Overall


TKI
crizotinib
(ISH)
(ISH)



Benefit




T
Any



T




F or No
T



T




Data




F
F or No



F





Data




No Data
F or No



Indet.





Data



doxorubicin,


Anthracyclines
liposomal-
TOP2A
Her2
TOP2A
PGP


and related
doxorubicin,
Amplified
Amplified
Positive
Positive

Overall


substances
epirubicin
(ISH)
(ISH)
(IHC)
(IHC)

Benefit




T
Any
Any
Any

T




F or No
T or
Any
Any

T




Data
Equivocal





High




F or No
F,
T
Any

T




Data
Equivocal





Low or No





Data




F
F,
F or No
Any

F





Equivocal
Data





Low or No





Data




No Data
F,
F
Any

F





Equivocal





Low or No





Data




No Data
F or
No Data
Any

F





Equivocal





Low




No Data
No Data
No Data
T

F




No Data
No Data
No Data
F

T




No Data
No Data
No Data
No Data

Indet.




RET


TKI (RET-

Mutated




Overall


targeted)
vandetanib
(Seq.)




benefit




T




T




F or No




Indeterminate




Data




SPARC



paclitaxel,
Positive
SPARC
TLE3
TUBB3
PGP



docetaxel, nab-
(Mono
Positive
Positive
Positive
Positive
Overall


Taxanes
paclitaxel
IHC)
(Poly IHC)
(IHC)
(IHC)
(IHC)
BenefiT



paclitaxel,
Any
Any
T
Any
Any
T



docetaxel, nab-



paclitaxel



nab-paclitaxel
T
Any
F or No
T or No
Any
T






Data
Data



paclitaxel,
T
Any
F or No
F
Any
T



docetaxel, nab-


Data



paclitaxel



nab-paclitaxel
F or No
T
F or No
T or No
Any
T




Data

Data
Data



paclitaxel,
F or No
T
F or No
F
Any
T



docetaxel, nab-
Data

Data



paclitaxel



paclitaxel,
F or No
F
F
T or No
Any
F



docetaxel, nab-
Data


Data



paclitaxel



paclitaxel,
F or No
F
F or No
F
Any
T



docetaxel, nab-
Data

Data



paclitaxel



paclitaxel,
F or No
F
No Data
T
Any
F



docetaxel, nab-
Data



paclitaxel



nab-paclitaxel
F
F or No
No Data
No Data
Any
F





Data



paclitaxel,
F
No Data
F
T or No
Any
F



docetaxel, nab-



Data



paclitaxel



paclitaxel,
F or No
No Data
F or No
F
Any
T



docetaxel, nab-
Data

Data



paclitaxel



paclitaxel,
F or No
No Data
No Data
T
Any
F



docetaxel, nab-
Data



paclitaxel



nab-paclitaxel
No Data
F
No Data
No Data
Any
F



paclitaxel,
No Data
No Data
F
T or No
Any
F



docetaxel, nab-



Data



paclitaxel



paclitaxel,
No Data
No Data
No Data
No Data
Any
Indet.



docetaxel, nab-



paclitaxel









In an embodiment, the invention provides molecular intelligence (MI) profiles that can be used for any lineage of cancer, e.g., for any solid tumor. The MI molecular profiles can be based on assessing the biomarkers using the molecular profiling methods illustrated in FIGS. 33A-B or Table 21. In an embodiment, the molecular intelligence molecular profile for a cancer comprises one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 or 58, of: ABL1, AKT1, ALK, APC, AR, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ER, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HER2, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MGMT, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PGP, PIK3CA, PR, PTEN, PTPN11, RB1, RET, RRM1, SMAD4, SMARCB1, SMO, SPARC, STK11, TLE3, TOP2A, TOPO1, TP53, TS, TUBB3, VHL. The invention further provides a method of selecting a candidate treatment for a cancer comprising assessment of one or more members of the cancer molecular profile using one or more molecular profiling technique presented herein, e.g., ISH (e.g., FISH, CISH), IHC, RT-PCR, expression array, mutation analysis (e.g., NextGen sequencing, Sanger sequencing, pyrosequencing, Fragment analysis (FA, e.g., RFLP), PCR), etc. In one embodiment, ISH is used to assess one or more, e.g., 1 or 2, of: cMET, HER2. Any useful ISH technique can be used. For example, FISH can be used to assess cMET and/or HER2; or CISH can be used to assess cMET and/or HER2. In an embodiment, protein analysis such as IHC is used to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 of: AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3. “m” and “p” as in SPARC (m/p) refer to IHC performed with monoclonal (“m”) or polyclonal (“p”) primary antibodies. In some embodiments, sequence analysis is used to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 of: ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, STK11, TP53, VHL. For example, the sequence analysis can be performed on one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 of ABL1, APC, BRAF, EGFR, FLT3, GNAQ, IDH1, JAK2, cKIT, KRAS, MPL, NRAS, PDGFRA, VHL. The sequence analysis can also be performed on one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 of ABL1, APC, BRAF, EGFR, FLT3, GNAQ, IDH1, JAK2, cKIT, KRAS, MPL, NPM1, NRAS, PDGFRA, VHL. The sequencing may be performed using Next Generation sequencing technology or other technologies as described herein. For example, methylation of the MGMT promoter region can be assessed using pyrosequencing. The molecular profile can be based on assessing the biomarkers as illustrated in FIGS. 33A-B or Table 21.


In an embodiment, the invention provides a molecular intelligence molecular profile for a cancer comprising analysis of the biomarkers in FIG. 33A, which may be assessed as indicated in the paragraph above and/or as in FIG. 33A or Table 21. For example, the MI profile for a cancer such as a solid tumor may comprise: 1) ISH to assess one or more, e.g., 1 or 2, of: cMET, HER2; 2) IHC to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 of: AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3; and/or 3) sequence analysis to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 of: ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL. In another embodiment, the invention provides a molecular intelligence (MI) PLUS profile for a cancer comprising analysis of the biomarkers in the molecular intelligence (MI) profile and the additional biomarker in FIG. 33B, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1, PTPN11, RB1, SMAD4, SMARCB1 and STK11, which may be assessed as indicated this paragraph and/or as in FIG. 33B or Table 21 below. The invention further provides a report comprising results of the molecular profiling and corresponding candidate treatments that are identified as likely beneficial or likely not beneficial, as further described herein.


Table 21 below presents a view of the information that is reported for a molecular intelligence molecular profile for any cancer, including without limitation a solid tumor, which can be interpreted as described for Table 7 above. The biomarker-treatment associations for the molecular profile for the cancer may comprise those associations in Table 22, which can generally be interpreted as described for Table 8 above.









TABLE 21







Molecular Profile and Report Parameters: Any Solid Tumor


(including Glioma)









Agent(s)/Biomarker Status Reported
Biomarker
Platform





docetaxel, paclitaxel, nab-paclitaxel
TLE3
IHC



TUBB3
IHC



Pgp
IHC



SPARCm
IHC



SPARCp
IHC


capecitabine, fluorouracil, pemetrexed
TS
IHC


doxorubicin, liposomal-doxorubicin,
HER2
FISH/CISH


epirubicin
TOP2A
IHC



Pgp
IHC


irinotecan, top otecan
TOPO1
IHC


gemcitabine
RRM1
IHC


temozolomide, dacarbazine
MGMT
IHC




(all lineages




EXCEPT Glioma)



MGMT-Me
Pyrosequencing




(Glioma ONLY)



IDH1*
NGS


abiraterone, bicalutamide, flutamide
AR
IHC


fulvestrant, tamoxifen, toremifene,
ER
IHC


anastrozole, exemestane, letrozole,
PR
IHC


megestrol acetate, leuprolide, goserelin




trastuzumab, lapatinib, pertuzumab,
HER2
IHC, FISH/CISH


T-DM1, clinical trials




imatinib
cKIT
NGS



PDGFRA
NGS


sunitinib (GIST only)
cKIT
NGS


everolimus, temsirolimus, clinical trials
PIK3CA
NGS


vandetanib
RET
NGS


Clinical Trials
EGFRvIII
Fragment




Analysis (FA)




(Glioma ONLY)


Clinical Trials
IDH2
Sanger Sequencing




(Glioma ONLY)


clinical trials
PTEN
IHC


clinical trials
cMET
IHC, FISH/CISH


clinical trials
BRAF
NGS


clinical trials
KRAS
NGS


clinical trials
NRAS
NGS


clinical trials
VHL
NGS


clinical trials
PTEN
NGS


clinical trials
ABL1
NGS


clinical trials
AKT1
NGS


clinical trials
ALK
NGS


clinical trials
APC
NGS


clinical trials
ATM
NGS


clinical trials
CSF1R
NGS


clinical trials
CTNNB1
NGS


clinical trials
EGFR
NGS


clinical trials
ERBB2
NGS



(HER2)



clinical trials
FGFR1
NGS


clinical trials
FGFR2
NGS


clinical trials
FLT3
NGS


clinical trials
GNAQ
NGS


clinical trials
GNA11
NGS


clinical trials
GNAS
NGS


clinical trials
HRAS
NGS


clinical trials
JAK2
NGS


clinical trials
KDR
NGS



(VEGFR2)



clinical trials
cMET
NGS


clinical trials
MLH1
NGS


clinical trials
MPL
NGS


clinical trials
NOTCH1
NGS


clinical trials
SMO
NGS


clinical trials
TP53
NGS





*IDH1 will only associate with temozolomide, dacarbazine in High Grade Glioma lineage.






In addition to the columns in the tables above, Table 22 provides a predicted benefit level and an evidence level, and list of references for each biomarker-drug association rule in the table. The benefit level is ranked from 1-5, wherein the levels indicate the predicted strength of the biomarker-drug association based on the indicated evidence. All relevant published studies were evaluated using the U.S. Preventive Services Task Force (“USPSTF”) grading scheme for study design and validity. See, e.g., www.uspreventiveservicestaskforce.org/uspstf/grades.htm. The benefit level in the table (“Bene. Level”) corresponds to the following:


1: Expected benefit.


2: Expected reduced benefit.


3: Expected lack of benefit.


4: No data is available.


5: Data is available but no expected benefit or lack of benefit reported because the biomarker in this case is the not principal driver of that specific rule.


The evidence level in the table (“Evid. Level”) corresponds to the following:


1: Very high level of evidence. For example, the treatment comprises the standard of care.


2: High level of evidence but perhaps insufficient to be considered for standard of care.


3: Weaker evidence—fewer publications or clinical studies, or perhaps some controversial evidence.


Abbreviations used in Table 22 include: Bene. (Benefit); Evid. (Evidence); Indet. (Indeterminate); Equiv. (Equivocal); Seq. (Sequencing). In the column “Drugs,” under the section for Taxanes, the following abbreviations are used: PDN (paclitaxel, docetaxel, nab-paclitaxel) and N (nab-paclitaxel).


The column “Partial Report Overall Benefit” in Table 22 is to make drug association in a preliminary molecular profiling report when all the biomarker assessment results may not be ready. For example, a preliminary report may be produced when requested by the treating physician. Interpretation of benefit of lack of benefit of the various drugs is more cautious in these scenarios to avoid potential change in drug association from benefit or lack of benefit or vice versa between the preliminary report and a final report that is produced when all biomarker results become available. Hence you will see some indeterminate scenarios.









TABLE 22







Solid Tumor Drug - Biomarker Associations





















































Partial












Bio-



Bio-








Report




Biomarker
Bene.
Evid.
Ref.
Biomarker
Bene.
Evid.
Ref.
marker
Bene.
Evid.
Ref.
marker
Bene.
Evid.
Ref.
Biomarker
Bene.
Evid.
Ref.
Overall
Overall


Class of Drugs
Drugs
Result
Level
Level
No.
Result
Level
Level
No.
Result
Level
Level
No.
Result
Level
Level
No.
Result
Level
Level
No.
Bene.
Bene.




























Partial




RRM1




















Report




Negative
Bene.
Evid.

















Overall
Overall


Antimetabolites
gemcitabine
(IHC)
Level
Level
1
















Bene.
Bene.




T
1
2

















T
T




F
3
2

















F
F




No Data
4


















Indet.
Indet.

























Partial



fluorouracil,
TS




















Report



capecitabine,
Negative
Bene.
Evid.

















Overall
Overall


Antimetabolites
pemetrexed
(IHC)
Level
Level
2
















Bene.
Bene.




T
1
2

















T
T




F
3
2

















F
F




No Data
4


















Indet.
Indet.

























Partial




TOPO1




















Report


Topo1
irinotecan,
Positive
Bene.
Evid.

















Overall
Overall


inhibitors
topotecan
(IHC)
Level
Level
3
















Bene.
Bene.




T
1
2

















T
T




F
3
2

















F
F




No Data
4


















Indet.
Indet.

























Partial




MGMT




















Report


Alkylating
temozolomide,
Negative
Bene.
Evid.

















Overall
Overall


agents
dacarbazine
(IHC)
Level
Level
4
















Bene.
Bene.




T
1
2

















T
T




F
3
2

















F
F




No Data
4


















Indet.
Indet.

























Partial



bicalutamide,
AR




















Report



flutamide,
Positive
Bene.
Evid.

















Overall
Overall


Anti-androgens
abiraterone
(IHC)
Level
Level
5
















Bene.
Bene.




T
1
2

















T
T




F
3
2

















F
F




No Data
4


















Indet.
Indet.



tamoxifen,



toremifene,



fulvestrant,



letrozole,



anastrozole,



exemestane,



megestrol





















Partial



acetate,
ER



PR
















Report


Hormonal
leuprolide,
Positive
Bene.
Evid.

Positive
Bene.
Evid.













Overall
Overall


Agents
goserelin
(IHC)
Level
Level
6
(IHC)
Level
Level
7












Bene.
Bene.




T
1
1

T
1
1













T
T




T
1
1

F
2
1













T
T




T
1
1

No Data
4














T
T




F
2
1

T
1
1













T
T




F
3
1

F
3
1













F
F




F
3
1

No Data
4














Indet.
Indet.




No Data
4


T
1
1













T
T




No Data
4


F
3
1













Indet.
Indet.




No Data
4


No Data
4














Indet.
Indet.

























Pending




HER2



HER2
















Report




Positive
Bene.
Evid.

Amplified
Bene.
Evid.













Overall
Overall


TKI
lapatinib
(IHC)
Level
Level
8
(ISH)
Level
Level
9












Bene.
Bene.




T
1
1

T
1
1













T
T




T
1
1

F
5














T
T




T
1
1

Equiv.
1
1













T
T








High




T
1
1

Equiv.
5














T
T








Low




T
1
1

No Data
4














T
T




F
5


T
1
1













T
T




F
3
1

F
3
1













F
F




F
5


Equiv.
1
1













T
T








High




F
3
1

Equiv.
3
1













F
F








Low




F
3
1

No Data
4














Indet.
Indet.




Equiv.
5


T
1
1













T
T




Equiv.
5


F
3
1













F
F




Equiv.
5


Equiv.
1
1













T
T








High




Equiv.
5


Equiv.
3
1













F
F








Low




Equiv.
5


No Data
4














Indet.
Indet.




No Data
4


T
1
1













T
T




No Data
4


F
3
1













Indet.
Indet.




No Data
4


Equiv.
1
1













T
T








High




No Data
4


Equiv.
3
1













Indet.
Indet.








Low




No Data
4


No Data
4














Indet.
Indet.



trastuzumab,



pertuzumab,


Monoclonal
ado-





















Partial


antibodies
trastuzumab
HER2



HER2
















Report


(Her2-
emtansine (T-
Positive
Bene.
Evid.

Amplified
Bene.
Evid.













Overall
Overall


Targeted)
DM1)
(IHC)
Level
Level
10
(ISH)
Level
Level
11












Bene.
Bene.




T
1
1

T
1
1













T
T




T
1
1

F
5














T
T




T
1
1

Equiv. low
5














T
T




T
1
1

Equiv. high
1
1













T
T




T
1
1

No Data
4














T
T




F
5


T
1
1













T
T




F
3
1

F
3
1













F
F




F
3
1

Equiv. low
3
1













F
F




F
5


Equiv. high
1
1













T
T




F
3
1

No Data
4














Indet.
Indet.




Equiv.
5


T
1
1













T
T




Equiv.
5


F
3
1













F
F




Equiv.
5


Equiv. low
3
1













F
F




Equiv.
5


Equiv. high
1
1













T
T




Equiv.
5


No Data
4














Indet.
Indet.




No Data
4


T
1
1













T
T




No Data
4


F
3
1













Indet.
Indet.




No Data
4


Equiv. low
3
1













Indet.
Indet.




No Data
4


Equiv. high
1
1













T
T




No Data
4


No Data
4














Indet.
Indet.



doxorubicin,





















Partial


Anthracyclines
liposomal-
TOP2A



Her2



TOP2A



PGP








Report


and related
doxorubicin,
Amplified
Bene.
Evid.

Amplified
Bene.
Evid.

Positive
Bene.
Evid.

Positive
Bene.
Evid.





Overall
Overall


substances
epirubicin
(ISH)
Level
Level
12
(ISH)
Level
Level
13
(IHC)
Level
Level
14
(IHC)
Level
Level
15




Bene.
Bene.




T
1
1

T
1
1

T
1
2

T
2
2





T
T




T
1
1

T
1
1

T
1
2

F
1
2





T
T




T
1
1

T
1
1

T
1
2

No Data
4






T
T




T
1
1

T
1
1

F
2
2

T
2
2





T
T




T
1
1

T
1
1

F
2
2

F
1
2





T
T




T
1
1

T
1
1

F
2
2

No Data
4






T
T




T
1
1

T
1
1

No Data
4


T
2
2





T
T




T
1
1

T
1
1

No Data
4


F
1
2





T
T




T
1
1

T
1
1

No Data
4


No Data
4






T
T




T
1
1

F
2
2

T
1
2

T
2
2





T
T




T
1
1

F
2
2

T
1
2

F
1
2





T
T




T
1
1

F
2
2

T
1
2

No Data
4






T
T




T
1
1

F
2
1

F
2
2

T
2
2





T
T




T
1
1

F
2
1

F
2
2

F
1
2





T
T




T
1
1

F
2
1

F
2
2

No Data
4






T
T




T
1
1

F
2
1

No Data
4


T
2
2





T
T




T
1
1

F
2
1

No Data
4


F
1
2





T
T




T
1
1

F
2
1

No Data
4


No Data
4






T
T




T
1
1

No Data
4


T
1
2

T
2
2





T
T




T
1
1

No Data
4


T
1
2

F
1
2





T
T




T
1
1

No Data
4


T
1
2

No Data
4






T
T




T
1
1

No Data
4


F
2
2

T
2
2





T
T




T
1
1

No Data
4


F
2
2

F
1
2





T
T




T
1
1

No Data
4


F
2
2

No Data
4






T
T




T
1
1

No Data
4


No Data
4


T
2
2





T
T




T
1
1

No Data
4


No Data
4


F
1
2





T
T




T
1
1

No Data
4


No Data
4


No Data
4






T
T




T
1
1

Equiv. high
1
1

T
1
2

T
2
2





T
T




T
1
1

Equiv. high
1
1

T
1
2

F
1
2





T
T




T
1
1

Equiv. high
1
1

T
1
2

No Data
4






T
T




T
1
1

Equiv. high
1
1

F
2
2

T
2
2





T
T




T
1
1

Equiv. high
1
1

F
2
2

F
1
2





T
T




T
1
1

Equiv. high
1
1

F
2
2

No Data
4






T
T




T
1
1

Equiv. high
1
1

No Data
4


T
2
2





T
T




T
1
1

Equiv. high
1
1

No Data
4


F
1
2





T
T




T
1
1

Equiv. high
1
1

No Data
4


No Data
4






T
T




T
1
1

Equiv. low
2
2

T
1
2

T
2
2





T
T




T
1
1

Equiv. low
2
2

T
1
2

F
1
2





T
T




T
1
1

Equiv. low
2
2

T
1
2

No Data
4






T
T




T
1
1

Equiv. low
2
1

F
2
2

T
2
2





T
T




T
1
1

Equiv. low
2
1

F
2
2

F
1
2





T
T




T
1
1

Equiv. low
2
1

F
2
2

No Data
4






T
T




T
1
1

Equiv. low
2
1

No Data
4


T
2
2





T
T




T
1
1

Equiv. low
2
1

No Data
4


F
1
2





T
T




T
1
1

Equiv. low
2
1

No Data
4


No Data
4






T
T




F
2
2

T
1
1

T
1
2

T
2
2





T
T




F
2
2

T
1
1

T
1
2

F
1
2





T
T




F
2
2

T
1
1

T
1
2

No Data
4






T
T




F
2
1

T
1
1

F
2
2

T
2
2





T
T




F
2
1

T
1
1

F
2
2

F
1
2





T
T




F
2
1

T
1
1

F
2
2

No Data
4






T
T




F
2
1

T
1
1

No Data
4


T
2
2





T
T




F
2
1

T
1
1

No Data
4


F
1
2





T
T




F
2
1

T
1
1

No Data
4


No Data
4






T
T




F
2
2

F
2
2

T
1
2

T
2
2





T
T




F
2
2

F
2
2

T
1
2

F
1
2





T
T




F
2
2

F
2
2

T
1
2

No Data
4






T
T




F
3
1

F
3
1

F
3
2

T
3
2





F
F




F
3
1

F
3
1

F
3
2

F
2
2





F
F




F
3
1

F
3
1

F
3
2

No Data
4






F
F




F
3
1

F
3
1

No Data
4


T
3
2





F
Indet.




F
3
1

F
3
1

No Data
4


F
2
2





F
Indet.




F
3
1

F
3
1

No Data
4


No Data
4






F
Indet.




F
2
2

No Data
4


T
1
2

T
2
2





T
T




F
2
2

No Data
4


T
1
2

F
1
2





T
T




F
2
2

No Data
4


T
1
2

No Data
4






T
T




F
3
1

No Data
4


F
3
2

T
3
2





F
Indet.




F
3
1

No Data
4


F
3
2

F
2
2





F
Indet.




F
3
1

No Data
4


F
3
2

No Data
4






F
Indet.




F
3
1

No Data
4


No Data
4


T
3
2





F
Indet.




F
3
1

No Data
4


No Data
4


F
2
2





F
Indet.




F
3
1

No Data
4


No Data
4


No Data
4






F
Indet.




F
2
2

Equiv. high
1
1

T
1
2

T
2
2





T
T




F
2
2

Equiv. high
1
1

T
1
2

F
1
2





T
T




F
2
2

Equiv. high
1
1

T
1
2

No Data
4






T
T




F
2
1

Equiv. high
1
1

F
2
2

T
2
2





T
T




F
2
1

Equiv. high
1
1

F
2
2

F
1
2





T
T




F
2
1

Equiv. high
1
1

F
2
2

No Data
4






T
T




F
2
1

Equiv. high
1
1

No Data
4


T
2
2





T
T




F
2
1

Equiv. high
1
1

No Data
4


F
1
2





T
T




F
2
1

Equiv. high
1
1

No Data
4


No Data
4






T
T




F
2
2

Equiv. low
2
2

T
1
2

T
2
2





T
T




F
2
2

Equiv. low
2
2

T
1
2

F
1
2





T
T




F
2
2

Equiv. low
2
2

T
1
2

No Data
4






T
T




F
3
1

Equiv. low
3
1

F
3
2

T
3
2





F
F




F
3
1

Equiv. low
3
1

F
3
2

F
2
2





F
F




F
3
1

Equiv. low
3
1

F
3
2

No Data
4






F
F




F
3
1

Equiv. low
3
1

No Data
4


T
3
2





F
Indet.




F
3
1

Equiv. low
3
1

No Data
4


F
2
2





F
Indet.




F
3
1

Equiv. low
3
1

No Data
4


No Data
4






F
Indet.




No Data
4


T
1
1

T
1
2

T
2
2





T
T




No Data
4


T
1
1

T
1
2

F
1
2





T
T




No Data
4


T
1
1

T
1
2

No Data
4






T
T




No Data
4


T
1
1

F
2
2

T
2
2





T
T




No Data
4


T
1
1

F
2
2

F
1
2





T
T




No Data
4


T
1
1

F
2
2

No Data
4






T
T




No Data
4


T
1
1

No Data
4


T
2
2





T
T




No Data
4


T
1
1

No Data
4


F
1
2





T
T




No Data
4


T
1
1

No Data
4


No Data
4






T
T




No Data
4


F
2
2

T
1
2

T
2
2





T
T




No Data
4


F
2
2

T
1
2

F
1
2





T
T




No Data
4


F
2
2

T
1
2

No Data
4






T
T




No Data
4


F
3
1

F
3
2

T
3
2





F
Indet.




No Data
4


F
3
1

F
3
2

F
2
2





F
Indet.




No Data
4


F
3
1

F
3
2

No Data
4






F
Indet.




No Data
4


F
3
1

No Data
4


T
3
2





F
Indet.




No Data
4


F
3
1

No Data
4


F
2
2





F
Indet.




No Data
4


F
3
1

No Data
4


No Data
4






F
Indet.




No Data
4


No Data
4


T
1
2

T
2
2





T
T




No Data
4


No Data
4


T
1
2

F
1
2





T
T




No Data
4


No Data
4


T
1
2

No Data
4






T
T




No Data
4


No Data
4


F
3
2

T
3
2





F
Indet.




No Data
4


No Data
4


F
3
2

F
2
2





F
Indet.




No Data
4


No Data
4


F
3
2

No Data
4






F
Indet.




No Data
4


No Data
4


No Data
4


T
3
2





F
Indet.




No Data
4


No Data
4


No Data
4


F
1
2





T
Indet.




No Data
4


No Data
4


No Data
4


No Data
4






Indet.
Indet.




No Data
4


Equiv. high
1
1

T
1
2

T
2
2





T
T




No Data
4


Equiv. high
1
1

T
1
2

F
1
2





T
T




No Data
4


Equiv. high
1
1

T
1
2

No Data
4






T
T




No Data
4


Equiv. high
1
1

F
2
2

T
2
2





T
T




No Data
4


Equiv. high
1
1

F
2
2

F
1
2





T
T




No Data
4


Equiv. high
1
1

F
2
2

No Data
4






T
T




No Data
4


Equiv. high
1
1

No Data
4


T
2
2





T
T




No Data
4


Equiv. high
1
1

No Data
4


F
1
2





T
T




No Data
4


Equiv. high
1
1

No Data
4


No Data
4






T
T




No Data
4


Equiv. low
2
2

T
1
2

T
2
2





T
T




No Data
4


Equiv. low
2
2

T
1
2

F
1
2





T
T




No Data
4


Equiv. low
2
2

T
1
2

No Data
4






T
T




No Data
4


Equiv. low
3
1

F
3
2

T
3
2





F
Indet.




No Data
4


Equiv. low
3
1

F
3
2

F
2
2





F
Indet.




No Data
4


Equiv. low
3
1

F
3
2

No Data
4






F
Indet.




No Data
4


Equiv. low
3
1

No Data
4


T
3
2





F
Indet.




No Data
4


Equiv. low
3
1

No Data
4


F
2
2





F
Indet.




No Data
4


Equiv. low
3
1

No Data
4


No Data
4






F
Indet.








PDGFRA




c-KIT



exon 12|
















Partial




exon11|



exon 14|
















Report




exon13
Bene.
Evid.

exon 18
Bene.
Evid.













Overall
Overall


TKI
imatinib
(Seq.)
Level
Level
16
(Seq.)
Level
Level
17












Bene.
Bene.




T
1
2

T
1
2













T
T




T
1
2

F
5














T
T




T
2
2

D842V
3
2













F
F




T
1
2

No Data
4














T
Indet.




F
2
2

T
1
2













T
T




F
3
2

F
3
2













Indet.
Indet.




F
3
2

D842V
3
2













F
F




F
3
2

No Data
4














Indet.
Indet.




V654A
3
2

T
2
2













F
F




V654A
3
2

F
3
2













F
F




V654A
3
2

D842V
3
2













F
F




V654A
3
2

No Data
4














F
F




exon 14
5


T
1
2













T
T




exon 14
5


F
3
2













Indet.
Indet.




exon 14
5


D842V
3
2













F
F




exon 14
5


No Data
4














Indet.
Indet.




exon 17 or
5


T
1
2













T
T




18




exon 17 or
5


F
3
2













Indet.
Indet.




18




exon 17 or
5


D842V
3
2













F
F




18




exon 17 or
5


No Data
4














Indet.
Indet.




18




No Data
4


T
1
2













T
Indet.




No Data
4


F
3
2













Indet.
Indet.




No Data
4


D842V
3
2













F
F




No Data
4


No Data
4














Indet.
Indet.

























Pending




ALK



ROS1
















Report


TKI

Positive
Bene.
Evid.

Positive
Bene.
Evid.













Overall
Overall


(crizotinib)
crizotinib
(ISH)
Level
Level
18
(ISH)
Level
Level
19












Bene.
Bene.




T
1
2

T
1
2













T
T




F
5


T
1
2













T
T




No Data
4


T
1
2













T
T




T
1
2

F
5














T
T




F
3
2

F
3
2













F
F




No Data
4


F
3
2













Indet.
Indet.




T
1
2

No Data
4














T
T




F
3
2

No Data
4














F
Indet.




No Data
4


No Data
4














Indet.
Indet.

























Partial




PIK3CA




















Report


mTOR
everolimus,
exon20
Bene.
Evid.

















Overall
Overall


inhibitors
temsirolimus
(Seq.)
Level
Level
20
















Bene.
Bene.




T
1
2

















T
T




F
3
2

















Indet.
Indet.




No Data
4


















Indet.
Indet.

























Partial




RET




















Report


TKI (RET-

Mutated
Bene.
Evid.

















Overall
Overall


targeted)
vandetanib
(Seq.)
Level
Level
21
















Bene.
Bene.




T
1
1

















T
T




F
5


















Indet.
Indet.




No Data
4


















Indet.
Indet.




SPARC




















Partial



paclitaxel,
Positive



SPARC



TLE3



TUBB3



PGP




Report



docetaxel,
(Mono
Bene.
Evid.

Positive
Bene.
Evid.

Positive
Bene.
Evid.

Positive
Bene.
Evid.

Positive
Bene.
Evid.

Overall
Overall


Taxanes
nab-paclitaxel
IHC)
Level
Level
22
(Poly IHC)
Level
Level
22
(IHC)
Level
Level
23
(IHC)
Level
Level
24
(IHC)
Level
Level
25
Bene.
Benefft



PDN
T
1
2

T
1
2

T
1
2

T
2
2

T
2
3

T
T



PDN
T
1
2

T
1
2

T
1
2

T
2
2

F
1
3

T
T



PDN
T
1
2

T
1
2

T
1
2

T
2
2

No Data
4


T
T



PDN
T
1
2

T
1
2

T
1
2

F
1
2

T
2
3

T
T



PDN
T
1
2

T
1
2

T
1
2

F
1
2

F
1
3

T
T



PDN
T
1
2

T
1
2

T
1
2

F
1
2

No Data
4


T
T



PDN
T
1
2

T
1
2

T
1
2

No Data
4


T
2
3

T
T



PDN
T
1
2

T
1
2

T
1
2

No Data
4


F
1
3

T
T



PDN
T
1
2

T
1
2

T
1
2

No Data
4


No Data
4


T
T



N
T
1
2

T
1
2

F
2
2

T
2
2

T
2
3

T
T



N
T
1
2

T
1
2

F
2
2

T
2
2

F
1
3

T
T



N
T
1
2

T
1
2

F
2
2

T
2
2

No Data
4


T
T



PDN
T
1
2

T
1
2

F
2
2

F
1
2

T
2
3

T
T



PDN
T
1
2

T
1
2

F
2
2

F
1
2

F
1
3

T
T



PDN
T
1
2

T
1
2

F
2
2

F
1
2

No Data
4


T
T



N
T
1
2

T
1
2

F
2
2

No Data
4


T
2
3

T
Indet.



N
T
1
2

T
1
2

F
2
2

No Data
4


F
1
3

T
Indet.



N
T
1
2

T
1
2

F
2
2

No Data
4


No Data
4


T
Indet.



N
T
1
2

T
1
2

No Data
4


T
2
2

T
2
3

T
Indet.



N
T
1
2

T
1
2

No Data
4


T
2
2

F
1
3

T
Indet.



N
T
1
2

T
1
2

No Data
4


T
2
2

No Data
4


T
Indet.



PDN
T
1
2

T
1
2

No Data
4


F
1
2

T
2
3

T
T



PDN
T
1
2

T
1
2

No Data
4


F
1
2

F
1
3

T
T



PDN
T
1
2

T
1
2

No Data
4


F
1
2

No Data
4


T
T



N
T
1
2

T
1
2

No Data
4


No Data
4


T
2
3

T
Indet.



N
T
1
2

T
1
2

No Data
4


No Data
4


F
1
3

T
Indet.



N
T
1
2

T
1
2

No Data
4


No Data
4


No Data
4


T
Indet.



PDN
T
1
2

F
2
2

T
1
2

T
2
2

T
2
3

T
T



PDN
T
1
2

F
2
2

T
1
2

T
2
2

F
1
3

T
T



PDN
T
1
2

F
2
2

T
1
2

T
2
2

No Data
4


T
T



PDN
T
1
2

F
2
2

T
1
2

F
1
2

T
2
3

T
T



PDN
T
1
2

F
2
2

T
1
2

F
1
2

F
1
3

T
T



PDN
T
1
2

F
2
2

T
1
2

F
1
2

No Data
4


T
T



PDN
T
1
2

F
2
2

T
1
2

No Data
4


T
2
3

T
T



PDN
T
1
2

F
2
2

T
1
2

No Data
4


F
1
3

T
T



PDN
T
1
2

F
2
2

T
1
2

No Data
4


No Data
4


T
T



N
T
1
2

F
2
2

F
2
2

T
2
2

T
2
3

T
T



N
T
1
2

F
2
2

F
2
2

T
2
2

F
1
3

T
T



N
T
1
2

F
2
2

F
2
2

T
2
2

No Data
4


T
T



PDN
T
1
2

F
2
2

F
2
2

F
1
2

T
2
3

T
T



PDN
T
1
2

F
2
2

F
2
2

F
1
2

F
1
3

T
T



PDN
T
1
2

F
2
2

F
2
2

F
1
2

No Data
4


T
T



N
T
1
2

F
2
2

F
2
2

No Data
4


T
2
3

T
Indet.



N
T
1
2

F
2
2

F
2
2

No Data
4


F
1
3

T
Indet.



N
T
1
2

F
2
2

F
2
2

No Data
4


No Data
4


T
Indet.



N
T
1
2

F
2
2

No Data
4


T
2
2

T
2
3

T
Indet.



N
T
1
2

F
2
2

No Data
4


T
2
2

F
1
3

T
Indet.



N
T
1
2

F
2
2

No Data
4


T
2
2

No Data
4


T
Indet.



PDN
T
1
2

F
2
2

No Data
4


F
1
2

T
2
3

T
T



PDN
T
1
2

F
2
2

No Data
4


F
1
2

F
1
3

T
T



PDN
T
1
2

F
2
2

No Data
4


F
1
2

No Data
4


T
T



N
T
1
2

F
2
2

No Data
4


No Data
4


T
2
3

T
Indet.



N
T
1
2

F
2
2

No Data
4


No Data
4


F
1
3

T
Indet.



N
T
1
2

F
2
2

No Data
4


No Data
4


No Data
4


T
Indet.



PDN
T
1
2

No Data
4


T
1
2

T
2
2

T
2
3

T
T



PDN
T
1
2

No Data
4


T
1
2

T
2
2

F
1
3

T
T



PDN
T
1
2

No Data
4


T
1
2

T
2
2

No Data
4


T
T



PDN
T
1
2

No Data
4


T
1
2

F
1
2

T
2
3

T
T



PDN
T
1
2

No Data
4


T
1
2

F
1
2

F
1
3

T
T



PDN
T
1
2

No Data
4


T
1
2

F
1
2

No Data
4


T
T



PDN
T
1
2

No Data
4


T
1
2

No Data
4


T
2
3

T
T



PDN
T
1
2

No Data
4


T
1
2

No Data
4


F
1
3

T
T



PDN
T
1
2

No Data
4


T
1
2

No Data
4


No Data
4


T
T



N
T
1
2

No Data
4


F
2
2

T
2
2

T
2
3

T
T



N
T
1
2

No Data
4


F
2
2

T
2
2

F
1
3

T
T



N
T
1
2

No Data
4


F
2
2

T
2
2

No Data
4


T
T



PDN
T
1
2

No Data
4


F
2
2

F
1
2

T
2
3

T
T



PDN
T
1
2

No Data
4


F
2
2

F
1
2

F
1
3

T
T



PDN
T
1
2

No Data
4


F
2
2

F
1
2

No Data
4


T
T



N
T
1
2

No Data
4


F
2
2

No Data
4


T
2
3

T
Indet.



N
T
1
2

No Data
4


F
2
2

No Data
4


F
1
3

T
Indet.



N
T
1
2

No Data
4


F
2
2

No Data
4


No Data
4


T
Indet.



N
T
1
2

No Data
4


No Data
4


T
2
2

T
2
3

T
Indet.



N
T
1
2

No Data
4


No Data
4


T
2
2

F
1
3

T
Indet.



N
T
1
2

No Data
4


No Data
4


T
2
2

No Data
4


T
Indet.



PDN
T
1
2

No Data
4


No Data
4


F
1
2

T
2
3

T
T



PDN
T
1
2

No Data
4


No Data
4


F
1
2

F
1
3

T
T



PDN
T
1
2

No Data
4


No Data
4


F
1
2

No Data
4


T
T



N
T
1
2

No Data
4


No Data
4


No Data
4


T
2
3

T
Indet.



N
T
1
2

No Data
4


No Data
4


No Data
4


F
1
3

T
Indet.



N
T
1
2

No Data
4


No Data
4


No Data
4


No Data
4


T
Indet.



PDN
F
2
2

T
1
2

T
1
2

T
2
2

T
2
3

T
T



PDN
F
2
2

T
1
2

T
1
2

T
2
2

F
1
3

T
T



PDN
F
2
2

T
1
2

T
1
2

T
2
2

No Data
4


T
T



PDN
F
2
2

T
1
2

T
1
2

F
1
2

T
2
3

T
T



PDN
F
2
2

T
1
2

T
1
2

F
1
2

F
1
3

T
T



PDN
F
2
2

T
1
2

T
1
2

F
1
2

No Data
4


T
T



PDN
F
2
2

T
1
2

T
1
2

No Data
4


T
2
3

T
T



PDN
F
2
2

T
1
2

T
1
2

No Data
4


F
1
3

T
T



PDN
F
2
2

T
1
2

T
1
2

No Data
4


No Data
4


T
T



N
F
2
2

T
1
2

F
2
2

T
2
2

T
2
3

T
T



N
F
2
2

T
1
2

F
2
2

T
2
2

F
1
3

T
T



N
F
2
2

T
1
2

F
2
2

T
2
2

No Data
4


T
T



PDN
F
2
2

T
1
2

F
2
2

F
1
2

T
2
3

T
T



PDN
F
2
2

T
1
2

F
2
2

F
1
2

F
1
3

T
T



PDN
F
2
2

T
1
2

F
2
2

F
1
2

No Data
4


T
T



N
F
2
2

T
1
2

F
2
2

No Data
4


T
2
3

T
Indet.



N
F
2
2

T
1
2

F
2
2

No Data
4


F
1
3

T
Indet.



N
F
2
2

T
1
2

F
2
2

No Data
4


No Data
4


T
Indet.



N
F
2
2

T
1
2

No Data
4


T
2
2

T
2
3

T
Indet.



N
F
2
2

T
1
2

No Data
4


T
2
2

F
1
3

T
Indet.



N
F
2
2

T
1
2

No Data
4


T
2
2

No Data
4


T
Indet.



PDN
F
2
2

T
1
2

No Data
4


F
1
2

T
2
3

T
T



PDN
F
2
2

T
1
2

No Data
4


F
1
2

F
1
3

T
T



PDN
F
2
2

T
1
2

No Data
4


F
1
2

No Data
4


T
T



N
F
2
2

T
1
2

No Data
4


No Data
4


T
2
3

T
Indet.



N
F
2
2

T
1
2

No Data
4


No Data
4


F
1
3

T
Indet.



N
F
2
2

T
1
2

No Data
4


No Data
4


No Data
4


T
Indet.



PDN
F
2
2

F
2
2

T
1
2

T
2
2

T
2
3

T
T



PDN
F
2
2

F
2
2

T
1
2

T
2
2

F
1
3

T
T



PDN
F
2
2

F
2
2

T
1
2

T
2
2

No Data
4


T
T



PDN
F
2
2

F
2
2

T
1
2

F
1
2

T
2
3

T
T



PDN
F
2
2

F
2
2

T
1
2

F
1
2

F
1
3

T
T



PDN
F
2
2

F
2
2

T
1
2

F
1
2

No Data
4


T
T



PDN
F
2
2

F
2
2

T
1
2

No Data
4


T
2
3

T
T



PDN
F
2
2

F
2
2

T
1
2

No Data
4


F
1
3

T
T



PDN
F
2
2

F
2
2

T
1
2

No Data
4


No Data
4


T
T



PDN
F
3
2

F
3
2

F
3
2

T
3
2

T
3
3

F
F



PDN
F
3
2

F
3
2

F
3
2

T
3
2

F
2
3

F
F



PDN
F
3
2

F
3
2

F
3
2

T
3
2

No Data
4


F
F



PDN
F
2
2

F
2
2

F
2
2

F
1
2

T
2
3

T
T



PDN
F
2
2

F
2
2

F
2
2

F
1
2

F
1
3

T
T



PDN
F
2
2

F
2
2

F
2
2

F
1
2

No Data
4


T
T



PDN
F
3
2

F
3
2

F
3
2

No Data
4


T
3
3

F
Indet.



PDN
F
3
2

F
3
2

F
3
2

No Data
4


F
2
3

F
Indet.



PDN
F
3
2

F
3
2

F
3
2

No Data
4


No Data
4


F
Indet.



PDN
F
3
2

F
3
2

No Data
4


T
3
2

T
3
3

F
Indet.



PDN
F
3
2

F
3
2

No Data
4


T
3
2

F
2
3

F
Indet.



PDN
F
3
2

F
3
2

No Data
4


T
3
2

No Data
4


F
Indet.



PDN
F
2
2

F
2
2

No Data
4


F
1
2

T
2
3

T
T



PDN
F
2
2

F
2
2

No Data
4


F
1
2

F
1
3

T
T



PDN
F
2
2

F
2
2

No Data
4


F
1
2

No Data
4


T
T



N
F
3
2

F
3
2

No Data
4


No Data
4


T
3
3

F
Indet.



N
F
3
2

F
3
2

No Data
4


No Data
4


F
2
3

F
Indet.



N
F
3
2

F
3
2

No Data
4


No Data
4


No Data
4


F
Indet.



PDN
F
2
2

No Data
4


T
1
2

T
2
2

T
2
3

T
T



PDN
F
2
2

No Data
4


T
1
2

T
2
2

F
1
3

T
T



PDN
F
2
2

No Data
4


T
1
2

T
2
2

No Data
4


T
T



PDN
F
2
2

No Data
4


T
1
2

F
1
2

T
2
3

T
T



PDN
F
2
2

No Data
4


T
1
2

F
1
2

F
1
3

T
T



PDN
F
2
2

No Data
4


T
1
2

F
1
2

No Data
4


T
T



PDN
F
2
2

No Data
4


T
1
2

No Data
4


T
2
3

T
T



PDN
F
2
2

No Data
4


T
1
2

No Data
4


F
1
3

T
T



PDN
F
2
2

No Data
4


T
1
2

No Data
4


No Data
4


T
T



PDN
F
3
2

No Data
4


F
3
2

T
3
2

T
3
3

F
Indet.



PDN
F
3
2

No Data
4


F
3
2

T
3
2

F
2
3

F
Indet.



PDN
F
3
2

No Data
4


F
3
2

T
3
2

No Data
4


F
Indet.



PDN
F
2
2

No Data
4


F
2
2

F
1
2

T
2
3

T
T



PDN
F
2
2

No Data
4


F
2
2

F
1
2

F
1
3

T
T



PDN
F
2
2

No Data
4


F
2
2

F
1
2

No Data
4


T
T



PDN
F
3
2

No Data
4


F
3
2

No Data
4


T
3
3

F
Indet.



PDN
F
3
2

No Data
4


F
3
2

No Data
4


F
2
3

F
Indet.



PDN
F
3
2

No Data
4


F
3
2

No Data
4


No Data
4


F
Indet.



PDN
F
3
2

No Data
4


No Data
4


T
3
2

T
3
3

F
Indet.



PDN
F
3
2

No Data
4


No Data
4


T
3
2

F
2
3

F
Indet.



PDN
F
3
2

No Data
4


No Data
4


T
3
2

No Data
4


F
Indet.



PDN
F
2
2

No Data
4


No Data
4


F
1
2

T
2
3

T
T



PDN
F
2
2

No Data
4


No Data
4


F
1
2

F
1
3

T
T



PDN
F
2
2

No Data
4


No Data
4


F
1
2

No Data
4


T
T



N
F
3
2

No Data
4


No Data
4


No Data
4


T
3
3

F
Indet.



N
F
3
2

No Data
4


No Data
4


No Data
4


F
2
3

F
Indet.



N
F
3
2

No Data
4


No Data
4


No Data
4


No Data
4


F
Indet.



PDN
No Data
4


T
1
2

T
1
2

T
2
2

T
2
3

T
T



PDN
No Data
4


T
1
2

T
1
2

T
2
2

F
1
3

T
T



PDN
No Data
4


T
1
2

T
1
2

T
2
2

No Data
4


T
T



PDN
No Data
4


T
1
2

T
1
2

F
1
2

T
2
3

T
T



PDN
No Data
4


T
1
2

T
1
2

F
1
2

F
1
3

T
T



PDN
No Data
4


T
1
2

T
1
2

F
1
2

No Data
4


T
T



PDN
No Data
4


T
1
2

T
1
2

No Data
4


T
2
3

T
T



PDN
No Data
4


T
1
2

T
1
2

No Data
4


F
1
3

T
T



PDN
No Data
4


T
1
2

T
1
2

No Data
4


No Data
4


T
T



N
No Data
4


T
1
2

F
2
2

T
2
2

T
2
3

T
T



N
No Data
4


T
1
2

F
2
2

T
2
2

F
1
3

T
T



N
No Data
4


T
1
2

F
2
2

T
2
2

No Data
4


T
T



PDN
No Data
4


T
1
2

F
2
2

F
1
2

T
2
3

T
T



PDN
No Data
4


T
1
2

F
2
2

F
1
2

F
1
3

T
T



PDN
No Data
4


T
1
2

F
2
2

F
1
2

No Data
4


T
T



N
No Data
4


T
1
2

F
2
2

No Data
4


T
2
3

T
Indet.



N
No Data
4


T
1
2

F
2
2

No Data
4


F
1
3

T
Indet.



N
No Data
4


T
1
2

F
2
2

No Data
4


No Data
4


T
Indet.



N
No Data
4


T
1
2

No Data
4


T
2
2

T
2
3

T
Indet.



N
No Data
4


T
1
2

No Data
4


T
2
2

F
1
3

T
Indet.



N
No Data
4


T
1
2

No Data
4


T
2
2

No Data
4


T
Indet.



PDN
No Data
4


T
1
2

No Data
4


F
1
2

T
2
3

T
T



PDN
No Data
4


T
1
2

No Data
4


F
1
2

F
1
3

T
T



PDN
No Data
4


T
1
2

No Data
4


F
1
2

No Data
4


T
T



N
No Data
4


T
1
2

No Data
4


No Data
4


T
2
3

T
Indet.



N
No Data
4


T
1
2

No Data
4


No Data
4


F
1
3

T
Indet.



N
No Data
4


T
1
2

No Data
4


No Data
4


No Data
4


T
Indet.



PDN
No Data
4


F
2
2

T
1
2

T
2
2

T
2
3

T
T



PDN
No Data
4


F
2
2

T
1
2

T
2
2

F
1
3

T
T



PDN
No Data
4


F
2
2

T
1
2

T
2
2

No Data
4


T
T



PDN
No Data
4


F
2
2

T
1
2

F
1
2

T
2
3

T
T



PDN
No Data
4


F
2
2

T
1
2

F
1
2

F
1
3

T
T



PDN
No Data
4


F
2
2

T
1
2

F
1
2

No Data
4


T
T



PDN
No Data
4


F
2
2

T
1
2

No Data
4


T
2
3

T
T



PDN
No Data
4


F
2
2

T
1
2

No Data
4


F
1
3

T
T



PDN
No Data
4


F
2
2

T
1
2

No Data
4


No Data
4


T
T



PDN
No Data
4


F
3
2

F
3
2

T
3
2

T
3
3

F
Indet.



PDN
No Data
4


F
3
2

F
3
2

T
3
2

F
2
3

F
Indet.



PDN
No Data
4


F
3
2

F
3
2

T
3
2

No Data
4


F
Indet.



PDN
No Data
4


F
2
2

F
2
2

F
1
2

T
2
3

T
T



PDN
No Data
4


F
2
2

F
2
2

F
1
2

F
1
3

T
T



PDN
No Data
4


F
2
2

F
2
2

F
1
2

No Data
4


T
T



PDN
No Data
4


F
3
2

F
3
2

No Data
4


T
3
3

F
Indet.



PDN
No Data
4


F
3
2

F
3
2

No Data
4


F
2
3

F
Indet.



PDN
No Data
4


F
3
2

F
3
2

No Data
4


No Data
4


F
Indet.



PDN
No Data
4


F
3
2

No Data
4


T
3
2

T
3
3

F
Indet.



PDN
No Data
4


F
3
2

No Data
4


T
3
2

F
2
3

F
Indet.



PDN
No Data
4


F
3
2

No Data
4


T
3
2

No Data
4


F
Indet.



PDN
No Data
4


F
2
2

No Data
4


F
1
2

T
2
3

T
T



PDN
No Data
4


F
2
2

No Data
4


F
1
2

F
1
3

T
T



PDN
No Data
4


F
2
2

No Data
4


F
1
2

No Data
4


T
T



N
No Data
4


F
3
2

No Data
4


No Data
4


T
3
3

F
Indet.



N
No Data
4


F
3
2

No Data
4


No Data
4


F
2
3

F
Indet.



N
No Data
4


F
3
2

No Data
4


No Data
4


No Data
4


F
Indet.



PDN
No Data
4


No Data
4


T
1
2

T
2

2
T
2
3

T
T



PDN
No Data
4


No Data
4


T
1
2

T
2

2
F
1
3

T
T



PDN
No Data
4


No Data
4


T
1
2

T
2
2

No Data
4


T
T



PDN
No Data
4


No Data
4


T
1
2

F
1
2

T
2
3

T
T



PDN
No Data
4


No Data
4


T
1
2

F
1
2

F
1
3

T
T



PDN
No Data
4


No Data
4


T
1
2

F
1
2

No Data
4


T
T



PDN
No Data
4


No Data
4


T
1
2

No Data
4


T
2
3

T
T



PDN
No Data
4


No Data
4


T
1
2

No Data
4


F
1
3

T
T



PDN
No Data
4


No Data
4


T
1
2

No Data
4


No Data
4


T
T



PDN
No Data
4


No Data
4


F
3
2

T
3
2

T
3
3

F
Indet.



PDN
No Data
4


No Data
4


F
3
2

T
3
2

F
2
3

F
Indet.



PDN
No Data
4


No Data
4


F
3
2

T
3
2

No Data
4


F
Indet.



PDN
No Data
4


No Data
4


F
2
2

F
1
2

T
2
3

T
T



PDN
No Data
4


No Data
4


F
2
2

F
1
2

F
1
3

T
T



PDN
No Data
4


No Data
4


F
2
2

F
1
2

No Data
4


T
T



PDN
No Data
4


No Data
4


F
3
2

No Data
4


T
3
3

F
Indet.



PDN
No Data
4


No Data
4


F
3
2

No Data
4


F
2
3

F
Indet.



PDN
No Data
4


No Data
4


F
3
2

No Data
4


No Data
4


F
Indet.



PDN
No Data
4


No Data
4


No Data
4


T
3
2

T
3
3

F
Indet.



PDN
No Data
4


No Data
4


No Data
4


T
3
2

F
2
3

F
Indet.



PDN
No Data
4


No Data
4


No Data
4


T
3
2

No Data
4


F
Indet.



PDN
No Data
4


No Data
4


No Data
4


F
1
2

T
2
3

T
T



PDN
No Data
4


No Data
4


No Data
4


F
1
2

F
1
3

T
T



PDN
No Data
4


No Data
4


No Data
4


F
1
2

No Data
4


T
T



PDN
No Data
4


No Data
4


No Data
4


No Data
4


T
3
3

Indet.
Indet.



PDN
No Data
4


No Data
4


No Data
4


No Data
4


F
1
3

Indet.
Indet.



PDN
No Data
4


No Data
4


No Data
4


No Data
4


No Data
4


Indet.
Indet.









Table 23 contains the references used to predict benefit level and provide an evidence level as shown in Table 22 above. The “Ref. No.” column in Table 23 corresponds to the “Ref. No.” columns in Table 22. Specifically, the reference numbers in Table 22 include those references indicated in Table 23.









TABLE 23







References for Comprehensive Cancer Molecular Profile








Ref.



No.
References











1
Gong, W., J. Dong, et. al. (2012). “RRM1 expression and clinical



outcome of gemcitabine-containing chemotherapy for advanced



non-small-cell lung cancer: A meta-analysis.” Lung Cancer. 75:374-



380.


2
Qiu, L. X., M. H. Zheng, et. al. (2008). “Predictive value of



thymidylate synthase expression in advanced colorectal cancer



patients receiving fluoropyrimidine-based chemotherapy: Evidence



from 24 studies.” Int. J. Cancer: 123, 2384-2389.



Chen, C.-Y., P.-C. Yang, et al. (2011). “Thymidylate synthase and



dihydrofolate reductase expression in non-small cell lung carcinoma:



The association with treatment efficacy of pemetrexed.” Lung



Cancer 74(1): 132-138.



Lee, S. J., Y. H. Im, et. al. (2010). “Thymidylate synthase and



thymidine phosphorylase as predictive markers of capecitabine



monotherapy in patients with anthracycline- and taxane- pretreated



metastatic breast cancer.” Cancer Chemother. Pharmacol. DOI



10.1007/s00280-010-1545-0.


3
Braun, M. S., M. T. Seymour, et. al. (2008). “Predictive biomarkers



of chemotherapy efficacy in colorectal cancer: results from the UK



MRC FOCUS trial.” J. Clin. Oncol. 26:2690-2698.



Kostopoulos, I., G. Fountzilas, et. al. (2009). ” Topoisomerase I but



not thymidylate synthase is associated with improved outcome in



patients with resected colorectal cancer treated with irinotecan



containing adjuvant chemotherapy.” BMC Cancer. 9:339.



Ataka, M., K. Katano, et. al. (2007). “Topoisomerase I protein



expression and prognosis of patients with colorectal cancer.”



Yonago Acta medica. 50:81-87.


4
Chinot, O. L., M. Barrie, et al. (2007). “Correlation between O6-



methylguanine-DNA methyltransferase and survival in inoperable



newly diagnosed glioblastoma patients treated with neoadjuvant



temozolomide.” J Clin Onco125(12): 1470-5.



Kulke, M. H., M. S. Redston, et al. (2008). “O6-Methylguanine



DNA Methyltransferase Deficiency and Response to Temozolomide-



Based Therapy in Patients with Neuroendocrine Tumors.” Clin



Cancer Res 15(1): 338-345.


5
El Sheikh, S. S., H. M. Romanska, et. al. (2008). “Predictive value



of PTEN and AR coexpression of sustained responsiveness to



hormonal therapy in prostate cancer—a pilot study.” Neoplasia.



10(9): 949-53.


6
Lewis, J. D., M. J. Edwards, et al. (2010). “Excellent outcomes with



adjuvant toremifene or tamoxifen in early stage breast cancer.”



Cancer116:2307-15.



Bartlett, J. M. S., D. Rea, et al. (2011). “Estrogen receptor and



progesterone receptor as predictive biomarkers of response to



endocrine therapy: a prospectively powered pathology study in the



Tamoxifen and Exemestane Adjuvant Multinational trial.” J Clin



Oncol 29 (12):1531-1538.



Dowsett, M., C. Allred, et al. (2008). “Relationship between



quantitative estrogen and progesterone receptor expression and



human epidermal growth factor receptor 2 (HER-2) status with



recurrence in the Arimidex, Tamoxifen, Alone or in Combination



trial.” J Clin Oncol 26(7): 1059-65.



Viale, G., M. M. Regan, et al. (2008). “Chemoendocrine compared



with endocrine adjuvant therapies for node-negative breast cancer:



predictive value of centrally reviewed expression of estrogen and



progesterone receptors—International Breast Cancer Study Group.”



J Clin Oncol 26(9): 1404-10.



Anderson, H., M. Dowsett, et. al. (2011). “Relationship between



estrogen receptor, progesterone receptor, HER-2 and Ki67 expression



and efficacy of aromatase inhibitors in advanced breast cancer.



Annals of Oncology. 22:1770-1776.



Coombes, R. C., J. M. Bliss, et al. (2007). “Survival and safety of



exemestane versus tamoxifen after 2-3 years' tamoxifen treatment



(Intergroup Exemestane Study): a randomized controlled trial.”



The Lancet 369:559-570.



Stuart, N. S. A., H. Earl, et. al. (1996). “A randomized phase III



cross-over study of tamoxifen versus megestrol acetate in advanced



and recurrent breast cancer.” European Journal of Cancer.



32(11):1888-1892.



Thurlimann, B., A. Goldhirsch, et al. (1997). “Formestane versus



Megestrol Acetate in Postmenopausal Breast Cancer Patients After



Failure of Tamoxifen: A Phase III Prospective Randomised Cross



Over Trial of Second-line Hormonal Treatment (SAKK 20/90). E J



Cancer 33 (7): 1017-1024.



Cuzick J, LHRH-agonists in Early Breast Cancer Overview group.



(2007). “Use of luteinising-hormone-releasing hormone agonists as



adjuvant treatment in premenopausal patients with hormone-



receptor-positive breast cancer: a meta-analysis of individual patient



data from randomised adjuvant trials.” The Lancet 369: 1711-1723.


7
Lewis, J. D., M. J. Edwards, et al. (2010). “Excellent outcomes with



adjuvant toremifene or tamoxifen in early stage breast cancer.”



Cancer116:2307-15.



Stendahl, M., L. Ryden, et al. (2006). “High progesterone receptor



expression correlates to the effect of adjuvant tamoxifen in



premenopausal breast cancer patients.” Clin Cancer Res 12(15):



4614-8.



Bartlett, J. M. S., D. Rea, et al. (2011). “Estrogen receptor and



progesterone receptor as predictive biomarkers of response to



endocrine therapy: a prospectively powered pathology study in the



Tamoxifen and Exemestane Adjuvant Multinational trial.” J Clin



Oncol 29 (12): 1531-1538.



Dowsett, M., C. Allred, et al. (2008). “Relationship between



quantitative estrogen and progesterone receptor expression and



human epidermal growth factor receptor 2 (HER-2) status with



recurrence in the Arimidex, Tamoxifen, Alone or in Combination



trial.” J Clin Oncol 26(7): 1059-65.



Coombes, R. C., J. M. Bliss, et al. (2007). “Survival and safety of



exemestane versus tamoxifen after 2-3 years' tamoxifen treatment



(Intergroup Exemestane Study): a randomized controlled trial.”



The Lancet 369:559-570.



Yamashita, H., Y. Yando, et al. (2006). “Immunohistochemical



evaluation of hormone receptor status for predicting response to



endocrine therapy in metastatic breast cancer.” Breast Cancer



13(1): 74-83.



Stuart, N. S. A., H. Earl, et. al. (1996). “A randomized phase III



cross-over study of tamoxifen versus megestrol acetate in advanced



and recurrent breast cancer.” European Journal of Cancer.



32(11):1888-1892.



Thurlimann, B., A. Goldhirsch, et al. (1997). “Formestane versus



Megestrol Acetate in Postmenopausal Breast Cancer Patients After



Failure of Tamoxifen: A Phase III Prospective Randomised Cross



Over Trial of Second-line Hormonal Treatment (SAKK 20/90). E J



Cancer 33 (7): 1017-1024.



Cuzick J, LHRH-agonists in Early Breast Cancer Overview group.



(2007). “Use of luteinising-hormone-releasing hormone agonists as



adjuvant treatment in premenopausal patients with hormone-receptor-



positive breast cancer: a meta-analysis of individual patient data from



randomised adjuvant trials.” The Lancet 369: 1711-1723.


8
Amir, E. et. al. (2010). “Lapatinib and HER2 status: results of a meta-



analysis of randomized phase III trials in metastatic breast cancer.”



Cancer Treatment Reviews. 36:410-415.



Johnston, S., Pegram M., et. al. (2009). “Lapatinib combined with



letrozole versus letrozole and placebo as first-line therapy for



postmenopausal hormone receptor-positive metastatic breast cancer.



Journal of Clinical Oncology. Published ahead of print on Sep. 28,



2009 as 10.1200/JCO.2009.23.3734.



Press, M. F., R. S. Finn, et al. (2008). “HER-2 gene amplification,



HER-2 and epidermal growth factor receptor mRNA and protein



expression, and lapatinib efficacy in women with metastatic breast



cancer.” Clin Cancer Res 14(23): 7861-70.


9
Amir, E. et. al. (2010). “Lapatinib and HER2 status: results of a meta-



analysis of randomized phase III trials in metastatic breast cancer.”



Cancer Treatment Reviews. 36:410-415.



Johnston, S., Pegram M., et. al. (2009). “Lapatinib combined with



letrozole versus letrozole and placebo as first-line therapy for



postmenopausal hormone receptor-positive metastatic breast cancer.



Journal of Clinical Oncology. Published ahead of print on Sep. 28,



2009 as 10.1200/JCO.2009.23.3734.



Press, M. F., R. S. Finn, et al. (2008). “HER-2 gene amplification,



HER-2 and epidermal growth factor receptor mRNA and protein



expression, and lapatinib efficacy in women with metastatic breast



cancer.” Clin Cancer Res 14(23): 7861-70.



Bartlett, J. M. S., K. Miller, et. al. (2011). “A UK NEQAS ISH



multicenter ring study using the Ventana HER2 dual-color ISH



assay.” Am. J. Clin. Pathol. 135:157-162.


10
Slamon, D., M. Buyse, et. al. (2011). “Adjuvant trastuzumab in



HER2-positive breast cancer.” N. Engl. J. Med. 365:1273-83.



Yin, W., J. Lu, et. al. (2011). “Trastuzumab in adjuvant treatment



HER2-positive early breast cancer patients: A meta-analysis of



published randomized controlled trials.” PLoS ONE 6(6): e21030.



doi:10.1371/journal.pone.0021030.



Cortes, J., J. Baselga, et. al. (2012). “Pertuzumab monotherapy



after trastuzumab-based treatment and subsequent reintroduction



of trastuzumab: activity and tolerability in patients with advanced



human epidermal growth factor receptor-2-positive breast cancer.”



J. Clin. Oncol. 30. DOI: 10.1200/JCO.2011.37.4207.



Bang, Y-J., Y-K. Kang, et. al. (2010). “Trastuzumab in combination



with chemotherapy versus chemotherapy alone for treatment of



HER2-positive advanced gastric or gastro-oesophageal junction



cancer (ToGA): a phase 3, open-label, randomised controlled trial.”



Lancet. 376:687-97.



Baselga, J., S. M. Swain, et. al. (2012). “Pertuzumab plus



trastuzumab plus docetaxel for metastatic breast cancer”. N. Engl.



J. Med. 36:109-119.



Verma, S., K. Blackwell, et. al. (2012) “Trastuzumab Emtansine for



HER2-Positive Advanced Breast Cancer” N Engl J Med.



367(19):1783-91.



Hurvitz, S. A., E. A. Perez, et. al. (2013) “Phase II randomized study



of trastuzumab emtansine versus trastuzumab plus docetaxel in



patients with human epidermal growth factor receptor 2-positive



metastatic breast cancer.” J Clin Oncol.31(9):1157-63


11
Slamon, D., M. Buyse, et. al. (2011). “Adjuvant trastuzumab in



HER2-positive breast cancer.” N. Engl. J. Med. 365:1273-83.



Yin, W., J. Lu, et. al. (2011). “Trastuzumab in adjuvant treatment



HER2-positive early breast cancer patients: A meta-analysis of



published randomized controlled trials.” PLoS ONE 6(6): e21030.



doi:10.1371/journal.pone.0021030.



Cortes, J., J. Baselga, et. al. (2012). “Pertuzumab monotherapy



after trastuzumab-based treatment and subsequent reintroduction of



trastuzumab: activity and tolerability in patients with advanced



human epidermal growth factor receptor-2-positive breast cancer.”



J. Clin. Oncol. 30. DOI: 10.1200/JCO.2011.37.4207.



Bang, Y-J., Y-K. Kang, et. al. (2010). “Trastuzumab in combination



with chemotherapy versus chemotherapy alone for treatment of



HER2-positive advanced gastric or gastro-oesophageal junction



cancer (ToGA): a phase 3, open-label, randomised controlled trial.”



Lancet. 376:687-97.



Bartlett, J. M. S., K. Miller, et. al. (2011). “A UK NEQAS ISH



multicenter ring study using the Ventana HER2 dual-color ISH



assay.” Am. J. Clin. Pathol. 135:157-162.



Baselga, J., S. M. Swain, et. al. (2012). “Pertuzumab plus



trastuzumab plus docetaxel for metastatic breast cancer”. N. Engl.



J. Med. 36:109-119.



Verma, S., K. Blackwell, et. al. (2012) “Trastuzumab Emtansine for



HER2-Positive Advanced Breast Cancer” N Engl J Med.



367(19):1783-91.



Hurvitz, S. A., E. A. Perez, et. al. (2013) “Phase II randomized study



of trastuzumab emtansine versus trastuzumab plus docetaxel in



patients with human epidermal growth factor receptor 2-positive



metastatic breast cancer.” J Clin Oncol.31(9):1157-63


12
Press, M. F., Slamon, D. J., et. al. (2011). “Alteration of



topoisomerase II-alpha gene in human breast cancer: association with



responsiveness to anthracycline based chemotherapy.” J. Clin. Oncol,



29(7):859-67.



Du, Y., J. Lu, et. al. (2011). “The role of topoisomerase II a in



predicting sensitivity to anthracyclines in breast cancer patients: a



meta-analysis of published literatures.” Breast Can Res Treat.



129(3):839-848.



O'Malley, F. P., K. I. Pritchard, et. al (2009) “Topoisomerase II alpha



and responsiveness of breast cancer to adjuvant chemotherapy.” J



Natl Can Inst. 101: 644-650.



Tanner, M., J. Bergh, et al. (2006). “Topoisomerase II-α Gene



Amplification Predicts Favorable Treatment Response to Tailored and



Dose-Escalated Anthracycline-Based Adjuvant Chemotherapy in



HER-2/neu-Amplified Breast Cancer: Scandinavian Breast Group



Trial 9401.” J Clin Oncol 24(16):2428-2436.


13
Press, M. F., Slamon, D. J., et. al. (2011).“Alteration of



topoisomerase II-alpha gene in human breast cancer: association with



responsiveness to anthracycline based chemotherapy.” J. Clin. Oncol,



29(7):859-67.



Gennari, A., P. Bruzzi, et. al (2008) “HER2 status and efficacy of



adjuvant anthracyclines in early breast cancer: a pooled analysis of



randomized trials.” J Natl Can Inst. 100:14-20.


14
O'Malley, F. P., K. I. Pritchard, et al. (2011). “Topoisomerase II alpha



protein and resposiveness of breast cancer to adjuvant chemotherapy



with CEF compared to CMF in the NCIC CTG randomized MA.5



adjuvant trial.” Breast Can Res Treat. 128, 401-409.



Rodrigo, R. S., C. Axel le, et. al. (2011). “Topoisomerase II-alpha



protein expression and histological response following doxorubicin-



based induction chemotherapy predict survival of locally advanced



soft tissues sarcomas.” Eur J of Can. 47, 1319-1327.


15
Chintamani, J. P., Singh, et. al. (2005). “Role of p-glycoprotein



expression in predicting response to neoadjuvant chemotherapy in



breast cancer—a prospective clinical study.” World J. Surg. Oncol.



3:61.



Akimoto, M., H, Saisho, et al. (2006). “Relationship between



therapeutic efficacy of arterial infusion chemotherapy and expression



of P-glycoprotein and p53 protein in advanced hepatocellular



carcinoma.” World J of Gastroenterol, 12(6), 868-873.


16
Carvajal, R. D., G. K. Schwartz, et. al. (2011). “KIT as a therapeutic



target in metastatic melanoma.” JAMA. 305(22):2327-2334.



Guo, Q. Z., Z. J. Wang, et. al. (2010). “High expression of ERCC1



is a poor prognostic factor in Chinese patients with non-small cell



lung cancer receiving cisplatin-based therapy.” Chin. J. Cancer Res.



22(4):296-302.


17
Cassier, P. A., P. Hohenberger, et al. (2012). “Outcome of Patients



with Platelet-Derived Growth Factor Receptor Alpha-Mutated



Gastrointestinal Stromal Tumors in the Tyrosine Kinase Inhibitor



Era.” Clin Cancer Res 18:4458-4464.



Heinrich, M. C., J. A. Fletcher, et. al. (2008). “Correlation of kinase



genotype and clinical outcome in North American Intergroup phase



III trial of imatinib mesylate for treatment of advanced



gastrointestinal stromal tumor: CALGB 150105 study by Cancer and



Leukemia Group B and Southwest Oncology Group.” J Clin Oncol.



26(33): 5360-5367.



Debiec-Rychter, M., I. Judson, et al. (2006). “KIT mutations and



dose selection for imatinib in patients with advanced gastrointestinal



stromal tumours.” Eur J Cancer 42:1093-1103.


18
Kwak, E. L., A. J. Iafrate, et. al. (2010). “Anaplastic lymphoma



kinase inhibition in non-small cell lung cancer.” N. Engl. J. Med.



363:1693-703.



Lin, E., Modrusan, Z., (2009). Exon array profiling detects EML4-



ALK fusion in breast, colorectal and non-small lung cancers, Mol.



Cancer Res. 7(9):1466-76.


19
Bergethon, K., A. J. Iafrate, et. al. (2012) “ROS1 Rearrangements



Define a Unique Molecular Class of Lung Cancers.” J. Clin. Oncol.



30(8):863-70.



Davies, K. D., R. C. Deobele, et. al. (2012) “Identifying and



Targeting ROS1 Gene Fusions in Non-Small Cell Lung Cancer.”



Clin. Cancer Res. 18(17) : 4570-9.



Shaw, A. T., S. I. Ou, et. al. (2012) “Clinical activity of crizotinib



in advanced non-small cell lung cancer (NSCLC) harboring ROS1



gene rearrangement.” J Clin Oncol 30 (suppl; abstr 7508).



National Comprehensive Cancer Network. NCCN Clinical



Practice Guidelines in Oncology. Non-Small Cell Lung Cancer



2.2013. 2013; National Comprehensive Cancer Network.


20
Janku, F., R. Kurzrock, et. al. (2011). “PIK3CA mutations in



patients with advanced cancers treated with PI3K/AKT/mTOR



axis inhibitors.” Molecular Cancer Therapeutics. 10(3):558-65.



Janku, F., R. Kurzrock, et. al. (2012). “PI3K/Akt/mTOR inhibitors



in patients with breast and gynecologic malignancies harboring



PIK3CA mutations.” Journal of Clinical Oncology. DOI:



10.1200/JCO.2011.36.1196.



Moroney, J.W., R. Kurzrock, et. al. (2011). “A phase I trial of



liposomal doxorubicin, bevacizumab, and temsirolimus in patients



with advanced gynecologic and breast malignancies.” Clin. Cancer



Res. 17:6840-6846.


21
Wells, S. A., M. J. Schlumberger, et al. (2012). “Vandetanib in



Patients with Locally Advanced or Metastatic Medullary Thyroid



Cancer: A Randomized, Double-Blind Phase III Trial.” J Clin



Oncol 30: 134-141.


22
Desai, N., Soon-Shiong, P., et al. (2009). “SPARC Expression



Correlates with Tumor Response to Albumin-Bound Paclitaxel



in Head and Neck Cancer Patients.” Translational Oncology 2(2):



59-64.



Von Hoff, D. D., M. Hidalgo, et. al. (2011). “Gemcitabine plus



nab-paclitaxel is an active regimen in patients with advanced



pancreatic cancer: a phase I/II trial.” J. Clin. Oncol. DOI:



10.1200/JCO.2011.36.5742.


23
Kulkarni, S. A., D. T. Ross, et. al. (2009). “TLE3 as a candidate



biomarker of response to taxane therapy”. Breast Cancer Research.



11:R17 (doi:10.1186/bcr2241).


24
Zhang, H.-L., X.-W. Zhou, et al. (2012). “Association between class



III β-tubulin expression and response to paclitaxel/vinorelbine-based



chemotherapy for non-small cell lung cancer: A meta-analysis.” Lung



Cancer 77: 9-15.



Seve, P., C. Dumontet, et al. (2005). “Class III β-tubulin expression



in tumor cells predicts response and outcome in patients with non-



small cell lung cancer receiving paclitaxel.” Mol Cancer Ther 4(12):



2001-2007.



Gao, S., J. Gao, et al. (2012). “Clinical implications of REST and



TUBB3 in ovarian cancer and its relationship to paclitaxel



resistance.” Tumor Biol 33:1759-1765.



Ploussard, G., A. de la Taille, et al. (2010). “Class III β-Tubulin



Expression Predicts Prostate Tumor Aggressiveness and Patient



Response to Docetaxel-Based Chemotherapy.” Clin Cancer Res



70(22): 9253-9264.


25
Penson, R. T., M. V. Seiden, et al. (2004). “Expression of multidrug



resistance-1 protein inversely correlates with paclitaxel response and



survival in ovarian cancer patients: a study in serial samples.”



Gynecologic Oncology 93:98-106.



Yeh, J. J., A. Kao, et al. (2003). “Predicting Chemotherapy Response



to Paclitaxel-Based Therapy in Advanced Non-Small-Cell Lung



Cancer with P-Glycoprotein Expression.” Respiration 70:32-35.









The PLUS profiles described above and shown in the appropriate panels in FIGS. 33A-33Q include additional sequencing as in Table 24.









TABLE 24





PLUS Sequencing panel



















ABL1
ERBB2 (Her2)
HRAS
NOTCH1
SMARCB1


AKT1
ERBB4
IDH1
NPM1
SMO


ALK
FBXW7
JAK2
NRAS
STK11


APC
FGFR1
JAK3
PDGFRA
TP53


ATM
FGFR2
KDR (VGFR2)
PIK3CA
VHL


BRAF
FLT3
cKIT
PTEN



CDH1
GNA11
KRAS
PTPN11



CSF1R
GNAQ
cMET
RB1



CTNNB1
GNAS
MLH1
RET



EGFR
HNF1A
MPL
SMAD4









Any of the biomarker assays herein, e.g., as shown in FIGS. 33A-33Q or Tables 7-24 can be performed individually as desired. One of skill will appreciate that any combination of the individual biomarker assays could be performed. For example, a treating physician may choose to order one or more of the following to profile a particular patient's tumor: IHC for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 of AR, cMET, EGFR (including H-score for lung cancer such as NSCLC), ER, HER2, MGMT, Pgp, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOPO1, TOP2A, TS, TUBB3; FISH or CISH for 1, 2, 3, 4, or 5 of ALK, cMET, HER2, ROS1, TOP2A; Mutational Analysis of 1, 2, 3 or 4 of BRAF (e.g., cobas R PCR), IDH2 (e.g., Sanger Sequencing), MGMT-Me (e.g., by PyroSequencing); EGFR (e.g., fragment analysis to detect EGFRvIII); and/or Mutational Analysis (e.g., by Next-Generation Sequencing) of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 of ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CSF1R, CTNNB1, EGFR, ERBB2 (HER2), ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, STK11, TP53, VHL. In some embodiments, a selection of individual tests is made when insufficient tumor sample is available for performing all molecular profiling tests in FIGS. 33A-33Q or Tables 7-24.



FIGS. 34A-34C illustrate biomarkers assessed using a molecular profiling approach as outlined in FIGS. 33A-33Q or Tables 7-24, and accompanying text herein. FIG. 34A illustrates biomarkers that are assessed. The biomarkers that are assessed according to the Next Generation sequencing panel are shown in FIG. 34B. FIG. 34C illustrates sample requirements that can be used to perform molecular profiling on a patient tumor sample according to the panels in FIGS. 34A-34B.


In certain embodiments, ERCC1 is assessed according to the profiles described below and in FIGS. 33A-Q and Tables 7-24. Lack of ERCC1 expression, e.g., as determined by IHC, can indicate positive benefit for platinum compounds (cisplatin, carboplatin, oxaliplatin), and conversely positive expression of ERCC1 can indicate lack of benefit of these drugs. Additional biomarkers that can be assessed according to the molecular profiles include EGFRvIII, IDH2, and PD1. The presence of EGFRvIII may be assessed using expression analysis at the protein or mRNA level, e.g., by either IHC or PCR, respectively. Expression of EGFRvIII can suggest treatment with EGFR inhibitors. Mutational analysis can be performed for IDH2, e.g., by Sanger sequencing, pyrosequencing or by next generation sequencing approaches. IDH2 mutations suggest the same therapy indications as IDH1 mutations, e.g., for decarbazine and temozolomide as described herein. PD1 (programmed death-1, PD-1) can be assessed at the protein level, e.g., by IHC. Monoclonal antibodies targeting PD-1 that boost the immune system are being developed for the treatment of cancer. See, e.g., Flies et al, Blockade of the B7-H1/PD-1 pathway for cancer immunotherapy. Yale J Biol Med. 2011 December; 84(4):409-21.


Lab Technique Substitution

One of skill will appreciate that the laboratory techniques of the molecular profiles herein can be substituted by alternative techniques if appropriate, including alternative techniques as disclosed herein or known in the art. For example, FISH and CISH are generally interchangeable methods so that one can often be used in place of the other. Similarly, Dual ISH methods such as described herein can be substituted for conventional ISH methods. In an embodiment, the FDA approved INFORM HER2 Dual ISH DNA Probe Cocktail kit from Ventana Medical Systems, Inc. (Tucson, Ariz.) is used for FISH/CISH analysis of HER2. This kit allows the determination of the HER2 gene status by enumeration of the ratio of the HER2 gene to Chromosome 17. The HER2 and Chromosome 17 probes are detected using two color chromogenic in situ hybridization (CISH) reactions. A number of methods can be used to assess nucleic acid sequences, and any alterations thereof, including without limitation point mutations, insertions, deletions, translocations, rearrangements. Nucleic acid analysis methods include Sanger sequencing, next generation sequencing, polymerase chain reaction (PCR), real-time PCR (qPCR; RT-PCR), a low density microarray, a DNA microarray, a comparative genomic hybridization (CGH) microarray, a single nucleotide polymorphism (SNP) microarray, fragment analysis, RFLP, pyrosequencing, methylation specific PCR, mass spec, Southern blotting, hybridization, and related methods such as described herein. Similarly, a number of methods can be used to assess gene expression, including without limitation next generation sequencing, polymerase chain reaction (PCR), real-time PCR (qPCR; RT-PCR), a low density microarray, a DNA microarray, a comparative genomic hybridization (CGH) microarray, a single nucleotide polymorphism (SNP) microarray, proteomic arrays, antibody arrays or mass spec. The presence or level of a protein can also be assessed using multiple methods as appropriate, including without limitation IHC, immunocapture, immunoblotting, Western analysis, ELISA, immunoprecipitation, flow cytometry, and the like. The desired laboratory technique can be chosen based of multiple criteria, including without limitation accuracy, precision, reproduceability, cost, amount of sample available, type of sample available, time to perform the technique, regulatory approval status of the technique platform, regulatory approval status of the particular test, and the like.


In some embodiments, more than one technique is used to assess a same biomarker. For example, results of profiling both gene expression and protein expression can provide confirmatory results. In other cases, a certain method may provide optimal results depending on the available sample. In some embodiments, sequencing is used to assess EGFR if the sample is more than 50% tumor. Fragment analysis (FA) can also be used to assess EGFR. In some embodiments, FA, e.g., RFLP, is used to assess EGFR if the sample is less than 50% tumor. In still other cases, one technique may indicate a desire to perform another technique, e.g., a less expensive technique or one that requires lesser sample quantity may indicate a desire to perform a more expensive technique or one that consumes more sample. In an embodiment, FA of ALK is performed first, and then FISH or PCR is performed if the FA indicates the presence of a particular ALK alteration such as an ALK fusion. The FISH and/or PCR assay can be designed such that only certain fusion products are detected, e.g., EML4-ALK. The alternate methods may also provide different information about the biomarker. For example, sequence analysis may reveal the presence of a mutant protein, whereas IHC of the protein may reveal its level and/or cellular location. As another example, gene copy number or gene expression at the RNA level may be elevated, but the presence of interfering RNAs may still downregulate protein expression. As still another example, a biomarker can be assessed using a same technique but with different reagents that provide actionable results. As an example, SPARC can be assessed by IHC using either a polyclonal or a monoclonal antibody. This context is identified herein, e.g., as SPARCp, SPARC poly, or variants thereof for SPARC detected using a polyclonal antibody), and as SPARCm, SPARC mono, or variants thereof, for SPARC detected using a monoclonal antibody). SPARC (m/p) and similar derivations can be used to refer to IHC performed using both polyclonal and monoclonal antibodies.


One of skill will appreciate that molecular profiles of the invention can be updated as new evidence becomes available. For example, new evidence may appear in the literature describing an association between a treatment and potential benefit for cancer or a certain lineage of cancer. This information can be incorporated into an appropriate molecular profile. As another example, new evidence may be presented for a biomarker that is already assessed according to the invention. Consider the BRAF V600E mutation that is currently FDA approved for directed treatment with vemurafenib for melanoma. If the treatment is determined to be effective in another setting, e.g., for another lineage of cancer, BRAF V600E can be added to an appropriate molecular profile for that setting.


Mutational Analysis (4.4+, 4.5, 4.6, 4.7, 5.0)

Mutational or sequence analysis can be performed using any number of techniques described herein or known in the art, including without limitation sequencing (e.g., Sanger, Next Generation, pyrosequencing), PCR, variants of PCR such as RT-PCR, fragment analysis, and the like. Table 25 describes a number of genes bearing mutations that have been identified in various cancer lineages. In an aspect, the invention provides a molecular profile comprising one or more genes in Table 25. In one embodiment, the genes are assessed using Next Generation sequencing methods, e.g., using a TruSeq system offered by Illumina Corporation or an Ion Torrent system from Life Technologies. One of skill will appreciate that the profiling may be used to identify candidate treatments for cancer lineages other than those described in Table 25. Clinical trials in the table can be found at www.clinicaltrials.gov using the indicated identifiers.









TABLE 25







Exemplary Mutated Genes and Gene Products and Related Therapies








Biomarker
Description





ABL1
Most CML patients have a chromosomal abnormality due to



a fusion between Abelson (Abl) tyrosine kinase gene at



chromosome 9 and break point cluster (Bcr) gene at



chromosome 22 resulting in constitutive activation of the



Bcr-Abl fusion gene. Imatinib is a Bcr-Abl tyrosine kinase



inhibitor commonly used in treating CML patients. Mutations



in the ABL1 gene are common in imatinib resistant CML



patients which occur in 30- 90% of the patients. However,



more than 50 different point mutations in the ABL1 kinase



domain may be inhibited by the second generation kinase



inhibitors, dasatinib, bosutinib and nilotinib. The gatekeeper



mutation, T315I that causes resistance to all currently



approved TKIs accounts for about 15% of the mutations



found in patients with imatinib resistance. BCR-ABL1



mutation analysis is recommended to help facilitate selection



of appropriate therapy for patients with CML after treatment



with imatinib fails. Agents that target this biomarker are in



clinical trials, e.g.: NCT01528085.


AKT1
AKT1 gene (v-akt murine thymoma viral oncogene



homologue 1) encodes a serine/threonine kinase which is a



pivotal mediator of the PI3K-related signaling pathway,



affecting cell survival, proliferation and invasion.



Dysregulated AKT activity is a frequent genetic defect



implicated in tumorigenesis and has been indicated to be



detrimental to hematopoiesis. Activating mutation E17K



has been described in breast (2-4%), endometrial (2-4%),



bladder cancers (3%), NSCLC (1%), squamous cell



carcinoma of the lung (5%) and ovarian cancer (2%). This



mutation in the pleckstrin homology domain facilitates the



recruitment of AKT to the plasma membrane and



subsequent activation by altering phosphoinositide binding.



A mosaic activating mutation E17K has also been



suggested to be the cause of Proteus syndrome. Mutation



E49K has been found in bladder cancer, which enhances



AKT activation and shows transforming activity in cell lines.



Agents targeting AKT1 are in clinical trials, e.g., the AKT



inhibitor MK-2206 is in trials for patients carrying AKT



mutations (see NCT01277757, NCT01425879).


ALK
APC, or adenomatous polyposis coli, is a key tumor



suppressor gene that encodes for a large multi-domain



protein. This protein exerts its tumor suppressor function



in the Wnt/β-catenin cascade mainly by controlling the



degradation of β-catenin, the central activator of transcrip-



tion in the Wnt signaling pathway. The Wnt signaling path-



way mediates important cellular functions including inter-



cellular adhesion, stabilization of the cytoskeleton, and cell



cycle regulation and apoptosis, and it is important in



embryonic development and oncogenesis. Mutation in APC



results in atruncated protein product with abnormal function,



lacking the domains involved in β-catenin degradation.



Somatic mutation in the APC gene can be detected in the



majority of colorectal tumors (80%) and it is an early event



in colorecta ltumorigenesis. APC wild type patients have



shown better disease control rate in the metastatic setting



when treated with oxaliplatin, while when treated with



fluoropyrimidine regimens, APC wild type patients



experience more hematological toxicities. APC mutation



has also been identified in oral squamous cell carcinoma,



gastric cancer as well as hepatoblastoma and may contribute



to cancer formation. Agents that target this gene and/or its



downstream or upstream effectors are in clinical trials, e.g.:



NCT01198743.



In addition, germline mutation in APC causes familial



adenomatous polyposis, which is an autosomal dominant



inherited disease that will inevitably develop to colorectal



cancer if left untreated. COX-2 inhibitors including



celecoxib may reduce the recurrence of adenomas and



incidence of advanced adenomas in individuals with an



increased risk of CRC. Turcot syndrome and Gardner's



syndrome have also been associated with germline APC



defects. Germline mutations of the APC have also been



associated with an increased risk of developing desmoid



disease, papillary thyroid carcinoma and hepatoblastoma.


APC
APC, or adenomatous polyposis coli, is a key tumor



suppressor gene that encodes for a large multi-domain



protein. This protein exerts its tumor suppressor function



in the Wnt/β-catenin cascade mainly by controlling the



degradation of β-catenin, the central activator of transcrip-



tion in the Wnt signaling pathway. Wnt signaling pathway



mediates important cellular functions including intercellular



adhesion, stabilization of the cytoskeleton and cell cycle



regulation and apoptosis, and is important in embryonic



development and oncogenesis. Mutation in APC results



in a truncated protein product with abnormal function,



lacking the domains involved in β-catenin degradation.



Germline mutation is APC causes familial adenomatous



polyposis, which is an autosomal dominant inherited



disease that will inevitably develop to colorectal cancer



if left untreated. Somatic mutation in APC gene can be



detected in the majority of colorectal tumors (~80%) and is



an early event in colorectal tumorigenesis. APC mutation



has been identified in about 12.5% of oral squamous cell



carcinoma and may contribute to the genesis of the cancer.



Chemoprevention studies in preclinical models show APC



deficient pre-malignant cells respond to a combination of



TRAIL (tumor necrosis factor-related apoptosis-inducing



ligand, or Apo2L) and RAc (9-cis-retinyl acetate) in vitro



without normal cells being affected.


ATM
ATM, or ataxia telangiectasia mutated, is activated by DNA



double-strand breaks and DNA replication stress. It encodes a



protein kinase that acts as a tumor suppressor and regulates



various biomarkers involved in DNA repair, e.g., p53,



BRCA1, CHK2, RAD17, RAD9, and NBS1. ATM is



associated with hematologic malignancies, and somatic



mutations have also been found in colon (18.2%), head and



neck (14.3%), and prostate (11.9%) cancers. Inactivating



ATM mutations may make patients more susceptible to



PARP inhibitors. Agents that target ATM and/or its down-



stream or upstream effectors are in clinical trials, e.g.:



NCT01311713.



In addition, germline mutations in ATM are associated with



ataxia-telangiectasia (also known as Louis-Bar syndrome) and



a predisposition to malignancy.


BRAF
BRAF encodes a protein belonging to the raf/mil family of



serine/threonine protein kinases. This protein plays a role in



regulating the MAP kinase/ERK signaling pathway initiated



by EGFR activation, which affects cell division, differen-



tiation, and secretion. BRAF somatic mutations have been



found in melanoma (43%), thyroid (39%), biliary tree (14%),



colon (12%), and ovarian tumors (12%). Patients with



mutated BRAF genes have a reduced likelihood of response



to EGFR targeted monoclonal antibodies in colorectal cancer.



In melanoma, BRAF-mutated patients are responsive to the



BRAF inhibitors, vemurafenib and dabrafenib, and MEK1/2



inhibitor, trametinib. Various clinical trials (on



www.clinicaltrials.gov) investigating agents which target this



gene may be available, which include the following:



NCT01543698, NCT01709292.



BRAF inherited mutations are associated with Noonan/



Cardio-Facio-Cutaneous (CFC) syndrome, syndromes



associated with short stature, distinct facial features, and



potential heart/skeletal abnormalities.


CDH1
CDH1 (epithelial cadherin/E-cad) encodes a transmembrane



calcium dependent cell adhesion glycoprotein that plays a



major role in epithelial architecture, cell adhesion and cell



invasion. Loss of function of CDH1 contributes to cancer



progression by increasing proliferation, invasion, and/or



metastasis. Various somatic mutations in CDH1 have been



identified in diffuse gastric, lobular breast, endometrial and



ovarian carcinomas; the resultant loss of function of E-cad



can contribute to tumor growth and progression.



In addition, germline mutations in CDH1 cause hereditary



diffuse gastric cancer and colorectal cancer; affected women



are predisposed to lobular breast cancer with a risk of about



50%. CDH1 mutation carriers have an estimated cumulative



risk of gastric cancer of 67% for men and 83% for women,



by age of 80 years.


CDKN2A
CDKN2A or cyclin-dependent kinase inhibitor 2A is a tumor



suppressor gene that encodes two cell cycle regulatory



proteins p16INK4A and p14ARF. As upstream regulators of



the retinoblastoma (RB) and p53 signaling pathways,



CDKN2A controls the induction of cell cycle arrest in



damaged cells that allows for repair of DNA. Loss of



CDKN2A through whole-gene deletion, point mutation, or



promoter methylation leads to disruption of these regulatory



proteins and consequently dysregulation of growth control.



Somatic CDKN2A mutations are documented to occur in



squamous cell lung cancers, head and neck cancer, colorectal



cancer, chronic myelogenous leukemia and malignant pleural



mesothelioma. Currently, there are agents that target down-



stream of CDKN2A such as CDK4/6 inhibitors which



function by restoring the cell's ability to induce cell cycle



arrest. CDK4/6 inhibitors are in clinical trials for advanced



solid tumors, including LEE011 (NCT01237236) and



PD0332991 (NCT01522989, NCT01536743, NCT01037790).



In addition, germline CDKN2A mutations are associated with



melanoma-pancreatic carcinoma syndrome, which increases



the risk for familial malignant melanoma and pancreatic



cancer.


c-Kit
c-Kit is a cytokine receptor expressed on the surface of



hematopoietic stem cells as well as other cell types. This



receptor binds to stem cell factor (SCF, a cell growth factor).



As c-Kit is a receptor tyrosine kinase, ligand binding causes



receptor dimerization and initiates a phosphorylation cascade



resulting in changes in gene expression. These changes affect



proliferation, apoptosis, chemotaxis and adhesion. C-KIT



mutation has been identified in various cancer types including



gastrointestinal stromal tumors (GIST) (up to 85%) and



melanoma (7%). c-Kit is inhibited by multi-targeted agents



including imatinib, sunitinib and sorafenib. Agents which



target c-KIT and/or its downstream or upstream effectors are



also in clinical trials for patients carrying c-KIT mutation,



e.g.: NCT01028222, NCT01092728.



In addition, germline mutations in c-KIT have been associated



with multiple gastrointestinal stromal tumors (GIST) and



Piebald trait.


C-Met
C-Met is a proto-oncogene that encodes the tyrosine kinase



receptor of hepatocyte growth factor (HGF) or scatter factor



(SF). c-Met mutation causes aberrant MET signaling in



various cancer types including renal papillary, hepatocellular,



head and neck squamous, gastric carcinomas and non-small



cell lung cancer. Activating point mutations of MET kinase



domain can cause cancer of various types, and may also



decrease endocytosis and/or degradation of the receptor,



resulting in enhanced tumor growth and metastasis.



Mutations in the juxtamembrane domain (exon 14, 15) results



in the constitutive activation and show enhanced tumor-



igenicity. c-MET inhibitors are in clinical trials for patients



carrying MET mutations, e.g.: NCT01121575,



NCT00813384.



Germline mutations in c-MET have been associated with



hereditary papillary renal cell carcinoma.


CSF1R
CSF1R or colony stimulating factor 1 receptor gene encodes a



transmembrane tyrosine kinase, a member of the CSF1/PDGF



receptor family. CSF1R mediates the cytokine (CSF-1)



responsible for macrophage production, differentiation, and



function. Mutations of this gene are associated with hemato-



logic malignancies, as well as cancers of the liver (21.4%),



colon (12.5%), prostate (3.3%), endometrium (2.4%), and



ovary (2.4%). Patients with CSF1R mutations may respond to



imatinib. Agents that target CSF1R and/or its downstream or



upstream effectors are in clinical trials, e.g.: NCT01346358,



NCT01440959.



In addition, germline mutations in CSF1R are associated with



diffuse leukoencephalopathy, a rapidly progressive neuro-



degenerative disorder.


CTNNB1
CTNNB1 or cadherin-associated protein, beta 1, encodes for



β-catenin, a central mediator of the Wnt signaling pathway



which regulates cell growth, migration, differentiation and



apoptosis. Mutations in CTNNB1 (often occurring in exon 3)



avert the breakdown of β-catenin, which allows the protein to



accumulate resulting in persistent transactivation of target



genes including c-myc and cyclin-D1. Somatic CTNNB1



mutations account for 1-4% of colorectal cancers, 2-3% of



melanomas, 25-38% of endometrioid ovarian cancers,



84-87% of sporadic desmoid tumors, as well as the pediatric



cancers, hepatoblastoma, medulloblastoma and Wilms'



tumors. Compounds that suppress the Wnt/β-catenin pathway



are available in clinical trials including PRI-724 for advanced



solid tumors (NCT01302405) and LGK974 for melanoma



and lobular breast cancer.


EGFR
EGFR or epidermal growth factor receptor, is a trans-



membrane receptor tyrosine kinase belonging to the ErbB



family of receptors. Upon ligand binding, the activated



receptor triggers a series of intracellular pathways (Ras/



MAPK, PI3K/Akt, JAK-STAT) that result in cell



proliferation, migration and adhesion. Dysregulation of



EGFR through mutation leads to ligand-independent



activation and constitutive kinase activity, which results in



uncontrolled growth and proliferation of many human cancers.



EGFR mutations have been observed in 20-25% of non-small



cell lung cancer (NSCLC), 10% of endometrial and peritoneal



cancers. Somatic gain-of-function EGFR mutations, including



in-frame deletions in exon 19 or point mutations in exon 21,



confer sensitivity to first-generation EGFR-targeted tyrosine



kinase inhibitors, whereas the secondary mutation, T790M



in exon 20, confers resistance to tyrosine kinase inhibitors.



New agents and combination therapies that include EGFR



TKIs are in clinical trials for primary treatment of EGFR-



mutated patients, including second-generation tyrosine



kinase inhibitors such as icotinib (NCT01665417) for NSCLC



or afatinib for advanced solid tumors (NCT00809133) and



lung neoplasms (NCT01466660). In addition, new therapies



and combination therapies are being explored for patients that



have progressed on EGFR-targeted agents including afatinib



(NCT01647711) for NSCLC.



Germline mutations and polymorphisms of EGFR have been



associated with familial lung adeocarcinomas.


ERBB2
ERBB2 (HER2) or v-erb-b2 erythroblastic leukemia viral



oncogene homolog 2, neuro/glioblastoma derived oncogene



homolog (avian) encodes a member of the epidermal growth



factor (EGF) receptor family of receptor tyrosine kinases.



This gene binds to other ligand-bound EGF receptor family



members to form a heterodimer and enhances kinase-



mediated activation of downstream signaling pathways,



leading to cell proliferation. The most common mechanism



for activation of HER2 is gene amplification, seen in



approximately 15% of breast cancers. Somatic mutations



have been found in colon (3.8%), endometrium (3.7%),



prostate (3.0%), ovarian (2.5%), breast (1.7%) gastric (1.9%)



cancers and 2-4% of lung adenocarcinomas. HER2 activated



patients may respond to trastuzumab, afatinib, or lapatinib.



Agents that target HER2 are in clinical trials, e.g.:



NCT01306045.


ERBB4
ERBB4 is a member of the Erbb receptor family known to



play a pivotal role in cell-cell signaling and signal



transduction regulating cell growth and development. The



most commonly affected signaling pathways are the PI3K-



Akt and MAP kinase pathways. Erbb4 was found to be



somatically mutated in 19% of melanomas and Erbb4



mutations may confer “oncogene addiction” on melanoma



cells. Erbb4 mutations have also been observed in various



other cancer types, including, gastric carcinomas (1.7%),



colorectal carcinomas (0.68-2.9%), non-small cell lung



cancer (2.3-4.7%) and breast carcinomas (1.1%), however,



their biological impact is not uniform or consistent across



these cancers. Agents that target ERBB4 are in clinical trials,



e.g.: NCT0126408.


FBXW7
FBXW7, or E3 ligase F-box and WD repeat domain



containing 7, also known as Cdc4, encodes three protein



isoforms which constitute a component of the ubiquitin-



proteasome complex. Mutation of FBXW7 occurs in



hotspots and disrupts the recognition of and binding with



substrates which inhibits the proper targeting of proteins



for degradation (e.g. Cyclin E, c-Myc, SREBP1, c-Jun,



Notch-1 and mTOR). Mutation frequencies identified in



cholangiocarcinomas, T-ALL, and carcinomas of



endometrium, colon and stomach are 35%, 31%, 9%, 9%,



and 6%, respectively. Therapeutic strategies comprise



targeting an oncoprotein downstream of FBXW7, such as



mTOR or c-Myc. Tumor cells with mutated FBXW7 are



particularly sensitive to rapamycin treatment, indicating



FBXW7 loss (mutation) can be a predictive biomarker



for treatment with inhibitors of the mTOR pathway.


FGFR1
FGFR1, or fibroblast growth factor receptor 1, encodes for



FGFR1 which is important for cell division, regulation of cell



maturation, formation of blood vessels, wound healing and



embryonic development. Somatic activating mutations have



been documented in melanoma, glioblastoma, and lung



tumors. Other aberrations of FGFR1 including protein over-



expression and gene amplification are common in breast



cancer, squamous cell lung cancer, colorectal cancer, and, to



some extent in adenocarcinoma of the lung. Recently, it has



been shown that osteosarcoma and advanced solid tumors that



exhibit FGFR1 amplification are sensitive to the pan-FGFR



inhibitor, NVP-BGJ398. Other FGFR1-targeted agents under



clinical investigation include dovitinib (NCT01440959).



In addition, germline, gain-of-function mutations in FGFR1



result in developmental disorders including Kallmann



syndrome and Pfeiffer syndrome.


FGFR2
FGFR2 is a receptor for fibroblast growth factor. Activation of



FGFR2 through mutation and amplification has been noted in



a number of cancers. Somatic mutations of the FGFR2



tyrosine kinase have been observed in endometrial carcinoma,



lung squamous cell carcinoma, cervical carcinoma, and



melanoma. In the endometrioid histology of endometrial



cancer, the frequency of FGFR2 mutation is 16% and the



mutation is associated with shorter disease free survival in



patients diagnosed with early stage disease. Loss of function



FGFR2 mutations occur in about 8% melanomas and



contribute to melanoma pathogenesis. Functional poly-



morphisms in the FGFR2 promoter are associated with breast



cancer susceptibility. Agents that target FGFR2 are in clinical



trials, e.g.: NCT01379534.



In addition, germline mutations in FGFR2 are associated with



numerous medical conditions that include congenital



craniofacial malformation disorders, Apert syndrome and the



related Pfeiffer and Crouzon syndromes.


FGFR3
FGFR3 or fibroblast growth factor receptor type 3 gene



encodes a member of the FGFR tyrosine kinase family, which



include FGFR1, 2, 3, and 4. Dysregulation of FGFR3 has



been implicated in activating the RAS-ERK pathway. FGFR3



has been found in various malignancies, including bladder



cancer and multiple myeloma. Somatic mutations of this gene



have also been found in skin (25.8%), head and neck (20.0%),



and testicular (4.3%) cancers. Studies indicate FGFR3 and



PIK3CA mutations occur together. FGFR3 mutations could



serve as a strong prognostic indicator of a low recurrence rate



in bladder cancer.



Agents that target FGFR3 and/or its downstream or upstream



effectors are in clinical trials, e.g.: NCT01004224.



In addition, germline mutations in FGFR3 are associated with



achondroplasia, hypochondroplasia, and Muenke syndrome,



disorders involving but not limited to craniosynostosis and



shortened extremities. FGFR3 is also associated with Crouzon



syndrome with acanthosis nigricans.


FLT3
FLT3, or Fms-like tyrosine kinase 3 receptor, is a member of



class III receptor tyrosine kinase family, which includes



PDGFRA/B and KIT. Signaling through FLT3 ligand-receptor



complex regulates hematopoiesis, specifically lymphocyte



development. The FLT3 internal tandem duplication (FLT3-



ITD) is the most common genetic lesion in acute myeloid



leukemia (AML), occurring in 25% of cases. FLT3 mutations



are as common in solid tumors but have been documented in



breast cancer. Several small molecule multikinase inhibitors



targeting the RTK-III family are in clinical trials, including



phase II trials for crenolanib in AML (NCT01657682),



famitinib for nasopharyngeal carcinoma (NCT01462474),



dovitinib for GIST (NCT01440959), and phase I trial for



PLX108-01 in solid tumors (NCT01004861).


GNA11
GNA11 is a proto-oncogene that belongs to the Gq family of



the G alpha family of G protein coupled receptors. Known



downstream signaling partners of GNA11 are phospholipase



C beta and RhoA and activation of GNA11 induces MAPK



activity. Over half of uveal melanoma patients lacking a



mutation in GNAQ exhibit somatic mutations in GNA11.



Agents that target GNA11 are in clinical trials, e.g.:



NCT01587352, NCT01390818, NCT01143402.


GNAQ
GNAQ encodes the Gq alpha subunit of G proteins. G



proteins are a family of heterotrimeric proteins coupling



seven-transmembrane domain receptors. Oncogenic



mutations in GNAQ result in a loss of intrinsic GTPase



activity, resulting in a constitutively active Galpha subunit.



This results in increased signaling through the MAPK



pathway. Somatic mutations in GNAQ have been found in



50% of primary uveal melanoma patients and up to 28% of



uveal melanoma metastases. Agents that target GNAQ are



in clinical trials, e.g.: NCT01587352, NCT01390818,



NCT01143402.


GNAS
GNAS (or GNAS complex locus) encodes a stimulatory G



protein alpha-subunit. These guanine nucleotide binding



proteins (G proteins) are a family of heterotrimeric proteins



which couple seven-transmembrane domain receptors to



intracellular cascades. Stimulatory G-protein alpha-subunit



transmits hormonal and growth factor signals to effector



proteins and is involved in the activation of adenylate



cyclases. Mutations of GNAS gene at codons 201 or 227



lead to constitutive cAMP signaling. GNAS somatic



mutations have been found in pituitary (27.9%), pancreatic



(19.2%), ovarian (11.4%), adrenal gland (6.2%), and colon



(6.0%) cancers. SNPs in GNAS1 are a predictive marker



for tumor response in cisplatin/fluorouracil-based radio-



chemotherapy in esophageal cancer.



In addition, germline mutations of GNAS have been shown to



be the cause of McCune-Albright syndrome (MAS), a



disorder marked by endocrine, dermatologic, and bone



abnormalities. GNAS is usually found as a mosaic mutation



in patients. Loss of function mutations are associated with



pseudohypoparathyroidism and



pseudopseudohypoparathyroidism.


HNF1A
HNF1A, or hepatocyte nuclear factor 1 homeobox A, encodes



a transcription factor that is highly expressed in the liver,



found on chromosome 12. It regulates a large number of



genes, including those for albumin, alphal-antitrypsin, and



fibrinogen. HNF1A has been associated with an increased risk



of pancreatic cancer. HNF1A somatic mutations are found in



liver (30.1%), colon (14.5%), endometrium (11.1%), and



ovarian (2.5%) cancers.



In addition, germline mutations of HNF1A are associated



with maturity-onset diabetes of the young type 3.


HRAS
HRAS (homologous to the oncogene of the Harvey rat



sarcoma virus), together with KRAS and NRAS, belong to



the superfamily of RAS GTPase. RAS protein activates



RAS-MEK-ERK/MAPK kinase cascade and controls intra-



cellular signaling pathways involved in fundamental cellular



processes such as proliferation, differentiation, and apoptosis.



Mutant Ras proteins are persistently GTP-bound and active,



causing severe dysregulation of the effector signaling. HRAS



mutations have been identified in cancers from the urinary



tract (10%-40%), skin (6%) and thyroid (4%) and they



account for 3% of all RAS mutations identified in cancer.



RAS mutations (especially HRAS mutations) occur (5%) in



cutaneous squamous cell carcinomas and keratoacanthomas



that develop in patients treated with BRAF inhibitor



vemurafenib, likely due to the paradoxical activation of the



MAPK pathway. Agents that target HRAS and/or its down-



stream or upstream effectors are in clinical trials, e.g.:



NCT01306045.



In addition, germline mutation in HRAS has been associated



with Costello syndrome, a genetic disorder that is



characterized by delayed development and mental retardation



and distinctive facial features and heart abnormalities.


IDH1
IDH1 encodes for isocitrate dehydrogenase in cytoplasm and



is found to be mutated in ~5% of primary gliomas and 60-



90% of secondary gliomas, as well as in 12-18% of patients



with acute myeloid leukemia. Mutated IDH1 results in



impaired catalytic function of the enzyme, thus altering



normal physiology of cellular respiration and metabolism.



Furthermore, this mutation results in tumorigenesis. In



gliomas, IDH1 mutations are associated with lower-grade



astrocytomas and oligodendrogliomas (grade II/III). IDH



gene mutations are associated with markedly better survival



in patients diagnosed with malignant astrocytoma; and



clinical data support a more aggressive surgery for IDH1



mutated patients because these individuals may be able to



achieve long-term survival. In contrast, IDH1 mutation is



associated with a worse prognosis in AML. In low-grade



glioma patients receiving temozolomide before anaplastic



transformation, IDH mutations (IDH1 and IDH2) have



been shown to predict response to temozolomide. Agents



that target IDH and/or its downstream or upstream effectors



are in clinical trials, e.g.: NCT01534845.


JAK2
JAK2 or Janus kinase 2 is a part of the JAK/STAT pathway



which mediates multiple cellular responses to cytokines



and growth factors including proliferation and cell survival.



It is also essential for numerous developmental and homeo-



static processes, including hematopoiesis and immune cell



development. Mutations in the JAK2 kinase domain result in



constitutive activation of the kinase and the development of



chronic myeloproliferative neoplasms such as polycythemia



vera (95%), essential thrombocythemia (50%) and



myelofibrosis (50%). JAK2 mutations were also found in



BCR-ABL1-negative acute lymphoblastic leukemia patients



and the mutated patients show a poor outcome. Agents that



target JAK2 and/or its downstream or upstream effectors are



in clinical trials for patients carrying JAK2 mutations, e.g.:



NCT00668421, NCT01038856.



In addition, germline mutations in JAK2 have been



associated with myeloproliferative neoplasms and



thrombocythemia.


JAK3
JAK3 or Janus activated kinase 3 is an intracellular tyrosine



kinase involved in cytokine signaling, while interacting with



members of the STAT family. Like JAK1, JAK2, and TYK2,



JAK3 is a member of the JAK family of kinases. When



activated, kinase enzymes phosphorylate one or more signal



transducer and activator of transcription (STAT) factors,



which translocate to the cell nucleus and regulate the



expression of genes associated with survival and proliferation.



JAK3 signaling is related to T cell development and



proliferation. This biomarker is found in malignancies like



head and neck (20.8%) colon (7.2%), prostate (4.8%), ovary



(3.5%), breast (1.7%), lung (1.2%), and stomach (0.6%)



cancer.



In addition, germline mutations of JAK3 are associated with



severe, combined immunodeficiency disease (SCID).


KDR
KDR (VEGFR2) or Kinase insert domain receptor gene, also



known as vascular endothelial growth factor receptor-2



(VEGFR2), is involved with angiogenesis and is expressed



on almost all endothelial cells. VEGF ligands bind to KDR,



which leads to receptor dimerization and signal transduction.



Somatic mutations in KDR have been observed in



angiosarcoma (10.0%), and colon (12.7%), skin (12.7%),



gastric (5.3%), lung (3.2%), renal (2.3%), and ovarian



(1.9%) cancers. VEGFR antagonists that are FDA-approved



or in clinical trials include bevacizumab, regorafenib,



pazopanib, and vandetanib. Additional agents that target KDR



and/or its downstream or upstream effectors are in clinical



trials, e.g.: NCT01068587.


KRAS
KRAS, or V-Ki-ras2 Kirsten rat sarcoma viral oncogene



homolog, encodes a signaling intermediate involved in many



signaling cascades including the EGFR pathway. KRAS



somatic mutations have been found in pancreatic (57.4%),



colon (34.9%), lung (16.0%), biliary tract (28.2%), and



endometrial (14.6%) cancers. Mutations at activating hotspots



are associated with resistance to EGFR tyrosine kinase



inhibitors (e.g., erlotinib, gefitinib) and monoclonal



antibodies (e.g., cetuximab, panitumumab). Agents that target



KRAS are in clinical trials, e.g.: NCT01248247,



NCT01229150.



In addition, germline mutations of KRAS (V141, T58I, and



D153V amino acid substitutions) are associated with



Noonan syndrome.


MLH1
MLH1 or mutL homolog 1, colon cancer, nonpolyposis type



2 (E. coli) gene encodes a mismatch repair (MMR) protein



which repairs DNA mismatches that occur during replication.



Although the frequency is higher in colon cancer (10.4%),



MLH1 somatic mutations have been found in esophageal



(6.4%), ovarian (5.4%), urinary tract (5.3%), pancreatic



(5.2%), and prostate (4.7%) cancers. Germline mutations of



MLH1 are associated with Lynch syndrome, also known as



hereditary non-polyposis colorectal cancer (HNPCC).



Patients with Lynch syndrome are at increased risk for



various malignancies, including intestinal, gynecologic, and



upper urinary tract cancers and in its variant, Muir-Tone



syndrome, with sebaceous tumors.


MPL
MPL or myeloproliferative leukemia gene encodes the



thrombopoietin receptor, which is the main humoral



regulator of thrombopoiesis in humans. MPL mutations



cause constitutive activation of JAK-STAT signaling and



have been detected in 5-7% of patients with primary



myelofibrosis (PMF) and 1% of those with essential



thrombocythemia (ET).



In addition, germline mutations in MPL (S505N) have been



associated with familial thrombocythemia.


NOTCH1
NOTCH1, or notch homolog 1, translocation-associated,



encodes a member of the Notch signaling network, an



evolutionary conserved pathway that regulates developmental



processes by regulating interactions between physically



adjacent cells. Notch signaling modulates interplay between



tumor cells, stromal matrix, endothelial cells and immune



cells, and mutations in NOTCH1 play a central role in



disruption of microenvironmental communication, potentially



leading to cancer progression. Due to the dual, bi-directional



signaling of NOTCH1, activating mutations have been found



in ALL and CLL, however loss of function mutations in



NOTCH1 are prevalent in 11-15% of HNSCC. NOTCH1



mutations have also been found in 2% of glioblastomas, ~1%



of ovarian cancers, 10% lung adenocarcinomas, 8% of



squamous cell lung cancers and 5% of breast cancers. Notch



pathway-directed therapy approaches differ depending on



whether the tumor harbors gain or loss of function mutations,



thus are classified as Notch pathway inhibitors or activators,



respectively. Notch pathway modulators are being



investigated in clinical trials, including MK0752 for



advanced solid tumors (NCT01295632) and panobinostat



(LBH589) for various refractory hematologic malignancies



and many types of solid tumors including thyroid cancer



(NCT01013597) and melanoma (NCT01065467).


NPM1
NPM1, or nucleophosmin, is a nucleolar phosphoprotein



belonging to a family of nuclear chaperones with proliferative



and growth-suppressive roles. In several hematological



malignancies, the NPM locus is lost or translocated, leading



to expression of oncogenic proteins. NPM1 is mutated in



one-third of patients with adult AML and leads to aberrant



localization in the cytoplasm leading to activation of down-



stream pathways including JAK/STAT, RAS/ERK, and PI3K,



leading to cell proliferation, survival and cytoskeletal



rearrangements. In addition, the most common translocation



in anaplastic large cell lymphoma (ALCL) is the NPM-ALK



translocation which leads to expression of an oncogenic



fusion protein with constitutive to some kinase activity. AML



cells with mutant NPM are more sensitive chemotherapeutic



agents including daunorubicin and camptothecin. ALK-



targeted therapies such as crizotinib are under clinical



investigation for ALK-NPM positive ALCL (NCT00939770).


NRAS
NRAS is an oncogene and a member of the (GTPase) ras



family, which includes KRAS and HRAS. This biomarker has



been detected in multiple cancers including melanoma (15%),



colorectal cancer (4%), AML (10%) and bladder cancer (2%).



Acquired mutations in NRAS may be associated with



resistance to vemurafenib in melanoma patients. In colorectal



cancer patients NRAS mutation is associated with resistance



to EGFR-targeted monoclonal antibodies. Agents which



target this gene and/or its downstream or upstream effectors



are in clinical trials, e.g.: NCT01306045, NCT01320085



In addition, germline mutations in NRAS have been



associated with Noonan syndrome, autoimmune



lymphoproliferative syndrome and juvenile myelomonocytic



leukemia.


PDGFRA
PDGFRA is the alpha subunit of platelet-derived growth



factor receptor, a surface tyrosine kinase receptor, which



can activate multiple signaling pathways including PIK3CA/



AKT, RAS/MAPK and JAK/STAT. PDGFRA mutations are



found in 5-8% of gastrointestinal stromal tumor cases, and in



40-50% of KIT wild type GISTs. Gain of function PDGFRA



mutations confer imatinib sensitivity, while substitution



mutation in exon 18 (D842V) shows resistance to the drug.



A PDGFRA mutation in the extracellular domain was shown



to identify a subgroup of DIPG (diffuse intrinsic pontine



glioma) patients with significantly worse outcome PDGFRA



inhibitors (e.g., crenolanib, pazopanib) are in clinical trials



for patients carrying PDGFRA mutations, e.g.:



NCT01243346, NCT01524848, NCT01478373.



In addition, germline mutations in PDGFRA have been



associated with Familial gastrointestinal stromal tumors and



Hypereosinophillic Syndrome (HES).


PIK3CA
PIK3CA or phosphoinositide-3-kinase catalytic alpha poly-



peptide encodes a protein in the PI3 kinase pathway. This



pathway is an active target for drug development. PIK3CA



somatic mutations have been found in breast (26.1%),



endometrial (23.3%), urinary tract (19.3%), colon (13.0%),



and ovarian (10.8%) cancers. Somatic mosaic activating



mutations in PIK3CA may cause CLOVES syndrome.



PIK3CA mutations have been associated with benefit



from mTOR inhibitors (e.g., everolimus, temsirolimus).



Breast cancer patients with activation of the PI3K



pathway due to PTEN loss or PIK3CA mutation/



amplification may have a shorter survival following



trastuzumab treatment. PIK3CA mutated (exon 20)



colorectal cancer patients are less likely to respond to EGFR



targeted monoclonal antibody therapy. Agents that target



PIK3CA are in clinical trials, e.g.: NCT00877773,



NCT01277757, NCT01219699, NCT01501604.


PTEN
PTEN, or phosphatase and tensin homolog, is a tumor



suppressor gene that prevents cells from proliferating. PTEN



is an important mediator in signaling downstream of EGFR,



and loss of PTEN gene function/expression due to gene



mutations or allele loss is associated with reduced benefit to



EGFR-targeted monoclonal antibodies. Mutation in PTEN is



found in 5-14% of colorectal cancer and 7% of breast cancer.



PTEN mutation is generally related to loss of function of the



encoded phosphatase, and an upregulation of the PIK3CA/



AKT pathway. The role of PTEN loss in response to



PIK3CA and mTOR inhibitors has been evaluated in some



clinical studies. Agents that target PTEN and/or its down-



stream or upstream effectors are in clinical trials, including



the following: NCT01430572, NCT01306045.



In addition, germline PTEN mutations associate with Cowden



disease and Bannayan-Riley-Ruvalcaba syndrome. These



dominantly inherited disorders belong to a family of



hamartomatous polyposis syndromes which feature multiple



tumor-like growths (hamartomas) accompanied by an



increased risk of breast carcinoma, follicular carcinoma of the



thyroid, glioma, prostate and endometrial cancer.



Trichilemmoma, a benign, multifocal neoplasm of the skin is



also associated with PTEN germline mutations.


PTPN11
PTPN11, or tyrosine-protein phosphatase non-receptor type



11, is a proto-oncogene that encodes a signaling molecule,



Shp-2, which regulates various cell functions like mitogenic



activation and transcription regulation. PTPN11 gain-of-



function somatic mutations have been found to induce



hyperactivation of the Ala and MAPK networks. Because



of this hyperactivation, Ras effectors such as Mek and PI3K



are targets for candidate therapies in those with PTPN11



gain-of-function mutations. PTPN11 somatic mutations are



found in hematologic and lymphoid malignancies (8%),



gastric (2.4%), colon (2%), ovarian (1.7%), and soft tissue



(1.6%) cancers.



In addition, germline mutations of PTPN11 are associated



with Noonan syndrome, which itself is associated with



juvenile myelomonocytic leukemia (JMML). PTPN11 is



also associated with LEOPARD syndrome, which is



associated with neuroblastoma and myeloid leukemia.


RB1
RB1, or retinoblastoma-1, is a tumor suppressor gene whose



protein regulates the cell cycle by interacting with various



transcription factors, including the E2F family (which



controls the expression of genes involved in the transition



of cell cycle checkpoints). RB1 mutations have also been



detected in ocular and other malignancies, such as ovarian



(10.4%), bladder (41.3%), prostate (8.2%), breast (6.1%),



brain (5.6%), colon (5.3%), and renal (1.5%) cancers. RB1



status, along with other mitotic checkpoints, has been



associated with the prognosis of GIST patients.



In addition, germline mutations of RB1 are associated with



the pediatric tumor, retinoblastoma. Inherited retinoblastoma



is usually bilateral. Patients with a history of retinoblastoma



are at increased risk for secondary malignancies.


RET
RET or rearranged during transfection gene, located on



chromosome 10, activates cell signaling pathways involved in



proliferation and cell survival. RET mutations are mostly



found in papillary thyroid cancers and medullary thyroid



cancers (MTC), but RET fusions have also been found in 1%



of lung adenocarcinomas. A 10-year study notes that



medullary thyroid cancer patients with somatic mutations of



RET correlate with a poor prognosis. Approximately 50% of



patients with sporadic MTC have somatic RET mutations;



85% of these involve the M918T mutation, which is



associated with a higher response rate to vandetanib in



comparison to M918T negative patients. Agents that target



RET are in clinical trials, e.g.: NCT00514046,



NCT01582191.



Germline activating mutations of RET are associated with



multiple endocrine neoplasia type 2 (MEN2), which is



characterized by the presence of medullary thyroid carcinoma,



bilateral pheochromocytoma, and primary hyper-



parathyroidism. Germline inactivating mutations of RET are



associated with Hirschsprung's disease.


SMAD4
SMAD4, or mothers against decapentaplegic homolog 4, is



one of eight proteins in the SMAD family, whose members



are involved in multiple signaling pathways and are key



modulators of the transcriptional responses to the



transforming growth factor-β (TGFβ) receptor kinase



complex. SMAD4 resides on chromosome 18q21, one of



the most frequently deleted chromosomal regions in



colorectal cancer. Smad4 stabilizes Smad DNA-binding



complexes and also recruits transcriptional coactivators



such as histone acetyltransferases to regulatory elements.



Dysregulation of SMAD4 may occur late in tumor



development, and can occur through mutations of the MH1



domain which inhibits the DNA-binding function, thus



dysregulating TGFβR signaling. Mutated (inactivated)



SMAD4 is found in 50% of pancreatic cancers and 10-35%



of colorectal cancers. Studies have shown that preservation



of SMAD4 through retention of the 18q21 region, leads to



clinical benefit from 5-fluorouracil-based therapy. In addition,



various clinical trials investigating agents which target the



TGFβR signaling axis are available including PF-03446962



for advanced solid tumors including NCT00557856.



In addition, germline mutations in SMAD4 are associated



with juvenile polyposis (JP) and combined syndrome of JP



and hereditary hemorrhagic teleangiectasia (JP-HHT).


SMARCB1
SMARCB1 also known as SWI/SNF related, matrix



associated, actin dependent regulator of chromatin, subfamily



b, member 1, is a tumor suppressor gene implicated in cell



growth and development. Loss of expression of SMARCB1



has been observed in tumors including epithelioid sarcoma,



renal medullary carcinoma, undifferentiated pediatric



sarcomas, and a subset of hepatoblastomas.



In addition, germline mutation in SMARCB1 causes about



20% of all rhabdoid tumors which makes it important for



clinicians to facilitate genetic testing and refer families for



genetic counseling.



Germline SMARCB1 mutations have also been identified as



the pathogenic cause of a subset of schwannomas and



meningiomas.


SMO
SMO (smoothened) is a G protein-coupled receptor which



plays an important role in the Hedgehog signaling pathway. It



is a key regulator of cell growth and differentiation during



development, and is important in epithelial and mesenchymal



interaction in many tissues during embryogenesis.



Dysregulation of the Hedgehog pathway is found in cancers



including basal cell carcinomas (12%) and medulloblastoma



(1%). A gain-of-function mutation in SMO results in



constitutive activation of hedgehog pathway signaling,



contributing to the genesis of basal cell carcinoma. SMO



mutations have been associated with the resistance to SMO



antagonist GDC-0449 in medulloblastoma patients. SMO



mutation may also contribute to resistance to SMO antagonist



LDE225 in BCC. SMO antagonists are in clinical trials, e.g.:



NCT01529450.


SRC
SRC, or c-Src is a non-receptor tyrosine kinase, plays a



critical role in cellular growth, proliferation, adhesion and



angiogenesis. Normally maintained in a repressed state by



intramolecular interactions involving the SH2 and SH3



domains, Src mutation prevents these restrictive intra-



molecular interactions, conferring a constitutively active



state. Mutations are found in 12% of colon cancers (especially



those metastatic to the liver) and 1-2% of endometrial cancers.



Agents that target SRC are in clinical trials, e.g.: dasatinib for



treatment of GIST (NCT01643278), endometrial cancer



(NCT01440998), and other solid tumors (NCT01445509);



saracatinib (AZD0530) for breast (NCT01216176) and



pancreatic (NCT00735917) ancers; and bosutinib (SKI-606)



for glioblastoma (NCT01331291).


STK11
STK11, also known as LKB1, is a serine/threonine kinase. It



is thought to be a tumor suppressor gene which acts by



interacting with p53 and CDC42. It modulates the activity of



AMP-activated protein kinase, causes inhibition of mTOR,



regulates cell polarity, inhibits the cell cycle, and activates



p53. Somatic mutations in STK11are associated with a



history of smoking and KRAS mutation in NSCLC patients.



The frequency of STK11 mutation in lung adenocarcinomas



ranges from 7%-30%. STK11 loss may play a role in



development of metastatic disease in lung cancer patients.



Mutations of this gene also drive progression of HPV-



induced dysplasia to invasive, cervical cancer and hence



STK11 status may be exploited clinically to predict the



likelihood of disease recurrence. Agents that target STK11



are in clinical trials, e.g.: NCT01578551.



In addition, germline mutations in STK11 are associated with



Peutz-Jeghers syndrome which is characterized by early onset



hamartomatous gastro-intestinal polyps and increased risk of



breast, colon, gastric and ovarian cancer.


TP53
TP53, or p53, plays a central role in modulating response to



cellular stress through transcriptional regulation of genes



involved in cell-cycle arrest, DNA repair, apoptosis, and



senescence. Inactivation of the p53 pathway is essential for



the formation of the majority of human tumors. Mutation in



p53 (TP53) remains one of the most commonly described



genetic events in human neoplasia, estimated to occur in 30-



50% of all cancers with the highest mutation rates occurring



in head and neck squamous cell carcinoma and colorectal



cancer. Generally, presence of a disruptive p53 mutation is



associated with a poor prognosis in all types of cancers, and



diminished sensitivity to radiation and chemotherapy. Agents



are in clinical trials which target p53's downstream or up-



stream effectors. Utility may depend on the p53 status. For



p53 mutated patients, Chk1 inhibitors in advanced cancer



(NCT01115790) and Weel inhibitors in refractory ovarian



cancer (NCT01164995) are being investigated. For p53



wildtype patients with sarcoma, mdm2 inhibitors



(NCT01605526) are being investigated.



In addition, germline p53 mutations are associated with the



Li-Fraumeni syndrome (LFS) which may lead to early-onset



of several forms of cancer currently known to occur in the



syndrome, including sarcomas of the bone and soft tissues,



carcinomas of the breast and adrenal cortex (hereditary



adrenocortical carcinoma), brain tumors and acute leukemias.


VHL
VHL or von Hippel-Lindau gene encodes for tumor



suppressor protein pVHL, which polyubiquitylates



hypoxia-inducible factor in an oxygen dependent manner.



Absence of pVHL causes stabilization of HIF and expression



of its target genes, many of which are important in regulating



angiogenesis, cell growth and cell survival. VHL somatic



mutation has been seen in 20-70% of patients with sporadic



clear cell renal cell carcinoma (ccRCC) and the mutation may



imply a poor prognosis, adverse pathological features, and



increased tumor grade or lymph-node involvement. Renal cell



cancer patients with a ‘loss of function’ mutation in VHL



show a higher response rate to therapy (bevacizumab or



sorafenib) than is seen in patients with wild type VHL. Agents



which target VHL and/or its downstream or upstream



effectors are in clinical trials, e.g.: NCT01538238.



In addition, germline mutations in VHL cause von Hippel-



Lindau syndrome, associated with clear-cell renal-cell



carcinomas, central nervous system hemangioblastomas,



pheochromocytomas and pancreatic tumors.









In an aspect, the invention provides a molecular profile for a cancer which comprises mutational analysis of a panel of genes, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45 or at least 50 genes. As described herein, the molecular profile can be used to identify a candidate agent that is likely to benefit the cancer patient. The molecular profile can also be used to identify a candidate agent that is not likely to benefit the cancer patient. Further as described, a report can be generated that describes results of the molecular profile. The report may include a summary of the mutational analysis for the genes assessed. The report may also provide a linkage of the mutational analysis with the predicted efficacy of various treatments based on the mutational analysis. Such rules for mutation drug association are provided herein, e.g., in Table 25 or any of Tables 7-24. The report may also comprise one or more clinical trials associated with one or more identified mutation in the patient. Mutational analysis can also be used to detect mutations of genes that are known to affect a prognosis or provide other characterization of a cancer.


The molecular profile may comprise mutational analysis of one or more gene in Table 25. For example, the molecular profile may include the mutational analysis of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or at least 50 genes in Table 25. The molecular profile may include the mutational analysis of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, c-Kit, C-Met, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR, KRAS, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, VHL. In an embodiment, the molecular profile comprises mutational analysis of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 of ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CSF1R, CTNNB1, EGFR, ERBB2 (HER2), ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, STK11, TP53, VHL. For example, the molecular profile may comprise mutational analysis of ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CSF1R, CTNNB1, EGFR, ERBB2 (HER2), ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, STK11, TP53, and VHL. In an embodiment, the mutational analysis molecular profile is performed in concert with another molecular profile provided herein. For example, the analysis of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 of ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CSF1R, CTNNB1, EGFR, ERBB2 (HER2), ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, STK11, TP53 and VHL can be reported together with the molecular profiling described in any of FIGS. 33A-Q, FIGS. 35A-I and/or Tables 7-25. In an embodiment, the mutational analysis of ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CSF1R, CTNNB1, EGFR, ERBB2 (HER2), ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, STK11, TP53 and VHL genes is reported together with the molecular profiling described in any of FIGS. 33A-Q, FIGS. 35A-I and/or Tables 7-25.


In an embodiment, the molecular profile comprises mutational analysis of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 of ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL. For example, ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL may be assessed. As desired, additional biomarkers may be assessed for mutational analysis including at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1, PTPN11, RB1, SMAD4, SMARCB1, STK11. For example, CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1, PTPN11, RB1, SMAD4, SMARCB1, STK11 may be assessed in addition to the biomarkers above. In an embodiment, the molecular profile comprises mutational analysis of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 of ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, STK11, TP53, VHL. For example, the molecular profile may comprise or consist of mutational analysis of ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, STK11, TP53, VHL.


In still other embodiments, the molecular profile comprises mutational analysis of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 of ALK, BRAF, BRCA1, BRCA2, EGFR, ERRB2, GNA11, GNAQ, IDH1, IDH2, KIT, KRAS, MET, NRAS, PDGFRA, PIK3CA, PTEN, RET, SRC, TP53. The molecular profile may comprise mutational analysis of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 of AKT1, HRAS, GNAS, MEK1, MEK2, ERK1, ERK2, ERBB3, CDKN2A, PDGFRB, IFG1R, FGFR1, FGFR2, FGFR3, ERBB4, SMO, DDR2, GRB1, PTCH, SHH, PD1, UGTIA1, BIM, ESR1, MLL, AR, CDK4, SMAD4. The molecular profile may also comprise mutational analysis of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 of ABL, APC, ATM, CDH1, CSFRI, CTNNB1, FBXW7, FLT3, HNF1A, JAK2, JAK3, KDR, MLH1, MPL, NOTCH1, NPM1, PTPN11, RB1, SMARCB1, STK11, VHL. The genes assessed by mutational analysis may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, at least 200 genes, or all genes, selected from the group consisting of ABL1, AKT1, AKT2, AKT3, ALK, APC, AR, ARAF, ARFRP1, ARID1A, ARID2, ASXL1, ATM, ATR, ATRX, AURKA, AURKB, AXL, BAPI, BARD1, BCL2, BCL2L2, BCL6, BCOR, BCORL1, BLM, BRAF, BRCA1, BRCA2, BRIP1, BTK, CARD11, CBFB, CBL, CCND1, CCND2, CCND3, CCNE1, CD79A, CD79B, CDC73, CDH1, CDK12, CDK4, CDK6, CDK8, CDKN1B, CDKN2A, CDKN2B, CDKN2C, CEBPA, CHEK1, CHEK2, CIC, CREBBP, CRKL, CRLF2, CSF1R, CTCF, CTNNA1, CTNNB1, DAXX, DDR2, DNMT3A, DOTIL, EGFR, EMSY (Cllorf30), EP300, EPHA3, EPHA5, EPHB1, ERBB2, ERBB3, ERBB4, ERG, ESR1, EZH2, FAM123B (WTX), FAM46C, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCL, FBXW7, FGF10, FGF14, FGF19, FGF23, FGF3, FGF4, FGF6, FGFR1, FGFR2, FGFR3, FGFR4, FLT1, FLT3, FLT4, FOXL2, GATA1, GATA2, GATA3, GID4 (C17orf39), GNA11, GNA13, GNAQ, GNAS, GPR124, GRIN2A, GSK3B, HGF, HRAS, IDH1, IDH2, IGFIR, IKBKE, IKZF1, IL7R, INHBA, IRF4, IRS2, JAK1, JAK2, JAK3, JUN, KAT6A (MYST3), KDM5A, KDM5C, KDM6A, KDR, KEAP1, KIT, KLHL6, KRAS, LRP1B, MAP2K1, MAP2K2, MAP2K4, MAP3K1, MCL1, MDM2, MDM4, MED12, MEF2B, MEN1, MET, MITF, MLH1, MLL, MLL2, MPL, MRE11A, MSH2, MSH6, MTOR, MUTYH, MYC, MYCL1, MYCN, MYD88, NF1, NF2, NFE2L2, NFKBIA, NKX2-1, NOTCH1, NOTCH2, NPM1, NRAS, NTRK1, NTRK2, NTRK3, NUP93, PAK3, PALB2, PAX5, PBRM1, PDGFRA, PDGFRB, PDK1, PIK3CA, PIK3CG, PIK3R1, PIK3R2, PPP2R1A, PRDM1, PRKAR1A, PRKDC, PTCH1, PTEN, PTPN11, R, RAD50, RAD51, RAF1, RARA, RB1, RET, RICTOR, RNF43, RPTOR, RUNX1, SETD2, SF3BI, SMAD2, SMAD4, SMARCA4, SMARCB1, SMO, SOCS1, SOX10, SOX2, SPEN, SPOP, SRC, STAG2, STAT4, STK11, SUFU, TET2, TGFBR2, TNFAIP3, TNFRSF14, TOP1, TP53, TSC1, TSC2, TSHR, VHL, WISP3, WT 1, XPO 1, ZNF217, ZNF703. The mutational analysis may be performed to detect a gene rearrangement, e.g., a rearrangement in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 of ALK, BCR, BCL2, BRAF, EGFR, ETV1, ETV4, ETV5, ETV6, EWSR1, MLL, MYC, NTRK1, PDGFRA, RAF1, RARA, RET, ROS1, TMPRSS2.


Molecular Profiling with Prioritized Sequencing (4.6, 4.7)


The invention further provides molecular profiles that use IHC for expression profiling and Next Generation sequencing for mutational analysis. Such profiles are described in FIGS. 35A-I and Table 26.


The profiling is performed using the rules for the biomarker-drug associations for the various cancer lineages as described for FIGS. 33A-Q and Tables 7-24 above. An expanded set of genes may be assessed by mutational analysis for each molecular profile, as described further below.


Table 26 presents a view of the information that is reported for the molecular profiles. Modifications made dependent on cancer lineage are indicated in the table. The columns headed “Agent/Biomarker Status Reported” provide either candidate agents (e.g., drugs) or biomarker status to be included in the report. Where agents are indicated, the association of the agent with the indicated biomarker is included in the report. Where a status is indicated (e.g., mutational status, protein expression status, gene copy number status), the biomarker status is indicated in the report instead of drug associations. The candidate agents may comprise those undergoing clinical trials, as indicated.









TABLE 26







Molecular Profile and Report Parameters









Agent(s)/Biomarker Status




Reported
Biomarker
Platform





docetaxel, paclitaxel, nab-
Pgp
IHC


paclitaxel, protein expression
SPARCm
IHC



SPARCp
IHC



TLE3
IHC



TUBB3
IHC


capecitabine, fluorouracil,
TS
IHC


pemetrexed




doxorubicin, liposomal-
HER2
FISH/CISH


doxorubicin, epirubicin,
TOP2A
IHC (excluding Breast)


protein expression

FISH/CISH (Breast only)



Pgp
IHC


irinotecan, topotecan
TOPO1
IHC


gemcitabine
RRM1
IHC


imatinib
cKIT
NextGen Sequencing



PDGFRA
NextGen Sequencing


temozolomide, dacarbazine†
MGMT (excluding Glioma)
IHC (excluding Glioma)



MGMT-Me (Glioma ONLY)
Pyrosequencing (Glioma ONLY)



IDH1
NextGen Sequencing



(assoc. in High Grade




Glioma only)



vandetanib
RET
NextGen Sequencing


abiraterone, bicalutamide,
AR
IHC


flutamide, protein expression




anastrozole, exemestane,
ER
IHC


fulvestrant, goserelin,
PR
IHC


megestrol acetate, letrozole,




leuprolide, tamoxifen,




toremifene, protein




expression




trastuzumab
HER2
IHC; FISH/CISH



PTEN (assoc. in Breast only)
IHC



PIK3CA (assoc. in Breast only)
NextGen Sequencing


lapatinib, pertuzumab, T-
HER2
IHC, FISH/CISH


DM1, clinical trials




everolimus, temsirolimus,
ER (assoc. in Breast only)
IHC


clinical trials
HER2 (assoc. in Breast only)
IHC; FISH/CISH



PIK3CA
NextGen Sequencing


cetuximab, panitumumab†
BRAF
NextGen Sequencing


(assoc. in CRC only)
KRAS
NextGen Sequencing



NRAS
NextGen Sequencing



PIK3CA
NextGen Sequencing



PTEN
IHC


cetuximab† (assoc. in
EGFR (NSCLC only)
IHC (H-score)


NSCLC only)

(NSCLC only)


erlotinib, gefitinib†
EGFR (NSCLC only)
NextGen Sequencing


(assoc. in NSCLC only)

(NSCLC only)



KRAS
NextGen Sequencing



PIK3CA
NextGen Sequencing



cMET
FISH/CISH



PTEN
IHC


crizotinib†
ALK (assoc. in NSCLC only)
FISH



ROS1 (assoc. in NSCLC only)
(NSCLC only)


vemurafenib† (assoc. in
BRAF
NextGen Sequencing


Melanoma and Uveal

PCR (cobas ®)


Melanoma only)




dabrafenib†,trametinib*†
BRAF
NextGen Sequencing


(assoc. in Melanoma only)

PCR (cobas ®)


sunitinib† (assoc. in GIST
cKIT
NextGen Sequencing


only)




clinical trials (HDAC and
GNA11 (assoc. in Uveal Melanoma only)
NextGen Sequencing


MEK inhibitors)

(Uveal Melanoma only)


(assoc. in Uveal Melanoma




only)




clinical trials (cMET
cMET
IHC, FISH/CISH


inhibitors)




clinical trials (MEK and
BRAF
NextGen Sequencing


BRAF inhibitors)
KRAS
NextGen Sequencing



NRAS
NextGen Sequencing


clinical trials (angiogenesis
VHL
NextGen Sequencing


inhibitors)




clinical trials (PIK3CA,
PTEN
NextGen Sequencing


mTOR, MEK, angiogenesis,




and IGF pathway inhibitors)





†Assay and therapy will only be performed and reported for specific tumor types.


*Trametinib association will include BRAF by Next-Generation Sequencing testing for V600K mutations.






The molecular profile in Table 26 can be used to profile any cancer for selected a candidate treatment, e.g., by assessing a solid tumor sample as described herein. The biomarkers used for associations with specific cancer lineages are indicated in Table 26. FIGS. 35A-I further illustrate lineage specific profiling that can be performed. FIG. 35A illustrates a molecular profile for any solid tumor. FIG. 35B illustrates a molecular profile for an ovarian cancer. FIG. 35C illustrates a molecular profile for a melanoma. FIG. 35D illustrates a molecular profile for a uveal melanoma. FIG. 35E illustrates a molecular profile for a non-small cell lung cancer (NSCLC). FIG. 35F illustrates a molecular profile for a breast cancer. FIG. 35G illustrates a molecular profile for a colorectal cancer (CRC). FIG. 35H illustrates a molecular profile for a glioma. FIG. 35I illustrates individual marker profiling that can be added to any of the molecular profiles in FIGS. 35A-35G. As described, each of the molecular profiles in FIGS. 35A-I and Table 26 can be performed in conjunction with expanded mutational analysis as described above. See, e.g., Table 25 and accompanying text.


Sample-Dependent Molecular Profiling (4.2)

The molecular profiling that is performed may depend on the amount and quality of sample that is available. For example, certain molecular profiling techniques can be performed with lesser amount of quality sample than other techniques. Thus, in some aspects the invention provides a molecular profile wherein the techniques performed depend on the amount and/or quality of the sample. For example, RT-PCR can be used to measure gene expression if sufficient sample is available; otherwise, IHC is performed to measure protein expression of the same biomarker. Such substitution may require that the evidence is available to support the substitution in order for the alternatively biomarker to be used to assess the likely benefit or not of a candidate agent. Sample dependent molecular profiles are described in more detail in this Section.


Consider an exemplary comprehensive molecular profile for any cancer comprising assessment of the biomarkers as illustrated in FIG. 36A and FIG. 36B in order to determine whether treatments in FIG. 36C are likely beneficial or not. The molecular profile uses RT-PCR to determine gene expression. As shown in FIG. 36A, the profiling may comprise: 1) RT-PCR to assess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of AREG, BRCA1, EGFR, ERBB3, ERCC1, EREG, PGP (MDR-1), RRM1, TOPO1, TOPO2A, TS, TUBB3; 2) sequencing to assess 1, 2, 3 4, or 5 of BRAF, c-KIT, KRAS, NRAS, PIK3CA; 3) ISH to assess 1, 2, or 3 of ALK, cMET, HER2; 4) IHC to assess 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of AR, cMET, ER, HER2, MGMT, PR, PTEN, SPARC (m/p), TLE3; and/or fragment analysis (e.g., RFLP) to assess ALK. As shown in FIG. 36B, certain additional biomarkers are assessed depending on tumor lineage, including: 1) BRAF by PCR (e.g., cobas PCR) and/or sequencing of GNAQ and/or GNA11 for melanoma; 2) sequencing or fragment analysis of EGFR, ISH analysis of ROS1, and/or IHC H-score analysis of EGFR for lung cancer; and 3) ISH analysis of TOPO2A for breast cancer. The biomarker-treatment associations for this molecular profile may comprise those associations in FIG. 36C and determination of likely benefit or not of the treatments based on the profiling results can be according to the rules in Table 27. Table 27 indicates whether the indicated markers are profiled for gastrointestinal stromal tumor (GIST) and/or profiling of any cancer. See column headed “GIST, Comprehensive, or Both.” The class of drug and illustrative drugs of the indicated class are indicated in the columns “Class of Drugs” and “Drugs,” respectively. The columns headed “Biomarker Result” illustrate illustrative methods of profiling the indicated biomarkers, generally as true (“T”) or false (“F”) or any. One of skill will appreciate that alternative methods can be used to analyze the biomarkers as appropriate. For example, expression analysis performed by RT-PCR could be performed by microarray or other expression analysis method such as those described herein or known in the art. The joint result of the indicated biomarker results combined to predict a benefit or not of the indicated candidate drugs. As an example of the logic used to select a drug treatment in Table 27, consider the first rules concerning ERCC1 and BRCA1 to assess the efficacy of platinum compounds. If gene expression of ERCC1 is found to be low by RT-PCR (bERCC 1 low=T), then platinum compounds are predicted to have treatment benefit (T). However, if low expression of ERCC1 is determined to be false, then the expression of BRCA1 will determine the expected benefit with platinum compounds: if expression of ERCC1 is not low (i.e., ERCC1 low=F) and expression of BRCA1 is low (i.e., BRCA1 low=T), then platinum compounds are expected to be of benefit (i.e., overall benefit=T); if expression of ERCC1 is not low (i.e., ERCC1 low=F) and expression of BRCA1 is not low or is not determined (i.e., BRCA1 low=F or No Data), then platinum compounds are not expected to be of benefit (i.e., overall benefit=F).


The molecular profile for GIST can comprise a comprehensive profile with the additional molecular profiling indicated for a GIST in Table 27, namely differential sequence analysis of cKIT in GIST versus other cancers to predict treatment benefit with tyrosine kinase inhibitors (TKI). In GIST, imatinib associates with mutations in exons 9, 11 and/or 13 of cKIT, sunitinib associates with mutations in exon 9 of cKIT, and sorafenib associates with mutations in exons 9 and/or 11 of cKIT. In all other lineages, imatinib and sunitinib associate with mutations in exon 11 and/or 13 of cKIT.









TABLE 27







Comprehensive Molecular Profile using RT-PCR













GIST,








Comprehensive,
Class of

Biomarker
Biomarker
Biomarker
Treatment


or Both
Drugs
Drugs
Result
Result
Result
Benefit





Both
Platinum
cisplatin,
ERCC1 Low
BRCA1 Low

Overall



compounds
carboplatin,
(RT-PCR)
(RT-PCR)

Benefit




oxaliplatin





T
Any

T





F
Any

F





No Data
T

T





No Data
F

F





No Data
No Data

Indeterminate


Both
Anthracyclines
doxorubicin,
TOP2A High
PGP Low

Overall



and
liposomal-
(RT-PCR)
(RT-PCR)

Benefit



related
doxorubicin,



substances
epirubicin





T
T or No Data

T





T
F

F





F
Any

F





No Data
T

T





No Data
F

F





No Data
No Data

Indeterminate


Both
Taxanes
docetaxel,
TLE3
TUBB3 Low

Overall




paclitaxel
Positive
(RT-PCR)

Benefit





(IHC)





T
Any

T





F
Any

F





No Data
T

T





No Data
F

F





No Data
No Data

Indeterminate


Both
Taxanes
nab-
SPARC
SPARC

Overall




paclitaxel
MONO
POLY

Benefit





Positive
Positive





(IHC)
(IHC)





T
Any

T





F
T

T





F
F or No Data

F





No Data
T

T





No Data
F

F





No Data
No Data

Indeterminate


Both
Antimetabolites
gemcitabine
RRM1 Low


Overall





(RT-PCR)


benefit





T


T





F


F





No Data


Indeterminate


Both
Fluoropyrimidines/
pemetrexed,
TS Low (RT-


Overall



Antimetabolites
fluorouracil,
PCR)


benefit




capecitabine





T


T





F


F





No Data


Indeterminate


Both
TOPO1
irinotecan,
TOPO1


Overall



inhibitors
topotecan
High (RT-


benefit





PCR)





T


T





F


F





No Data


Indeterminate


Both
Alkylating
temozolomide,
MGMT


Overall



agents
dacarbazine
Negative


benefit





(IHC)





T


T





F


F





No Data


Indeterminate


Both
mTOR
everolimus,
PIK3CA
PTEN

Overall



inhibitors
temsirolimus
Mutated
Negative

Benefit





(Sequencing)
(IHC)





T
Any

T





F
T

T





F
F

F





F
No Data

Indeterminate





No Data
T

T





No Data
F or No Data

Indeterminate


Both
Anti-
bicalutamide,
AR Positive


Overall



androgens
flutamide,
(IHC)


Benefit




abiraterone





T


T





F


F





No Data


Indeterminate


Both
Anti-
tamoxifen,
ER Positive
PR Positive

Overall



estrogens
toremifene,
(IHC)
(IHC)

Benefit




fulvestrant





T
Any

T





F
T

T





F
F

F





F
No Data

Indeterminate





No Data
T

T





No Data
F or No Data

Indeterminate


Both
Endocrine
letrozole,
ER Positive
PR Positive

Overall



therapy -
anastrozole,
(IHC)
(IHC)

Benefit



enzyme
exemestane



inhibitor





T
Any

T





F
T

T





F
F

F





F
No Data

Indeterminate





No Data
T

T





No Data
F or No Data

Indeterminate


Both
Progestogens
medroxyprogesterone,
ER Positive
PR Positive

Overall




megestrol
(IHC)
(IHC)

Benefit




acetate





T
Any

T





F
T

T





F
F

F





F
No Data

Indeterminate





No Data
T

T





No Data
F or No Data

Indeterminate


Both
Gonadotropin
leuprolide,
ER Positive
PR Positive

Overall



releasing
goserelin
(IHC)
(IHC)

Benefit



hormone



analogs





T
Any

T





F
T

T





F
F

F





F
No Data

Indeterminate





No Data
T

T





No Data
F or No Data

Indeterminate


Both
TKI
lapatinib
HER2
HER2

Overall





Positive
Amplified

Benefit





(IHC)
(FISH)





T
Any

T





F
T or

T






Equivocal






High





F
F or

F






Equivocal






Low





F
No Data

Indeterminate





Equivocal
T or

T






Equivocal






High





Equivocal
F or

F






Equivocal






Low





Equivocal
No Data

Indeterminate





No Data
T or

T






Equivocal






High





No Data
F, Equivocal

Indeterminate






Low or No






Data


Both
Monoclonal
trastuzumab
HER2
HER2

Overall



antibodies

Positive
Amplified

Benefit



(Her2-

(IHC)
(FISH)



targeted -



trastuzumab)





T
Any

T





F
T or

T






Equivocal






High





F
F or

F






Equivocal






Low





F
No Data

Indeterminate





Equivocal
T or

T






Equivocal






High





Equivocal
F or

F






Equivocal






Low





Equivocal
No Data

Indeterminate





No Data
T or

T






Equivocal






High





No Data
F, Equivocal

Indeterminate






Low or No






Data


Both
TKI
erlotinib,
EGFR High
cMET
cMET
Overall




gefitinib
(RT-PCR)
Positive
Amplified
Benefit






(IHC)
(FISH)





T
Any
Any
T





F
Any
Any
F





No Data
Any
Any
Indeterminate


Both
TKI
crizotinib
ALK
ALK

Overall





Positive
Positive (FA)

benefit





(FISH)





T
Any

T





F
Any

F





No Data
Any

Indeterminate


GIST
TKI
imatinib
c-KIT


Overall





Mutated


Benefit





(Sequencing)





T


T





F


F





No Data


Indeterminate


GIST
TKI
sunitinib
c-KIT


Overall





Mutated


Benefit





(Sequencing)





T


T





F


F





No Data


Indeterminate


GIST
TKI
sorafenib
c-KIT


Overall





Mutated


Benefit





(Sequencing)





T


T





F


F





No Data


Indeterminate


Comprehensive
TKI
imatinib,
c-KIT


Overall




sunitinib
Mutated


Benefit





(Sequencing)





T


T





F


F





No Data


Indeterminate









In an embodiment, the invention provides a comprehensive molecular profile for cancer comprising one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 of: ALK, AR, AREG, BRAF, BRCA1, c-KIT, cMET, EGFR, ER, ERBB3, ERCC1, EREG, HER2, KRAS, MGMT, NRAS, PGP (MDR-1), PIK3CA, PR, PTEN, RRM1, SPARC, TLE3, TOPO1, TOPO2A, TS, TUBB3. The invention further provides a method of selecting a candidate treatment for a cancer comprising assessment of one or more members of the comprehensive cancer profile using one or more molecular profiling method presented herein, e.g., FISH/CISH, IHC, RT-PCR, expression array, sequencing, FA such as RFLP, etc. In one embodiment, FISH/CISH is used to assess one or more, e.g., 1 or 2, of: cMET and HER2. In an embodiment, protein analysis such as IHC is used to assess one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9, of: AR, cMET, ER, HER2, MGMT, PR, PTEN, SPARC, TLE3. The IHC can be used to ascertain an IHC score (H-score), which takes into account the percentage of cells (0-100%) as well as each staining intensity category (0-3+) to compute a semi-quantitative score between 0 and 300. In another embodiment, expression analysis, e.g., by RT-PCR (qPCR) or microarray, is used to assess one or more of, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, of: AREG, BRCA1, EGFR, ERBB3, ERCC1, EREG, PGP (MDR-1), RRM1, TOPO1, TOPO2A, TS, TUBB3. In still another embodiment, sequence analysis is used to assess one or more, e.g., 1, 2, 3, 4, or 5, of: BRAF, KRAS, NRAS, PIK3CA, c-KIT. The comprehensive cancer profile can also comprise assessment of the presence of ALK or an ALK mutation/translocation/rearrangement, e.g., an EML4-ALK fusion, e.g., by FISH, RT-PCR, sequencing or fragment analysis (FA). In an embodiment, the molecular profile further comprises detection of the presense of VEGFR2, e.g., by RT-PCR. Any biomarker disclosed herein, e.g., in Table 2, Table 6 or Table 25, can be assessed as part of the comprehensive molecular profile. The comprehensive profile for a malignancy of any lineage can be as shown in FIGS. 36A-C. The profile can be used to identify drugs as likely beneficial or not based on rules in Table 27.


The comprehensive profile can further comprise molecular profiling of certain genes in the context of specific cancer lineage. For example, the comprehensive profile can comprise the molecular profiling described above and in addition one or more of the following markers. A comprehensive profile of melanoma can include molecular profiling of BRAF, GNA11 and/or GNAQ. For example, one or more of these biomarkers can be assessed for a mutation, e.g., by sequencing or PCR. In an embodiment, BRAF is assessed using the FDA approved cobas 4800 BRAF V600 Mutation Test from Roche Molecular Diagnostics (Roche Diagnostics, Indianapolis, Ind.). According to the manufacturer, the kit comprises a real-time PCR test to detect the BRAF V600E (1799 T>A) mutation in human melanoma, e.g., in formalin-fixed, paraffin-embedded (FFPE) tissue. It is designed to help select patients for treatment with vemurafenib, an oral medicine designed to treat patients whose melanoma tumors harbor a mutated form of the BRAF gene. The test may also detect other V600 mutations such as V600D and V600K. Vemurafenib is designed to target and inhibit some mutated forms of the BRAF protein found in about half of all cases of melanoma. GNAQ/GNA11 mutations can promote tumor growth and metastatis. MEK inhibitors may inhibit the GNAQ/GNA11 pathway. Similarly, a comprehensive profile of non-small cell lung cancer can include additional molecular profiling of EGFR and/or ALK. For example, and EGFR mutation can be detected by sequence analysis and/or fragment analysis. EGFR protein can be assessed by IHC, including by determining an H-score. ALK can be assessed using FISH and/or CISH. In an embodiment, ALK is assessed using the Vysis ALK Break Apart FISH Probe Kit from Abbott Molecular, Inc. (Des Plaines, Ill.). According to the manufacturer, this kit comprises a laboratory test that uses DNA probes with attached fluorescent dyes to detect the presence of chromosomal rearrangements of the ALK gene, located on chromosome 2, in a non-small cell lung cancer (NSCLC) tissue sample. If the test result indicates the presence of rearrangements (such as translocation) involving the ALK gene in the cancer cell, then a patient with NSCLC may be eligible for treatment with the cancer drug crizotinib. Crizotinib selectively interferes with the ALK gene and can benefit patients with ALK mutations. In addition, the comprehensive profile for a breast cancer can comprise further molecular profiling of TOPO2A, e.g., using FISH or CISH. In sum, embodiments of the comprehensive profile can be as shown in FIGS. 36A-36C with rules to identify drugs as likely beneficial or not based as shown in Table 27.


The molecular profiles of the invention can comprise further gene and gene products to identify additional biomarker-treatment associations. In an embodiment, the molecular profile comprises one or more additional gene or gene product listed in Table 2, Table 6 or Table 25. For example, the molecular profile may comprise one or more additional gene or gene product selected from the group consisting of MSH2, ERBB4, ROS1, MGMT, and a combination thereof. Any appropriate technique can be used to assess the gene and/or gene products. In a non-limiting example, the molecular profile can include one or more additional analysis selected from the group consisting of allele-specific PCR for BRAF and/or KRAS; RT-PCR for one or more of ER, HER2, MSH2 and PR; sequence analysis for ERBB4; FISH, fragment analysis and/or microsatellite instability for ROS1 rearrangements and/or HER2 exon 20 insertion; pyrosequencing for MGMT methylation status; and a combination thereof.


As noted above, different technologies used for molecular profiles can require different amounts of the input biological sample. In some embodiments of the invention, the precise technology used depends upon the amount of tumor sample that is available. A threshold amount of tumor sample can be set to perform certain tests. For example, a threshold amount of tumor can be set for determining whether or not to perform RT-PCR for gene expression analysis. If insufficient tumor sample is available, then another technique for measuring expression levels can be performed, such as IHC to measure protein expression. Alternately, if there is not enough sample to perform RT-PCR, then FISH is performed. As another example, a threshold amount of tumor can be set for determining whether or not to perform Sanger sequence analysis. If insufficient tumor sample is available, then another technique for detecting a gene mutation can be performed, such as fragment analysis (FA). The threshold can depend on factors such as molecular profiling technique to be performed, size of the tumor sample, and percentage of tumor in the sample. In some embodiments, the patient sample is subjected to microdissection to select areas enriched in tumor before performing molecular profiling. Thus, the threshold can be set after microdissection as desired. In an embodiment, the threshold takes into account the size of the tumor sample available. The size required can be at least 0.1 mm2, 0.5 mm2, 1.0 mm2, 1.5 mm2, 2.0 mm2, 2.5 mm2, 3.0 mm2, 3.5 mm2, 4.0 mm2, 4.5 mm2, 5.0 mm2, 6.0 mm2, 7.0 mm2, 8.0 mm2, 9.0 mm2, 10.0 mm2, 11.0 mm2, 12.0 mm2, 13.0 mm2, 14.0 mm2, 15.0 mm2, 16.0 mm2, 17.0 mm2, 18.0 mm2, 19.0 mm2, 20.0 mm2, 22.5 mm2, 25.0 mm2, 27.5 mm2, 30.0 mm2, 32.5 mm2, 35.0 mm2, 37.5 mm2, 40.0 mm2, 45.0 mm2, or at least 50.0 mm2. In another embodiment, the threshold takes into account the percentage of tumor in the sample. The percentage of tumor required can be at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The percentage can be expressed as the percentage of tumor nuclei. When the sample is cut into pathology slides, a minimum number of slides can be required. In still another embodiment, the threshold takes into account the number of sample slides available. The number of slides required can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or at least 50 slides.


Any useful combination of parameters can be used to determine the threshold. For example, the threshold to determine whether to run RT-PCR or IHC/FISH may comprise having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or at least 50 slides pathology slides each having at least 0.1 mm2, 0.5 mm2, 1.0 mm2, 1.5 mm2, 2.0 mm2, 2.5 mm2, 3.0 mm2, 3.5 mm2, 4.0 mm2, 4.5 mm2, 5.0 mm2, 6.0 mm2, 7.0 mm2, 8.0 mm2, 9.0 mm2, 10.0 mm2, 11.0 mm2, 12.0 mm2, 13.0 mm2, 14.0 mm2, 15.0 mm2, 16.0 mm2, 17.0 mm2, 18.0 mm2, 19.0 mm2, 20.0 mm2, 22.5 mm2, 25.0 mm2, 27.5 mm2, 30.0 mm2, 32.5 mm2, 35.0 mm2, 37.5 mm2, 40.0 mm2, 45.0 mm2, or at least 50.0 mm2 of tumor sample with at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% tumor nuclei in a sample after microdissection.


In an embodiment, if sufficient tumor is available, RT-PCR is performed; otherwise, IHC or FISH are performed. For example, RT-PCR can be performed if the sample after microdissection comprises at least 2.0 mm2, 2.5 mm2, 3.0 mm2, 3.5 mm2, 4.0 mm2, 4.5 mm2, 5.0 mm2, 6.0 mm2, 7.0 mm2, 8.0 mm2, 9.0 mm2, or 10.0 mm2 of tumor and at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% tumor nuclei; otherwise IHC or FISH is performed. At least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or at least 50 slides pathology slides can be required to perform RT-PCR. In an embodiment, RT-PCR is performed if the sample after microdissection comprises at least 15 slides having 5.0 mm2 of tumor and at least 80% tumor nuclei; otherwise IHC or FISH is performed. The threshold can be applied to any biomarkers assessed by molecular profiling. For example, the threshold can be performed to determine whether to perform RT-PCR or IHC/FISH to assess one or more of, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, of: AREG, BRCA1, EGFR, ERBB3, ERCC1, EREG, PGP (MDR-1), RRM1, TOPO1, TOPO2A, TS, and TUBB3. The threshold can be applied for any useful subset of these markers, including without limitation one or more of ERCC1, TS, TOPO1, TOP2A, RRM1 and PGP. In embodiments, if the threshold for performing RT-PCR is not met, IHC is performed for ERCC1, TS, TOPO1, RRM1 and PGP, and FISH is performed for TOP2A. If FISH is not possible, then IHC for both TOP2A and PGP may be performed instead.


In another embodiment, if sufficient tumor is available, nucleotide sequencing such as Sanger sequencing is performed; otherwise, fragment analysis such as RFLP is performed. For example, nucleotide sequencing can be performed if the sample after microdissection comprises at least 2.0 mm2, 2.5 mm2, 3.0 mm2, 3.5 mm2, 4.0 mm2, 4.5 mm2, 5.0 mm2, 6.0 mm2, 7.0 mm2, 8.0 mm2, 9.0 mm2, or 10.0 mm2 of tumor and at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% tumor nuclei; otherwise fragment analysis is performed. In an embodiment, nucleotide sequencing is performed if the sample after microdissection comprises at least 50% tumor nuclei; otherwise fragment analysis is performed. The threshold can be applied to any biomarkers assessed by molecular profiling. For example, the threshold can be performed to determine whether to perform nucleotide sequencing or fragment analysis to assess one or more, e.g., 1, 2, 3, 4, 5 or 6, of: BRAF, KRAS, NRAS, PIK3CA, c-KIT, EGFR. The threshold can be applied for any useful subset of these markers, including without limitation EGFR.


In an aspect, the invention provides a method comprising microdissecting a tumor sample from a tissue sample, determining a size of the microdissected tumor sample and an amount of the microdissected sample that comprises tumor nuclei, and performing RT-PCR on the microdissected tumor sample to detect an amount of one or more biomarker target if the size of microdissected tumor sample is greater than or equal to 5.0 mm2 and the microdissected tumor sample comprises 80% or more tumor nuclei, else performing IHC on the microdissected tumor sample to detect an amount of the one or more biomarker target. The one or more biomarker can be selected from the group consisting of ERCC1, TS, TOPO1, TOP2A, RRM1 and PGP. For example, the one or more biomarker can comprise ERCC1, TS, TOPO1, TOP2A, RRM1 and PGP. As noted above, the threshold size and percentage tumor nuclei can be adjusted as appropriate.


The comprehensive molecular profile in this Section (e.g., as shown in FIGS. 36A-C) can be adjusted to reflect such changes when the thresholds for running RT-PCR are not met. For example, if the sample after microdissection comprises at least 15 slides having 5.0 mm2 of tumor and at least 80% tumor nuclei, then the molecular profiles shown in FIGS. 36A-C are used to guide selection of the candidate treatment. If the conditions for running RT-PCR are not met, then the alternate molecular profile shown in FIG. 36D is used to guide selection of the candidate treatment/s. Biomarkers shown in bold in FIG. 36D indicate biomarkers whose molecular profiling technique was changed as the thresholds for RT-PCR were not met. Comparing then the molecular profiles shown in FIGS. 36A-C with the molecular profiles shown in FIG. 36D, it is observed that when the threshold for performing RT-PCR is not met, IHC is performed for ERCC1, TS, TOPO1, RRM1 and PGP, and FISH is performed for TOP2A. Furthermore, as shown in FIG. 36E, if FISH is not possible, then IHC for TOP2A and PGP may be performed instead.


The rules implemented for selection of the candidate treatment can be the same as those presented for RT-PCR, except that the expression results obtained using IHC are substituted. For example, overexpression observed with IHC can trigger the same rules as overexpression with RT-PCR and underexpression observed with IHC can trigger the same rules as underexpression with RT-PCR. With respect to the rules presented in Table 27, references to “Low (RT-PCR)” can be substituted with “Negative (IHC),” and references to “High (RT-PCR)” can be substituted with “Positive (IHC).” As a non-limiting example, associations between TOPO1 by RT-PCR and irinotecan can be substituted with associations between TOPO1 by IHC and irinotecan. Similarly, associations between ERCC1 by RT-PCR and platinum compounds can be substituted with associations between ERCC1 by IHC and platinum compounds. As still another example, associations between RRM1 by RT-PCR and gemcitabine can be substituted with associations between RRM1 by IHC and gemcitabine.


When the sample available is close to the threshold, multiple tests may be performed. For example, if any of the factors for performing RT-PCR or IHC/FISH are within 25% of the threshold value, e.g., 20%, 15%, 10%, 5%, both tests can be performed. In this case, the results of tests providing sufficient data will be applied to the rules above in order to select the candidate treatment. If both tests provide usable results a priority scheme can be used, e.g., when both RT-PCR and IHC are successfully performed on a sample. In an embodiment, results for IHC trump rules for RT-PCR in case of disagreement. Results for FISH can also trump rules for RT-PCR in this scenario. For example, IHC for any of TOPO1, TS, RRM1, TOPO2A, ERCC1, PGP can trump results of RT-PCR for TOPO1, TS, RRM1, TOPO2A, ERCC1, PGP, respectively. Inconsistent results can also depend on the particular biomarker-drug associations. In an embodiment, for TS and fluoropyrimidine rules, when TS PCR and IHC results are inconsistent, the overall benefit of fluoropyrimidine is deemed “Indeterminate.” In another embodiment, for RRM1 and gemcitabine rules, when RRM1 PCR and IHC results are inconsistent, the overall benefit of gemcitabine is deemed true when RRM1 PCR is low and false when RRM1 PCR is high. In still another embodiment, for TOPO1 rules, the benefit is “indeterminate” when Topo1 IHC does not provide results, regardless of whether the Topo1 RT-PCR has actionable data. When TOP2A FISH is used to replace TOP2A RT-PCR, when either TOP2A FISH or Her2 FISH show amplification, anthracyclines are considered to be of benefit.


As an alternative to, or in addition to, substituting laboratory techniques when lower amounts of sample are available, the invention contemplates that certain biomarker tests can be prioritized. FIG. 36F provides illustrative biomarker tests that can be prioritized for various lineages, e.g., when insufficient sample is available for comprehensive molecular profiling as provided herein (e.g., in FIGS. 33A-Q, 35A-I, 36A-E). The biomarkers can be prioritized by the strength of evidence of clinical utility and by standard of care practice guidelines, e.g., the NCCN compendia. Biomarkers followed by the symbol # in FIG. 36F indicate that the drug associated with that particular biomarker is not part of the NCCN compendia. FIG. 36Fi provides a priority panel for a breast cancer, wherein the panel comprises one or more of, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, of: ER assessed by IHC; PR assessed by IHC; HER2 assessed by IHC; TLE3 assessed by IHC; PTEN assessed by IHC; HER2 assessed by FISH or CISH; TOPO2A assessed by FISH; TS assessed by IHC; RRM1 assessed by IHC; TOPO1 assessed by IHC; PIK3CA assessed by Sequencing; KRAS assessed by Sequencing; and BRAF assessed by Sequencing. FIG. 36Fii provides a priority panel for a lung cancer, wherein the panel comprises one or more of, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, of: EGFR assessed by Sequencing; ALK assessed by FISH; ROS1 assessed by FISH; KRAS assessed by Sequencing; RRM1 assessed by IHC; TS assessed by IHC; EGFR assessed by IHC (H-Score); PTEN assessed by IHC; TUBB3 assessed by IHC; cMET assessed by FISH; HER2 assessed by FISH; BRAF assessed by Sequencing; PIK3CA assessed by Sequencing; cMET assessed by IHC. FIG. 36Fiii provides a priority panel for a colorectal cancer (CRC), wherein the panel comprises one or more of, e.g., 1, 2, 3, 4, 5, 6 or 7, of: KRAS assessed by Sequencing; BRAF assessed by Sequencing; TS assessed by IHC; TOPO1 assessed by IHC; PTEN assessed by IHC; PIK3CA assessed by Sequencing; NRAS assessed by Sequencing. FIG. 36Fiv provides a priority panel for a melanoma, wherein the panel comprises one or more of, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9, of: BRAF assessed by PCR; BRAF assessed by Sequencing; cKIT assessed by Sequencing; NRAS assessed by Sequencing; MGMT assessed by IHC; TUBB3 assessed by IHC; SPARC assessed by IHC using a monoclonal antibody; SPARC assessed by IHC using a polyclonal antibody; PIK3CA assessed by Sequencing. FIG. 36Fv provides a priority panel for a melanoma, wherein the panel comprises one or more of, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of: TUBB3 assessed by IHC; RRM1 assessed by IHC; TOPO1 assessed by IHC; TOP2A assessed by IHC; TS assessed by IHC; ER assessed by IHC; PR assessed by IHC; HER2 assessed by IHC; cMET assessed by IHC; PIK3CA assessed by Sequencing. The biomarkers assessed are linked to the likely benefit or lack of benefit of various chemotherapy agents using rules such as provided herein, e.g., in Tables 7-24 or 27. Priority panels can be constructed for other lineages also based on the available evidence.


Clinical Trial Connector

Thousands of clinical trials for therapies are underway in the United States, with several hundred of these tied to biomarker status. In an embodiment, the molecular intelligence molecular profiles of the invention include molecular profiling of markers that are associated with ongoing clinical trials. Thus, the molecular profile can be linked to clinical trials of therapies that are correlated to a subject's biomarker profile. The method can further comprise identifying trial location(s) to facilitate patient enrollment. The database of ongoing clinical trials can be obtained from www.clinicaltrials.gov in the United States, or similar source in other locations. The molecular profiles generated by the methods of the invention can be linked to ongoing clinical trials and updated on a regular basis, e.g., daily, bi-weekly, weekly, monthly, or other appropriate time period.


Although significant advances in cancer treatment have been made in recent years, not all patients can be effectively treated within the standard of care paradigm. Many patients are eligible for clinical trials participation, yet less than 3 percent are actually enrolled in a trial, according to recent National Cancer Institute (NCI) statistics. The Clinical Trials Connector allows caregivers such as physicians to quickly identify and review global clinical trial opportunities in real-time that are molecularly targeted to each patient. In embodiments, the Clinical Trials Connector has one or more of the following features: Examines thousands of open and enrolling clinical trials; Individualizes clinical trials based on molecular profiling as described herein; Includes interactive and customizable trial search filters by: Biomarker, Mechanism of action, Therapy, Phase of study, and other clinical factors (age, sex, etc.). The Clinical Trials Connector can be a computer database that is accessed once molecular profiling results are available. In some embodiments, the database comprises the EmergingMed database (EmergingMed, New York, N.Y.).


Tables 7, 9, 11, 13, 15, 17 and 21 herein indicates an association of certain biomarkers in the molecular profiles of the invention with ongoing clinical trials. Profiling of the specified markers can provide an indication that a subject is a candidate for a clinical trial, e.g., by suggesting that an agent in a clinical trial may benefit the subject. For example, Table 7 indicates that molecular profiling of HER2, PIK3CA, PTEN, cMET and the other indicated gene mutations (i.e., as profiled using NGS) can associate ovarian cancer with ongoing clinical trials. Table 9 indicates that molecular profiling of HER2, ER/HER2/PIK3CA, AR, cMET and the other indicated gene mutations (i.e., as profiled using NGS) can associate breast cancer with ongoing clinical trials. Table 11 indicates that molecular profiling of PIK3CA, PTEN, cMET and the other indicated gene mutations (i.e., as profiled using NGS) can associate melanoma with ongoing clinical trials. Table 13 indicates that molecular profiling of PIK3CA, PTEN, cMET and the other indicated gene mutations (i.e., as profiled using NGS) can associate melanoma with ongoing clinical trials. Table 15 indicates that molecular profiling of cMET and the other indicated gene mutations (i.e., as profiled using NGS) can associate colorectal cancer with ongoing clinical trials. Table 17 indicates that molecular profiling of HER2, PIK3CA, cMET and the indicated gene mutations (i.e., as profiled using NGS) can associate NSCLC with ongoing clinical trials. Table 21 indicates that molecular profiling of HER2, PIK3CA, PTEN, cMET, EGFRvIII, IDH2 and the indicated gene mutations (i.e., as profiled using NGS) can associate various solid tumors with ongoing clinical trials. An illustrative listing of such clinical trials is found in Table 28 below.



FIG. 36C and Table 26 herein further indicate associations of certain biomarkers in the respective molecular profiles with ongoing clinical trials. The clinical trial connections are interpreted as indicated above.


In an aspect, the invention provides a set of rules for matching of clinical trials to biomarker status as determined by the molecular profiling described herein. In some embodiments, the matching of clinical trials to biomarker status is performed using one or more pre-specified criteria: 1) Trials are matched based on the OFF NCCN Compendia drug/drug class associated with potential benefit by the molecular profiling rules; 2) Trials are matched based on biomarker driven eligibility requirement of the trial; and 3) Trials are matched based on the molecular profile of the patient, the biology of the disease and the associated signaling pathways. In the latter case, i.e. item 3, clinical trial matching may comprise further criteria as follows. First, for directly targetable markers, match trials with agents directly targeting the gene (e.g., FGFR results map to anti-FGFR therapy trials; ERBB2 results map to anti-HER2 agents, etc). In addition, for directly targetable markers, trial matching considers downstream markers under the following scenarios: a) a known resistance mechanism is available (e.g., cMET inhibitors for EGFR gene); b) clinical evidence associates the (mutated) biomarker with drugs targeting downstream pathways (e.g., mTOR inhibitors when PIK3CA is mutated); and c) active clinical trials are enrolling patients (with the biomarker aberration in the inclusion criteria) with drugs targeting the downstream pathways (e.g., SMO inhibitors for BCR-ABL mutation T315I). In the case of markers that are not directly targetable by a known therapeutic agent, trial matching may consider alternative, downstream markers (e.g., platinum agents for ATM gene; MEK inhibitors for GNAS/GNAQ/GNA11 mutation). The clinical trials that are matched may be identified based on results of “pathogenic,” “presumed pathogenic,” or variant of uncertain (or unknown) significance (“VUS”). In some embodiments, the decision to incorporate/associate a drug class with a biomarker mutation can further depend on one or more of the following: 1) Clinical evidence; 2) Preclinical evidence; 3) Understanding of the biological pathway affected by the biomarker; and 4) expert analysis. In some embodiments, the mutation of biomarkers in the above section “Mutational Analysis” is linked to clinical trials using one or more of these criteria.


The guiding principle above can be used to identify classes of drugs that are linked to certain biomarkers. The biomarkers can be linked to various clinical trials that are studying these biomarkers, including without limitation requiring a certain biomarker status for clinical trial inclusion. Table 28 presents an illustrative overview of biomarker statuses that are matched to classes of drugs. In the table, the column headed “Biomarker” identifies that biomarker that is assessed according to the molecular profiling technique specified in the column headed “Technique.” It will be appreciated that equivalent methods can be used as desired. For example, Next Generation Sequencing (NGS; Next Gen SEQ) is used to identify mutations, but alternate nucleic acid sequencing and analysis techniques (Sanger sequencing, PCR, RFLP, etc) can be used in the alternative or in the conjunction. Results that indicate a potential match (e.g., a potential benefit) to a class of drugs are indicated in the column “Result.” For sequencing methods, “Pathogenic/Presumed Pathogenic/Variant of Unknown Significance” refer to mutations that are detected and are known, presumed, or potentially pathogenic. As appropriate, particular mutations or other alterations in the biomarker that are potentially matched to the class of drugs are identified in the column headed “Mutation Type/Alteration.” The matched drug classes are identified in the column headed “Drug Class (Associated Agents).” Associated agents are illustrative drugs that are members of the class. Clinical trials studying the drug classes and/or specific agents listed can be matched to the biomarker. In an aspect, the invention provides a method of selecting a clinical trial for enrollment of a patient, comprising performing molecular profiling of one or more biomarker on a sample from the patient using the methods described herein. For example, the profiling can be performed for one on more biomarker in Table 28 using the technique indicated in the table. The results of the profiling are matched to classes of drugs using the above criteria. Clinical trials studying members of the classes of drugs are identified. The matching between the biomarkers and the clinical trials can follow the rules in Table 29, which is described in more detail below. The patient is a potential candidate for the so-identified clinical trials.









TABLE 28







Biomarker—Drug Associations for Drugs in Matched Clinical Trials















Drug Class (Associated





Mutation Type/
Agents) matched by


Biomarker
Technique
Result
Alteration
clinical trials










NGS tests











ATM
Next Gen
Pathogenic/Presumed

PARP inhibitors (ABT-767,



SEQ
Pathogenic/Variant

CEP9722, E7016, iniparib,




of Unknown

MK4827, olaparib, rucaparib,




Significance

veliparib),






HDAC inhibitors






(abexinostat, ACY-1215,






AR-42, belinostat, CUDC-






907, entinostat, FK228,






givinostat, JNJ26481585,






mocetinostat, panobinostat,






SHP-141, valproic acid,






vorinostat, 4SC-202)






Platinum compounds






(carboplatin, cisplatin,






oxaliplatin)


CSF1R
Next Gen
Pathogenic/Presumed

FGFR TKI (dovitinib),



SEQ
Pathogenic/Variant

anti-CSF1R monoclonal




of Unknown

antibody (IMC-CS4)




Significance




ERBB2
Next Gen
Pathogenic/Presumed

anti-HER2 monoclonal



SEQ
Pathogenic/Variant

antibody (pertuzumab,




of Unknown

trastuzumab)




Significance

HER2-targeted tyrosine






kinase inhibitors (afatinib,






dacomitinib, lapatinib,






neratinib)






anti-HER2 monoclonal






antibody—drug conjugate






(ado-trastuzumab emtansine






(T-DM1))


GNAS
Next Gen
Pathogenic/Presumed

MEK inhibitors (AZD8330,



SEQ
Pathogenic/Variant

BAY86-9766, CI-1040,




of Unknown

GDC-0623, GDC-0973,




Significance

MEK162, MSC1936369B,






MSC2015103B, PD0325901,






pimasertib (AS-703026),






selumetinib, TAK-733,






trametinib, XL518)


GNAQ
Next Gen
Pathogenic/Presumed

MEK inhibitors (AZD8330,



SEQ
Pathogenic/Variant

BAY86-9766, CI-1040,




of Unknown

GDC-0623, GDC-0973,




Significance

MEK162, MSC1936369B,






MSC2015103B, PD0325901,






pimasertib (AS-703026),






selumetinib, TAK-733,






trametinib, XL518)


GNA11
Next Gen
Pathogenic/Presumed

MEK inhibitors (AZD8330,



SEQ
Pathogenic/Variant

BAY86-9766, CI-1040,




of Unknown

GDC-0623, GDC-0973,




Significance

MEK162, MSC1936369B,






MSC2015103B, PD0325901,






pimasertib (AS-703026),






selumetinib, TAK-733,






trametinib, XL518)


KDR
Next Gen
Pathogenic/Presumed

VEGFR2-targeted tyrosine



SEQ
Pathogenic/Variant

kinase inhibitors (apatinib,




of Unknown

axitinib, cabozantinib,




Significance

famitinib, fruquintinib,






lenvatinib, motesanib,






ninedanib, pazopanib,






regorafenib, sorafenib,






sunitinib, tivozanib,






vandetanib, vatalanib)






anti-VEGFR2-targeted






monoclonal antibody






(ramucirumab, tanibirumab)


MLH1
Next Gen
Pathogenic/Presumed

PARP inhibitors (ABT-767,



SEQ
Pathogenic/Variant

CEP9722, E7016, iniparib,




of Unknown

MK4827, olaparib, rucaparib,




Significance

veliparib)


JAK3
Next Gen
Pathogenic/Presumed

no drugs



SEQ
Pathogenic/Variant






of Unknown






Significance




PTPN11
Next Gen
Pathogenic/Presumed

no drugs



SEQ
Pathogenic/Variant






of Unknown






Significance




RB1
Next Gen
Pathogenic/Presumed

no drugs



SEQ
Pathogenic/Variant






of Unknown






Significance




VHL
Next Gen
Pathogenic/Presumed

VEGF, VEGFR targeted



SEQ
Pathogenic/Variant

therapies: Aflibercept,




of Unknown

Axitinib, Bevacizumab,




Significance

Cabozantinib, Pazopanib,






Regorafenib, Sorafenib,






Sunitinib, Tivozanib,






Apatinib, Famitinib,






Fruquintinib, Lenvatinib,






Motesanib, Ninedanib,






Vandetanib, Vatalanib,






Ramucirumab, Tanibirumab,






IMC-3C5, IMC-18F1






PI3K/Akt/mTor






inhibitors:Temsirolimus,






Everolimus, CC-223,






Ridaforolimus, sirolimus,






MLN0128, GDC0941,






Deforolimus, BEZ235, DS-






7423, GDC-0980, PF-






04691502, PF-05212384,






SAR245409, BKM120,






BYL719, PX-866, GDC-






0068, MK2206,






GSK2131795, GSK2110183,






GSK2141795, XL147






(SAR245408), INK1117,






AZD5363, Perifosine,






ARQ092, AZD8055, OSI-






027, BAY80-6946


c-KIT
Next Gen
Pathogenic/Presumed
all mutations except
KIT inhibitiors: Sorafenib,



SEQ
Pathogenic/Variant
V654A, T670I, D820A,
Dasatinib, Sunitinib,




of Unknown
D820E, D820G,
Nilotinib, Imatinib,




Significance
D820Y, N822H,
Regorafenib, Vatalanib,





N822K, Y823D,
Masitinib, Pazopanib





D816A, D816G,






D816H, D816V, A829P



c-KIT
Next Gen
Pathogenic/Presumed
V654A, T6701, D820A,
KIT inhibitiors: Sorafenib,



SEQ
Pathogenic/Variant
D820E, D820G,
Dasatinib, Sunitinib,




of Unknown
D820Y, N822H,
Nilotinib, Regorafenib,




Significance
N822K, Y823D,
Vatalanib, Masitinib,





D816A, D816G,
Pazopanib





D816H, D816V, A829P



PDGFRA
Next Gen
Pathogenic/Presumed
all mutations except
PDGFRA inhibitors:



SEQ
Pathogenic/Variant
D842V
Sorafenib, Dasatinib,




of Unknown

Sunitinib, Nilotinib,




Significance

Imatinib, Crenolanib (CP






868-956), Masitinib,






Pazopanib


PDGFRA
Next Gen
Pathogenic/Presumed
D842V
PDGFRA inhibitors:



SEQ
Pathogenic/Variant

Sorafenib, Dasatinib,




of Unknown

Sunitinib, Nilotinib,




Significance

Crenolanib (CP 868-956),






Masitinib, Pazopanib


ABL1
Next Gen
Pathogenic/Presumed
T315I
PI3K/Akt/mTor



SEQ
Pathogenic/Variant

inhibitors:Temsirolimus,




of Unknown

Everolimus, CC-223,




Significance

Ridaforolimus, sirolimus,






MLN0128, GDC0941,






Deforolimus, BEZ235, DS-






7423, GDC-0980, PF-






04691502, PF-05212384,






SAR245409, BKM120,






BYL719, PX-866, GDC-






0068, MK2206,






GSK2131795, GSK2110183,






GSK2141795,






XL147(SAR245408),






INK1117, AZD5363,






Perifosine, ARQ092,






AZD8055, OSI-027,






BAY80-6946






SMO antagonists: GDC-






0449, LDE225, BMS833923


ABL1
Next Gen
Pathogenic/Presumed
all mutations except
PI3K/Akt/mTor



SEQ
Pathogenic/Variant
T315I
inhibitors:Temsirolimus,




of Unknown

Everolimus, CC-223,




Significance

Ridaforolimus, sirolimus,






MLN0128, GDC0941,






Deforolimus, BEZ235, DS-






7423, GDC-0980, PF-






04691502, PF-05212384,






SAR245409, BKM120,






BYL719, PX-866, GDC-






0068, MK2206,






GSK2131795, GSK2110183,






GSK2141795, XL147






(SAR245408), INK1117,






AZD5363, Perifosine,






ARQ092, AZD8055, OSI-






027, BAY80-6946






SMO antagonists: GDC-






0449, LDE225, BMS833923






BCR-ABL inhibitors:






nilotinib, dasatinib,






ponatinib, bosutinib


cMET
Next Gen
Pathogenic/Presumed

anti-HGF monoclonal



SEQ
Pathogenic/Variant

antibody (Ficlatuzumab,




of Unknown

Rilotumumab, TAK-701)




Significance

cMET-targeted inhibitors






(AMG-208, BMS-777607,






Compound 1 (Amgen), EMD






1214063/EMD 1204831,






INC280, JNJ38877605,






Onartuzumab (MetMAb),






MK-2461, MK-8033, NK4,






PF4217903, PHA665752,






SGX126, Tivantinib (ARQ






197), cabozantinib,






crizotinib, foretenib,






MGCD265)


FGFR1
Next Gen
Pathogenic/Presumed

Small molecule tyrosine



SEQ
Pathogenic/Variant

kinase inhibitors (TKI258,




of Unknown

BIBF1120, BMS-




Significance

582,664(Brivanib), E7080,






TSU-68, AZD4547,






Dovitinib, E-3810, BGJ398,






TKI258, FP-1039, Ponatinib,






JNJ-42756493)






FGFR antibodies and FGF






ligand traps: (1A6, FP-






1039)


FGFR2
Next Gen
Pathogenic/Presumed

Small molecule tyrosine



SEQ
Pathogenic/Variant

kinase inhibitors (TKI258,




of Unknown

BIBF1120, BMS-




Significance

582,664(Brivanib), E7080,






TSU-68, AZD4547,






Dovitinib, E-3810, BGJ398,






TKI258, FP-1039, Ponatinib,






JNJ-42756493)






FGFR antibodies and FGF






ligand traps: (1A6, FP-






1039)


RET
Next Gen
Pathogenic/Presumed

RET inhibitors (Sorafenib,



SEQ
Pathogenic/Variant

sunitinib, motesanib,




of Unknown

cabozantinib, vandetanib,




Significance

lenvatinib)


CDH1
Next Gen
Pathogenic/Presumed

no drugs



SEQ
Pathogenic/Variant






of Unknown






Significance




STK11
Next Gen
Pathogenic/Presumed

no drugs



SEQ
Pathogenic/Variant






of Unknown






Significance




ERBB4
Next Gen
Pathogenic/Presumed

no drugs



SEQ
Pathogenic/Variant






of Unknown






Significance




SMARCB1
Next Gen
Pathogenic/Presumed

no drugs



SEQ
Pathogenic/Variant






of Unknown






Significance




PIK3CA
Next Gen
Pathogenic/Presumed

PI3K/Akt/mTor



SEQ
Pathogenic/Variant

inhibitors:Temsirolimus,




of Unknown

Everolimus, CC-223,




Significance

Ridaforolimus, sirolimus,






MLN0128, GDC0941,






Deforolimus, BEZ235, DS-






7423, GDC-0980, PF-






04691502, PF-05212384,






SAR245409, BKM120,






BYL719, PX-866, GDC-






0068, MK2206,






GSK2131795, GSK2110183,






GSK2141795, XL147






(SAR245408), INK1117,






AZD5363, Perifosine,






ARQ092, AZD8055, OSI-






027, BAY80-6946






Aspirin: aspirin


PTEN
Next Gen
Pathogenic/Presumed

PI3K/Akt/mTor



SEQ
Pathogenic/Variant

inhibitors:Temsirolimus,




of Unknown

Everolimus, CC-223,




Significance

Ridaforolimus, sirolimus,






MLN0128, GDC0941,






Deforolimus, BEZ235, DS-






7423, GDC-0980, PF-






04691502, PF-05212384,






SAR245409, BKM120,






BYL719, PX-866, GDC-






0068, MK2206,






GSK2131795, GSK2110183,






GSK2141795, XL147






(SAR245408), INK1117,






AZD5363, Perifosine,






ARQ092, AZD8055, OSI-






027, BAY80-6946






Parp inhibitors: ABT-767,






CEP9722, E7016, iniparib,






MK4827, olaparib, rucaparib,






veliparib, ABT-888


AKT1
Next Gen
Pathogenic/Presumed

Akt inhibitors: AZD5363,



SEQ
Pathogenic/Variant

GDC-0068, MK2206,




of Unknown

Perifosine, ARQ092




Significance




ALK
Next Gen
Pathogenic/Presumed

ALK inhibitors: crizotinib,



SEQ
Pathogenic/Variant

AP26113, X-396,




of Unknown

CH5424802(AF-802),




Significance

ASP3026, CEP-28122, CEP-






37440, LDK378


SMO
Next Gen
Pathogenic/Presumed

SMO inhibitors:



SEQ
Pathogenic/Variant

Vismodegib, Erismodegib




of Unknown

(LDE255), IPI-926, BMS-




Significance

838923, PF-04449913,






LEQ506, TAK441,






LY2940680.


KRAS
Next Gen
Pathogenic/Presumed

MEK inhibitors: AZD8330,



SEQ
Pathogenic/Variant

BAY86-9766, CI-1040,




of Unknown

GDC-0623, GDC-0973,




Significance

MEK162, MSC1936369B,






MSC2015103B, PD0325901,






pimasertib (AS-703026),






selumetinib, TAK-733,






trametinib, XL518, ARRY-






438162






ERK inhibitors:






LY2228820, LY3007113,






BVD-523, BAY86-9766,






ARRY-614






Regorafenib: regorafenib


NRAS
Next Gen
Pathogenic/Presumed

MEK inhibitors: AZD8330,



SEQ
Pathogenic/Variant

BAY86-9766, CI-1040,




of Unknown

GDC-0623, GDC-0973,




Significance

MEK162, MSC1936369B,






MSC2015103B, PD0325901,






pimasertib (AS-703026),






selumetinib, TAK-733,






trametinib, XL518, ARRY-






438162






ERK inhibitors:






LY2228820, LY3007113,






BVD-523, BAY86-9766,






ARRY-614


HRAS
Next Gen
Pathogenic/Presumed

MEK inhibitors: AZD8330,



SEQ
Pathogenic/Variant

BAY86-9766, CI-1040,




of Unknown

GDC-0623, GDC-0973,




Significance

MEK162, MSC1936369B,






MSC2015103B, PD0325901,






pimasertib (AS-703026),






selumetinib, TAK-733,






trametinib, XL518, ARRY-






438162






ERK inhibitors:






LY2228820, LY3007113,






BVD-523, BAY86-9766,






ARRY-614


SMAD4
Next Gen
Pathogenic/Presumed

no drugs



SEQ
Pathogenic/Variant






of Unknown






Significance




IDH1
Next Gen
Pathogenic/Presumed

Alkylating agents:



SEQ
Pathogenic/Variant

temozolomide, dacarbazine




of Unknown

Hypomethylating agents:




Significance

azacitidine, decitabine


JAK2
Next Gen
Pathogenic/Presumed

JAK2 inhibitors:



SEQ
Pathogenic/Variant

ruxolitinib, tg101348




of Unknown

(panolosetron), CEP-701




Significance

(lestaurtinib), NS-018,






LY278544


MPL
Next Gen
Pathogenic/Presumed

JAK2 inhibitors:



SEQ
Pathogenic/Variant

ruxolitinib, tg101348




of Unknown

(panolosetron), CEP-701




Significance

(lestaurtinib), NS-018,






LY278544


FLT3
Next Gen
Pathogenic/Presumed

FLT3 inhibitors: CEP-701



SEQ
Pathogenic/Variant

(lestaurtinib), sunitinib,




of Unknown

MLN518 (tandutinib),




Significance

PKC412 (midostaurin)


NPM1
Next Gen
Pathogenic/Presumed

no drugs



SEQ
Pathogenic/Variant






of Unknown






Significance




APC
Next Gen
Pathogenic/Presumed

Wnt pathway inhibitors:



SEQ
Pathogenic/Variant

PRI-724




of Unknown






Significance




CTNNB1
Next Gen
Pathogenic/Presumed

Wnt pathway inhibitors:



SEQ
Pathogenic/Variant

PRI-724




of Unknown






Significance




FBXW7
Next Gen
Pathogenic/Presumed

no drugs



SEQ
Pathogenic/Variant






of Unknown






Significance




BRAF
Next Gen
Pathogenic/Presumed

BRAF inhibitors: sorafenib,



SEQ
Pathogenic/Variant

vemurafenib, RAF-265,




of Unknown

XL281, LGX818,




Significance

GSK2118436 (dabrafenib),






ARQ736, R05212054






MEK inhibitors: AZD8330,






BAY86-9766, CI-1040,






GDC-0623, GDC-0973,






MEK162, MSC1936369B,






MSC2015103B, PD0325901,






pimasertib (AS-703026),






selumetinib, TAK-733,






trametinib, XL518, ARRY-






438162






ERK inhibitors:






LY2228820, LY3007113,






BVD-523, BAY86-9766,






ARRY-614


HNF1A
Next Gen
Pathogenic/Presumed

no drugs



SEQ
Pathogenic/Variant






of Unknown






Significance




EGFR
Next Gen
Pathogenic/Presumed
T790M; exon20 insert
Pan HER inhibitors:



SEQ
Pathogenic/Variant
(A763_Y764insFQEA,
(afatinib, dacomitinib, CO-




of Unknown
A767_D770dup,
1686, XL647, neratinib,




Significance
A767_V769dup,
BMS-690514, Icotinib,





D770delinsGY,
poziotinib)





D770dup,






D770_N771insG,






D770_N771insGF,






D770_N771insGT,






D770_N771insGY,






D770_N771insNPH,






D770_P772delinsKG,






H770dup, H773dup,






H773_V774dup,






H773_V774insAH,






H773_V774insY,






N771delinsGF,






N771delinsGY,






N771delinsKG,






N771delinsRY,






N771_H773delinsTGG,






N771_H773dup,






N771_P772insH,






P772_H773insGNP,






S768_D770dup,






V769_D770dup,






V769_D770insDNP,






V769_D770insGG,






V769_D770insVTW,






Y764_V765insHH)



EGFR
Next Gen
Pathogenic/Presumed
all mutations except
Pan HER inhibitors:



SEQ
Pathogenic/Variant
T790M and exon20
(afatinib, dacomitinib, CO-




of Unknown
insert
1686, XL647, neratinib,




Significance
(A763_Y764insFQEA,
BMS-690514, Icotinib,





A767_D770dup,
poziotinib)





A767_V769dup,
EGFR TKIs: (erlotinib,





D770delinsGY,
gefitinib)





D770dup,






D770_N771insG,






D770_N771insGF,






D770_N771insGT,






D770_N771insGY,






D770_N771insNPH,






D770_P772delinsKG,






H770dup, H773dup,






H773_V774dup,






H773_V774insAH,






H773_V774insY,






N771delinsGF,






N771delinsGY,






N771delinsKG,






N771delinsRY,






N771 _H773delinsTGG,






N771 _H773dup,






N771_P772insH,






P772_H773insGNP,






S768_D770dup,






V769_D770dup,






V769_D770insDNP,






V769_D770insGG,






V769_D770insVTW,






Y764_V765insHH)



EGFR
Next Gen
Present

Pan HER inhibitors:


T790M
SEQ


(afatinib, dacomitinib, CO-






1686, XL647, neratinib,






BMS-690514, Icotinib,






poziotinib)


NOTCH1
Next Gen
Pathogenic/Presumed

HDAC inhibitors: HDAC



SEQ
Pathogenic/Variant

inhibitors (abexinostat,




of Unknown

ACY-1215, AR-42,




Significance

belinostat, CUDC-907,






entinostat, FK228, givinostat,






JNJ26481585, mocetinostat,






panobinostat, SHP-141,






valproic acid, vorinostat,






4SC-202)






GSI: (MK0752, R04929097,






R4733, BMS-906024, PF-






03084014, MEDI0639)


TP53
Next Gen
Pathogenic/Presumed

WEE1 inhibitors: MK-1775



SEQ
Pathogenic/Variant

CHKI inhibitors:




of Unknown

LY2606368, SCH 900776




Significance

Biologicals (gene therapy,






vaccines): rAd-p53, P53-






SLP, Ad5CMV-p53,






adenovirus-p53 transduced






dendritic cell vaccine






(Ad.p53-DC vaccines),






modified vaccinia virus






ankara vaccine expressing






p53, ALT-801






p53 activators: PRIMA


TP53
Next Gen
Wild Type

P53-MDM2 interaction



SEQ


inhibitors: CGM097,






RO5503781, R05045337,






Kevetrin






(thioureidobutyronitrile), DS-






3032







Sanger SEQ











PIK3CA
Sanger
Exon 20; Exon 9;

PI3K/Akt/mTor



SEQ
Mutated—Other

inhibitors:Temsirolimus,






Everolimus, CC-223,






Ridaforolimus, sirolimus,






MLN0128, GDC0941,






Deforolimus, BEZ235, DS-






7423, GDC-0980, PF-






04691502, PF-05212384,






SAR245409, BKM120,






BYL719, PX-866, GDC-






0068, MK2206,






GSK2131795, GSK2110183,






GSK2141795, XL147






(SAR245408), INK1117,






AZD5363, Perifosine,






ARQ092, AZD8055, OSI-






027, BAY80-6946






Aspirin: aspirin


KRAS
Sanger
G12, G13; G13D;

MEK inhibitors: AZD8330,



SEQ
Q61;

BAY86-9766, CI-1040,




Mutated—Other

GDC-0623, GDC-0973,






MEK162, MSC1936369B,






MSC2015103B, PD0325901,






pimasertib (AS-703026),






selumetinib, TAK-733,






trametinib, XL518, ARRY-






438162






ERK inhibitors:






LY2228820, LY3007113,






BVD-523, BAY86-9766,






ARRY-614






Regorafenib: regorafenib


KRAS
Sanger
Present

MEK inhibitors: AZD8330,


G13D
SEQ


BAY86-9766, CI-1040,






GDC-0623, GDC-0973,






MEK162, MSC1936369B,






MSC2015103B, PD0325901,






pimasertib (AS-703026),






selumetinib, TAK-733,






trametinib, XL518, ARRY-






438162






ERK inhibitors:






LY2228820, LY3007113,






BVD-523, BAY86-9766,






ARRY-614






Regorafenib: regorafenib


NRAS
Sanger
G12, G13; Q61;

MEK inhibitors: AZD8330,



SEQ
Mutated—Other

BAY86-9766, CI-1040,






GDC-0623, GDC-0973,






MEK162, MSC1936369B,






MSC2015103B, PD0325901,






pimasertib (AS-703026),






selumetinib, TAK-733,






trametinib, XL518, ARRY-






438162






ERK inhibitors:






LY2228820, LY3007113,






BVD-523, BAY86-9766,






ARRY-614


BRAF
Sanger
V600D; V600E;

BRAF inhibitors: sorafenib,



SEQ
V600K; V600R;

vemurafenib, RAF-265,




Exonl 1;

XL281, LGX818,




Mutated—Other;

GSK2118436 (dabrafenib),




SEQ-MUT/PCR-

ARQ736, R05212054




WT; SEQ-WT/

MEK inhibitors: AZD8330,




PCR-MUT;

BAY86-9766, CI-1040,






GDC-0623, GDC-0973,






MEK162, MSC1936369B,






MSC2015103B, PD0325901,






pimasertib (AS-703026),






selumetinib, TAK-733,






trametinib, XL518, ARRY-






438162






ERK inhibitors:






LY2228820, LY3007113,






BVD-523, BAY86-9766,






ARRY-614


EGFR
Sanger
Exon 18 G719A;

EGFR TKIs: (erlotinib,



SEQ and
Exon 19 del; Exon

gefitinib)



RFLP
20 R776; Exon 21

Pan HER inhibitors:




L858R; Exon 21

(afatinib, dacomitinib, CO-




L861;

1686, XL647, neratinib,






BMS-690514, Icotinib,






poziotinib)


EGFR
Sanger
Present

Pan HER inhibitors:


T790M
SEQ and


(afatinib, dacomitinib, CO-



RFLP


1686, XL647, neratinib,






BMS-690514, Icotinib,






poziotinib)


EGFR
Sanger
Present

Pan HER inhibitors:


Exon 20
SEQ and


(afatinib, dacomitinib, CO-


ins
RFLP


1686, XL647, neratinib,






BMS-690514, Icotinib,






poziotinib)


IDH2
Sanger
Mutated-Other,

Alkylating agents:



SEQ
R140, R172

temozolomide, dacarbazine






Hypomethylating agents:






azacitidine, decitabine







IHC Tests











Her2/Neu
IHC
Positive

anti-HER2 monoclonal






antibody (pertuzumab,






trastuzumab)






HER2-targeted tyrosine






kinase inhibitors (afatinib,






dacomitinib, lapatinib,






neratinib)






anti-HER2 monoclonal






antibody—drug conjugate






(ado-trastuzumab emtansine






(T-DM1))


cMET
IHC
Positive

anti-HGF monoclonal






antibody (Ficlatuzumab,






Rilotumumab, TAK-701)






cMET-targeted inhibitors






(AMG-208, BMS-777607,






Compound 1 (Amgen), EMD






1214063/EMD 1204831,






INC280, JNJ38877605,






Onartuzumab (MetMAb),






MK-2461, MK-8033, NK4,






PF4217903, PHA665752,






SGX126, Tivantinib (ARQ






197), cabozantinib,






crizotinib, foretenib,






MGCD265)






cMET antibody: ABT-700


PTEN
IHC
Negative

PI3K/Akt/mTor






inhibitors:Temsirolimus,






Everolimus, CC-223,






Ridaforolimus, sirolimus,






MLN0128, GDC0941,






Deforolimus, BEZ235, DS-






7423, GDC-0980, PF-






04691502, PF-05212384,






SAR245409, BKM120,






BYL719, PX-866, GDC-






0068, MK2206,






G5K2131795, GSK2110183,






GSK2141795, XL147






(SAR245408), INK1117,






AZD5363, Perifosine,






ARQ092, AZD8055, OSI-






027, BAY80-6946






Parp inhibitors: ABT-767,






CEP9722, E7016, iniparib,






MK4827, olaparib, rucaparib,






veliparib, ABT-888


Androgen
IHC
positive

Anti androgens:


Receptor



(Bicalutamide, flutamide,






abiraterone, enzalutamide,






TAK-700, ARN-509) GnRH






agonists/antagonists:






(goserelin, leuprolide,






degarelix, abarelix);


EGFR
IHC
Positive

EGFR monoclonal






antibody: cetuximab,






nimotuzumab







CISH/FISH Tests











Her2/Neu
CISH/FISH
Amplified

anti-HER2 monoclonal






antibody (pertuzumab,






trastuzumab)






HER2-targeted tyrosine






kinase inhibitors (afatinib,






dacomitinib, lapatinib,






neratinib)






anti-HER2 monoclonal






antibody—drug conjugate






(ado-trastuzumab emtansine






(T-DMI))


cMET
CISH/FISH
Amplified

anti-HGF monoclonal






antibody (Ficlatuzumab,






Rilotumumab, TAK-701)






cMET-targeted inhibitors






(AMG-208, BMS-777607,






Compound 1 (Amgen), EMD






1214063/EMD 1204831,






INC280, JNJ38877605,






Onartuzumab (MetMAb),






MK-2461, MK-8033, NK4,






PF4217903, PHA665752,






SGX126, Tivantinib (ARQ






197), cabozantinib,






crizotinib, foretenib,






MGCD265)






cMET antibody: ABT-700


ALK
FISH
Positive

ALK inhibitors: crizotinib,






AP26113, X-396,






CH5424802(AF-802),






ASP3026, CEP-28122, CEP-






37440, LDK378






HSP90 inhibitors: AUY922,






Ganetespib, 17-AGG







Cobas PCR











BRAF
Cobas PCR
V600E

BRAF inhibitors: sorafenib,



(qPCR)


vemurafenib, RAF-265,






XL281, LGX818,






GSK2118436 (dabrafenib),






ARQ736, R05212054






MEK inhibitors: AZD8330,






BAY86-9766, CI-1040,






GDC-0623, GDC-0973,






MEK162, MSC1936369B,






MSC2015103B, PD0325901,






pimasertib (AS-703026),






selumetinib, TAK-733,






trametinib, XL518, ARRY-






438162






ERK inhibitors:






LY2228820, LY3007113,






BVD-523, BAY86-9766,






ARRY-614







Fragment Analysis











EGFRvIII
Fragment
present

EGFRvIII targeted peptide



Analysis


vaccine: rindopepimut






(CDX-110; PEP-3-KLH )






EGFRvIII targeted






antibodies and antibody






conjugates: ABT-806






(mAb806), ABT-414, AMG






595






EGFR TKIs:erlotinib,






gefitinib






Pan HER inhibitors:






afatinib, dacomitinib, CO-






1686, XL647, neratinib,






BMS-690514, Icotinib,






poziotinib









As noted herein, the status of various biomarkers assessed by molecular profiling of the invention can be used to match a patient with a given biomarker status to a clinical trial. Table 29 provides illustrative rules that can be followed to match various clinical trials. In the table, the column headed “NCT Number” provides a unique identifier for each trial ongoing in the United States at clinicaltrials.gov. Every study in ClinicalTrials.gov is assigned a unique number called the ClinicalTrials.gov Identifier (NCT Number). For example, a trial can be accessed on the internet by following the URL convention “clinicaltrials.gov/show/[NCT Number]” where “[NCT Number]” is replaced by the NCT Number of the trial, such as indicated in the table. The column “Tumor Type” indicates what lineages or lineages of tumor are being studied in the clinical trial. The column “Drug” indicates what drug or drugs are being studied in the clinical trial. The column “Biomarker” indicates the biomarkers that are considered by the trial. The various columns “Test” and “Result” indicate the test results for the one or more biomarkers whose status is used to determine eligibility for the clinical trial. If the test results are matched in a given row in Table 29, then the patient is indicated as a candidate for a clinical trial. By way of non-limiting example, consider the first row in Table 29 underneath the headers. A colorectal adenocarcinoma is assessed according to the systems and methods of the invention. A V600E or V600K mutation in BRAF is identified by sequencing. The patient may be eligible for inclusion in the clinical trial identified by the NCT Number NCT00326495, which trial is studying the drug sorafenib. The same patient may also be eligible for the clinical trial identified by the NCT Number NCT00625378, which trial is studying the drug sorafenib in all cancer lineages having a V600E or V600K mutation in BRAF. Similar logic is followed when multiple biomarkers are implicated in the rule set in Table 29. In such cases, all of the biomarkers and test results thereof must be matched in order to suggest the patient as eligible for enrollment in the clinical trial.


Abbreviations in Table 29 are as found elsewhere herein, e.g.: Seq. (sequencing); BAC (bronchioloalveolar cancer); NSCLC (non-small cell lung cancer); IHC (immunohistochemistry); ISH (in situ hybridization).


The rule set in Table 29 can be updated, as new trials linking various biomarkers to enrollment eligibility become available. If the biomarkers are not already assessed as part of the molecular profiles provided by the invention, then the biomarkers can be added to the molecular profiles.









TABLE 29







Rules for Clinical Trial Selection based on Biomarker Assessment


















NCT Number
Tumor Type
Drug
Biomarker
Test
Result
Test
Result
Test
Result
Test
Result





NCT00326495
Colorectal
sorafenib
BRAF
Seq. BRAF
Mutated|









Adenocarcinoma



V600E|







V600K


NCT00326495
Colorectal
sorafenib
BRAF
PCR BRAF
V600E



Adenocarcinoma


NCT00574769
Prostatic
everolimus
PIK3CA,
Seq.
Mutated|



Adenocarcinoma

PTEN
PIK3CA
exon20


NCT00574769
Prostatic
everolimus
PIK3CA,
Seq. PTEN
Mutated



Adenocarcinoma

PTEN


NCT00574769
Prostatic
bevacizumab
VHL
Seq. VHL
Mutated



adenocarcinoma


NCT00585195
All
crizotinib
ALK
ISH ALK
Positive


NCT00585195
All
crizotinib
ROS1
ISH ROS1
Positive


NCT00610948
All
everolimus
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT00610948
All
everolimus
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT00622466
Breast Carcinoma
sorafenib
VHL
Seq. VHL
Mutated


NCT00625378
All
sorafenib
BRAF
Seq. BRAF
Mutated|







V600E|







V600K


NCT00625378
All
sorafenib
BRAF
PCR BRAF
V600E


NCT00756340
All
everolimus
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT00756340
All
everolimus
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT00770263
All
temsirolimus
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT00770263
All
temsirolimus
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT00780494
Gastric
bevacizumab
VHL
Seq. VHL
Mutated



adenocarcinoma,



Esophageal and



Esophagogastric



Junction Carcinoma


NCT00912340
Breast Carcinoma
everolimus
ER, PR,
IHC ER
Positive
IHC Her2
No Data
ISH
No Data





Her2




Her2


NCT00912340
Breast Carcinoma
everolimus
ER, PR,
IHC ER
Positive
IHC Her2
No Data
ISH
Equiv. Low





Her2




Her2


NCT00912340
Breast Carcinoma
everolimus
ER, PR,
IHC ER
Positive
IHC Her2
No Data
ISH
Not





Her2




Her2
Amplified


NCT00912340
Breast Carcinoma
everolimus
ER, PR,
IHC ER
Positive
IHC Her2
Equiv.
ISH
No Data





Her2




Her2


NCT00912340
Breast Carcinoma
everolimus
ER, PR,
IHC ER
Positive
IHC Her2
Equiv.
ISH
Equiv. Low





Her2




Her2


NCT00912340
Breast Carcinoma
everolimus
ER, PR,
IHC ER
Positive
IHC Her2
Equiv.
ISH
Not





Her2




Her2
Amplified


NCT00912340
Breast Carcinoma
everolimus
ER, PR,
IHC ER
Positive
IHC Her2
Negative
ISH
No Data





Her2




Her2


NCT00912340
Breast Carcinoma
everolimus
ER, PR,
IHC ER
Positive
IHC Her2
Negative
ISH
Equiv. Low





Her2




Her2


NCT00912340
Breast Carcinoma
everolimus
ER, PR,
IHC ER
Positive
IHC Her2
Negative
ISH
Not





Her2




Her2
Amplified


NCT00912340
Breast Carcinoma
everolimus
ER, PR,
IHC PR
Positive
IHC Her2
No Data
ISH
No Data





Her2




Her2


NCT00912340
Breast Carcinoma
everolimus
ER, PR,
IHC PR
Positive
IHC Her2
No Data
ISH
Equiv. Low





Her2




Her2


NCT00912340
Breast Carcinoma
everolimus
ER, PR,
IHC PR
Positive
IHC Her2
No Data
ISH
Not





Her2




Her2
Amplified


NCT00912340
Breast Carcinoma
everolimus
ER, PR,
IHC PR
Positive
IHC Her2
Equiv.
ISH
No Data





Her2




Her2


NCT00912340
Breast Carcinoma
everolimus
ER, PR,
IHC PR
Positive
IHC Her2
Equiv.
ISH
Equiv. Low





Her2




Her2


NCT00912340
Breast Carcinoma
everolimus
ER, PR,
IHC PR
Positive
IHC Her2
Equiv.
ISH
Not





Her2




Her2
Amplified


NCT00912340
Breast Carcinoma
everolimus
ER, PR,
IHC PR
Positive
IHC Her2
Negative
ISH
No Data





Her2




Her2


NCT00912340
Breast Carcinoma
everolimus
ER, PR,
IHC PR
Positive
IHC Her2
Negative
ISH
Not





Her2




Her2
Amplified


NCT00912340
Breast Carcinoma
everolimus
ER, PR,
IHC PR
Positive
IHC Her2
Negative
ISH
Negative





Her2




Her2


NCT00934895
Breast Carcinoma
everolimus
Her2
IHC Her2
No Data
ISH Her2
Equiv. Low


NCT00934895
Breast Carcinoma
everolimus
Her2
IHC Her2
No Data
ISH Her2
Negative


NCT00934895
Breast Carcinoma
everolimus
Her2
IHC Her2
Equiv.
ISH Her2
Equiv. Low


NCT00934895
Breast Carcinoma
everolimus
Her2
IHC Her2
Equiv.
ISH Her2
Negative


NCT00934895
Breast Carcinoma
everolimus
Her2
IHC Her2
Negative
ISH Her2
No Data


NCT00934895
Breast Carcinoma
everolimus
Her2
IHC Her2
Negative
ISH Her2
Equiv. Low


NCT00934895
Breast Carcinoma
everolimus
Her2
IHC Her2
Negative
ISH Her2
Negative


NCT00936858
Thyroid Carcinoma
everolimus
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT00936858
Thyroid Carcinoma
everolimus
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT00976573
Melanoma
everolimus
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT00976573
Melanoma
everolimus
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01006369
Colorectal
bevacizumab
VHL
Seq. VHL
Mutated



Adenocarcinoma


NCT01014936
All
EMD 1214063
cMET
ISH CMET
Amplified


NCT01014936
All
EMD 1214063
cMET
IHC CMET
Positive


NCT01031381
Ovarian Surface
everolimus
PIK3CA,
Seq.
Mutated|



Epithelial

PTEN
PIK3CA
exon20



Carcinomas


NCT01031381
Ovarian Surface
everolimus
PIK3CA,
Seq. PTEN
Mutated



Epithelial

PTEN



Carcinomas


NCT01047293
Colorectal
bevacizumab
VHL
Seq. VHL
Mutated



Adenocarcinoma


NCT01061788
All
everolimus
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01061788
All
everolimus
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01087554
All
sirolimus
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01087554
All
sirolimus
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01087983
All
sirolimus,
PIK3CA,
Seq.
Mutated|




metformin
PTEN
PIK3CA
exon20


NCT01087983
All
sirolimus,
PIK3CA,
Seq. PTEN
Mutated




metformin
PTEN


NCT01089101
Glioblastoma
AZD6244
KRAS,
Seq. KRAS
Mutated|





NRAS,

G13D





BRAF


NCT01089101
Glioblastoma
AZD6244
KRAS,
Seq. BRAF
Mutated|





NRAS,

V600E|





BRAF

V600K


NCT01089101
Glioblastoma
AZD6244
KRAS,
Seq. NRAS
Mutated





NRAS,





BRAF


NCT01111825
Breast Carcinoma
temsirolimus
Her2
IHC Her2
Positive


NCT01111825
Breast Carcinoma
temsirolimus
Her2
ISH Her2
Amplified


NCT01111825
Breast Carcinoma
temsirolimus
Her2
ISH Her2
Equiv.







High


NCT01111825
Breast Carcinoma
temsirolimus
ER, PR,
IHC ER
Negative
IHC PR
Negative
IHC
No Data
ISH Her2
Equiv. Low





Her2




Her2


NCT01111825
Breast Carcinoma
temsirolimus
ER, PR,
IHC ER
Negative
IHC PR
Negative
IHC
No Data
ISH Her2
Negative





Her2




Her2


NCT01111825
Breast Carcinoma
temsirolimus
ER, PR,
IHC ER
Negative
IHC PR
Negative
IHC
Equiv.
ISH Her2
Equiv. Low





Her2




Her2


NCT01111825
Breast Carcinoma
temsirolimus
ER, PR,
IHC ER
Negative
IHC PR
Negative
IHC
Equiv.
ISH Her2
Negative





Her2




Her2


NCT01111825
Breast Carcinoma
temsirolimus
ER, PR,
IHC ER
Negative
IHC PR
Negative
IHC
Negative
ISH Her2
No Data





Her2




Her2


NCT01111825
Breast Carcinoma
temsirolimus
ER, PR,
IHC ER
Negative
IHC PR
Negative
IHC
Negative
ISH Her2
Equiv. Low





Her2




Her2


NCT01111825
Breast Carcinoma
temsirolimus
ER, PR,
IHC ER
Negative
IHC PR
Negative
IHC
Negative
ISH Her2
Negative





Her2




Her2


NCT01121575
BAC, NSCLC
crizotinib
cMET
ISH CMET
Amplified


NCT01121575
BAC, NSCLC
crizotinib
cMET
IHC CMET
Positive


NCT01122199
All
everolimus
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01122199
All
everolimus
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01132664
Breast carcinoma
BKM120
PIK3CA,
Seq.
Mutated|
IHC
Positive





PTEN
PIK3CA
exon20
HER2/Neu


NCT01132664
Breast carcinoma
BKM120
PIK3CA,
Seq.
Mutated|
ISH
Amplified|





PTEN
PIK3CA
exon20
HER2/Neu
Equiv.









High


NCT01132664
Breast carcinoma
BKM120
PIK3CA,
IHC PTEN
Negative
IHC
Positive





PTEN


HER2/Neu


NCT01132664
Breast carcinoma
BKM120
PIK3CA,
IHC PTEN
Negative
ISH
Amplified|





PTEN


HER2/Neu
Equiv.









High


NCT01132664
Breast carcinoma
BKM120
PIK3CA,
Seq. PTEN
Mutated
IHC
Positive





PTEN


HER2/Neu


NCT01132664
Breast carcinoma
BKM120
PIK3CA,
Seq. PTEN
Mutated
ISH
Amplified|





PTEN


HER2/Neu
Equiv.









High


NCT01141244
All
temsirolimus
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01141244
All
temsirolimus
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01143402
Uveal Melanoma
AZD6244
GNAQ,
Seq.
Mutated





GNA11
GNAQ


NCT01143402
Uveal Melanoma
AZD6244
GNAQ,
Seq.
Mutated





GNA11
GNA11


NCT01148849
Breast carcinoma,
MGAH22
Her2
ISH
Amplified|



Gastric


Her2/Neu
Equiv.



adenocarcinoma,



High



Non epithelial ovarian



cancer (non-EOC),



Ovarian surface epithelial



carcinomas, BAC, NSCLC


NCT01148849
Breast carcinoma,
MGAH22
Her2
IHC
Positive



Gastric


Her2/Neu



adenocarcinoma,



Non epithelial ovarian



cancer (non-EOC),



Ovarian surface epithelial



carcinomas, BAC, NSCLC


NCT01158651
Glioblastoma
everolimus
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01158651
Glioblastoma
everolimus
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01174199
Prostatic
temsirolimus
PIK3CA,
Seq.
Mutated|



Adenocarcinoma

PTEN
PIK3CA
exon20


NCT01174199
Prostatic
temsirolimus
PIK3CA,
Seq. PTEN
Mutated



Adenocarcinoma

PTEN


NCT01182168
All
everolimus
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01182168
All
everolimus
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01187199
All
temsirolimus
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01187199
All
temsirolimus
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01191697
Gastric
bevacizumab
VHL
Seq. VHL
Mutated



adenocarcinoma,



Esophageal and



Esophagogastric



Junction Carcinoma


NCT01191697
Gastric
bevacizumab
VHL
Seq. VHL
Mutated
IHC Her2
Positive



adenocarcinoma,



Esophageal and



Esophagogastric



Junction Carcinoma


NCT01191697
Gastric
bevacizumab
VHL
Seq. VHL
Mutated
ISH
Amplified|



adenocarcinoma,




HER2/Neu
Equiv.



Esophageal and





High



Esophagogastric



Junction Carcinoma


NCT01194869
Breast carcinoma
sorafenib
VHL
Seq. VHL
Mutated
IHC ER
Negative
IHC PR
Negative
IHC Her2












Negative


NCT01194869
Breast carcinoma
sorafenib
VHL
Seq. VHL
Mutated
IHC ER
Negative
IHC PR
Negative
ISH
Not












HER2/Neu
Amplified|













Equiv. Low


NCT01195922
Head and neck
rapamycin
PIK3CA,
Seq.
Mutated|



Squamous

PTEN
PIK3CA
exon20



Carcinoma


NCT01195922
Head and neck
rapamycin
PIK3CA,
Seq. PTEN
Mutated



Squamous

PTEN



Carcinoma


NCT01196429
Ovarian Surface
temsirolimus
PIK3CA,
Seq.
Mutated|



Epithelial

PTEN
PIK3CA
exon20



Carcinomas


NCT01196429
Ovarian Surface
temsirolimus
PIK3CA,
Seq. PTEN
Mutated



Epithelial

PTEN



Carcinomas


NCT01204099
BAC, NSCLC,
PX-866
PIK3CA,
Seq.
Mutated|



Head and neck

PTEN
PIK3CA
exon20



Squamous



Carcinoma


NCT01204099
BAC, NSCLC,
PX-866
PIK3CA,
IHC PTEN
Negative



Head and neck

PTEN



Squamous



Carcinoma


NCT01204099
BAC, NSCLC,
PX-866
PIK3CA,
Seq. PTEN
Mutated



Head and neck

PTEN



Squamous



Carcinoma


NCT01206530
Colorectal
bevacizumab
VHL
Seq. VHL
Mutated



Adenocarcinoma


NCT01212822
Gastric
bevacizumab
VHL
Seq. VHL
Mutated



adenocarcinoma,



Esophageal and



Esophagogastric



Junction Carcinoma


NCT01218555
All
everolimus
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01218555
All
everolimus
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01219699
All
BYL719
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01219699
All
BYL719
PIK3CA,
IHC PTEN
Negative





PTEN


NCT01219699
All
BYL719
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01222715
Soft tissue tumors
temsirolimus
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01222715
Soft tissue tumors
temsirolimus
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01231399
Gastric
everolimus
PIK3CA,
Seq.
Mutated|



Adenocarcinoma,

PTEN
PIK3CA
exon20



Esophageal and



Esophagogastric



Junction Carcinoma


NCT01231399
Gastric
everolimus
PIK3CA,
Seq. PTEN
Mutated



Adenocarcinoma,

PTEN



Esophageal and



Esophagogastric



Junction Carcinoma


NCT01231594
All
GSK2118436
BRAF
Seq. BRAF
Mutated|







V600E|







V600K


NCT01231594
All
GSK2118436
BRAF
PCR BRAF
V600E


NCT01235897
All
MK2206
PIK3CA,
Seq.
Mutated|
IHC
Positive





PTEN
PIK3CA
exon20
HER2/Neu


NCT01235897
All
MK2206
PIK3CA,
IHC PTEN
Negative
IHC
Positive





PTEN


HER2/Neu


NCT01235897
All
MK2206
PIK3CA,
Seq.
Mutated|
ISH
Amplified|





PTEN
PIK3CA
exon20
HER2/Neu
Equiv.









High


NCT01235897
All
MK2206
PIK3CA,
IHC PTEN
Negative
ISH
Amplified|





PTEN


HER2/Neu
Equiv.









High


NCT01235897
All
MK2206
PIK3CA,
Seq. PTEN
Mutated
IHC
Positive





PTEN


HER2/Neu


NCT01235897
All
MK2206
PIK3CA,
Seq. PTEN
Mutated
ISH
Amplified|





PTEN


HER2/Neu
Equiv.









High


NCT01245205
All
MK2206
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01245205
All
MK2206
PIK3CA,
IHC PTEN
Negative





PTEN


NCT01245205
All
MK2206
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01248247
BAC, NSCLC,
AZD6244
KRAS,
Seq. KRAS
Mutated|



Lung Small Cell

NRAS,

G13D



Cancer (SCLC)

BRAF


NCT01248247
BAC, NSCLC,
AZD6244
KRAS,
Seq. BRAF
Mutated|



Lung Small Cell

NRAS,

V600E|



Cancer (SCLC)

BRAF

V600K


NCT01248247
BAC, NSCLC,
AZD6244
KRAS,
Seq. NRAS
Mutated



Lung Small Cell

NRAS,



Cancer (SCLC)

BRAF


NCT01251861
Prostatic
MK2206
PIK3CA,
Seq.
Mutated|



adenocarcinoma

PTEN
PIK3CA
exon20


NCT01251861
Prostatic
MK2206
PIK3CA,
IHC PTEN
Negative



adenocarcinoma

PTEN


NCT01251861
Prostatic
MK2206
PIK3CA,
Seq. PTEN
Mutated



adenocarcinoma

PTEN


NCT01252251
Uveal Melanoma
everolimus
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01252251
Uveal Melanoma
everolimus
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01256268
Female Genital
ridaforolimus
PIK3CA,
Seq. PTEN
Mutated



Tract Malignancy,

PTEN



Ovarian Surface



Epithelial



Carcinomas


NCT01256268
Female Genital
ridaforolimus
PIK3CA,
Seq.
Mutated|



Tract Malignancy,

PTEN
PIK3CA
exon20



Ovarian Surface



Epithelial



Carcinomas


NCT01256385
Head and neck
temsirolimus
PIK3CA,
Seq.
Mutated|



Squamous

PTEN
PIK3CA
exon20



Carcinoma


NCT01256385
Head and neck
temsirolimus
PIK3CA,
Seq. PTEN
Mutated



Squamous

PTEN



Carcinoma


NCT01270321
Thyroid Carcinoma
everolimus
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01270321
Thyroid Carcinoma
everolimus
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01276210
All
sorafenib
BRAF
Seq. BRAF
Mutated|







V600E|







V600K


NCT01276210
All
sorafenib
BRAF
PCR BRAF
V600E


NCT01277757
Breast carcinoma
MK2206
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01277757
Breast carcinoma
MK2206
PIK3CA,
IHC PTEN
Negative





PTEN


NCT01277757
Breast carcinoma
MK2206
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01279681
Colorectal
bevacizumab
VHL
Seq. VHL
Mutated



Adenocarcinoma


NCT01281163
Breast carcinoma
MK2206
PIK3CA,
Seq.
Mutated|
IHC
Positive





PTEN
PIK3CA
exon20
HER2/Neu


NCT01281163
Breast carcinoma
MK2206
PIK3CA,
Seq.
Mutated|
ISH
Amplified|





PTEN
PIK3CA
exon20
Her2/Neu
Equiv.









High


NCT01281163
Breast carcinoma
MK2206
PIK3CA,
IHC PTEN
Negative
IHC
Positive





PTEN


HER2/Neu


NCT01281163
Breast carcinoma
MK2206
PIK3CA,
IHC PTEN
Negative
ISH
Amplified|





PTEN


Her2/Neu
Equiv.









High


NCT01281163
Breast carcinoma
MK2206
PIK3CA,
Seq. PTEN
Mutated
IHC
Positive





PTEN


HER2/Neu


NCT01281163
Breast carcinoma
MK2206
PIK3CA,
Seq. PTEN
Mutated
ISH
Amplified|





PTEN


Her2/Neu
Equiv.









High


NCT01281514
Ovarian Surface
everolimus
PIK3CA,
Seq.
Mutated|



Epithelial

PTEN
PIK3CA
exon20



Carcinomas


NCT01281514
Ovarian Surface
everolimus
PIK3CA,
Seq. PTEN
Mutated



Epithelial

PTEN



Carcinomas


NCT01283789
Breast Carcinoma
everolimus
Her2
IHC Her2
Positive


NCT01283789
Breast Carcinoma
everolimus
Her2
ISH Her2
Amplified


NCT01283789
Breast Carcinoma
everolimus
Her2
ISH Her2
Equiv.







High


NCT01297452
All
BKM120
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01297452
All
BKM120
PIK3CA,
IHC PTEN
Negative





PTEN


NCT01297452
All
BKM120
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01297491
BAC, NSCLC
BKM120
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01297491
BAC, NSCLC
BKM120
PIK3CA,
IHC PTEN
Negative





PTEN


NCT01297491
BAC, NSCLC
BKM120
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01298570
Colorectal
regorafenib
KRAS,
Seq. KRAS
Mutated|



Adenocarcinoma

BRAF

G13D


NCT01298570
Colorectal
regorafenib
KRAS,
Seq. BRAF
Mutated|



Adenocarcinoma

BRAF

V600E|







V600K


NCT01298570
Colorectal
regorafenib
VHL
Seq. VHL
Mutated



Adenocarcinoma


NCT01300429
BAC, NSCLC
crizotinib
ALK
ISH ALK
Positive


NCT01300962
Breast carcinoma
BKM120,
PIK3CA,
Seq.
Mutated|




BEZ235
PTEN
PIK3CA
exon20


NCT01300962
Breast carcinoma
BKM120,
PIK3CA,
IHC PTEN
Negative




BEZ235
PTEN


NCT01300962
Breast carcinoma
BKM120,
PIK3CA,
Seq. PTEN
Mutated




BEZ235
PTEN


NCT01301716
All
GDC-0980
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01301716
All
GDC-0980
PIK3CA,
IHC PTEN
Negative





PTEN


NCT01301716
All
GDC-0980
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01304602
Colorectal
BKM120
PIK3CA,
Seq.
Mutated|



adenocarcinoma

PTEN
PIK3CA
exon20


NCT01304602
Colorectal
BKM120
PIK3CA,
IHC PTEN
Negative



adenocarcinoma

PTEN


NCT01304602
Colorectal
BKM120
PIK3CA,
Seq. PTEN
Mutated



adenocarcinoma

PTEN


NCT01305941
Breast Carcinoma
everolimus
Her2
IHC Her2
Positive


NCT01305941
Breast Carcinoma
everolimus
Her2
ISH Her2
Amplified


NCT01305941
Breast Carcinoma
everolimus
Her2
ISH Her2
Equiv.







High


NCT01306045
BAC, NSCLC,
AZD6244
KRAS,
Seq. KRAS
Mutated|



Thymic Carcinoma,

NRAS,

G13D



Lung Small Cell

BRAF



Cancer (SCLC)


NCT01306045
BAC, NSCLC,
AZD6244
KRAS,
Seq. BRAF
Mutated|



Thymic Carcinoma,

NRAS,

V600E|



Lung Small Cell

BRAF

V600K



Cancer (SCLC)


NCT01306045
BAC, NSCLC,
AZD6244
KRAS,
Seq. NRAS
Mutated



Thymic Carcinoma,

NRAS,



Lung Small Cell

BRAF



Cancer (SCLC)


NCT01307631
Female genital tract
MK2206
PIK3CA,
Seq.
Mutated|



malignancy

PTEN
PIK3CA
exon20


NCT01307631
Female genital tract
MK2206
PIK3CA,
IHC PTEN
Negative



malignancy

PTEN


NCT01307631
Female genital tract
MK2206
PIK3CA,
Seq. PTEN
Mutated



malignancy

PTEN


NCT01313039
Breast Carcinoma
AZD6244
KRAS,
Seq. KRAS
Mutated|
IHC ER
Negative





NRAS,

G13D





BRAF


NCT01313039
Breast Carcinoma
AZD6244
KRAS,
Seq. BRAF
Mutated|
IHC ER
Negative





NRAS,

V600E|





BRAF

V600K


NCT01313039
Breast Carcinoma
AZD6244
KRAS,
Seq. NRAS
Mutated
IHC ER
Negative





NRAS,





BRAF


NCT01319539
Breast Carcinoma
MK2206
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01319539
Breast Carcinoma
MK2206
PIK3CA,
IHC PTEN
Negative





PTEN


NCT01319539
Breast Carcinoma
MK2206
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01320085
Melanoma
MEK162
KRAS,
Seq. KRAS
Mutated|





NRAS,

G13D





BRAF


NCT01320085
Melanoma
MEK162
KRAS,
Seq. BRAF
Mutated|





NRAS,

V600E|





BRAF

V600K


NCT01320085
Melanoma
MEK162
KRAS,
Seq. NRAS
Mutated





NRAS,





BRAF


NCT01322815
Colorectal
bevacizumab
VHL
Seq. VHL
Mutated



Adenocarcinoma


NCT01331135
All
sirolimus
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01331135
All
sirolimus
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01333475
Colorectal
AZD6244
KRAS,
Seq. KRAS
Mutated|



adenocarcinoma

NRAS,

G13D





BRAF


NCT01333475
Colorectal
AZD6244
KRAS,
Seq. BRAF
Mutated|



adenocarcinoma

NRAS,

V600E|





BRAF

V600K


NCT01333475
Colorectal
AZD6244
KRAS,
Seq. NRAS
Mutated



adenocarcinoma

NRAS,





BRAF


NCT01339052
Glioblastoma
BKM120
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01339052
Glioblastoma
BKM120
PIK3CA,
IHC PTEN
Negative





PTEN


NCT01339052
Glioblastoma
BKM120
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01339442
Breast Carcinoma
BKM120
PIK3CA,
Seq.
Mutated|
IHC ER
Positive





PTEN
PIK3CA
exon20


NCT01339442
Breast Carcinoma
BKM120
PIK3CA,
IHC PTEN
Negative
IHC ER
Positive





PTEN


NCT01339442
Breast Carcinoma
BKM120
PIK3CA,
Seq. PTEN
Mutated
IHC ER
Positive





PTEN


NCT01344031
Breast Carcinoma
MK2206
PIK3CA,
Seq.
Mutated|
IHC ER
Positive





PTEN
PIK3CA
exon20


NCT01344031
Breast Carcinoma
MK2206
PIK3CA,
IHC PTEN
Negative
IHC ER
Positive





PTEN


NCT01344031
Breast Carcinoma
MK2206
PIK3CA,
Seq. PTEN
Mutated
IHC ER
Positive





PTEN


NCT01347866
All
PF-04691502,
PIK3CA,
Seq.
Mutated|




PF-05212384
PTEN
PIK3CA
exon20


NCT01347866
All
PF-04691502,
PIK3CA,
IHC PTEN
Negative




PF-05212384
PTEN


NCT01347866
All
PF-04691502,
PIK3CA,
Seq. PTEN
Mutated




PF-05212384
PTEN


NCT01349660
All
BKM120
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01349660
All
BKM120
PIK3CA,
IHC PTEN
Negative





PTEN


NCT01349660
All
BKM120
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01349933
Head and neck
MK2206
PIK3CA,
Seq.
Mutated|



squamous

PTEN
PIK3CA
exon20



carcinoma


NCT01349933
Head and neck
MK2206
PIK3CA,
IHC PTEN
Negative



squamous

PTEN



carcinoma


NCT01349933
Head and neck
MK2206
PIK3CA,
Seq. PTEN
Mutated



squamous

PTEN



carcinoma


NCT01362374
All
GDC-0068
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01362374
All
GDC-0068
PIK3CA,
IHC PTEN
Negative





PTEN


NCT01362374
All
GDC-0068
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01363232
All
MEK162
KRAS,
Seq. KRAS
Mutated|





NRAS,

G13D





BRAF


NCT01363232
All
MEK162
KRAS,
Seq. BRAF
Mutated|





NRAS,

V600E|





BRAF

V600K


NCT01363232
All
MEK162
KRAS,
Seq. NRAS
Mutated





NRAS,





BRAF


NCT01364051
Melanoma
AZD6244
KRAS,
Seq. KRAS
Mutated|





NRAS,

G13D





BRAF


NCT01364051
Melanoma
AZD6244
KRAS,
Seq. BRAF
Mutated|





NRAS,

V600E|





BRAF

V600K


NCT01364051
Melanoma
AZD6244
KRAS,
Seq. NRAS
Mutated





NRAS,





BRAF


NCT01374425
Colorectal
bevacizumab
VHL
Seq. VHL
Mutated



Adenocarcinoma


NCT01375829
All
temsirolimus
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01375829
All
temsirolimus
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01376310
All
GSK1120212
KRAS,
Seq. KRAS
Mutated|





NRAS,

G13D





BRAF


NCT01376310
All
GSK1120212
KRAS,
Seq. BRAF
Mutated|





NRAS,

V600E|





BRAF

V600K


NCT01376310
All
GSK1120212
KRAS,
Seq. NRAS
Mutated





NRAS,





BRAF


NCT01376453
Colorectal
sorafenib
BRAF
Seq. BRAF
Mutated|



Adenocarcinoma



V600E|







V600K


NCT01376453
Colorectal
sorafenib
BRAF
PCR BRAF
V600E



Adenocarcinoma


NCT01385228
Prostatic
pazopanib
VHL
Seq. VHL
Mutated



adenocarcinoma


NCT01385293
Prostatic
BKM120
PIK3CA,
Seq.
Mutated|



adenocarcinoma

PTEN
PIK3CA
exon20


NCT01385293
Prostatic
BKM120
PIK3CA,
IHC PTEN
Negative



adenocarcinoma

PTEN


NCT01385293
Prostatic
BKM120
PIK3CA,
Seq. PTEN
Mutated



adenocarcinoma

PTEN


NCT01386450
Glioblastoma
AZD6244
KRAS,
Seq. KRAS
Mutated|





NRAS,

G13D





BRAF


NCT01386450
Glioblastoma
AZD6244
KRAS,
Seq. BRAF
Mutated|





NRAS,

V600E|





BRAF

V600K


NCT01386450
Glioblastoma
AZD6244
KRAS,
Seq. NRAS
Mutated





NRAS,





BRAF


NCT01390818
All
MSC1936369B,
GNAQ,
Seq.
Mutated




SAR245409
GNA11
GNAQ


NCT01390818
All
MSC1936369B,
GNAQ,
Seq.
Mutated




SAR245409
GNA11
GNA11


NCT01392521
All
BAY 86-9766
KRAS,
Seq. KRAS
Mutated|





NRAS,

G13D





BRAF


NCT01392521
All
BAY 86-9766
KRAS,
Seq. BRAF
Mutated|





NRAS,

V600E|





BRAF

V600K


NCT01392521
All
BAY 86-9766
KRAS,
Seq. NRAS
Mutated





NRAS,





BRAF


NCT01396148
Gastrointestinal
Sunitinib
VHL
Seq. VHL
Mutated



Stromal Tumors



(GIST)


NCT01409200
Prostatic
axitinib
VHL
Seq. VHL
Mutated



adenocarcinoma


NCT01420081
Female genital tract
PF-04691502,
PIK3CA,
Seq.
Mutated|



malignancy
PF-05212384
PTEN
PIK3CA
exon20


NCT01420081
Female genital tract
PF-04691502,
PIK3CA,
IHC PTEN
Negative



malignancy
PF-05212384
PTEN


NCT01420081
Female genital tract
PF-04691502,
PIK3CA,
Seq. PTEN
Mutated



malignancy
PF-05212384
PTEN


NCT01427946
BAC, NSCLC
everolimus
KRAS
Seq. KRAS
Mutated|







G13D


NCT01434602
Glioblastoma
sorafenib
BRAF
Seq. BRAF
Mutated|







V600E|







V600K


NCT01434602
Glioblastoma
sorafenib
BRAF
PCR BRAF
V600E


NCT01437566
Breast Carcinoma
GDC-0980
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01437566
Breast Carcinoma
GDC-0980
PIK3CA,
IHC PTEN
Negative





PTEN


NCT01437566
Breast Carcinoma
GDC-0980
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01441947
Breast Carcinoma
cabozantinib
VHL
Seq. VHL
Mutated


NCT01449058
All
BYL719
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01449058
All
MEK162
KRAS,
Seq. KRAS
Mutated|





NRAS,

G13D





BRAF


NCT01449058
All
MEK162
KRAS,
Seq. BRAF
Mutated|





NRAS,

V600E|





BRAF

V600K


NCT01449058
All
MEK162
KRAS,
Seq. NRAS
Mutated





NRAS,





BRAF


NCT01449058
All
BYL719
PIK3CA,
IHC PTEN
Negative





PTEN


NCT01449058
All
BYL719
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01450384
All
sorafenib
BRAF
Seq. BRAF
Mutated|







V600E|







V600K


NCT01450384
All
sorafenib
BRAF
PCR BRAF
V600E


NCT01465802
BAC, NSCLC
PF-00299804
Her2
ISH
Amplified|






Her2/Neu
Equiv.







High


NCT01465802
BAC, NSCLC
PF-00299804
Her2
IHC
Positive






Her2/Neu


NCT01466244
Head and neck
cetuximab
PTEN
Seq. PTEN
Mutated



squamous



carcinoma


NCT01466244
Head and neck
cetuximab
PTEN
IHC PTEN
Negative



squamous



carcinoma


NCT01466972
Breast Carcinoma
Pazopanib
VHL
Seq. VHL
Mutated


NCT01468688
Gastrointestinal
BKM120
PIK3CA,
Seq.
Mutated|



stromal tumors

PTEN
PIK3CA
exon20



(GIST)


NCT01468688
Gastrointestinal
BKM120
PIK3CA,
IHC PTEN
Negative



stromal tumors

PTEN



(GIST)


NCT01468688
Gastrointestinal
BKM120
PIK3CA,
Seq. PTEN
Mutated



stromal tumors

PTEN



(GIST)


NCT01469572
Neuroendocrine
everolimus
PIK3CA,
Seq.
Mutated|



tumors

PTEN
PIK3CA
exon20


NCT01469572
Neuroendocrine
everolimus
PIK3CA,
Seq. PTEN
Mutated



tumors

PTEN


NCT01470209
All
BKM120
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01470209
All
BKM120
PIK3CA,
IHC PTEN
Negative





PTEN


NCT01470209
All
BKM120
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01471353
Colorectal
sorafenib
VHL
Seq. VHL
Mutated



Adenocarcinoma


NCT01473901
Glioblastoma
BKM120
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01473901
Glioblastoma
BKM120
PIK3CA,
IHC PTEN
Negative





PTEN


NCT01473901
Glioblastoma
BKM120
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01480154
Prostatic
MK2206
PIK3CA,
Seq.
Mutated|



adenocarcinoma

PTEN
PIK3CA
exon20


NCT01480154
Prostatic
MK2206
PIK3CA,
IHC PTEN
Negative



adenocarcinoma

PTEN


NCT01480154
Prostatic
MK2206
PIK3CA,
Seq. PTEN
Mutated



adenocarcinoma

PTEN


NCT01482156
All
BEZ235
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01482156
All
BEZ235
PIK3CA,
IHC PTEN
Negative





PTEN


NCT01482156
All
BEZ235
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01488487
Liver
everolimus
PIK3CA,
Seq.
Mutated|



Hepatocellular

PTEN
PIK3CA
exon20



Carcinoma


NCT01488487
Liver
everolimus
PIK3CA,
Seq. PTEN
Mutated



Hepatocellular

PTEN



Carcinoma


NCT01490749
Esophageal and
everolimus
PIK3CA,
Seq.
Mutated|



Esophagogastric

PTEN
PIK3CA
exon20



Junction Carcinoma


NCT01490749
Esophageal and
everolimus
PIK3CA,
Seq. PTEN
Mutated



Esophagogastric

PTEN



Junction Carcinoma


NCT01490866
Colorectal
Axitinib
VHL
Seq. VHL
Mutated



Adenocarcinoma


NCT01495247
Breast carcinoma
BEZ235
PIK3CA,
Seq.
Mutated|
IHC
Negative





PTEN
PIK3CA
exon20
HER2/Neu


NCT01495247
Breast carcinoma
BEZ235
PIK3CA,
Seq.
Mutated|
ISH
Not





PTEN
PIK3CA
exon20
HER2/Neu
Amplified


NCT01495247
Breast carcinoma
BEZ235
PIK3CA,
IHC PTEN
Negative
IHC
Negative





PTEN


HER2/Neu


NCT01495247
Breast carcinoma
BEZ235
PIK3CA,
IHC PTEN
Negative
ISH
Not





PTEN


HER2/Neu
Amplified


NCT01495247
Breast carcinoma
BEZ235
PIK3CA,
Seq. PTEN
Mutated
IHC
Negative





PTEN


HER2/Neu


NCT01495247
Breast carcinoma
BEZ235
PIK3CA,
Seq. PTEN
Mutated
ISH
Not





PTEN


HER2/Neu
Amplified


NCT01499160
Breast Carcinoma
everolimus
ER, PR
IHC ER
Positive


NCT01499160
Breast Carcinoma
everolimus
ER, PR
IHC PR
Positive


NCT01508104
All
BEZ235
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01508104
All
BEZ235
PIK3CA,
IHC PTEN
Negative





PTEN


NCT01508104
All
BEZ235
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01512251
Melanoma
BKM120
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01512251
Melanoma
BKM120
PIK3CA,
IHC PTEN
Negative





PTEN


NCT01512251
Melanoma
BKM120
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01516216
Colorectal
bevacizumab
VHL
Seq. VHL
Mutated



Adenocarcinoma


NCT01519414
Prostatic
ARQ 197
cMET
ISH CMET
Amplified



adenocarcinoma


NCT01519414
Prostatic
ARQ 197
cMET
IHC CMET
Positive



adenocarcinoma


NCT01522768
Gastric
afatinib
Her2
ISH
Amplified|



adenocarcinoma,


Her2/Neu
Equiv.



Esophageal and



High



Esophagogastric



Junction Carcinoma


NCT01522768
Gastric
afatinib
Her2
IHC
Positive



adenocarcinoma,


Her2/Neu



Esophageal and



Esophagogastric



Junction Carcinoma


NCT01524783
Neuroendocrine
everolimus
PIK3CA,
Seq.
Mutated|



tumors

PTEN
PIK3CA
exon20


NCT01524783
Neuroendocrine
everolimus
PIK3CA,
Seq. PTEN
Mutated



tumors

PTEN


NCT01529593
All
temsirolimus,
PIK3CA,
Seq.
Mutated|




metformin
PTEN
PIK3CA
exon20


NCT01529593
All
temsirolimus,
PIK3CA,
Seq. PTEN
Mutated




metformin
PTEN


NCT01531361
All
vemurafenib,
BRAF
Seq. BRAF
Mutated|




sorafenib


V600E|







V600K


NCT01531361
All
vemurafenib,
BRAF
PCR BRAF
V600E




sorafenib


NCT01536054
Ovarian Surface
sirolimus
PIK3CA,
Seq.
Mutated|



Epithelial

PTEN
PIK3CA
exon20



Carcinomas


NCT01536054
Ovarian Surface
sirolimus
PIK3CA,
Seq. PTEN
Mutated



Epithelial

PTEN



Carcinomas


NCT01538680
Colorectal
regorafenib
KRAS,
Seq. KRAS
Mutated|



Adenocarcinoma

BRAF

G13D


NCT01538680
Colorectal
regorafenib
KRAS,
Seq. BRAF
Mutated|



Adenocarcinoma

BRAF

V600E|







V600K


NCT01540253
All
BKM120
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01540253
All
BKM120
PIK3CA,
IHC PTEN
Negative





PTEN


NCT01540253
All
BKM120
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01542996
Breast Carcinoma
ARQ 197
cMET
ISH CMET
Amplified
IHC ER
Negative
IHC PR
Negative
IHC
Negative












HER2/Neu


NCT01542996
Breast Carcinoma
ARQ 197
cMET
IHC CMET
Positive
IHC ER
Negative
IHC PR
Negative
IHC
Negative












HER2/Neu


NCT01542996
Breast Carcinoma
ARQ 197
cMET
ISH CMET
Amplified
IHC ER
Negative
IHC PR
Negative
ISH
Negative












HER2/Neu


NCT01542996
Breast Carcinoma
ARQ 197
cMET
IHC CMET
Positive
IHC ER
Negative
IHC PR
Negative
ISH
Negative












HER2/Neu


NCT01543698
All
MEK162
KRAS,
Seq. KRAS
Mutated|





NRAS,

G13D





BRAF


NCT01543698
All
MEK162
KRAS,
Seq. BRAF
Mutated|





NRAS,

V600E|





BRAF

V600K


NCT01543698
All
MEK162
KRAS,
Seq. NRAS
Mutated





NRAS,





BRAF


NCT01545947
BAC, NSCLC
CC-223
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01545947
BAC, NSCLC
CC-223
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01548144
All
crizotinib
ALK
ISH ALK
Positive


NCT01548144
All
crizotinib
ROS1
ISH ROS1
Positive


NCT01548807
Prostatic
everolimus
PIK3CA,
Seq.
Mutated|



Adenocarcinoma

PTEN
PIK3CA
exon20


NCT01548807
Prostatic
everolimus
PIK3CA,
Seq. PTEN
Mutated



Adenocarcinoma

PTEN


NCT01550380
Female genital tract
BKM120
PIK3CA,
Seq.
Mutated|



malignancy

PTEN
PIK3CA
exon20


NCT01550380
Female genital tract
BKM120
PIK3CA,
IHC PTEN
Negative



malignancy

PTEN


NCT01550380
Female genital tract
BKM120
PIK3CA,
Seq. PTEN
Mutated



malignancy

PTEN


NCT01552434
All
temsirolimus
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01552434
All
temsirolimus
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01553851
Head and neck
GSK1120212
KRAS,
Seq. KRAS
Mutated|



squamous

NRAS,

G13D



carcinoma

BRAF


NCT01553851
Head and neck
GSK1120212
KRAS,
Seq. BRAF
Mutated|



squamous

NRAS,

V600E|



carcinoma

BRAF

V600K


NCT01553851
Head and neck
GSK1120212
KRAS,
Seq. NRAS
Mutated



squamous

NRAS,



carcinoma

BRAF


NCT01553942
BAC, NSCLC
afatinib
EGFR
Seq. EGFR
Activating







Mutation|







Exon 21







L858R|







Exon 19 del


NCT01562275
All
GDC-0068
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01562275
All
GDC-0068
PIK3CA,
IHC PTEN
Negative





PTEN


NCT01562275
All
GDC-0068
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01562899
Colorectal
MEK162
KRAS,
Seq. KRAS
Mutated|



adenocarcinoma,

NRAS,

G13D



Melanoma,

BRAF



Pancreatic



adenocarcinoma


NCT01562899
Colorectal
MEK162
KRAS,
Seq. BRAF
Mutated|



adenocarcinoma,

NRAS,

V600E|



Melanoma,

BRAF

V600K



Pancreatic



adenocarcinoma


NCT01562899
Colorectal
MEK162
KRAS,
Seq. NRAS
Mutated



adenocarcinoma,

NRAS,



Melanoma,

BRAF



Pancreatic



adenocarcinoma


NCT01563354
Neuroendocrine
everolimus
PIK3CA,
Seq.
Mutated|



tumors

PTEN
PIK3CA
exon20


NCT01563354
Neuroendocrine
everolimus
PIK3CA,
Seq. PTEN
Mutated



tumors

PTEN


NCT01567930
Liver
temsirolimus
PIK3CA,
Seq.
Mutated|



Hepatocellular

PTEN
PIK3CA
exon20



Carcinoma


NCT01567930
Liver
temsirolimus
PIK3CA,
Seq. PTEN
Mutated



Hepatocellular

PTEN



Carcinoma


NCT01571024
Colorectal
BKM120
PIK3CA,
Seq.
Mutated|



adenocarcinoma,

PTEN
PIK3CA
exon20



Pancreatic



adenocarcinoma


NCT01571024
Colorectal
BKM120
PIK3CA,
IHC PTEN
Negative



adenocarcinoma,

PTEN



Pancreatic



adenocarcinoma


NCT01571024
Colorectal
BKM120
PIK3CA,
Seq. PTEN
Mutated



adenocarcinoma,

PTEN



Pancreatic



adenocarcinoma


NCT01571284
Colorectal
aflibercept
VHL
Seq. VHL
Mutated



Adenocarcinoma


NCT01576406
All
crizotinib
ALK
ISH ALK
Positive


NCT01576666
All
BKM120
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01576666
All
BKM120
PIK3CA,
IHC PTEN
Negative





PTEN


NCT01576666
All
BKM120
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01579994
BAC, NSCLC
crizotinib
ALK
ISH ALK
Positive


NCT01582191
All
everolimus
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01582191
All
everolimus
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01587040
All
SAR245409
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01587040
All
SAR245409
PIK3CA,
IHC PTEN
Negative





PTEN


NCT01587040
All
SAR245409
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01587352
Uveal Melanoma
vorinostat
GNAQ,
Seq.
Mutated





GNA11
GNAQ


NCT01587352
Uveal Melanoma
vorinostat
GNAQ,
Seq.
Mutated





GNA11
GNA11


NCT01596270
All
SAR245409
PIK3CA,
Seq.
Mutated|





PTEN
PIK3CA
exon20


NCT01596270
All
SAR245409
PIK3CA,
IHC PTEN
Negative





PTEN


NCT01596270
All
SAR245409
PIK3CA,
Seq. PTEN
Mutated





PTEN


NCT01661972
Colorectal
aflibercept
VHL
Seq. VHL
Mutated



Adenocarcinoma


NCT01693068
Melanoma
MSBC1936369B
NRAS
Seq. NRAS
Mutated


NCT01745367
Breast Carcinoma
tivozanib
VHL
Seq. VHL
Mutated
IHC ER
Negative
IHC PR
Negative
IHC Her2 Negative


NCT01745367
Breast Carcinoma
tivozanib
VHL
Seq. VHL
Mutated
IHC ER
Negative
IHC PR
Negative
ISH
Not Amplified|












HER2/Neu
Equiv. Low


NCT01456325
BAC, NSCLC
onartuzumab
cMET
IHC CMET
Positive
Seq. EGFR
Mutated


NCT01662869
Esophageal and
onartuzumab
cMET
IHC CMET
Positive
IHC HER2
Negative
ISH
Negative



Esophagogastric






HER2



Junction Carcinoma


NCT01121575
BAC, NSCLC
crizotinib
cMET
Seq.
Mutated






CMET


NCT01548144
All
crizotinib
cMET
Seq.
Mutated






CMET


NCT01744652
All
crizotinib
cMET
Seq.
Mutated






CMET


NCT01276041
Breast Carcinoma
pertuzumab
Her2
IHC HER2
Positive


NCT01276041
Breast Carcinoma
pertuzumab
Her2
ISH HER2
Amplified|







Equiv.







High


NCT01774786
Esophageal and
pertuzumab
Her2
IHC HER2
Positive



Esophagogastric



Junction



Carcinoma, Gastric



adenocarcinoma


NCT01774786
Esophageal and
pertuzumab
Her2
ISH HER2
Amplified|



Esophagogastric



Equiv.



Junction



High



Carcinoma, Gastric



adenocarcinoma


NCT01565083
Breast Carcinoma
pertuzumab
Her2
IHC HER2
Positive


NCT01565083
Breast Carcinoma
pertuzumab
Her2
ISH HER2
Amplified|







Equiv.







High


NCT01491737
Breast Carcinoma
pertuzumab
Her2
IHC HER2
Positive
IHC ER
Positive


NCT01491737
Breast Carcinoma
pertuzumab
Her2
ISH HER2
Amplified|
IHC ER
Positive







Equiv.







High


NCT01491737
Breast Carcinoma
pertuzumab
Her2
IHC HER2
Positive
IHC PR
Positive


NCT01491737
Breast Carcinoma
pertuzumab
Her2
ISH HER2
Amplified|
IHC PR
Positive







Equiv.







High


NCT01042379
Breast Carcinoma
T-DM1,
Her2
IHC HER2
Positive




pertuzumab


NCT01042379
Breast Carcinoma
T-DM1,
Her2
ISH HER2
Amplified|




pertuzumab


Equiv.







High


NCT01641939
Gastric
T-DM1
Her2
IHC HER2
Positive



adenocarcinoma


NCT01641939
Gastric
T-DM1
Her2
ISH HER2
Amplified|



adenocarcinoma



Equiv.







High


NCT01912963
Breast Carcinoma
pertuzumab
Her2
IHC HER2
Positive


NCT01912963
Breast Carcinoma
pertuzumab
Her2
ISH HER2
Amplified|







Equiv.







High


NCT01796197
Breast Carcinoma
pertuzumab
Her2
IHC HER2
Positive


NCT01796197
Breast Carcinoma
pertuzumab
Her2
ISH HER2
Amplified|







Equiv.







High


NCT01855828
Breast Carcinoma
pertuzumab
Her2
IHC HER2
Positive


NCT01855828
Breast Carcinoma
pertuzumab
Her2
ISH HER2
Amplified|







Equiv.







High


NCT01904903
Breast Carcinoma
pertuzumab, T-
Her2
IHC HER2
Positive




DM1


NCT01904903
Breast Carcinoma
pertuzumab, T-
Her2
ISH HER2
Amplified|




DM1


Equiv.







High


NCT01480479
Glioblastoma
Rindopepimut
EGFRvIII
FA
Present






EGFRvIII


NCT01498328
Glioblastoma
Rindopepimut
EGFRvIII
FA
Present






EGFRvIII


NCT01800695
Glioblastoma
ABT-414
EGFRvIII
FA
Present






EGFRvIII


NCT01475006
Glioblastoma
AMG 595
EGFRvIII
FA
Present






EGFRvIII


NCT01257594
Glioblastoma
erlotinib
EGFRvIII
FA
Present






EGFRvIII


NCT00301418
Glioblastoma
erlotinib
EGFRvIII
FA
Present






EGFRvIII


NCT01465802
BAC, NSCLC
dacomitinib
EGFRvIII
FA
Present






EGFRvIII


NCT01858389
BAC, NSCLC
dacomitinib
EGFRvIII
FA
Present






EGFRvIII


NCT01454596
Glioblastoma
Anti-EGFRvIII
EGFRvIII
FA
Present




CAR

EGFRvIII


NCT01112527
Glioblastoma
PF-00299804
EGFRvIII
FA
Present






EGFRvIII


NCT01732640
Head and neck
afatinib
EGFRvIII
FA
Present



squamous


EGFRvIII



carcinoma


NCT01783587
Head and neck
afatinib
EGFRvIII
FA
Present



squamous


EGFRvIII



carcinoma


NCT01345682
Head and neck
afatinib
EGFRvIII
FA
Present



squamous


EGFRvIII



carcinoma


NCT01721525
Head and neck
afatinib
EGFRvIII
FA
Present



squamous


EGFRvIII



carcinoma


NCT01646125
BAC, NSCLC
AUY922
EGFR
Seq. EGFR
Activating







Mutation|







Exon 21







L858R|







Exon 19 del


NCT01526928
BAC, NSCLC
CO-1686
EGFR
Seq. EGFR
Activating







Mutation|







Exon 21







L858R|







Exon 19 del


NCT01620190
BAC, NSCLC
nab-paclitaxel
EGFR
Seq. EGFR
Activating







Mutation|







Exon 21







L858R|







Exon 19 del


NCT01532089
BAC, NSCLC
erlotinib
EGFR
Seq. EGFR
Activating







Mutation|







Exon 21







L858R|







Exon 19 del


NCT01746251
BAC, NSCLC
afatinib
EGFR
Seq. EGFR
Activating







Mutation|







Exon 21







L858R|







Exon 19 del


NCT01866410
BAC, NSCLC
cabozantinib,
EGFR
Seq. EGFR
Activating




erlotinib


Mutation|







Exon 21







L858R|







Exon 19 del


NCT01553942
BAC, NSCLC
afatinib
EGFR
Seq. EGFR
Activating







Mutation|







Exon 21







L858R|







Exon 19 del


NCT01836341
BAC, NSCLC
afatinib
EGFR
Seq. EGFR
Activating







Mutation|







Exon 21







L858R|







Exon 19 del


NCT01465802
BAC, NSCLC
dacomitinib
EGFR
Seq. EGFR
Activating







Mutation|







Exon 21







L858R|







Exon 19 del


NCT01324479
All
INC280
cMET
Seq.
Mutated






CMET


NCT01324479
All
INC280
cMET
IHC CMET
Positive


NCT01324479
All
INC280
cMET
ISH CMET
Amplified


NCT00585195
All
crizotinib
cMET
Seq.
Mutated






CMET


NCT00585195
All
crizotinib
cMET
IHC CMET
Positive


NCT00585195
All
crizotinib
cMET
ISH CMET
Amplified


NCT01755767
Liver
tivanitinib
cMET
IHC CMET
Positive



Hepatocellular



Carcinoma


NCT00697632
All
MGCD265
cMET
Seq.
Mutated






CMET


NCT00697632
All
MGCD265
cMET
IHC CMET
Positive


NCT00697632
All
MGCD265
cMET
ISH CMET
Amplified


NCT01654965
All
tivanitinb
cMET
Seq.
Mutated






CMET


NCT01654965
All
tivanitinb
cMET
IHC CMET
Positive


NCT01654965
All
tivanitinb
cMET
ISH CMET
Amplified


NCT01822522
All
cabozantinib
cMET
Seq.
Mutated






CMET


NCT01822522
All
cabozantinib
cMET
IHC CMET
Positive


NCT01822522
All
cabozantinib
cMET
ISH CMET
Amplified


NCT01611857
All
tivantinib
cMET
Seq.
Mutated






CMET


NCT01611857
All
tivantinib
cMET
IHC CMET
Positive


NCT01611857
All
tivantinib
cMET
ISH CMET
Amplified


NCT01625156
All
tivantinib
cMET
Seq.
Mutated






CMET


NCT01625156
All
tivantinib
cMET
IHC CMET
Positive


NCT01625156
All
tivantinib
cMET
ISH CMET
Amplified


NCT01749384
All
tivanitinb
cMET
Seq.
Mutated






CMET


NCT01749384
All
tivanitinb
cMET
IHC CMET
Positive


NCT01749384
All
tivanitinb
cMET
ISH CMET
Amplified









Report

In an embodiment, the methods of the invention comprise generating a molecular profile report. The report can be delivered to the treating physician or other caregiver of the subject whose cancer has been profiled. The report can comprise multiple sections of relevant information, including without limitation: 1) a list of the genes and/or gene products in the molecular profile; 2) a description of the molecular profile of the genes and/or gene products as determined for the subject; 3) a treatment associated with one or more of the genes and/or gene products in the molecular profile; and 4) and an indication whether each treatment is likely to benefit the patient, not benefit the patient, or has indeterminate benefit. The list of the genes and/or gene products in the molecular profile can be those presented herein for the molecular intelligence profiles of the invention. The description of the molecular profile of the genes and/or gene products as determined for the subject may include such information as the laboratory technique used to assess each biomarker (e.g., RT-PCR, FISH/CISH, IHC, PCR, FA/RFLP, sequencing, etc) as well as the result and criteria used to score each technique. By way of example, the criteria for scoring a protein as positive or negative for IHC may comprise the amount of staining and/or percentage of positive cells, the criteria for scoring a nucleic acid RT-PCR may be a cycle number indicating whether the level of the appropriate nucleic acid is differentially regulated as compared to a control sample, or criteria for scoring a mutation may be a presence or absence. The treatment associated with one or more of the genes and/or gene products in the molecular profile can be determined using a rule set as described herein, e.g., in any of Tables 7-23. The indication whether each treatment is likely to benefit the patient, not benefit the patient, or has indeterminate benefit may be weighted. For example, a potential benefit may be a strong potential benefit or a lesser potential benefit. Such weighting can be based on any appropriate criteria, e.g., the strength of the evidence of the biomarker-treatment association, or the results of the profiling, e.g., a degree of over- or underexpression.


Various additional components can be added to the report as desired. In an embodiment, the report comprises a list having an indication of whether one or more of the genes and/or gene products in the molecular profile are associated with an ongoing clinical trial. The report may include identifiers for any such trials, e.g., to facilitate the treating physician's investigation of potential enrollment of the subject in the trial. In some embodiments, the report provides a list of evidence supporting the association of the genes and/or gene products in the molecular profile with the reported treatment. The list can contain citations to the evidentiary literature and/or an indication of the strength of the evidence for the particular biomarker-treatment association. In still another embodiment, the report comprises a description of the genes and/or gene products in the molecular profile. The description of the genes and/or gene products in the molecular profile may comprise without limitation the biological function and/or various treatment associations.



FIGS. 37-39 herein present illustrative patient reports. FIGS. 37A-Y provide an illustrative report for molecular profiling of high grade glioma (see FIGS. 33O-P and Tables 19, 21 and accompanying text) with expanded mutational analysis using Next Generation sequencing as described above (see, e.g., Tables 24-25 and accompanying text). FIG. 38 provides an illustrative report for molecular profiling of a lung adenocarcinoma (see FIGS. 33I-J and Tables 17-18 and accompanying text) with expanded mutational analysis using Next Generation sequencing as described above (see, e.g., Tables 24-25 and accompanying text). FIG. 39 provides an illustrative report for molecular profiling via mutational analysis of a non-small cell lung cancer using Next Generation sequencing as described above (see, e.g., Table 25 and accompanying text).


As noted herein, the same biomarker may be assessed by one or more technique. In such cases, the results of the different analysis may be prioritized in case of inconsistent results. For example, the different methods may detect different aspects of a single biomarker (e.g., expression level versus mutation), or one method may be more sensitive than another. In the profiles presented above in Tables 11-14, BRAF mutations for melanoma and uveal melanoma samples are assessed by both PCR and Next Generation sequencing. Results obtained using the FDA approved cobas PCR (Roche Diagnostics) may be prioritized over the Next Generation results. However, if the sequencing detects a mutation, e.g., V600E, V600E2 or V600K, when PCR either detects wild type or is not determinable, the report may contain a note describing both sets of results including any therapy that may be implicated. In the case of melanoma, when the result of BRAF cobas PCR is “Wild type” or “no data” whereas BRAF sequencing is “V600E” or “V600E2”, the report may comprise a note that BRAF mutation was not detected by the FDA-approved Cobas PCR test, however, a V600E/E2 mutation was detected by alternative methods (next generation/Sanger sequencing) and that evidence suggests that the presence of a V600E mutation associates with potential clinical benefit from vemurafenib, dabrafenib or trametinib therapy. Similarly, when the result of BRAF cobas PCR is “Wild type” or “no data” and BRAF sequencing is “V600K”, the report may comprise a note that BRAF mutation was not detected by the FDA-approved Cobas PCR test, however, a V600K mutation was detected by alternative methods (next generation/Sanger sequencing) and that evidence suggests that the presence of a V600K mutation associates with potential clinical benefit from trametinib therapy. In the case of uveal melanoma, when the result of BRAF cobas PCR is “Wild type” or “no data” and BRAF sequencing is “V600E”, or “V600E2” or “V600K”, the report may comprise a note that BRAF mutation was not detected by the FDA-approved Cobas PCR test, however, a V600E/E2 or a V600K mutation was detected by alternative methods (next generation/Sanger sequencing) and that evidence suggests that the presence of a V600E or V600K mutation associates with potential clinical benefit from vemurafenib.


Androgen Receptor Profiling

The androgen receptor (AR) is a type of nuclear receptor that is activated by binding of either of the androgenic hormones testosterone or dihydrotestosterone in the cytoplasm and then translocating into the nucleus. AR is related to the progesterone receptor, and progestins in higher dosages can block the androgen receptor. The main function of AR is as a DNA-binding transcription factor that regulates gene expression. AR is expressed in multiple cell types and plays a role in various cancers, including without limitation prostate, bladder, kidney, lung, breast and liver. See Chang et al., Androgen receptor (AR) differential roles in hormone-related tumors including prostate, bladder, kidney, lung, breast and liver. Oncogene. 2013 Jul. 22. doi: 10.1038/onc.2013.274.


To counteract cancer cell proliferation, antiandrogenic drugs are used for hormone therapy called androgen deprivation therapy (ADT). Antiandrogens, or androgen antagonists, prevent androgens from expressing their biological effects on responsive tissues, including without limitation abarelix, bicalutamide, flutamide, gonadorelin, goserelin, leuprolide. Some antiandrogenic drugs suppress androgen production whereas others inhibit androgens from binding to the cancer cells' androgen receptors. Flutamide, nilutamide and bicalutamide are nonsteroidal antiandrogens. 5-alpha-reductase inhibitors such as finasteride, dutasteride, bexlosteride, izonsteride, turosteride, and epristeride are antiandrogenic as they prevent the conversion of testosterone to dihydrotestosterone (DHT).


Antiandrogens can be used to treat various AR expressing cancers and is most commonly associated with protstate cancer. Androgen-deprivation therapy (ADT) has been shown to cause initial reduction of prostate tumors. However, antiandrogenic treatment can cause prostate cancer tumors to become androgen independent. Androgen independence occurs when cells that are not reliant on androgen proliferate and spread while cells that require androgen for survival undergo apoptosis. The cells that do not require androgen become the basis of the tumors, causing reoccurring tumors a few years after the initial disappearance of the prostate cancer. Once prostate cancer becomes androgen independent, hormone therapy will most likely no longer benefit the individual and a new treatment approach is needed. Because the cancer can proliferate despite castrate levels of androgen, it is referred to as a castration-resistant prostate cancer (CRPC). Treatments for CRPC include the CYP17 inhibitor abiraterone, CYP17A1 inhibitors orteronel and galeterone, chemotherapeutic cabazitaxel, antiandrogens enzalutamide and ARN-509, endocrine disruptor abiraterone acetate, immunotherapy sipuleucel-T, and bone-targeting radiopharmaceutical alpharadin. See, e.g., Acar et al., New therapeutics to treat castrate-resistant prostate cancer. Scientific World Journal. 2013 May 27; 2013:379641; Mitsiades. A Road Map to Comprehensive Androgen Receptor Axis Targeting for Castration-Resistant Prostate Cancer. Cancer Res. 2013 Aug. 1; 73(15):4599-605. doi: 10.1158/0008-5472.CAN-12-4414. Enzalutamide is an androgen receptor antagonist drug developed for the treatment of metastatic castration-resistant prostate cancer. Molecular profiling according to the invention can be used to identify candidate treatments for castrate-resistant prostate cancer.


In an aspect, the invention provides a method of molecular profiling a cancer, comprising determining a level of the androgen receptor (AR) in a cancer cell. The cancer cell can be in a sample from a subject having or suspected of having the cancer. Any appropriate sample such as described herein can be used. The cancer can be treated with an antiandrogen therapy if the androgen receptor is expressed. The antiandrogen can suppress androgen production and/or inhibit androgens from binding to androgen receptors. For example, the antiandrogen can be one or more of abarelix, bicalutamide, flutamide, gonadorelin, goserelin, leuprolide, nilutamide, a 5-alpha-reductase inhibitor, finasteride, dutasteride, bexlosteride, izonsteride, turosteride, and epristeride. The cancer may be androgen independent. The expression can be assessed at the gene or gene product (e.g., protein) level. The cancer can be any appropriate type of cancer, including without limitation an acute myeloid leukemia (AML), breast carcinoma, cholangiocarcinoma, colorectal adenocarcinoma, extrahepatic bile duct adenocarcinoma, female genital tract malignancy, gastric adenocarcinoma, gastroesophageal adenocarcinoma, gastrointestinal stromal tumors (GIST), glioblastoma, head and neck squamous carcinoma, leukemia, liver hepatocellular carcinoma, low grade glioma, lung bronchioloalveolar carcinoma (BAC), lung non-small cell lung cancer (NSCLC), lung small cell cancer (SCLC), lymphoma, male genital tract malignancy, malignant solitary fibrous tumor of the pleura (MSFT), melanoma, multiple myeloma, neuroendocrine tumor, nodal diffuse large B-cell lymphoma, non epithelial ovarian cancer (non-EOC), ovarian surface epithelial carcinoma, pancreatic adenocarcinoma, pituitary carcinomas, oligodendroglioma, prostatic adenocarcinoma, retroperitoneal or peritoneal carcinoma, retroperitoneal or peritoneal sarcoma, small intestinal malignancy, soft tissue tumor, thymic carcinoma, thyroid carcinoma, or uveal melanoma. For example, the cancer can be a prostate, bladder, kidney, lung, breast, or liver cancer. In embodiments, the treatment for an AR expressing cancer comprises one or more of a CYP17 inhibitor (e.g., abiraterone), CYP17A1 inhibitor (e.g., orteronel, galeterone), chemotherapeutic agent (e.g., cabazitaxel), antiandrogen (e.g., enzalutamide, ARN-509), an endocrine disruptor (e.g., abiraterone acetate), immunotherapy (e.g., sipuleucel-T), and bone-targeting radiopharmaceutical (e.g., alpharadin).


EXAMPLES
Example 1
Molecular Profiling to Find Targets and Select Treatments for Refractory Cancers

The primary objective was to compare progression free survival (PFS) using a treatment regimen selected by molecular profiling with the PFS for the most recent regimen the patient progressed on (e.g. patients are their own control) (FIG. 40). The molecular profiling approach was deemed of clinical benefit for the individual patient who had a PFS ratio (PFS on molecular profiling selected therapy/PFS on prior therapy) of ≧1.3.


The study was also performed to determine the frequency with which molecular profiling by IHC, FISH and microarray yielded a target against which there is a commercially available therapeutic agent and to determine response rate (RECIST) and percent of patients without progression or death at 4 months.


The study was conducted in 9 centers throughout the United States. An overview of the method is depicted in FIG. 41. As can be seen in FIG. 41, the patient was screened and consented for the study. Patient eligibility was verified by one of two physician monitors. The same physicians confirmed whether the patients had progressed on their prior therapy and how long that PFS (TTP) was. A tumor biopsy was then performed, as discussed below. The tumor was assayed using IHC, FISH (on paraffin-embedded material) and microarray (on fresh frozen tissue) analyses.


The results of the IHC/FISH and microarray were given to two study physicians who in general used the following algorithm in suggesting therapy to the physician caring for the patient: 1) IHC/FISH and microarray indicated same target was first priority; 2) IHC positive result alone next priority; and 3) microarray positive result alone the last priority.


The patient's physician was informed of the suggested treatment and the patient was treated with the suggested agent(s) (package insert recommendations). The patient's disease status was assessed every 8 weeks and adverse effects were assessed by the NCI CTCAE version 3.0.


To be eligible for the study, the patient was required to: 1) provide informed consent and HIPAA authorization; 2) have any histologic type of metastatic cancer; 3) have progressed by RECIST criteria on at least 2 prior regimens for advanced disease; 4) be able to undergo a biopsy or surgical procedure to obtain tumor samples; 5) be >18 years, have a life expectancy >3 months, and an Eastern Cooperative Oncology Group (ECOG) Performance Status or 0-1; 6) have measurable or evaluable disease; 7) be refractory to last line of therapy (documented disease progression under last treatment; received >6 weeks of last treatment; discontinued last treatment for progression); 8) have adequate organ and bone marrow function; 9) have adequate methods of birth control; and 10) if CNS metastases then adequately controlled. The ECOG performance scale is described in Oken, M. M., Creech, R. H., Tormey, D. C., Horton, J., Davis, T. E., McFadden, E. T., Carbone, P. P.: Toxicity And Response Criteria Of The Eastern Cooperative Oncology Group. Am J Clin Oncol 5:649-655, 1982, which is incorporated by reference in its entirety. Before molecular profiling was performed, the principal investigator at the site caring for the patient must designate what they would treat the patient with if no molecular profiling results were available.


Methods


All biopsies were performed at local investigators' sites. For needle biopsies, 2-3 18 gauge needle core biopsies were performed. For DNA microarray (MA) analysis, tissue was immediately frozen and shipped on dry ice via FedEx to a central CLIA certified laboratory, Caris MPI in Phoenix, Ariz. For IHC, paraffin blocks were shipped on cold packs. IHC was considered positive for target if 2+ in ≧30% of cells. The MA was considered positive for a target if the difference in expression for a gene between tumor and control organ tissue was at a significance level of p<0.001.


Ascertainment of the Time to Progression to Document the Progression-Free Survival Ratio


Time to progression under the last line of treatment was documented by imaging in 58 patients (88%). Among these 58 patients, documentation by imaging alone occurred in 49 patients (74%), and documentation by imaging with tumor markers occurred in nine patients (14%; ovarian cancer, n 3; colorectal, n 1; pancreas, n 1; prostate, n 3; breast, n 1). Patients with clinical proof of progression were accepted when the investigator reported the assessment of palpable and measurable lesions (i.e., inflammatory breast cancer, skin/subcutaneous nodules, or lymph nodes), which occurred in six patients (9%). One patient (2%) with prostate cancer was included with progression by tumor marker. In one patient (2%) with breast cancer, the progression was documented by increase of tumor marker and worsening of bone pain. The time to progression achieved with a treatment based on molecular profiling was documented by imaging in 44 patients (67%) and by clinical events detected between two scheduled tumor assessments in 20 patients. These clinical events were reported as serious adverse events related to disease progression (e.g., death, bleeding, bowel obstruction, hospitalization), and the dates of reporting were censored as progression of disease. The remaining two patients were censored at the date of last follow-up.


IHC/FISH


For IHC studies, the formalin fixed, paraffin embedded tumor samples had slices from these blocks submitted for IHC testing for the following proteins: EGFR, SPARC, C-kit, ER, PR, Androgen receptor, PGP, RRM1, TOPO1, BRCP1, MRP1, MGMT, PDGFR, DCK, ERCC1, Thymidylate synthase, Her2/neu and TOPO2A. IHCs for all proteins were not carried out on all patients' tumors.


Formalin-fixed paraffin-embedded patient tissue blocks were sectioned (4 μm thick) and mounted onto glass slides. After deparaffination and rehydration through a series of graded alcohols, pretreatment was performed as required to expose the targeted antigen.


Human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor (EGFR) were stained as specified by the vendor (DAKO, Denmark). All other antibodies were purchased from commercial sources and visualized with a DAB biotin-free polymer detection kit. Appropriate positive control tissue was used for each antibody. Negative control slides were stained by replacing the primary antibody with an appropriately matched isotype negative control reagent. All slides were counterstained with hematoxylin as the final step and cover slipped. Tissue microarray sections were analyzed by FISH for EGFR and HER-2/neu copy number per the manufacturer's instructions. FISH for HER-2/neu (was done with the PathVysion HER2 DNA Probe Kit (Abbott Molecular, Abbott Park, Ill.). FISH for EGFR was done with the LSI EGFR/CEP 7 Probe (Abbott Molecular).


All slides were evaluated semi-quantitatively by a first pathologist, who confirmed the original diagnosis as well as read each of the immunohistochemical stains using a light microscope. Some lineage immunohistochemical stains were performed to confirm the original diagnosis, as necessary. Staining intensity and extent of staining were determined; both positive, tumor-specific staining of tumor cells and highly positive (≧2+), pervasive (≧30%) tumor specific staining results were recorded. IHC was considered positive for target if staining was ≧2+ in ≧30% of cells. Rather than look for a positive signal without qualification, this approach raises the stringency of the cut point such that it would be a significant or more demonstrative positive. A higher positive is more likely to be associated with a therapy that would affect the time to progression. The cut point used (i.e., staining was ≧2+ in ≧30% of cells) is similar to some cut points used in breast cancer for HER2/neu. When IHC cut points were compared with evidence from the tissue of origin of the cancer, the cut points were equal to or higher (more stringent) than the evidence cut points. A standard 10% quality control was performed by a second pathologist.


Microarray


Tumor samples obtained for microarray were snap frozen within 30 minutes of resection and transmitted to Caris-MPI on dry ice. The frozen tumor fragments were placed on a 0.5 mL aliquot of frozen 0.5M guanidine isothiocyanate solution in a glass tube, and simultaneously thawed and homogenized with a Covaris S2 focused acoustic wave homogenizer (Covaris, Woburn, Mass.). A 0.5 mL aliquot of TriZol was added, mixed and the solution was heated to 65° C. for 5 minutes then cooled on ice and phase separated by the addition of chloroform followed by centrifugation. An equal volume of 70% ethanol was added to the aqueous phase and the mixture was chromatographed on a Qiagen RNeasy column (Qiagen, Germantown, Md.). RNA was specifically bound and then eluted. The RNA was tested for integrity by assessing the ratio of 28S to 18S ribosomal RNA on an Agilent BioAnalyzer (Agilent, Santa Clara, Calif.). Two to five micrograms of tumor RNA and two to five micrograms of RNA from a sample of a normal tissue representative of the tumor's tissue of origin were separately converted to cDNA and then labeled during T7 polymerase amplification with contrasting fluor tagged (Cy3, Cy5) cytidine triphosphate. The labeled tumor and its tissue of origin reference were hybridized to an Agilent H1Av2 60-mer olio array chip with 17,085 unique probes.


The arrays contain probes for 50 genes for which there is a possible therapeutic agent that would potentially interact with that gene (with either high expression or low expression). Those 50 genes included: ADA, AR, ASNA, BCL2, BRCA2, CD33, CDW52, CES2, DNMT1, EGFR, ERBB2, ERCC3, ESR1, FOLR2, GART, GSTP1, HDAC1, HIF1A, HSPCA, IL2RA, KIT, MLH1, MS4A1, MASH2, NFKB2, NFKBIA, OGFR, PDGFC, PDGFRA, PDGFRB, PGR, POLA, PTEN, PTGS2, RAF1, RARA, RXRB, SPARC, SSTR1, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGF, VHL, and ZAP70.


The chips were hybridized from 16 to 18 hours at 60° C. and then washed to remove non-stringently hybridized probe and scanned on an Agilent Microarray Scanner. Fluorescent intensity data were extracted, normalized, and analyzed using Agilent Feature Extraction Software. Gene expression was judged to be different from its reference based on an estimate of the significance of the extent of change, which was estimated using an error model that takes into account the levels of signal to noise for each channel, and uses a large number of positive and negative controls replicated on the chip to condition the estimate. Expression changes at the level of p≦0.001 were considered as significantly different.


Statistical Considerations


The protocol called for a planned 92 patients to be enrolled of which an estimated 64 patients would be treated with therapy assigned by molecular profiling. The other 28 patients were projected to not have molecular profiling results available because of (a) inability to biopsy the patient; (b) no target identified by the molecular profiling; or (c) deteriorating performance status. Sixty four patients were required to receive molecular profiling treatment in order to reject the null hypothesis (Ho) that: ≦15% of patients would have a PFS ratio of ≧1.3 (e.g. a non-promising outcome).


Treatment Selection


Treatment for the patients based on molecular profiling results was selected using the following algorithm: 1) IHC/FISH and microarray indicates same target; 2) IHC positive result alone; 3) microarray positive result alone. The patient's physician was informed of suggested treatment and the patient was treated based on package insert recommendations. Disease status was assessed every 8 weeks. Adverse effects were assessed by NCI CTCAE version 3.0.


The targets and associated drugs are listed in Table 30.









TABLE 30







Pairings of Targets and Drugs








Potential Target
Agents Suggested as Interacting With the Target





IHC



EGFR
Cetuximab, erlotinib, gefitinib


SPARC
Nanoparticle albumin-bound paclitaxel


c-KIT
Imatinib, sunitinib, sorafenib


ER
Tamoxifen, aromatase inhibitors, toremifene,



progestational agent


PR
Progestational agents, tamoxifen, aromatase



inhibitor, goserelin


Androgen receptor
Flutamide, abarelix, bicalutamide, leuprolide,



goserelin


PGP
Avoid natural products, doxorubicin, etoposide,



docetaxel, vinorelbine


HER2/NEU
Trastuzumab


PDGFR
Sunitinib, imatinib, sorafenib


CD52
Alemtuzumab


CD25
Denileukin diftitox


HSP90
Geldanamycin, CNF2024


TOP2A
Doxorubicin, epirubicin, etoposide


Microarray


ADA
Pentostatin, cytarabine


AR
Flutamide, abarelix, bicalutamide, leuprolide,



goserelin


ASNA
Asparaginase


BCL2
Oblimersen sodium†


BRCA2
Mitomycin


CD33
Gemtuzumab ozogamicin


CDW52
Alemtuzumab


CES-2
Irinotecan


DCK
Gemcitabine


DNMT1
Azacitidine, decitabine


EGFR
Cetuximab, erlotinib, gefitinib


ERBB2
Trastuzumab


ERCC1
Cisplatin, carboplatin, oxaliplatin


ESR1
Tamoxifen, aromatase inhibitors, toremifene,



progestational agent


FOLR2
Methotrexate, pemetrexed


GART
Pemetrexed


GSTP1
Platinum


HDAC1
Vorinostat


HIF1α
Bevacizumab, sunitinib, sorafenib


HSPCA
Geldanamycin, CNF2024


IL2RA
Aldesleukin


KIT
Imatinib, sunitinib, sorafenib


MLH-1
Gemcitabine, oxaliplatin


MSH1
Gemcitabine


MSH2
Gemcitabine, oxaliplatin


NFKB2
Bortezomib


NFKB1
Bortezomib


OGFR
Opioid growth factor


PDGFC
Sunitinib, imatinib, sorafenib


PDGFRA
Sunitinib, imatinib, sorafenib


PDGFRB
Sunitinib, imatinib, sorafenib


PGR
Progestational agents, tamoxifen, aromatase



inhibitors, goserelin


POLA
Cytarabine


PTEN
Rapamycin (if low)


PTGS2
Celecoxib


RAF1
Sorafenib


RARA
Bexarotene, all-trans-retinoic acid


RXRB
Bexarotene


SPARC
Nanoparticle albumin-bound paclitaxel


SSTR1
Octreotide


TK1
Capecitabine


TNF
Infliximab


TOP1
Irinotecan, topotecan


TOP2A
Doxorubicin, etoposide, mitoxantrone


TOP2B
Doxorubicin, etoposide, mitoxantrone


TXNRD1
Px12


TYMS
Fluorouracil, capecitabine


VDR
Calcitriol


VEGF
Bevacizumab, sunitinib, sorafenib


VHL
Bevacizumab, sunitinib, sorafenib


ZAP70
Geldanamycin, CNF2024









Results

The distribution of the patients is diagrammed in FIG. 42 and the characteristics of the patients shown in Tables 31 and 32. As can be seen in FIG. 42, 106 patients were consented and evaluated. There were 20 patients who did not proceed with molecular profiling for the reasons outlined in FIG. 42 (mainly worsening condition or withdrawing their consent or they did not want any additional therapy). There were 18 patients who were not treated following molecular profiling (mainly due to worsening condition or withdrawing consent because they did not want additional therapy). There were 68 patients treated, with 66 of them treated according to molecular profiling results and 2 not treated according to molecular profiling results. One of the two was treated with another agent because the clinician caring for the patient felt a sense of urgency to treat and the other was treated with another agent because the insurance company would not cover the molecular profiling suggested treatment.


The median time for molecular profiling results being made accessible to a clinician was 16 days from biopsy (range 8 to 30 days) and a median of 8 days (range 0 to 23 days) from receipt of the tissue sample for analysis. Some modest delays were caused by the local teams not sending the patients' blocks immediately (due to their need for a pathology workup of the specimen). Patient tumors were sent from 9 sites throughout the United States including: Greenville, S.C.; Tyler, Tex.; Beverly Hills, Calif.; Huntsville, Ala.; Indianapolis, Ind.; San Antonio, Tex.; Scottsdale, Ariz. and Los Angeles, Calif.


Table 31 details the characteristics of the 66 patients who had molecular profiling performed on their tumors and who had treatment according to the molecular profiling results. As seen in Table 32, of the 66 patients the majority were female, with a median age of 60 (range 27-75). The number of prior treatment regimens was 2-4 in 53% of patients and 5-13 in 38% of patients. There were 6 patients (9%), who had only 1 prior therapy because no approved active 2nd line therapy was available. Twenty patients had progressed on prior phase I therapies. The majority of patients had an ECOG performance status of 1.









TABLE 31







Patient Characteristics (n = 66)











Characteristic
n
%















Gender





Female
43
65



Male
23
35



Age



Median (range)
60
(27-75)



Number of Prior Treatments



2-4*
35
53



5-13
25
38



ECOG



0
18
27



1
48
73







*Note: 6 patients (9%) had 1 prior






As seen in Table 32, tumor types in the 66 patients included breast cancer 18 (27%), colorectal 11 (17%), ovarian 5 (8%), and 32 patients (48%) were in the miscellaneous categories. Many patients had the more rare types of cancers.









TABLE 32







Patient Tumor Types (n = 66)











Tumor Type
n
%















Breast
18
27



Colorectal
11
17



Ovarian
5
8



Miscellaneous
32
48



Prostate
4
6



Lung
3
5



Melanoma
2
3



Small cell (esopha/retroperit)
2
3



Cholangiocarcinoma
2
3



Mesothelioma
2
3



H&N (SCC)
2
3



Pancreas
2
3



Pancreas neuroendocrine
1
1.5



Unknown (SCC)
1
1.5



Gastric
1
1.5



Peritoneal pseudomyxoma
1
1.5



Anal Canal (SCC)
1
1.5



Vagina (SCC)
1
1.5



Cervis
1
1.5



Renal
1
1.5



Eccrine seat adenocarinoma
1
1.5



Salivary gland adenocarinoma
1
1.5



Soft tissue sarcoma (uterine)
1
1.5



GIST (Gastric)
1
1.5



Thyroid-Anaplastic
1
1.5










Primary Endpoint: PFS Ratio ≧1.3


As far as the primary endpoint for the study is concerned (PFS ratio of ≧1.3), in the 66 patients treated according to molecular profiling results, the number of patients with PFS ratio greater or equal to 1.3 was 18 out of the 66 or 27%, 95% CI 17-38% one-sided, one-sample non parametric test p=0.007. The null hypothesis was that ≦15% of this patient population would have a PFS ratio of ≧1.3. Therefore, the null hypothesis is rejected and our conclusion is that this molecular profiling approach is beneficial. FIG. 43 details the comparison of PFS on molecular profiling therapy (the bar) versus PFS (TTP) on the patient's last prior therapy (the boxes) for the 18 patients. The median PFS ratio is 2.9 (range 1.3-8.15).


If the primary endpoint is examined, as shown in Table 33, a PFS ratio ≧1.3 was achieved in 8/18 (44%) of patients with breast cancer, 4/11 (36%) patients with colorectal cancer, 1/5 (20%) of patients with ovarian cancer and 5/32 (16%) patients in the miscellaneous tumor types (note that miscellaneous tumor types with PFS ratio ≧1.3 included: lung 1/3, cholangiocarcinoma 1/3, mesothelioma 1/2, eccrine sweat gland tumor 1/1, and GIST (gastric) 1/1).









TABLE 33







Primary Endpoint - PFS Ratio ≧1.3 By Tumor Type










Tumor Type
Total Treated
Number with PFS Ratio ≧1.3
%













Breast
18
8
44


Colorectal
11
4
36


Ovarian
5
1
20


Miscellaneous*
32
5
16


Total
66
18
27





*lung ⅓, cholangiocarcinoma ½, mesothelioma ½, eccrine sweat 1/1, GIST (gastric) 1/1






The treatment that the 18 patients with the PFS ≧1.3 received based on profiling is detailed in Table 34. As can be seen in that table for breast cancer patients, the treatment ranged from diethylstibesterol to nab paclitaxel+gemcitabine to doxorubicin. Treatments for patients with other tumor types are also detailed in Table 34. The table further shows a comparison of the drugs that the responding patients received versus the drugs that would have been suggested without molecular profiling and indicates which targets were used to suggest the therapies. Overall, 14 were treated with combinations and 4 were treated with single agents.









TABLE 34







Targets Noted in Patients' Tumors, Treatment Suggested on the Basis of


These Results, and Treatment Investigator Would Use if No Target Was


Identified (in patients with PFS ratio ≧1.3)













Treatment the




Treatment Suggested
Investigator Would



Targets Used to
on Basis of Patient's
Have Used if No


Location of Primary
Suggest Treatment
Tumor Molecular
Results From


Tumor
and Method Used
Profiling
Molecular Profiling





Breast
ESR1: I; ESR1: M
DES 5 mg TID
Investigational


Cholangiocarcinoma
EGFR: I; TOP1: M
CPT-11 350 mg/m2
Investigational




every 3 weeks;




cetuximab 400 mg/m2




day 1, 250 mg/m2 every




week


Breast
SPARC: I; SPARC,
NAB paclitaxel 260 mg/m2
Docetaxel, trastuzumab



ERBB2: M
every 3 weeks;




trastuzumab 6 mg/kg




every 3 weeks


Eccrine sweat gland
c-KIT: I; c-KIT: M
Sunitinib 50 mg/d, 4
Best supportive care


(right forearm)

weeks on/2 weeks off


Ovary
HER2/NEU, ER: I;
Lapatinib 1,250 mg PO
Bevacizumab



HER2/NEU: M
days 1-21; tamoxifen 20 mg




PO


Colon/rectum
PDGFR, c-KIT: I I;
CPT-11 70 mg/m2
Cetuximab



PDGFR, TOP1: M
weekly for 4 weeks on/2




weeks off; sorafenib




400 mg BID


Breast
SPARC: I; DCK: M
NAB paclitaxel 90 mg/m2
Mitomycin




every 3 weeks;




gemcitabine 750 mg/m2




days 1, 8, 15, every 3




weeks


Breast
ER: I; ER, TYMS: M
Letrozole 2.5 mg daily;
Capecitabine




capecitabine 1,250 mg/m2




BID, 2 weeks




on/1 week off


Malignant mesothelioma
MLH1, MLH2: I;
Gemcitabine 1,000 mg/m2
Gemcitabine



RRM2B, RRM1, RRM2,
days 1 and 8,



TOP2B: M
every 3 weeks;




etoposide 50 mg/m2 3




days every 3 weeks


Breast
MSH2
Oxaliplatin 85 mg/m2
Investigational




every 2 weeks;




fluorouracil (5FU)




1,200 mg/m2 days 1 and




2, every 2 weeks;




trastuzumab 4 mg/kg




day 1, 2 mg/kg every




week


Non-small-cell lung
EGFR: I; EGFR
Cetuximab 400 mg/m2
Vinorelbine


cancer

day 1, 250 mg/m2 every




week; CPT-11 125 mg/m2




weekly for 4




weeks on/2 weeks off


Colon/rectum
MGMT
Temozolomide 150 mg/m2
Capecitabine




for 5 days every




4 weeks; bevacizumab 5 mg/kg




every 2 weeks


Colon/rectum
PDGFR, c-KIT: I;
Mitomycin 10 mg once
Capecitabine



PDGFR: KDR, HIF1A,
every 4-6 weeks;



BRCA2: M
sunitinib 37.5 mg/d, 4




weeks on/2 weeks off


Breast
DCK, DHFR: M
Gemcitabine 1,000 mg/m2
Best supportive care




days 1 and 8




every 3 weeks;




pemetrexed 500 mg/m2




days 1 and 8, every 3




weeks


Breast
TOP2A: I; TOP2A: M
Doxorubicin 50 mg/m2
Vinorelbine




every 3 weeks


Colon/rectum
MGMT, VEGFA,
Temozolomide 150 mg/m2
Panitumumab



HIF1A: M
for 5 days every




4 weeks; sorafenib 400 mg




BID


Breast
ESR1, PR: I; ESR1, PR: M
Exemestane 25 mg
Doxorubicin liposomal




every day


GIST (stomach)
EGFR: I; EGFR,
Gemcitabine 1,000 mg/m2
None



RRM2: M
days 1, 8, and 15




every 4 weeks;




cetuximab 400 mg/m2




day 1, 250 mg/m2 every




week





* Abbreviations used in Table 35:


I, immunohistochemistry;


M, microarray;


DES, diethylstilbestrol;


CPT-11, irinotecan;


TID, three times a day;


NAB, nanoparticle albumin bound;


PO, orally;


BID, twice a day;


GIST, GI stromal tumor.






Secondary Endpoints


The results for the secondary endpoint for this study are as follows. The frequency with which molecular profiling of a patients' tumor yielded a target in the 86 patients where molecular profiling was attempted was 84/86 (98%). Broken down by methodology, 83/86 (97%) yielded a target by IHC/FISH and 81/86 (94%) yielding a target by microarray. RNA was tested for integrity by assessing the ratio of 28S to 18S ribosomal RNA on an Agilent BioAnalyzer. 83/86 (97%) specimens had ratios of 1 or greater and gave high intra-chip reproducibility ratios. This demonstrates that very good collection and shipment of patients' specimens throughout the United States and excellent technical results can be obtained.


By RECIST criteria in 66 patients, there was 1 complete response and 5 partial responses for an overall response rate of 10% (one CR in a patient with breast cancer and PRs in breast, ovarian, colorectal and NSCL cancer patients). Patients without progression at 4 months included 14 out of 66 or 21%.


In an exploratory analysis, a waterfall plot for all patients for maximum % change of the summed diameters of target lesions with respect to baseline diameters was generated. The patients who had progression and the patients who had some shrinkage of their tumor sometime during their course along with those partial responses by RECIST criteria is demonstrated in FIG. 44. There is some shrinkage of patient's tumors in over 47% of the patients (where 2 or more evaluations were completed).


Other Analyses—Safety


As far as safety analyses there were no treatment related deaths. There were nine treatment related serious adverse events including anemia (2 patients), neutropenia (2 patients), dehydration (1 patient), pancreatitis (1 patient), nausea (1 patient), vomiting (1 patient), and febrile neutropenia (1 patient). Only one patient (1.5%) was discontinued due to a treatment related adverse event of grade 2 fatigue.


Other Analyses—Relationship Between What the Clinician Caring for the Patient would have Selected Versus What the Molecular Profiling Selected


The relationship between what the clinician selected to treat the patient before knowing what molecular profiling results suggested for treatment was also examined. As detailed in FIG. 45, there is no pattern between the two. More specifically, no matches for the 18 patients with PFS ratio ≧1.3 were noted.


The overall survival for the 18 patients with a PFS ratio of 11.3 versus all 66 patients is shown in FIG. 46. This exploratory analysis was done to help determine if the PFS ratio had some clinical relevance. The overall survival for the 18 patients with the PFS ratio of ≧1.3 is 9.7 months versus 5 months for the whole population—log rank 0.026. This exploratory analysis indicates that the PFS ratio is correlated with the clinical parameter of survival.


Conclusions


This prospective multi-center pilot study demonstrates: (a) the feasibility of measuring molecular targets in patients' tumors from 9 different centers across the US with good quality and sufficient tumor collection—and treat patients based on those results; (b) this molecular profiling approach gave a longer PFS for patients on a molecular profiling suggested regimen than on the regimen they had just progressed on for 27% of the patients (confidence interval 17-38%) p=0.007; and (c) this is a promising result demonstrating use and benefits of molecular profiling.


The results also demonstrate that patients with refractory cancer can commonly have simple targets (such as ER) for which therapies are available and can be beneficial to them. Molecular profiling for patients who have exhausted other therapies and who are perhaps candidates for phase I or II trials could have this molecular profiling performed.


Example 2
Molecular Profiling System

Molecular profiling is performed to determine a treatment for a disease, typically a cancer. Using a molecular profiling approach, molecular characteristics of the disease itself are assessed to determine a candidate treatment. Thus, this approach provides the ability to select treatments without regard to the anatomical origin of the diseased tissue, or other “one-size-fits-all” approaches that do not take into account personalized characteristics of a particular patient's affliction. The profiling comprises determining gene and gene product expression levels, gene copy number and mutation analysis. Treatments are identified that are indicated to be effective against diseased cells that overexpress certain genes or gene products, underexpress certain genes or gene products, carry certain chromosomal aberrations or mutations in certain genes, or any other measurable cellular alterations as compared to non-diseased cells. Because molecular profiling is not limited to choosing amongst therapeutics intended to treat specific diseases, the system has the power to take advantage of any useful technique to measure any biological characteristic that can be linked to a therapeutic efficacy. The end result allows caregivers to expand the range of therapies available to treat patients, thereby providing the potential for longer life span and/or quality of life than traditional “one-size-fits-all” approaches to selecting treatment regimens.


A molecular profiling system has several individual components to measure expression levels, chromosomal aberrations and mutations. The components are shown in FIG. 47. The input sample can be formalin fixed paraffin embedded (FFPE) cancer tissue.


Gene expression analysis is performed using an expression microarray or qPCR (RT-PCR). The qPCR can be performed using a low density microarray. In addition to gene expression analysis, the system can perform a set of immunohistochemistry assays on the input sample. Gene copy number is determined for a number of genes via FISH (fluorescence in situ hybridization) and mutation analysis is done by DNA sequencing (including sequence sensitive PCR assays and fragment analysis such as RFLP, as desired) for a several specific mutations. All of this data is stored for each patient case. Data is reported from the expression, IHC, FISH and DNA sequencing analysis. All laboratory experiments are performed according to Standard Operating Procedures (SOPs).


Expression can be measured using real-time PCR (qPCR, RT-PCR). The analysis can employ a low density microarray. The low density microarray can be a PCR-based microarray, such as a Taqman™ Low Density Microarray (Applied Biosystems, Foster City, Calif.).


Expression can be measured using a microarray. The expression microarray can be an Agilent 44K chip (Agilent Technologies, Inc., Santa Clara, Calif.). This system is capable of determining the relative expression level of roughly 44,000 different sequences through RT-PCR from RNA extracted from fresh frozen tissue. Alternately, the system uses the Illumina Whole Genome DASL assay (Illumina Inc., San Diego, Calif.), which offers a method to simultaneously profile over 24,000 transcripts from minimal RNA input, from both fresh frozen (FF) and formalin-fixed paraffin embedded (FFPE) tissue sources, in a high throughput fashion. The analysis makes use of the Whole-Genome DASL Assay with UDG (Illumina, cat#DA-903-1024/DA-903-1096), the Illumina Hybridization Oven, and the Illumina iScan System according to the manufacturer's protocols. FIG. 48 shows results obtained from microarray profiling of an FFPE sample. Total RNA was extracted from tumor tissue and was converted to cDNA. The cDNA sample was then subjected to a whole genome (24K) microarray analysis using the Illumina Whole Genome DASL process. The expression of a subset of 80 genes was then compared to a tissue specific normal control and the relative expression ratios of these 80 target genes indicated in the figure was determined as well as the statistical significance of the differential expression.


DNA for mutation analysis is extracted from formalin-fixed paraffin-embedded (FFPE) tissues after macrodissection of the fixed slides in an area that % tumor nuclei ≧10% as determined by a pathologist. Extracted DNA is only used for mutation analysis if % tumor nuclei ≧10%. DNA is extracted using the QIAamp DNA FFPE Tissue kit according to the manufacturer's instructions (QIAGEN Inc., Valencia, Calif.). DNA can also be extracted using the QuickExtract™ FFPE DNA Extraction Kit according to the manufacturer's instructions (Epicentre Biotechnologies, Madison, Wis.). The BRAF Mutector I BRAF Kit (TrimGen, cat#MH1001-04) is used to detect BRAF mutations (TrimGen Corporation, Sparks, Md.). The DxS KRAS Mutation Test Kit (DxS, #KR-03) is used to detect KRAS mutations (QIAGEN Inc., Valencia, Calif.). BRAF and KRAS sequencing of amplified DNA is performed using Applied Biosystems' BigDye® Terminator V1.1 chemistry (Life Technologies Corporation, Carlsbad, Calif.).


IHC is performed according to standard protocols. IHC detection systems vary by marker and include Dako's Autostainer Plus (Dako North America, Inc., Carpinteria, Calif.), Ventana Medical Systems Benchmark® XT (Ventana Medical Systems, Tucson, Ariz.), and the Leica/Vision Biosystems Bond System (Leica Microsystems Inc., Bannockburn, Ill.). All systems are operated according to the manufacturers' instructions. American Society of Clinical Oncology (ASCO) and College of American Pathologist (CAP) standards are followed for ER, PR, and HER2 testing. ER, PR and HER2 as well as Ki-67, p53, and E-cad IHCs analyzed by the ACIS® (Automated Cellular Imaging System). The ACIS system comprises a microscope that scans the slides and constructs an image of the entire tissue section. Ten areas of tumor are analyzed for percentage positive cells and staining intensity within the selected fields.


FISH is performed on formalin-fixed paraffin-embedded (FFPE) tissue. FFPE tissue slides for FISH must be Hematoxylin and Eosin (H&E) stained and given to a pathologist for evaluation. Pathologists will mark areas of tumor for FISH analysis. The pathologist report shows whether tumor is present and sufficient enough to perform a complete analysis. FISH is performed using the Abbott Molecular VP2000 according to the manufacturer's instructions (Abbott Laboratories, Des Plaines, Iowa).


Illustrative reports generated by the system are shown in International PCT Patent Application PCT/US2010/000407, filed Feb. 11, 2010; and International PCT Patent Application PCT/US2010/54366, filed Oct. 27, 2010, each of which application is incorporated herein by reference in its entirety.


Example 3
Workflow for Identifying a Therapeutic Agent


FIG. 49 illustrates a diagram that outlines a workflow for identifying a therapeutic agent by analyzing a sample from an individual with breast cancer (441). The sample is cut into a number of slides (442) and stained with hematoxylin and eosin (H&E) (443). The stained slides are read by a pathologist (444) to determine what panel of markers to test, e.g., whether to analyze the sample using a complete biomarker panel analysis or a tumor-specific biomarker panel analysis, e.g., for breast cancer sample analysis (445). The pathologist also identifies sections (446) for DNA microarray analysis (447), FISH analysis, e.g., to measure HER2 expression (448), or mutational analysis via sequencing (449). DNA microarray analysis can be performed on a whole genome scale, with focus on genes that are informative for therapeutic treatment options, including at least ABCC1, ABCG2, ADA, AR, ASNS, BCL2, BIRC5, BRCA1, BRCA2, CD33, CD52, CDA, CES2, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2, ERBB2, ERCC1, ERCC3, ESR1, FLT1, FOLR2, FYN, GART, GNRH1, GSTP1, HCK, HDAC1, HIF1A, HSP90AA1, IGFBP3, IGFBP4, IGFBP5, IL2RA, KDR, KIT, LCK, LYN, MET, MGMT, MLH1, MS4A1, MSH2, NFKB1, NFKB2, NFKBIA, OGFR, PARP1, PDGFC, PDGFRA, PDGFRB, PGP, PGR, POLA1, PTEN, PTGS2, PTPN12, RAF1, RARA, RRM1, RRM2, RRM2B, RXRB, RXRG, SIK2, SPARC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGFA, VHL, YES1, and ZAP70. IHC is run on selected sections to analyze expression of biomarkers including AR, c-kit, CAV-1, CK 5/6, CK14, CK17, ECAD, ER, Her2/Neu, Ki67, MRP1, P53, PDGFR, PGP, PR, PTEN, SPARC, TLE3 and TS (4410). Each marker can be analyzed using a single or multiple antibodies for IHC detection. For example, SPARC is detected using an anti-SPARC monoclonal antibody (referred to herein as SPARC MC, SPARC Mono, SPARC m or the like), and an anti-SPARC polyclonal antibody (referred to herein as SPARC PC, SPARC Poly, SPARC p or the like), Given the results of the previous analysis, the sample is further analyzed with relevant marker panels (4411). The sample is classified as HER2+(4412), Triple Negative (4416), or ER/PR+, HER2− (4418). Further analysis depends on whether prior analysis determined that the sample should undergo “complete” biomarker panel analysis or a “tumor-specific” biomarker panel analysis. Tumor-specific analysis is performed for any cancer with a primary diagnosis, or first line, second line or third line therapy. Complete biomarker analysis is indicated for cancers that are fourth line, metastatic or beyond. Complete is also performed if the therapeutic history of the cancer is unknown (and thus becomes the default). In this manner, unnecessary testing can be avoided. HER2+(4412) samples are further analyzed by FISH for CMYC and TOP2A (4413), by IHC for p95 for tumor-specific analysis or for BCRP, ERCC1, MGMT, P95, RRM1, TOP2A and TOPO1 for complete analysis (4414), and by sequencing for mutation analysis of PIK3CA (4415). Triple negative (4416) samples are analyzed by IHC for p95 for tumor-specific analysis or for BCRP, ERCC1, MGMT, P95, RRM1, TOP2A and TOPO1 for complete analysis (4417). ER/PR+, HER2− (4418) samples are further analyzed by FISH for CMYC (4419), by IHC for p95 for tumor-specific analysis or for BCRP, ERCC1, MGMT, P95, RRM1, TOP2A and TOPO1 for complete analysis (4420). The results of the analysis are used to identify a therapeutic for the individual. The workflow can be generalized for the analysis of other diseases and tumor types.



FIG. 50 and Table 35 illustrate a biomarker centric view of the workflow described above. In FIG. 34, initial IHC and FISH results on the indicated biomarkers is used to characterize the cancer as HER2+, Triple Negative, or ER/PR+, HER2−. The characterization guides the additional IHC, FISH and sequencing analysis that is performed. “DNA MA” indicates that a DNA microarray is performed on all samples that meet the quality threshold as described herein. DNA microarray analysis can be performed on a whole genome scale, with focus on genes that are informative for therapeutic treatment options, including at least ABCC1, ABCG2, ADA, AR, ASNS, BCL2, BIRC5, BRCA1, BRCA2, CD33, CD52, CDA, CES2, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2, ERBB2, ERCC1, ERCC3, ESR1, FLT1, FOLR2, FYN, GART, GNRH1, GSTP1, HCK, HDAC1, HIF1A, HSP90AA1, IGFBP3, IGFBP4, IGFBP5, IL2RA, KDR, KIT, LCK, LYN, MET, MGMT, MLH1, MS4A1, MSH2, NFKB1, NFKB2, NFKBIA, OGFR, PARP1, PDGFC, PDGFRA, PDGFRB, PGP, PGR, POLA1, PTEN, PTGS2, PTPN12, RAF1, RARA, RRM1, RRM2, RRM2B, RXRB, RXRG, SIK2, SPARC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGFA, VHL, YES1, and ZAP70. IHC is run on selected sections to analyze expression of biomarkers including AR, c-kit, CAV-1, CK 5/6, CK14, CK17, ECAD, ER, Her2/Neu, Ki67, MRP1, P53, PDGFR, PGP, PR, PTEN, SPARC, TLE3 and TS. Table 35 outlines shows the criteria used to perform additional assays. Tumor-specific analysis is used in the case of cancer with a primary diagnosis, or first line, second line or third line therapy. Complete biomarker analysis is indicated for cancers that are fourth line, metastatic or beyond.









TABLE 35







Additional Assays










Tumor-Specific
Complete













Criteria
Primary diagnosis
Fourth line therapy



First line therapy
Metastatic



Second line therapy
Therapeutic history unknown



Third line therapy


Additional Testing

IHC: BCRP, ERCC1,




MGMT, RRM1, TOPO1




FISH: EGFR









Table 35 indicates prognostic markers in the breast cancer profiling. The markers used in the profiling can be used for theranostic (e.g., to guide selection of a candidate therapeutic) and prognostic purposes. “Y” in the “Prognostic?” column indicates that the marker can indicate a prognosis. Further details are described herein.









TABLE 35







Prognostic Breast Cancer Profiling
















Triple
ER/PR+/





HER2+
Neg
HER2-


Biomarker
Method
Prognostic?
Profile
Profile
Profile





AR
IHC

Y
Y
Y


Caveolin-1
IHC
Y
Y
Y
Y


CK 14
IHC
Y
Y
Y
Y


CK 17
IHC
Y
Y
Y
Y


CK 5/6
IHC
Y
Y
Y
Y


c-Kit
IHC
Y
Y
Y
Y


cMYC
FISH
Y
Y

Y


Cyclin D1
IHC
Y


Y


ECAD
IHC
Y
Y
Y
Y


EGFR
IHC
Y


Y


ER (ESR1)
IHC

Y
Y
Y


HER2
IHC/FISH

Y
Y
Y


(ERBB2)


Ki67
IHC

Y
Y
Y


MRP1
IHC

Y
Y
Y


(ABCC1)


P53
IHC
Y
Y
Y
Y


P95
IHC

Y
Y
Y


PDGFR
IHC
Y
Y
Y
Y


PGP (ABCB1)
IHC

Y
Y
Y


PI3K
SEQ

Y


PR
IHC

Y
Y
Y


PTEN
IHC

Y
Y
Y


SPARC
IHC

Y
Y
Y


TLE3
IHC

Y
Y
Y


TOP2A
FISH

Y


TOP2A
IHC

Y
Y
Y


TS (TYMS)
IHC

Y
Y
Y









Table 37 provides illustrative candidate treatments corresponding to the molecular profiling described in this Example. In the table, a positive result for the indicated biomarker using the indicated technique guides selection of the corresponding therapeutic agent, or that of a related agent.









TABLE 37







Illustrative Drug-biomarker Associations









Drug
Method
Biomarker(s)





5-fluorouracil
DNA Microarray
TYMS



IHC
TS


aminoglutethimide
DNA Microarray
ESR1, PR



IHC
ER, PR


anastrozole
DNA Microarray
ESR1, PR



IHC
ER, PR


capecitabine
DNA Microarray
TYMS



IHC
TS


doxorubicin
DNA Microarray
ABCB1, TOP2A



FISH
HER2, TOP2A



IHC
PGP, TOP2A


epirubicin
DNA Microarray
ABCB1, TOP2A



FISH
HER2, TOP2A



IHC
PGP, TOP2A


exemestane
DNA Microarray
ESR1, PR



IHC
ER, PR


fulvestrant
DNA Microarray
ESR1, PR



IHC
ER, Ki67, PR


gonadorelin
DNA Microarray
PR


goserelin
DNA Microarray
PR


irinotecan
IHC
TOPO1


lapatinib
FISH
HER2



IHC
HER2


letrozole
DNA Microarray
ESR1, PR



IHC
ER, PR


leuprolide
DNA Microarray
PR


liposomal-doxorubicin
DNA Microarray
ABCB1, TOP2A



FISH
HER2, TOP2A



IHC
PGP, TOP2A


medroxyprogesterone
DNA Microarray
ESR1, PR



IHC
ER, PR


megestrol acetate
DNA Microarray
ESR1, PR



IHC
ER, PR


methotrexate
DNA Microarray
ABCC1, DHFR



IHC
MRP1


nab-paclitaxel
DNA Microarray
SPARC



IHC
SPARC mono, SPARC




poly


pemetrexed
DNA Microarray
DHFR, GART, TYMS



IHC
TS


tamoxifen
DNA Microarray
ESR1, PR



IHC
ER, Ki67, PR


taxanes
IHC
TLE3


toremifene
DNA Microarray
ESR1, PR



IHC
ER, Ki67, PR


trastuzumab
FISH
HER2



IHC
HER2, P95, PTEN



Mutation (sequence
PIK3CA



analysis)









An illustrative benefit of the molecular profiling approach is illustrated in FIG. 51. For every 100 HER2+ patients, only about 30 (30%) will be Responders to treatment with trastuzumab. Molecular profiling according to the Example identifies 50 (50%) out of the 70 patients (70%) not likely to respond, e.g., because of PIK3CA mutations (25%), lack of PTEN (15%) or a p95 HER2 truncation (10%). HER2 spans the cell membrane and trastuzumab binds the external portion of the protein. However, most HER2 tests, including the FDA approved tests available from Dako (Dako North America, Inc., Carpinteria, Calif.) and Ventana (Ventana Medical Systems, Inc., Tucson, Ariz.), target the internal domain of HER2. Profiling according to the invention uses two antibodies for HER2: one with affinity to the internal domain, another with affinity to both the internal and external domains. If the latter antibody is negative but the tests targeting the internal domain are positive (e.g., the FDA approved tests), then HER2 is “p95 truncated” and trastuzumab will not be effective. By identifying patients unlikely to respond, efficacy of trastuzumab for a selected population can be increased from 30% to 60%. Furthermore, the molecular profiling methods of the invention can identify candidate treatments that are more likely to be effective in the trastuzumab non-responders.


Illustrative reports generated by the system are shown in International PCT Patent Application PCT/US2010/000407, filed Feb. 11, 2010; and International PCT Patent Application PCT/US2010/54366, filed Oct. 27, 2010, each of which application is incorporated herein by reference in its entirety.


Example 4
Biomarker—Drug Associations and Lineage—Drug Associations

Table 38 lists exemplary associations between biomarkers and drugs associated with the biomarkers. When the biomarkers are found to be overexpressed in a patient sample, the drugs are indicated for use in treating the patient as described herein. For each drug, an indication is given of exemplary techniques that can be used to assess the corresponding biomarker. One of skill will appreciate that any technique can be used as described herein or known in the art, including without limitation microarray, PCR, IHC, ISH, FISH, and/or sequence analysis. Abbreviations in the table include the following: GE—Gene expression (e.g., RT-PCR; DNA microrarray); MA—Mutational analysis; IHC—Immunohistochemistry; FISH—Fluorescent in situ hybridization









TABLE 38







Biomarker - Drug Associations








Biomarker
Drug Associations





ABCC1 (MRP1)
doxorubicin (IHC and GE), epirubicin (IHC and GE), methotrexate (IHC and



GE), vincristine (IHC and GE), vinorelbine (IHC and GE), vinblastine (IHC



and GE), etoposide (IHC and GE)


ABCG2 (BCRP)
cisplatin (IHC and GE)), carboplatin (IHC and GE)


ADA
pentostatin (GE), cytarabine (GE)


ALK (e.g.,
crizotinib (FISH), pemetrexed (FISH)


EML4-ALK)


AR
bicalutamide (IHC and GE), flutamide (IHC and GE), abarelix (GE), goserelin



(GE), leuprolide (GE), gonadorelin (GE)


ASNS
asparaginase (GE), pegaspargase (GE)


BRCA1
mitomycin (GE), cisplatin (GE), carboplatin (GE)


BRCA2
mitomycin (GE), cisplatin (GE), carboplatin (GE)


CD52
alemtuzumab (IHC and GE)


CDA
cytarabine (GE)


CES2
irinotecan (GE)


DCK
gemcitabine (GE), cytarabine (GE)


DHFR
methotrexate (GE), pemetrexed (GE)


DNMT1
azacitidine (GE), decitabine (GE)


DNMT3A
azacitidine (GE), decitabine (GE)


DNMT3B
azacitidine (GE), decitabine (GE)


EGFR
gefitinib (FISH and MA), erlotinib (FISH and MA), cetuximab (FISH and



MA), panitumumab (FISH and MA)


EPHA2
dasatinib (GE)


ERBB2 (HER2)
trastuzumab (IHC and FISH), lapatinib (IHC and FISH), doxorubicin (FISH),



epirubicin (FISH), liposomal-doxorubicin (FISH)


ERCC1
cisplatin (IHC and GE), carboplatin (IHC and GE), oxaliplatin (IHC and GE)


ER
tamoxifen (IHC and GE), toremifene (GE), fulvestrant (GE), anastrozole (IHC



and GE), letrozole (IHC and GE), exemestane (GE), aminoglutethimide (GE),



megestrol (GE), medroxyprogesterone (GE)


FLT1 (VEGFR1)
bevacizumab (GE), sunitinib (GE), sorafenib (GE)


GART
pemetrexed (GE)


HIF1A
sunitinib (GE), sorafenib (GE)


IGFBP3
letrozole (GE)


IGFBP4
letrozole (GE)


IGFBP5
letrozole (GE)


KDR (VEGFR2)
sunitinib (GE), sorafenib (GE)


Ki67
“tamoxifen + chemotherapy” (IHC)—breast only


KIT (cKIT)
sunitinib (MA and GE), sorafenib (GE), imatinib (MA and GE), dasatinib (MA



and GE)


KRAS
gefitinib (MA), erlotinib (MA), cetuximab (MA), panitumumab (MA),



sorafenib (MA), combination therapy (VBMCP) (MA)


cMET/MET
gefitinib (FISH), erlotinib (FISH)


MGMT
temozolomide (IHC and GE)


PDGFRA
sunitinib (GE), sorafenib (GE)


PDGFRB
sunitinib (GE), sorafenib (GE)


PGP (ABCB1)
doxorubicin (IHC and GE), liposomal doxorubicin (IHC and GE), epirubicin



(IHC and GE), etoposide (IHC and GE), teniposide (GE), docetaxel (IHC and



GE), paclitaxel (IHC and GE), vincristine (IHC and GE), vinorelbine (IHC and



GE), vinblastine (IHC and GE)


PIK3CA/PI3K
cetuximab (MA), panitumumab (MA), trastuzumab (MA)


PR
tamoxifen (IHC and GE), toremifene (GE), fulvestrant (GE), anastrozole (IHC



and GE), letrozole (IHC and GE), exemestane (GE), aminoglutethimide (GE),



goserelin (GE), leuprolide (GE), gonadorelin (GE), megestrol (GE),



medroxyprogesterone (GE)


PTEN
erlotinib (IHC), gefitinib (IHC), cetuximab (IHC), panitumumab (IHC),



trastuzumab (IHC)


PTGS2 (COX2)
celecoxib (IHC and GE), aspirin (IHC)


BRAF1 (BRAF)
cetuximab (MA), panitumumab (MA)


RARA
ATRA (GE)


RRM1
gemcitabine (IHC and GE), hydroxyurea (GE)


RRM2
gemcitabine (GE), hydroxyurea (GE)


RRM2B
gemcitabine (GE), hydroxyurea (GE)


RXRB
bexarotene (GE)


SPARC
nab-paclitaxel (IHC and GE)


(mono/poly)


SRC
dasatinib (GE)


SSTR2
octreotide (GE)


SSTR5
octreotide (GE)


TLE3
paclitaxel (IHC), docetaxel (IHC)


TOPO1/TOP1
irinotecan (IHC and GE), topotecan (IHC and GE)


TOPO2A/TOP2A
doxorubicin (IHC, FISH and GE), liposomal doxorubicin (IHC, FISH and



GE), epirubicin (IHC, FISH and GE)


TOP2B
doxorubicin (GE), liposomal doxorubicin (GE), epirubicin (GE)


TUBB3
paclitaxel (IHC), docetaxel (IHC), vinorelbine (IHC)


TS/TYMS
pemetrexed (IHC and GE), capecitabine (GE), fluorouracil (IHC and GE)


VDR
choleciferol (GE), calcitriol (GE)


VHL
sunitinib (GE), sorafenib (GE)









Example 5
HER2 Overexpression in Various Tumors

Testing for HER2 assists in the management of breast cancer and gastro-esophageal junction (GEJ) cancer. The purpose of this Example was to capture the relative frequency and distribution of HER2 overexpression in other cancers.


In a cohort of 11,223 patient samples, HER2 was assayed by immunohistochemistry using the Ventana HER2 (4B5) antibody. Slides were read by pathologists using the cutoff of (≧=3+ and ≧=30% as positive) for HER2. In this same cohort, 2,246 patient samples underwent HER2 by FISH testing using Abbott's Pathvysion HER2 assay. FISH analysis was interpreted by a cytogeneticist based on ASCO/CAP guidelines for breast cancer. Prior to the analysis, 4,116 patient samples were excluded secondary to an unknown tumor lineage or other rarely observed lineages. In all, twenty-seven tumor lineages comprising 7,107 patient specimens were analyzed.


The frequency of HER2 by IHC was highest in breast carcinoma (68.6%), colorectal adenocarcinoma (7.2%), ovarian surface epithelial carcinoma (5.4%), gastroesophageal adenocarcinoma (4.5%), non-small cell lung cancer (3.1%), pancreatic adenocarcinoma (1.3%) and gastric adenocarcinoma (1.3%). In these same lineages, frequency of HER2 by FISH was highest in breast carcinoma (46.0%) followed by surface epithelial ovarian carcinoma (12.7%), non-small cell lung cancer (8.3%), gastroesophageal adenocarcinoma (6.7%), gastric adenocarcinoma (4.8%), pancreatic adenocarcinoma (4.0%), and colorectal adenocarcinoma (3.2%). Distributional differences in IHC versus FISH results were analyzed by Pearson's chi-square (×2) test, with statistical significance (p<0.05) achieved in breast carcinoma, gastric adenocarcinoma, gastroesophageal adenocarcinoma and surface epithelial ovarian carcinoma.


HER2 status was investigated in a large patient pool with advanced malignancies in a single clinical laboratory with standardized IHC and FISH. This study shows that HER2 is frequently expressed in multiple cancer types, which merits the inclusion of therapeutic strategies using HER2-targeted therapy in many types of tumors.


Example 6
Molecular Profiling of Metastatic Breast Cancer in Body Cavity Fluids

The diagnosis of malignant effusion signifies disease progression and is associated with a worse prognosis regardless of tumor origin. Cancer cells in fluids have genotypic and phenotypic characteristics that differ from the primary tumor. This Example reports the molecular profiling for breast cancer metastasis in pleural and peritoneal fluids.


Malignant fluid samples submitted for molecular profiling were retrospectively identified. A cell-block was prepared or available for testing for all samples. An H&E slide was prepared from the cell-block and reviewed by a pathologist before further testing. Malignant cell percentages were determined for purpose of DNA microarray and sequencing. The results were reviewed and data compiled to calculate the yield of various molecular predictive tests.


28 metastatic breast cancer fluid samples were identified. Of the 28 cases, 10 biomarkers by IHC could be performed in 20 samples (71.4%), 1-9 in 1 sample (3.5%), while 7 samples were insufficient quality for IHC (25%). DNA microarray analysis was performed for 10 cases (35.7%). FISH was performed for EGFR in 7 cases (25%), Her2 Neu FISH was performed for 11 cases (39%), cMYC FISH was performed for 5 samples (17.8%) and TOPO2a by FISH in was performed for 3 samples (10.7%). Combined IHC/FISH and MA data was available in 10 cases, IHC and FISH data in 11 cases and IHC and MA data in 10 cases. Combined results of predictive markers provided information on therapeutic guidance according to the workflow presented in Example 3.


Molecular profiling of malignant fluids offers additional opportunities for testing those patients where other tissue samples such as needle core biopsy or resection samples are not available. Molecular profiling of fluids provides insight into the molecular characteristics of malignant cells in body cavity fluids and associated expression of unique therapeutic targets.


Example 7
Molecular Profiling System

A system for carrying out molecular profiling according to the invention comprises the components used to perform molecular profiling on a patient sample, identify potentially beneficial and non-beneficial treatment options based on the molecular profiling, and return a report comprising the results of the analysis to the treating physician or other appropriate caregiver.


Formalin-fixed paraffin-embedded (FFPE) are reviewed by a pathologist for quality control before subsequent analysis. Nucleic acids (DNA and RNA) are extracted from FFPE tissues after microdissection of the fixed slides. Nucleic acids are extracted using phenol-chlorform extraction or a kit such as the QIAamp DNA FFPE Tissue kit according to the manufacturer's instructions (QIAGEN Inc., Valencia, Calif.).


Polymerase chain reaction (PCR) amplification is performed using the ABI Veriti Thermal Cycler (Applied Biosystems, cat#9902). PCR is performed using the Platinum Taq Polymerase High Fidelity Kit (Invitrogen, cat#11304-029). Amplified products can be purified prior to further analysis with Sanger sequencing, pyrosequencing or the like. Purification is performed using CleanSEQ reagent, (Beckman Coulter, cat#000121), AMPure XP reagent (Beckman Coulter, cat#A63881) or similar. Sequencing of amplified DNA is performed using Applied Biosystem's ABI Prism 3730xl DNA Analyzer and BigDye® Terminator V1.1 chemistry (Life Technologies Corporation, Carlsbad, Calif.). The BRAF V600E mutation is assessed using the FDA approved Cobas® 4800 BRAF V600 Mutation Test from Roche Molecular Diagnostics (Roche Diagnostics, Indianapolis, Ind.). NextGeneration sequencing is performed using the MiSeq platform from Illumina Corporation (San Diego, Calif., USA) according to the manufacturer's recommended protocols.


For RFLP, ALK fragment analysis is performed on reverse transcribed mRNA isolated from a formalin-fixed paraffin-embedded tumor sample using FAM-linked primers designed to flank and amplify EML4-ALK fusion products. The assay is designed to detect variants v1, v2, v3a, v3b, 4, 5a, 5b, 6, 7, 8a and 8b. Other rare translocations may be detected by this assay; however, detection is dependent on the specific rearrangement. This test does not detect ALK fusions to genes other than EML4.


IHC is performed according to standard protocols. IHC detection systems vary by marker and include Dako's Autostainer Plus (Dako North America, Inc., Carpinteria, Calif.), Ventana Medical Systems Benchmark® XT (Ventana Medical Systems, Tucson, Ariz.), and the Leica/Vision Biosystems Bond System (Leica Microsystems Inc., Bannockburn, Ill.). All systems are operated according to the manufacturers' instructions.


FISH is performed on formalin-fixed paraffin-embedded (FFPE) tissue. FFPE tissue slides for FISH must be Hematoxylin and Eosion (H & E) stained and given to a pathologist for evaluation. Pathologists will mark areas of tumor to be FISHed for analysis. The pathologist report must show tumor is present and sufficient enough to perform a complete analysis. FISH is performed using the Abbott Molecular VP2000 according to the manufacturer's instructions (Abbott Laboratories, Des Plaines, Iowa). ALK is assessed using the Vysis ALK Break Apart FISH Probe Kit from Abbott Molecular, Inc. (Des Plaines, Ill.). HER2 is assessed using the INFORM HER2 Dual ISH DNA Probe Cocktail kit from Ventana Medical Systems, Inc. (Tucson, Ariz.) and/or SPoT-Light® HER2 CISH Kit available from Life Technologies (Carlsbad, Calif.).


Example 8
Molecular Profiling Reports

Exemplary reports generated by the molecular profiling systems and methods of the invention are shown in FIGS. 37-39.



FIGS. 37A-37Y illustrate an exemplary patient report based on molecular profiling for an ovarian cancer. The molecular profile used was a molecular intelligence (MI) profile for a high grade glioma (see FIGS. 33O-P and Tables 19, 21 and accompanying text) and included mutational analysis on a panel of 34 genes performed by Next Generation sequencing. FIG. 37A illustrates a cover page of a report indicating patient and specimen information for the glioma patient. FIG. 37A also displays a summary of agents associated with potential benefit or potential lack of benefit. Agents associated with potential benefit are further annotated as on NCCN Compendium™ (i.e., recommended by NCCN guidelines for the particular tumor lineage) or off NCCN Compendium™ (i.e., not part of the NCCN guidelines for the particular tumor lineage). FIG. 37A also lists clinical trials which may be available given the molecular profiling results, here no trials were matched. FIG. 37B reports further patient and specimen information for the glioma patient. FIG. 37C illustrates more detailed information for biomarker profiling used to associate agents with potential benefit. FIG. 37D illustrates more detailed information for biomarker profiling used to associate agents with lack of potential benefit. FIG. 37E illustrates more detailed information for biomarker profiling used to associate agents with indeterminate benefit. FIG. 37F and FIG. 37G illustrate more detailed information for mutational analysis performed by Next Generation Sequencing. This section indicates mutations that were identified (FIG. 37F) as well as providing a list of genes that were tested without alterations (FIG. 37G). FIG. 37H, FIG. 37I, FIG. 37J and FIG. 37K provide a listing of published references used to provide evidence of the biomarker—agent association rules used to construct the report. FIG. 37L presents a disclaimer, e.g., that ultimate treatment decisions reside solely within the discretion of the treating physician. FIG. 37M provides a cover page for an Appendix to the report. FIG. 37N and FIG. 37O provide more information about the mutational analysis performed by Next Generation sequencing, Sanger sequencing, pyrosequencing, EGFR RFLP analysis, Cobas BRAF V600E analysis and MGMT Methyltion analysis. Depending on the tumor lineage, not all of these tests are necessarily performed. FIG. 37P provides more information about the IHC analysis performed on the patient sample, e.g., the staining results and threshold for each marker. FIG. 37Q provides more information about the ISH analysis performed on the patient sample, which comprised CISH for this tumor. FIG. 37R, FIG. 37S, FIG. 37T, FIG. 37U, FIG. 37V, FIG. 37W, and FIG. 37X provide a description of the biomarkers assessed per the molecular profiling. FIG. 37Y provides the framework used for the literature level of evidence as included in the report.


FIGS. 38A-38AA illustrate an exemplary patient report based on molecular profiling for a lung adenocarcinoma. The molecular profile used was a molecular intelligence (MI) profile for lung cancer (see FIGS. 33I-J, Table 17 and Table 18) and included mutational analysis on a panel of 34 genes performed by Next Generation sequencing (“NGS Panel,” see Table 25 and accompanying text). The sections of the report are the same as those described for FIGS. 37A-Y as adapted for lung cancer molecular profiling. FIG. 38A illustrates a cover page of a report indicating patient and specimen information for the lung cancer patient. FIG. 38A also displays a summary of agents associated with potential benefit or potential lack of benefit. Agents associated with potential benefit are further annotated as on NCCN Compendium™ (i.e., recommended by NCCN guidelines for the particular tumor lineage) or off NCCN Compendium™ (i.e., not part of the NCCN guidelines for the particular tumor lineage). FIG. 38A also lists clinical trials which may be available given the molecular profiling results, here trials were matched based on cMET status. FIG. 38B reports further patient and specimen information for the lung cancer patient. FIG. 38C and FIG. 38D illustrate more detailed information for biomarker profiling used to associate agents with potential benefit. FIG. 38E illustrates more detailed information for biomarker profiling used to associate agents with lack of potential benefit. FIG. 38F illustrates more detailed information for biomarker profiling used to associate agents with indeterminate benefit. FIG. 38G and FIG. 38H illustrate more detailed information for mutational analysis performed by Next Generation Sequencing. This section indicates mutations that were identified (FIGS. 38G-H) as well as providing a list of genes that were tested without alterations (FIG. 38H). FIG. 38I provides a listing of clinical trials matched to cMET. FIG. 38J, FIG. 38K, FIG. 38L and FIG. 38M provide a listing of published references used to provide evidence of the biomarker—agent association rules used to construct the report. FIG. 38N presents a disclaimer, e.g., that ultimate treatment decisions reside solely within the discretion of the treating physician. FIG. 38O provides a cover page for an Appendix to the report. FIG. 38P provides more information about the mutational analysis performed by Next Generation sequencing. FIG. 38Q provides more information about the IHC analysis performed on the patient sample, e.g., the staining results and threshold for each marker. FIG. 38R provides more information about the FISH analysis performed on the patient sample. FIG. 38S provides more information about the CISH analysis performed on the patient sample. FIG. 38T, FIG. 38U, FIG. 38V, FIG. 38W, FIG. 38X, FIG. 38Y, and FIG. 38Z provide a description of the biomarkers assessed per the molecular profiling. FIG. 38AA provides the framework used for the literature level of evidence as included in the report.



FIGS. 39A-39Y illustrate an exemplary patient report based on molecular profiling for a non-small cell lung cancer with stand alone mutational analysis performed by Next Generation sequencing (“NGS Panel,” see Table 25 and accompanying text). FIG. 39A presents an overview of the patient history and sample. FIG. 39B displays a summary of agents associated with potential benefit or potential lack of benefit. Agents associated with potential benefit are further annotated as on NCCN Compendium™ (i.e., recommended by NCCN guidelines for the particular tumor lineage) or off NCCN Compendium™ (i.e., not part of the NCCN guidelines for the particular tumor lineage). FIG. 39B also lists clinical trials which may be available given the molecular profiling results, here trials were matched based on status of PIK3CA, ALK, BRAF, KRAS, EGFR, and GNA11. FIG. 39C illustrates more detailed information for biomarker profiling used to associate agents with potential benefit. FIG. 39D illustrates more detailed information for biomarker profiling used to associate agents with lack of potential benefit. FIG. 39E, FIG. 39F and FIG. 39G illustrate more detailed information for mutations detected by Next Generation Sequencing. FIG. 39H provides a list of genes that were tested without finding alterations. FIG. 38I, FIG. 39J, FIG. 39K, FIG. 39L and FIG. 39M provide a listing of clinical trials matched to the gene alterations that were found. FIG. 39N and FIG. 39O provide a listing of published references used to provide evidence of the biomarker-agent association rules used to construct the report. FIG. 39P presents a disclaimer, e.g., that ultimate treatment decisions reside solely within the discretion of the treating physician. FIG. 38Q provides more information about the mutational analysis performed by Next Generation sequencing. FIG. 39R provides more information about the ISH analysis performed on the patient sample to assess gene rearrangements, which comprised FISH for this tumor. FIG. 39S, FIG. 39T, FIG. 39U, FIG. 39V, FIG. 39W, and FIG. 39X provide a description of the biomarkers assessed per the molecular profiling. FIG. 39Y provides the framework used for the literature level of evidence as included in the report.


Example 9
HER2 Status in Lung Non-Small Cell Carcinomas (NSCLC)

Between 10 and 30% of non-small cell lung cancers (NSCLC) harbor somatic activating mutations in the gene encoding the EGF receptor (EGFR). Tumors with the most common alterations, exon 19 deletions and exon 21 point mutations (L858R), are initially responsive to EGFR tyrosine kinase inhibitors (TKI) such as gefitinib or erlotinib, but eventually acquire resistance. Upon disease progression, more than half of the cases harbor a second-site mutation T790M in EGFR, which alters binding of drug to the ATP-binding pocket. Recently, HER2 amplification was recognized as a novel mechanism of acquired resistance that occurs in a subset of NSCLC lacking the EGFR T790M mutation. This Example investigated simultaneous occurence of EGFR mutations and HER2 gene amplifications.


Molecular profiles of 2271 cases of non-small cell lung cancers obtained according to the methods herein were reviewed for Her2 protein expression via immunohistochemistry, HER2 gene amplification via FISH, and EGFR and KRAS gene mutations via Sanger sequencing.


The molecular profiling revealed that EGFR was mutated in 12%, and KRAS in 32% of cases. HER2 gene amplification (HER2/CEP 17>2.2) was detected in 4% of tested cases (22/589) associated with 3+protein expression. Coexistence of HER2 gene amplification and EGFR mutation was identified in 3 cases (L858R, A859T and E746_A750del); while KRAS was mutated in 7 HER2-amplified cases. HER2 gene amplification is a rare event in non-small cell carcinomas (4%). In no case was HER2 amplification associated with T790M mutation. Double EGFR mutations (L858R/T790M and E746_A750del/T790M) were however found in only 2 cases. NSCLC with HER2 amplification were frequently (39%) associated with KRAS activating mutation. A rare A859T mutation was found in one case and was associated with HER2 gene amplification. This mutation was previously associated with TKI resistance (Han S et al. JCO 2005; no knowledge of HER2 status), but this may be the effect of HER2 gene amplification and not the intrinsic property of the EGFR mutated protein. Since earlier studies have suggested that HER2 amplification may cause resistance to erlotinib and gefitnib, NSCLC patients with HER2 amplification and activating EGFR mutation may respond better to afatinib which inhibits HER2 in addition to EGFR.


In this Example, molecular profiling identified a group of patients that can be expected to have no benefit from targeted therapy aimed at mutated EGFR (tumors with exon 21 point mutations and exon 19 deletions) because they have HER2 gene amplification (and concomitant protein over expression). Molecular profiling also identified association of HER2 amplification with a rare EGFR mutation that was previously considered to be resistance causing, but in the light of these findings and recent literature, this is most likely the result of HER2 amplification.


When devising a targeted treatment strategy in NSCLC, biomarkers evaluation should be comprehensive in order to maximize the benefit and minimize potential side effects of drugs without expected benefits.


Example 10
Integrating Molecular Profiling into Cancer Treatment Decision Making; Experience with Over 30,000 Cases

A limited number of well-defined genetic alterations determine cellular response to chemotherapy and these are the same across many cancer lineages. While these were all found in the common cancers, a limited amount of information exists that associates these geneti c alterations with rare cancers. Contrary to what their name implies, the “rare” cancers as a group constitute some ¼ of all cancer burden. Also, in common cancer types we find new genetic alterations that may have theranostic potential


A number of genetic alterations determine cellular response to chemotherapy. These changes are termed actionable as evidenced by clinical studies showing associations with improved survival or objective response in tumors carrying specific molecular characteristics. Molecular profiling of both common and rare cancer types provides for the identification of potentially actionable targets for chemotherapy with many unexpected associations.


Results of molecular profiling as described herein were stored in a database of >30,000 patients. At least the following biomarkers were assessed for members of the cohort: immunohistochemical results of 12 protein biomarkers (PTEN, AR, ER, PR, ERCC1, PGP, RRM1, SPARCm, SPARCp, TOP2A, TOPO1, TS), fluorescent in-situ hybridization of 8 genes (HER2, c-MET, c-MYC, EGFR, PIK3CA, TOP2A, ALK, ROS1) and sequencing for somatic mutations in 8 genes (BRAF, KRAS, EGFR, PIK3CA, c-KIT, NRAS, GNA11, GNAQ).


All data was deidentified and reviewed for well-established driver gene mutations, gene copy number alterations, and protein expression patterns that are potentially relevant for selection of targeted therapy. Selected genetic abnormalities in each patient's tumor were associated with potential benefit or lack of benefit with specific therapeutic agents based on evidence that exists for such an association in the peer-reviewed medical literature. All relevant published studies were evaluated using the U.S. Preventive Services Task Force (“USPSTF”) grading scheme for study design and validity. Assay methodologies to evaluate tumor genetic abnormalities and their potential theranostic associations included sequencing (Sanger, pyrosequencing), PCR, FISH, CISH, and immunohistochemistry.


All common malignancies (10 most common solid tumor types in men and women) and 10 rare cancer types were represented in the study cohort (minimum of 100 cases and maximum of >4,000 cases in each individual cancer type). Well established driver mutations and protein expression patterns were identified in common cancers with expected frequencies (e.g. HER2 amplification in breast, PIK3CA mutations in ER+ breast cancer, EGFR mutations in NSCLC, etc.). Importantly, unexpected new potentially actionable targets were identified in common cancers (e.g. 6.7% HER2 amplification in NSCLC, 1.6% KRAS mutation in prostatic adenocarcinoma) and rare cancers (e.g. 8.3% ALK alteration in soft tissue sarcomas, 10.5% c-MET and 26.4% EGFR gene amplification in melanomas, 16.3% KRAS mutation cholangiocarcinomas, 10% AR expression in soft tissue sarcomas), as well in cancers of unknown primary site (CUPS; approximately 4% of all tested cases). Thus, molecular profiling according to the invention identified biomarker statuses that can be linked to actionable therapeutic agents in the expected cancer lineages and also in non-standard lineages.


This review of a large referral cancer molecular profiling database provided unparalleled insight into the distribution of common and rare molecular alterations with potential treatment implications. Numerous targets were discovered that had a potential to be treated by the conventional chemotherapy as well as targeted therapy not usually considered for the cancer lineage. Comparison between an individual patient tumor profile and database for the matched cancer type provides additional level of support for targeted treatment choices.


Example 11
Biomarker Analysis of Glioblastoma and the Implications for Therapy

Glioblastoma multiform (GBM), or WHO grade IV malignant astrocytoma, represents the most prevalent and aggressive cancer from the central nervous system. GBM tumors are infiltrative in nature and often remain undetected until complete resection is impossible; therefore untreated patients usually die within 3 months of diagnosis. The challenges of GBM treatment are also presented by the involvement of multiple complex pathways underlying GBM cancer cell biology, which allows treated cancer cells to fast evolve and develop resistance; the drug delivery difficulty presented by the blood-brain barrier; and the regional heterogeneity of the tumor. Standard of care of GBM involves maximal safe surgical resection followed by a combination of radiation and chemotherapy with an oral DNA alkylating agent temozolomide, improving patient survival to approximately 14.6 months.


Almost all GBM patients experience recurrence, for which the standard treatment option is lacking, therefore novel treatment options are in great need. Research shows that distinct genetic events underlie the tumorigenesis and progression of symptomatically similar GBM subtypes, and thus full evaluation of patient's biomarker characteristics should guide treatment choices. A comprehensive genome-wide analysis for biomarkers in 206 GBMs by the Cancer Genome Atlas project identified key critical signaling pathways in GBM including RTK/Ras/PI3K s, p53 and RB, indicating that the genetic aberrations in these pathways may provide insight to guide molecularly targeted therapy. Recent GBM subtyping based on gene expression has shown importance in prognosis and predictivity of treatment response.


The DNA repair enzyme, O-6-methylguanine-DNA methyltransferase or MGMT, is one of the most important biomarkers in glioblastoma, the detection of which, through immunohistochemistry or promoter methylation analysis by pyrosequencing, is important in identifying GBM patients who can benefit from temozolomide. Patients with MGMT promoter methylation when treated with temozolomide have been reported to have median survivals of over 20 months. Clinical data show that 40% of GBM patients are >65 yrs old, presenting worse prognosis and that they are more frequently MGMT methylated. Evaluating MGMT status is particularly important in this patient cohort so that the non-responders identified can be spared the side effect of temozolomide. Presence of MGMT methylation also marks a mismatch repair (MMR)—deficient phenotype, and upon temozolomide treatment further selective pressure is introduced to lose mismatch repair function, causing a MMR-defective hypermutator phenotype, for which selective treatment strategy is needed to prevent the emergence of drug resistance. Thus, stratifying patients based on MGMT methylation status and profiling the biomarkers of each subgroup can suggest more effective combination therapy strategy.


Tumor profiling services using a multi-platform approach as described herein were used to provide a comprehensive analysis for glioblastoma patients and search for biomarker abnormalities in all key pathways identified. Biomarkers previously identified as important for GBM biology (MGMT, PTEN, BRAF) were interrogated through various techniques as described herein, along with over 30 additional biomarker abnormalities not typically suspected for GBM. See, e.g., FIGS. 33A-33B, Table 21 and Table 22. Biomarker analysis provides biomarker evidence to support drug usage that are common in treatment practice, but also proposes novel combination therapy strategies to tackle this challenging tumor type of glioblastoma. Through a thorough retrospective analysis on biomarker data, patient stratification are possible and trends of different treatment options tailored to patient's unique biomarker characteristics will be identified, thus shedding light on individualized medicine to treat this challenging disease.


Methodology and Results:


Biomarker data were analyzed from a cohort of 570 glioblastoma patients who received Tumor Profiling Services from 2009 to 2013 using the methods described herein. Test methodologies included IHC, FISH, CISH, Sanger sequencing, MGMT promoter methylation and NextGen Sequencing (Illumina TruSeq panel). Statistical tools including T-Test were used in analysis.


This study evaluated predictive biomarkers associated with NCCN-recommended therapeutic agents that provided clinicians with decision support in their selection of optimal chemotherapy. In our analysis, from the complete cohort of 570 patients, 492 had MGMT IHC testing, out of whom 344 (70%) patients had negative MGMT expression; 59% of a subgroup of 29 (17) patients were found to carry MGMT promoter methylation tested by pyrosequencing. This study thereby identified a patient subset that may respond better to alkylating agents including temozolomide. Negative ERCC1 expression was seen in 53% (201/376) patients and positive TOPO1 IHC was seen in 49% (178/367), indicating potential benefit from platinum agents cisplatin/carboplatin and irinotecan, respectively.


Biomarkers associated with other chemotherapies commonly used are also evaluated. TS was found to be under-expressed in 37% (132/360) patients, suggesting clinical benefit from fluorouracil. Drug pumps, PGP and MRP1, were overexpressed in 34/338 (10%) and 236/351 (67%) of patients, respectively, indicating possible resistance for their substrates, including etoposide, vinca alkaloids, and methotrexate.


Pathway assessment was performed by various molecular tests to stratify patients for targeted therapies. Ckit was overexpressed in 6.5% (5/77) patients and mutated in 5.6% (2/36), PDGFRA was overexpressed in 27% (57/211) of patients, indicating potential benefit from imatinib. Further, BRAF, KRAS, PIK3CA mutations and PTEN loss were found in 7.8% (11/142), 2.7% (4/149), 6.7% (8/120), and 9.6% (50/519) of patients, respectively, indicating activation of the RAS/RAF pathway and the PIK3CA/mTOR pathway.


Subgroup analysis showed that in patients with MGMT methylation (see Table 39), only 1 out of 19 patients (9%) overexpressed thymidylate synthase (TS), while in patients lacking MGMT methylation, 5 out of 8 patients (63%) overexpressed TS. The differential expression reached statistical significance (p=0.025) and indicated that fluoropyrimidines may be of potential benefit for patients presenting MGMT methylation. A similar trend was observed for RRM1 expression (36% vs. 75% for methylated vs. unmethylated), showing potential benefit of using gemcitabine for MGMT methylated patients.









TABLE 39







Subgroup analysis based on patient MGMT methylation status











MGMT methylated patients
MGMT un-methylated patients





















Positive



Positive
p value


Biomarker
N
+

Percentage
N
+

Percentage
(t-test)



















AR
15
2
13
0.13
12
0
12
0.00
0.16


cMET
15
1
14
0.07
13
1
12
0.08



ER
15
0
15
0.00
11
0
11
0.00



ERCC1
11
3
8
0.27
8
2
6
0.25



Her2
15
0
15
0.00
11
0
11
0.00



MGMT
1
0
1
0.00
1
0
1
0.00



PR
15
0
15
0.00
11
0
11
0.00



PTEN
15
13
2
0.87
12
12
0
1.00
0.16


RRM1
11
4
7
0.36
8
6
2
0.75
0.073


SPARC
15
1
14
0.07
12
4
8
0.33



mono











SPARC
15
4
11
0.27
12
2
10
0.17



poly











Sparc both
15
5
10
0.33
12
5
7
0.42



TLE3
15
6
9
0.40
12
5
7
0.42



TOPO1
15
6
9
0.40
8
5
3
0.63
0.3341


TS
11
1
10
0.09
8
5
3
0.63
0.025


ALK FA
5
0
5
0.00
5
0
5
0.00



BRAF
12
2
10
0.17
15
0
15
0.00
0.17


cKIT SEQ
11
0
11
0.00
11
0
11
0.00



KRAS
12
1
11
0.08
14
0
14
0.00
0.34


NRAS
10
0
10
0.00
11
1
10
0.09



PIK3CA
11
0
11
0.00
10
0
10
0.00









Conclusions:


A retrospective biomarker analysis of 570 glioblastoma patients who received tumor profiling services from 2009 to date was performed to search for clinical implications to support the usages of treatment regimens within and outside of standard of care.


From the robust biomarker evaluation performed using multiple platforms, these data show that 59% of patients are good responders to temozolomide, 53% to platinum agents and 49% to irinotecan. These agents all have shown to cross blood brain barrier and are recommended by NCCN; evaluating biomarker status will substantially assist the clinician in treatment selection.


Various targeted therapies are in different phases of clinical trials and our data provide biomarker information to stratify patients into trials where they can benefit. About 7% of patients show PIK3CA mutation and 10% shows a loss of PTEN, indicating a constitutive activated PIK3CA pathway, therefore may benefit from mTor inhibitors. About 8% of patients are shown to have a BRAF mutation and 3% to harbor KRAS mutation, indicating the dysregulation of RAS/RAF/MEK/ERK pathway, presenting a potential benefit of targeted agents including MEK inhibitors. Multi-targeted tyrosine kinase inhibitors including imatinib are being extensively studied in clinical trials, and these data indicate that 6.5% patients overexpress cKIT, 27% overexpress PDGFRA and 5.6% carry a cKIT mutation, and can potentially benefit.


The substrates of drug pumps PGP and MRP1 are different but overlap. PGP and MRP1 were overexpressed in 10% and 67% of patients in this study, showing that evaluation of drug pump level may be important when common drugs etoposide, vincristine (substrate of both) or methotrexate (substrate of MRP1) is applied.


From a subgroup analysis based on patient MGMT methylation status, differential biomarker expression pattern is noticeable and the most significant result is from a distinct expression of thymidylate synthase between the two groups (1 in 11, or 9% in MGMT methylated cohort vs. 5 in 8, or 63% in MGMT unmethylated cohort, p=0.025). See Table 39. This result provides biomarker evidence to propose a novel combination therapy to treat MGMT methylated GBM patients by alkylating agents and fluoropyrimidines. Synergistic treatment effect of temozolomide and 5-FU pro-drug capecitabine has been reported in pancreatic endocrine carcinomas. Fluorouracil and capecitabine can readily cross blood brain barrier, and the data here support their usage in conjunction with temozolomide in MGMT methylated GBM patient cohort. This finding shows great potential of using biomarker data and evidence-based association to provide guidance for clinical research and eventually for clinical practice.


Example 12
Use of Different Methodologies for Detecting EGFR Mutations in Selecting Chemotherapy for Patients with Lung Cancer

Mutation analysis of the kinase domain of EGFR (exons 18-21) is a standard recommended procedure for patients diagnosed with non-small cell lung cancer (NSCLC). NSCLC patients whose tumor harbors certain EGFR mutations have notable responses to EGFR inhibitors. Several different methodologies are available as published assays or commercially available kits and include Sanger Sequencing, allele specific PCR (ASP) and restriction fragment length polymorphism (RFLP). Differences in the design of these assays dictate which clinically relevant mutations will be detected.


Methods:


Sanger Sequencing of EGFR found 518 potentially clinically actionable mutations and 45 variants of unknown significance of the 4307 samples tested. To assess which clinically relevant EGFR mutations will be detected by the ASP and RFLP, we performed an in silico analysis of all observed mutations against the design specification of the ASP and RFLP assays. The RFLP assay used in this assessment was developed in our laboratory and is designed to detect all G719 mutations, all exon 19 deletions, all exon 20 insertions and the specific mutations T790M, L858R, L861R and L861Q. A commercially available ASP kit designed to detect 29 mutations in the kinase domain was also analyzed in this study.


Results:


Based on the performance characteristics assumed by the RFLP and ASP assays, the analytical sensitivities for detecting clinically actionable mutations of the two methodologies are 98.8% and 86.7% respectively, when compared to Sanger Sequencing. Among the 1.2% of mutations not detected by RFLP, the assay missed the S768I mutation (0% detected) and did not detect some G719 mutations, exon 19 deletions, and exon 20 insertions (76.3, 86.6 and 35.3% were detected, respectively). The ASP method did not detect 13.3% of the mutations detected by sequencing.


Conclusion:


Sequencing is still the preferred method of mutation detection in EGFR for NSCLC as it is the most comprehensive. However, if tumor nuclei are limited, then a more sensitive method than sequencing is required and, EGFR mutation detection by RFLP identies more potentially clinically relevant mutations than ASP as the ASP method would produce false negative results in 13.5% of patients expected to respond to EGFR inhibitors.


Example 13
Molecular Profiling Panels


FIGS. 34A-34C illustrate biomarkers assessed using a molecular profiling approach as outlined in FIGS. 33A-Q, Tables 7-25, and accompanying text herein. FIG. 34A illustrates biomarkers that are assessed. The row labeled MI Profile™ does not include the Next Generation sequencing panel. The row labeled MI Profile™ Plus includes the Next Generation sequencing panel. The biomarkers that are assessed according to the Next Generation sequencing panel are shown in FIG. 34B. FIG. 34C illustrates sample requirements that can be used to perform molecular profiling on a patient tumor sample according to the panels in FIGS. 34A-34B.


Example 14
Assessment of cMET by IHC, FISH, and Next Generation Sequencing

cMET overexpression and/or activation have been implicated in signaling pathways that promote cell proliferation, invasion, and survival. cMET is an oncogenic driver in various malignancies and is a potential therapeutic target. This Example assesses the distribution of cMET expression by immunohistochemistry (IHC), cMET amplification by FISH, and cMET mutation by next generation sequencing (NGS) across a variety of tumor types. This Example further assesses the correlation of cMET across technology platforms as performed in a CLIA-certified oncology reference laboratory.


In a cohort of 9161 patient samples, cMET protein expression was assayed by IHC (NCL-cMET and SP44, 9161 samples), FISH (BAC clone, 7435 samples) and NGS (Illumina Truseq Amplicon-Cancer Panel, 3163 samples).


This analysis found the highest cMET expression rates in the following tumor types: pancreatic cancer (56%, 231 out of 411), cholangiocarcinoma (51%, 63 out of 123), extrahepatic bile duct cancer (50%), small intestinal cancer (49%), colorectal cancer (46%), uveal melanoma (43%), gastroesophageal cancer (36%), gastric cancer (34%), and non-small cell lung cancer (33%), and head and neck cancer (32%). The lowest expression rates of cMET by IHC included non-epithelial ovarian cancer (6%, 5/84), glioblastoma (6%, 11/198), neuroendocrine tumors (6%, 18/296), prostate cancer (7%) and soft tissue malignancies (9%). Analysis of cMET by FISH identified the highest levels amplification in peritoneal/retroperitoneal sarcomas (9%, 2/23), non-small cell lung cancers (7%, 65/983), and melanoma (7%, 12/165). In 3163 samples tested by NGS platform, only 9 mutations were identified—all were variants of unknown significance and five were detected in non-small cell lung cancer specimens. The corresponding exon and protein changes in these five samples were as follows: D1028H (exon 14) in three samples; G391A (exon 2) and S203T (exon 2) in one sample each. The other four had the following mutations: S203T (exon 2) and G391E (exon 2) in melanoma; S203T (exon 2) in colorectal cancer; and K1121N (exon 16) in female genital tract malignancy. The highest percentage agreement between IHC and FISH was observed in the following lineages: pancreatic cancer (50.8%), cholangiocarcinoma (52.8%), colorectal cancer (64.5%), non-small cell lung cancer (66.1%), and gastric cancer (67.0%). Of those specimens with mutated cMET, five of nine were positive by IHC and none by FISH.


The data in the Example show that cMET overexpression and/or activation is prevalent in various malignancies. Ongoing clinical trials targeting cMET suggest that efforts should be made to accurately interrogate tumors for cMET testing. As shown by the FISH-IHC concordance data, cMET analysis is enhanced when assessed using multiple technologies.


Example 15
Molecular Profiling in Gastric Cancer

Current NCCN guidelines recommend perioperative epirubicin (E), cisplatin (C), and 5-fluorouracil (F) along with other triple agent derivations as first line therapeutic approaches for operable gastric adenocarcinoma (GC). In this Example, molecular profiling was used to evaluate chemotherapy targeted biomarkers associated with ECF therapy for GC.


Surgically obtained GC specimens were analyzed by immunohistochemistry for TOP2A, TS, and ERCC1 expression as described herein. Actionable gene targets were analyzed for mutually exclusive or simultaneous expression.


A total of 230 GC specimens were analyzed. The median age of patients was 61 (IQR: 50-72) years with the majority being male (n=139, 60%). IHC actionable targets included: 60% (n=138) high TOP2A, 63% (n=145) negative TS, and 55% (n=127) negative ERCC1, indicating potential benefit from E, C, and F respectively. Overall, over 90% of specimens showed expression of at least one of TOP2A, TS and ERCC1, indicating sensitivity to at least one of E, C and F. When analyzing for simultaneous expression profiles of the three genes, 24% (n=55) of patients had gene expression levels that suggested sensitivity to all three agents (ECF), whereas 6.5% (n=15) of patients expressed no actionable targets demonstrating a potential lack of sensitivity to first line ECF therapy. Overall, 61% (n=140) of patients had molecular profiles that indicated sensitivity to two or more agents.


Biomarker analysis of GC suggests that 76% of patients do not possess molecular profiles that reflect complete sensitivity to standard front-line ECF therapy. Further biomarker analysis to identify actionable targets associated with alternative chemotherapies is indicated.


Example 16
Molecular Profiling of Neuroendocrine Carcinomas Using Next Generation Sequencing

Neuroendocrine carcinomas are poorly understood and rare form of malignancies with highly variable clinical course. This Example presents a systematic analysis of 1250 cases that have been assessed by molecular profiling in a CLIA certified laboratory in order to identify biomarkers of drug sensitivity. The molecular profiling used a combination of immunohistochemistry (IHC), copy number analysis and sequencing of certain oncogenes based on their relevance to existing cancer therapies. Identification of a pathogenic pathway may provide for a druggable target in neuroendocrine tumors (NET), regardless of histologic classification or primary organ site.


Of 1250 cases, molecular profiling identified actionable alterations in 90% of analyzed cases (1130/1250). Low expression of MGMT, a potential marker of sensitivity to alkylating agents, was found in 100/219 pancreatic cases (46%). Sequencing of tumors showed mutations in: BRAF (4/369 (V600E in 3 and G596R in 1)), CTNNB1 (2/150), KIT (3/281), EGFR (1/178), FGFR2 (1/150), GNAS (1/150), HRAS (2/150), PIK3CA (6/343), RB (2/150) VHL (1/150), KRAS (10/125), NRAS (2/274), and APC (2/150). Gene amplifications found were: MET (4/236) and EGFR (46/686). Other biomarkers identified included high expression of RRM1 in 244/1100 tumors by IHC.


In several cases, dramatic responses to marker-guided therapy have been documented thus supporting the clinical relevance of molecular profiling in neuroendocrine carcinomas.


Assessment of neuroendocrine tumors with multiplatform molecular profiling revealed diverse biomarkers of drug response. Despite seemingly low frequency of individual biomarkers, the comprehensive evaluation of NET identified clinically relevant targets in the majority of patients.


Example 17
Molecular Profiling of Gynecological Tumors

In this Example, molecular profiling is used to determine biomarker status and predict drug response in various gynecological cancers, including primary, metastatic and recurrent endometrial, ovarian and cervical cancers.


Markers assessed according to the invention include without limitation phosphatidylinositide 3-kinases, HER 2, EGFR, CMET, K-RAS, BRCA, TUBB3, ER, PR, FBXW7. The markers are assessed for gene expression, protein expression, gene copy number, and/or mutational status as described herein.


Example 18
Molecular Profiling of Advanced Refractory Prostate Cancer

Prostate cancer is the second leading cause of cancer-related death among men in the U.S. Forty percent of men diagnosed will develop metastatic disease which has few treatment options. This Example describes molecular profiling of prostate cancer tumors and potential therapeutic options.


We reviewed profiling data of over 330 patients from a large referral laboratory (Caris Life Sciences, Phoenix, Ariz.) for information on biomarkers of drug response. Multiple methodologies were employed: sequencing (Next Generation (NGS), Sanger, pyrosequencing), in-situ hybridization (fluorescent (FISH) and chromogenic (CISH)) and immunohistochemistry (IHC). High expression was observed for AR, MRP1, TOPO1, TLE3 and EGFR, with positivity rates of 89%, 87%, 63%, 48% and 47%, respectively. Low expression was observed for TS, PGP, TUBB3, RRM1, PTEN and MGMT, with negativity rates of 94%, 87%, 75%, 69%, 54% and 45%, respectively. Gene copy number increases for EGFR and cMYC were observed in 13% of patients. Sequencing data showed 48% mutation rate for TP53, 18% for PTEN, 9% for CTNNB1, 8% for PIK3CA, 5% for RB1, ATM and cMET, and ˜2% for K/HRAS, ERBB4, ALK, BRAF and cKIT. Regarding targeted therapy options, imatinib may be considered for patients with high cKIT or PDGFRA (9-10%), and cetuximab for patients with EGFR positivity (13-47%). Promising agents may be considered, including cabozantinib, based on 4% of cohort with cMET aberrations or PAM pathway inhibitors (BEZ234, everolimus) based on ˜30% of cohort with PIK3CA pathway activation. Lastly, HDAC inhibitors have recently been linked to cMYC driven cancers (13% amplified). Chemotherapies including 5-FU, gemcitabine and temozolomide may be options based on ˜70% of cohort with low TS, RRM1 or MGMT. Biomarker guidance for common prostate cancer drugs is also provided, including cabazitaxel, based on ˜70% of cohort with low TUBB3 or PGP, or high TLE3. Finally, continued dependence on androgen signaling is exhibited by 89% of cohort with high AR, indicating potential utility of anti-androgen agents like enzalutamide.


Tumor profiling identified subsets of patients that may benefit from targeted agents approved for other solid tumors (e.g., imatinib, cetuximab), promising therapies in clinical trials (e.g., cabozantinib) or agents not routinely used for prostate cancer (e.g., gemcitabine).


Example 19
Therapeutic Implications of Ras-ERK and PI3k-mTOR Pathway Profiling in Solid Tumors

Ras-ERK and PI3K-mTOR pathways are key regulators of cell proliferation, differentiation, survival, migration and metabolism. Alterations of these pathways are commonly seen in cancer pathogenesis. As Next Generation Sequencing (NGS) platforms become more accessible to physicians in clinical care settings, the use of highly multiplexed mutational analysis for personalized medicine is on the rise. Molecular profiling of multiple signaling pathways can provide a basis for selecting targeted single agents or combination cancer therapy for treating cancer patients.


In this Example, biomarker components of the Ras-ERK pathway were tested by NGS in a cohort of tumor samples. Genes assessed by NGS included KRAS, NRAS, HRAS and BRAF. Genes involved in the PI3K-mTOR pathway tested by NGS included PIK3CA, PTEN, AKT1 and STK11. NGS was performed using the Trueseq Amplicon Cancer Panel using Illumina's Miseq platform (Illumina Corp., San Diego, Calif.). Formalin-fixed paraffin-embedded tissue sections from 2520 patients were subjected to DNA extraction and NGS. Immunohistochemistry (IHC) using anti-PTEN clone 6H2.1 (Dako North America, Inc., Carpinteria, Calif. 93013) was used to analyze PTEN protein expression.


Among 2520 cancer samples, a higher frequency of mutations in the mTOR pathway over that of ERK was observed for breast cancer (56% cases mutated in the mTOR pathway vs 0.70% cases mutated in the ERK pathway), endometrial cancer (52.8% mTOR vs 2.8% ERK), ovarian surface epithelial carcinoma (21.7% mTOR vs 6.8% ERK), which may explain the success of mTOR inhibitors in these female prevalent/restricted cancers. Significant bias towards ERK pathway was observed for pancreatic adenocarcinoma (4.9% mTOR vs 51.0% ERK), and a near significant trend towards the ERK pathway was seen for melanoma (12.9% mTOR vs 29.7% ERK). Colorectal adenocarcinoma and pancreatic adenocarcinoma were more likely to have alterations in both ERK and mTOR pathways compared with other tumor types. When NGS data was used instead of IHC for PTEN analysis, there were significantly fewer cases with PTEN alterations, highlighting the potential advantage of using both NGS and IHC to evaluate PTEN status.


Pathway profiling reveals mTOR bias in female prevalent/restricted tumors and ERK bias in colorectal adenocarcinoma. Colorectal adenocarcinoma and pancreatic adenocarcinoma have a tendency to have mutations in genes of both mTOR and ERK pathways, suggesting dual mTOR and ERK inhibitor therapy might be effective in these tumor types. Success of mTOR inhibitors in breast and endometrial cancers may also be a result of the low rate of ERK pathway activation.


Example 20
Concordance Between PTEN Protein Expression and Gene Mutations in a Large Cohort of Cancer Patients

PTEN is a tumor suppressor gene in the cancer signaling pathway downstream of EGFR. Loss of PTEN protein expression is one of the more common occurrences in human cancers, and its loss potentially reduces the benefit from trastuzumab, EGFR-targeted therapies, and mTOR inhibitors. Loss of PTEN is usually assessed with immunohistochemistry (IHC). Mutation analysis of PTEN gene has been recently introduced in clinical use. In this Example, we compared the concordance between PTEN by IHC and PTEN sequencing technologies in a large cohort of patients with various types of cancer.


1636 patients and 29 tumor types were utilized in this study. NGS was performed using the Trueseq Amplicon Cancer Panel using Illumina's Miseq platform (Illumina Corp., San Diego, Calif.) that employs 7 amplicons to sequence exons 1, 3, 6, 7, and 8 of PTEN gene. Immunohistochemistry was performed using the anti-PTEN clone 6H2.1 (Dako North America, Inc., Carpinteria, Calif. 93013).


Overall, 5% of the samples contained mutations in the PTEN gene. Of the 83 variations identified, 46% were frameshift, 29% nonsense, 23% missense, 1% inframe deletion, and 1% affecting splicing. When compared to IHC results, a significantly larger number (30% or 481 out of 1636) of patients lacked PTEN protein expression (defined as less than 50% tumor cells staining positive). 26% of the samples that were called wild type by sequencing did not show PTEN expression and 32% of the samples that contained a mutation in PTEN expressed PTEN by IHC. Among PTEN mutations, the largest discrepancy was seen with missense mutations at 31%. In contrast, of the negatively stained samples, only 13% were called mutant by sequencing whereas 96% of samples that stained positive by IHC were called wild type by sequencing.


These observations reveal low correlation between sequencing and IHC results for PTEN. These data suggest that neither the IHC nor sequencing alone have a full capability to predict PTEN status, but when combined they provide a more complete assessment of PTEN status. Additional methods (methylation assays, LOH assays) can be used to further assess PTEN status in patients with cancer.


Example 21
Practical Issues in Identifying and Communicating Incidental and Unexpected Findings Arising from Mutation Analysis Utilizing Next Generation Sequencing in Patients with Cancer

With the maturation of next generation sequencing (NGS) platforms in clinical diagnostics, there is a wealth of data that is generated in a time efficient and cost effective manner. One consequence of generating increased amounts of clinical data is the detection of incidental and/or unintended findings. A key consideration for many clinical labs is how to report or communicate these incidental findings to the ordering physician. Recently the ACMG released Guidelines for reporting incidental findings, however, these Guidelines may not meet the needs of a reference laboratory focused on molecular profiling of tumors. We report our experience with the identification of incidental and unexpected findings using NGS in over 3,000 specimens from patients with various types of cancer and we identify the need to consider modification of Guidelines on the reporting of such findings.


Mutation analysis was performed using the Truseq Amplicon Cancer Panel (Illumina) to determine the mutation status of select regions of 44 genes (detailed above, see, e.g., Table 25 and related disclosure). Ordering physicians have the ability to order mutation analysis for single genes or a combination of genes that the physician determined to be medically necessary. For all genes not reported, all mutation positive results are evaluated by a clinical geneticist to determine if the case merits further review. Mutations that have potential implications for clinical trials, potential germ line inheritance, therapeutic response guidance, or those that may help in determining a diagnosis are identified and discussed by a multidisciplinary team including geneticists, pathologists, and literature review scientists.


In order to appropriately identify patients with potential germ line inheritance of a mutation, we employed several criteria that included age at cancer diagnosis, allele frequency of the mutation, and the gene that is mutated.


In our analysis of over 3,000 samples that received mutation analysis by NGS, ˜75% of cases did not report results for all 44 genes. Of those cases, we identified 9 potentially eligible for clinical trial enrollment, 11 with potential germ line inheritance, 5 with diagnostic uncertainty, and 3 with potential FDA approved therapy implications. Two of the cases associated with diagnostic uncertainty resulted in a change of diagnosis following a consultation with the ordering physician and pathologist.


Establishing a standard procedure for addressing incidental or unexpected findings in oncology will be necessary as more reference laboratories adopt NGS platforms. Using our current method of identifying incidental findings ˜1% of cases are identified for review making this procedure tenable for high throughput oncology labs.


Example 22
Detecting EGFR Mutations in Selecting Chemotherapy for Patients with Lung Cancer

Mutation analysis of the kinase domain of EGFR (exons 18-21) is a standard recommended procedure for patients diagnosed with non-small cell lung cancer (NSCLC). NSCLC patients whose tumor harbors certain EGFR mutations have notable responses to EGFR inhibitors. Several different methodologies are available as published assays or commercially available kits and include Sanger Sequencing, allele specific PCR (ASP) and restriction fragment length polymorphism (RFLP). Differences in the design of these three assays dictate which clinically relevant mutations will be detected.


In this Example, tumor samples were assessed using various EGFR analysis methods. Sanger Sequencing of EGFR found 518 potentially clinically actionable mutations and 45 variants of unknown significance of 4307 samples tested. To assess which clinically relevant EGFR mutations will be detected by the ASP and RFLP, we performed an in silico analysis of all observed mutations against the design specification of the ASP and RFLP assays. The RFLP assay used in this assessment was developed in our laboratory and is designed to detect all G719 mutations, all exon 19 deletions, all exon 20 insertions and the specific mutations T790M, L858R, L861R and L861Q. A commercially available ASP kit designed to detect 29 mutations in the kinase domain was also analyzed in this study.


Based on the performance characteristics assumed by the RFLP and ASP assays, the analytical sensitivities for detecting clinically actionable mutations of the two methodologies are 98.8% and 86.7% respectively, when compared to Sanger Sequencing. It is notable that the RFLP assay missed the S7681 mutation (0% detected) whereas the assay did not detect some G719 mutations, exon 19 deletions, and exon 20 insertions (76.3, 86.6 and 35.3% detected respectively).


Sequencing is still the preferred method of mutation detection in EGFR for NSCLC as it is the most comprehensive. However, if tumor nuclei are limited, a more sensitive method than sequencing is required and, EGFR mutation detection by RFLP would identify more of the potentially clinically relevant mutations than ASP as the ASP method would produce false negative results in 13.5% of patients expected to respond to EGFR inhibitors.


Example 22
ERBB2 (HER2) Mutation Spectrum in Solid Tumors

The ERBB2 gene which encodes for Her2 is a major proliferative driver for several cancer types. Gene amplification and protein expression is associated with sensitivity to Her2-targeting drugs. In some types of cancer, ERBB2 mutations may be more clinically relevant than ERBB2 results measured by gene amplification or protein expression.


The mutation spectrum of ERBB2 in solid tumors is relatively unknown. The emergence of NGS methodology has enabled high throughput detection of both known and novel oncogenic mutations in human genome including the presence of activating mutations of ERBB2.


In this Example, comprehensive genomic profiling was performed on tumors from 2962 cancer patients. These include 319 breast, 346 colorectal (CRC), 358 lung (NSCLC), 299 uterine/cervical, 543 ovarian, 128 pancreatic cancers, 126 melanoma and 843 other solid tumors (e.g. glioblastoma, sarcomas, bladder carcinoma etc.) Direct sequence analysis of ERBB2 was performed on genomic DNA isolated from a formalin-fixed paraffin-embedded tumor sample using the Illumina MiSeq platform. Specific regions of the genome were amplified using the Illumina TruSeq Amplicon Cancer Hotspot panel. The HER2 protein expression and gene copy numbers were determined by immunohistochemistry and chromogenic in situ hybridization (CISH), respectively.


ERBB2 mutations in the kinase domain were detected in 30 patients (1% of all cases). These include previously published activating mutations (P780_Y781insGSP; V842I, L755S, V777L, D769Y) and several novel ones (such as 1767 F, R784C). 6 cases with coexisting HER2 amplification included: D769Y (breast), D769H (bladder), D769Y and T862A (ovary), and two cases with V777L (CRC). 25 of the 30 patients also had additional gene mutations (e.g. TP53, APC, PIK3CA, PTEN, KRAS). Five (17%) patients had ERBB2 mutation identified as the sole driver mutation, including L755S in CRC, breast and ovarian cancer, D769H in bladder cancer and D769E in NSCLC.


These data suggest that ERBB2 mutation might be a driver mutation in various solid tumors including breast, ovarian, CRC, NSCLC. Her2 protein overexpression was observed only when the ERBB2 gene was amplified (5/6 cases) but not in any of the ERBB2 mutated non-amplified cases (0/24). Activating ERBB2 mutations can coexist with ERBB2 gene amplification (6/30=20%) and with mutations in other key driver genes (24/30=80%).


Example 23
Androgen Receptor Profiling in Various Tumors

In this Example, expression of the androgen receptor (AR) was queried in various tumors and correlated to expression and/or mutation of other commonly assessed cancer biomarkers. AR expression was determined using IHC or gene expression profiling (microarray and/or RT-PCR) as described herein.


1) GIST: in ˜170 cases, observed 6% with both AR positivity and c-KIT mutation


2) Kidney: 14% AR positivity in ˜550 kidney cases


3) HCC: 16% AR positivity in ˜270 HCC cases


4) Non-epithelial ovarian cancer: 26% AR positivity (˜250 non-EOC cases; 26 Leydig cases)


Lower coincidence of AR expression was observed with the following: EGFR mutation in NSCLC, cKIT mutation in GIST, and Her2 mutation or overexpression in gastric cancer.


Example 24
EGFRvIII Mutation Detection by Fragment Analysis

EGFRvIII is a mutated form of the epidermal growth factor receptor protein (EGFR) that contains a deletion of exons 2 through 7 on the extracellular ligand binding domain, which confers ligand-independent activation of EGFR. The tumorigenicity of EGFRvIII and its tumor-specific expression make it an attractive therapeutic target, and various therapeutic agents targeting this variant are being investigated in different stages of clinical trials.


EGFRvIII Fragment Analysis uses RNA extracted from formalin-fixed paraffin-embedded (FFPE) tissues in a Reverse Transcription reaction, followed by Polymerase Chain Reaction (PCR) and subsequent capillary electrophoresis on the ABI 3500xL Genetic Analyzer.


Mutation Analysis of EGFRvIII using Fragment Analysis will be performed on FFPE tissue samples. This assay has the sensitivity to detect EGFRvIII deletions down to 20% mutation; therefore, patient tissue typically contains 20% or more, e.g., ≧50%, tumor nuclei for patient testing.


This assay detects mRNA with a deletion of EGFR exons 2-7 as well as wild-type EGFR transcripts within the exon 2-7 region. Two sets of primers in a single reaction are used to amplify cDNA of wild-type EGFR (89 base pair fragment) and EGFRvIII (98 base pair fragment). If the EGFRvIII fragment is not detected, the wild-type EGFR fragment confirms that the reaction was successful.


References, each of which is incorporated herein in its entirety:


1) Gan, H K., et al. 2009 “The EGFRvIII variant in glioblastoma multiforme” J Clin Neurosci 16(6):748-54


2) Sampson, J., et al. 2010 “Immunologic escape after prolonged progression-free survival with epidermal growth factor receptor variant III peptide vaccination in patients with newly diagnosed glioblastoma.” J Clin Oncol 28(31): 4722-9


3) Sampson, J., et al. 2011 “Greater chemotherapy-induced lymphopenia enhances tumor-specific immune responses that eliminate EGFRvIIJ-expressing tumor cells in patients with glioblastoma.” Neuro Oncol 13(3): 324-33


4) Scott, A A., et al. 2007 “A phase I clinical trial with monoclonal antibody ch806 targeting transitional state and mutant epidermal growth factor receptors” Proc Natl Acad Sci USA. 104(10):4071-6


5) Jeuken, J., et al. 2009 “Robust Detection of EGFR Copy Number Changes and EGFR Variant III: Technical Aspects and Relevance for Glioma Diagnostics” Brain Pathology 19: 661-671


Although preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims
  • 1. A method of identifying one or more candidate treatment for a cancer in a subject in need thereof, comprising: (a) determining a molecular profile for a sample from the subject by assessing a panel of gene or gene products, wherein the panel of gene or gene products are assessed as indicated in Table 21, FIG. 33A or FIG. 33B; and(b) identifying one or more treatment that is beneficially associated with the molecular profile of the subject, and optionally one or more treatment associated with lack of benefit, according to the determining in (a) and one or more rules in Table 22, thereby identifying the one or more candidate treatment.
  • 2. The method of claim 1, wherein the panel of gene or gene products comprises ABL1, AKT1, ALK, APC, AR, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ER, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HER2, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MGMT, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PGP, PIK3CA, PR, PTEN, PTPN11, RB1, RET, RRM1, SMAD4, SMARCB1, SMO, SPARC, STK11, TLE3, TOP2A, TOPO1, TP53, TS, TUBB3 and VHL.
  • 3. The method of claim 1, wherein assessing the panel of gene or gene products comprises using ISH to assess cMET and HER2.
  • 4. The method of claim 1, wherein assessing the panel of gene or gene products comprises using IHC to assess AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3.
  • 5. The method of claim 1, wherein assessing the panel of gene or gene products comprises using sequence analysis to assess ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL.
  • 6. The method of claim 1, wherein assessing the panel of gene or gene products comprises using ISH to assess cMET and HER2; using IHC to assess AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3; and/or comprises using sequence analysis to assess ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL.
  • 7. The method of claim 5 or 6, wherein assessing the panel of gene or gene products comprises using sequence analysis to assess CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1, PTPN11, RB1, SMAD4, SMARCB1 and STK11.
  • 8. The method of any of claims 5-7, wherein the sequence analysis comprises Next Generation Sequencing.
  • 9. The method of any preceding claim, wherein the panel of gene or gene products comprises the androgen receptor (AR).
  • 10. The method of claim 9, wherein the one or more candidate treatment comprises an antiandrogen.
  • 11. The method of claim 10, wherein the antiandrogen suppresses androgen production and/or inhibits androgens from binding to AR.
  • 12. The method of claim 10 or 11, wherein the antiandrogen comprises one or more of abarelix, bicalutamide, flutamide, gonadorelin, goserelin, leuprolide, nilutamide, a 5-alpha-reductase inhibitor, finasteride, dutasteride, bexlosteride, izonsteride, turosteride, and epristeride.
  • 13. The method of claim 9, wherein the cancer is androgen independent.
  • 14. The method of claim 13, wherein the one or more candidate treatment comprises one or more of a CYP17 inhibitor, CYP17A1 inhibitor, chemotherapeutic agent, antiandrogen, an endocrine disruptor, immunotherapy, and bone-targeting radiopharmaceutical.
  • 15. The method of any preceding claim, wherein the cancer comprises an acute lymphoblastic leukemia; acute myeloid leukemia; adrenocortical carcinoma; AIDS-related cancer; AIDS-related lymphoma; anal cancer; appendix cancer; astrocytomas; atypical teratoid/rhabdoid tumor; basal cell carcinoma; bladder cancer; brain stem glioma; brain tumor, brain stem glioma, central nervous system atypical teratoid/rhabdoid tumor, central nervous system embryonal tumors, astrocytomas, craniopharyngioma, ependymoblastoma, ependymoma, medulloblastoma, medulloepithelioma, pineal parenchymal tumors of intermediate differentiation, supratentorial primitive neuroectodermal tumors and pineoblastoma; breast cancer; bronchial tumors; Burkitt lymphoma; cancer of unknown primary site (CUP); carcinoid tumor; carcinoma of unknown primary site; central nervous system atypical teratoid/rhabdoid tumor; central nervous system embryonal tumors; cervical cancer; childhood cancers; chordoma; chronic lymphocytic leukemia; chronic myelogenous leukemia; chronic myeloproliferative disorders; colon cancer; colorectal cancer; craniopharyngioma; cutaneous T-cell lymphoma; endocrine pancreas islet cell tumors; endometrial cancer; ependymoblastoma; ependymoma; esophageal cancer; esthesioneuroblastoma; Ewing sarcoma; extracranial germ cell tumor; extragonadal germ cell tumor; extrahepatic bile duct cancer; gallbladder cancer; gastric (stomach) cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal cell tumor; gastrointestinal stromal tumor (GIST); gestational trophoblastic tumor; glioma; hairy cell leukemia; head and neck cancer; heart cancer; Hodgkin lymphoma; hypopharyngeal cancer; intraocular melanoma; islet cell tumors; Kaposi sarcoma; kidney cancer; Langerhans cell histiocytosis; laryngeal cancer; lip cancer; liver cancer; malignant fibrous histiocytoma bone cancer; medulloblastoma; medulloepithelioma; melanoma; Merkel cell carcinoma; Merkel cell skin carcinoma; mesothelioma; metastatic squamous neck cancer with occult primary; mouth cancer; multiple endocrine neoplasia syndromes; multiple myeloma; multiple myeloma/plasma cell neoplasm; mycosis fungoides; myelodysplastic syndromes; myeloproliferative neoplasms; nasal cavity cancer; nasopharyngeal cancer; neuroblastoma; Non-Hodgkin lymphoma; nonmelanoma skin cancer; non-small cell lung cancer; oral cancer; oral cavity cancer; oropharyngeal cancer; osteosarcoma; other brain and spinal cord tumors; ovarian cancer; ovarian epithelial cancer; ovarian germ cell tumor; ovarian low malignant potential tumor; pancreatic cancer; papillomatosis; paranasal sinus cancer; parathyroid cancer; pelvic cancer; penile cancer; pharyngeal cancer; pineal parenchymal tumors of intermediate differentiation; pineoblastoma; pituitary tumor; plasma cell neoplasm/multiple myeloma; pleuropulmonary blastoma; primary central nervous system (CNS) lymphoma; primary hepatocellular liver cancer; prostate cancer; rectal cancer; renal cancer; renal cell (kidney) cancer; renal cell cancer; respiratory tract cancer; retinoblastoma; rhabdomyosarcoma; salivary gland cancer; Sezary syndrome; small cell lung cancer; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma; squamous neck cancer; stomach (gastric) cancer; supratentorial primitive neuroectodermal tumors; T-cell lymphoma; testicular cancer; throat cancer; thymic carcinoma; thymoma; thyroid cancer; transitional cell cancer; transitional cell cancer of the renal pelvis and ureter; trophoblastic tumor; ureter cancer; urethral cancer; uterine cancer; uterine sarcoma; vaginal cancer; vulvar cancer; Waldenstrdm macroglobulinemia; or Wilm's tumor.
  • 16. The method of any preceding claim, wherein the cancer comprises an acute myeloid leukemia (AML), breast carcinoma, cholangiocarcinoma, colorectal adenocarcinoma, extrahepatic bile duct adenocarcinoma, female genital tract malignancy, gastric adenocarcinoma, gastroesophageal adenocarcinoma, gastrointestinal stromal tumor (GIST), glioblastoma, head and neck squamous carcinoma, leukemia, liver hepatocellular carcinoma, low grade glioma, lung bronchioloalveolar carcinoma (BAC), non-small cell lung cancer (NSCLC), lung small cell cancer (SCLC), lymphoma, male genital tract malignancy, malignant solitary fibrous tumor of the pleura (MSFT), melanoma, multiple myeloma, neuroendocrine tumor, nodal diffuse large B-cell lymphoma, non epithelial ovarian cancer (non-EOC), ovarian surface epithelial carcinoma, pancreatic adenocarcinoma, pituitary carcinomas, oligodendroglioma, prostatic adenocarcinoma, retroperitoneal or peritoneal carcinoma, retroperitoneal or peritoneal sarcoma, small intestinal malignancy, soft tissue tumor, thymic carcinoma, thyroid carcinoma, or uveal melanoma.
  • 17. The method of any preceding claim, wherein the cancer comprises a prostate, bladder, kidney, lung, breast, or liver cancer.
  • 18. A method of identifying one or more candidate treatment for an ovarian cancer in a subject in need thereof, comprising: (a) determining a molecular profile for a sample from the subject by assessing a panel of gene or gene products, wherein the panel of gene or gene products are assessed as indicated in Table 7, FIG. 33C or FIG. 33D; and(b) identifying one or more treatment that is beneficially associated with the molecular profile of the subject, and optionally one or more treatment associated with lack of benefit, according to the determining in (a) and one or more rules in Table 8, thereby identifying the one or more candidate treatment.
  • 19. The method of claim 18, wherein the panel of gene or gene products comprises ABL1, AKT1, ALK, APC, AR, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ER, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HER2, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MGMT, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PGP, PIK3CA, PR, PTEN, PTPN11, RB1, RET, RRM1, SMAD4, SMARCB1, SMO, SPARC, STK11, TLE3, TOP2A, TOPO1, TP53, TS, TUBB3, VHL.
  • 20. The method of claim 18, wherein assessing the panel of gene or gene products comprises using ISH to assess cMET and HER2.
  • 21. The method of claim 18, wherein assessing the panel of gene or gene products comprises using IHC to assess AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3.
  • 22. The method of claim 18, wherein assessing the panel of gene or gene products comprises using sequence analysis to assess ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL.
  • 23. The method of claim 18, wherein assessing the panel of gene or gene products comprises using ISH to assess cMET and HER2; using IHC to assess AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3; and/or using sequence analysis to assess ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL.
  • 24. The method of claim 22 or 23, wherein assessing the panel of gene or gene products comprises using sequence analysis to assess CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1, PTPN11, RB1, SMAD4, SMARCB1 and STK11.
  • 25. The method of any of claims 22-24, wherein the sequence analysis comprises Next Generation Sequencing.
  • 26. A method of identifying one or more candidate treatment for a breast cancer in a subject in need thereof, comprising: (a) determining a molecular profile for a sample from the subject by assessing a panel of gene or gene products, wherein the panel of gene or gene products are assessed as indicated in Table 9, FIG. 33K or FIG. 33L; and(b) identifying one or more treatment that is beneficially associated with the molecular profile of the subject, and optionally one or more treatment associated with lack of benefit, according to the determining in (a) and one or more rules in Table 10, thereby identifying the one or more candidate treatment.
  • 27. The method of claim 26, wherein the panel of gene or gene products comprises ABL1, AKT1, ALK, APC, AR, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ER, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HER2, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MGMT, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PGP, PIK3CA, PR, PTEN, PTPN11, RB1, RET, RRM1, SMAD4, SMARCB1, SMO, SPARC, STK11, TLE3, TOP2A, TOPO1, TP53, TS, TUBB3, VHL.
  • 28. The method of claim 26, wherein assessing the panel of gene or gene products comprises using ISH to assess cMET, HER2, TOP2A.
  • 29. The method of claim 26, wherein assessing the panel of gene or gene products comprises using IHC to assess AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOPO1, TS, TUBB3.
  • 30. The method of claim 26, wherein assessing the panel of gene or gene products comprises using sequence analysis to assess ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL.
  • 31. The method of claim 26, wherein assessing the panel of gene or gene products comprises using ISH to assess cMET, HER2, TOP2A; using IHC to assess AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOPO1, TS, TUBB3; and/or using sequence analysis to assess ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL.
  • 32. The method of claim 30 or 31, wherein assessing the panel of gene or gene products comprises using sequence analysis to assess CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1, PTPN11, RB1, SMAD4, SMARCB1 and STK11.
  • 33. The method of any of claims 30-32, wherein the sequence analysis comprises Next Generation Sequencing.
  • 34. A method of identifying one or more candidate treatment for a skin cancer (melanoma) in a subject in need thereof, comprising: (a) determining a molecular profile for a sample from the subject by assessing a panel of gene or gene products, wherein the panel of gene or gene products are assessed as indicated in Table 11, FIG. 33E or FIG. 33F; and(b) identifying one or more treatment that is beneficially associated with the molecular profile of the subject, and optionally one or more treatment associated with lack of benefit, according to the determining in (a) and one or more rules in Table 12, thereby identifying the one or more candidate treatment.
  • 35. The method of claim 34, wherein the panel of gene or gene products comprises ABL1, AKT1, ALK, APC, AR, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ER, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HER2, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MGMT, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PGP, PIK3CA, PR, PTEN, PTPN11, RB1, RET, RRM1, SMAD4, SMARCB1, SMO, SPARC, STK11, TLE3, TOP2A, TOPO1, TP53, TS, TUBB3, VHL.
  • 36. The method of claim 34, wherein assessing the panel of gene or gene products comprises using ISH to assess 1 or 2 of: cMET, HER2.
  • 37. The method of claim 34, wherein assessing the panel of gene or gene products comprises using IHC to assess AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3.
  • 38. The method of claim 34, wherein assessing the panel of gene or gene products comprises using sequence analysis to assess ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL.
  • 39. The method of claim 34, wherein assessing the panel of gene or gene products comprises using ISH to assess 1 or 2 of: cMET, HER2; using IHC to assess AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3; and/or using sequence analysis to assess ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL.
  • 40. The method of claim 38 or 39, wherein assessing the panel of gene or gene products comprises using sequence analysis to assess CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1, PTPN11, RB1, SMAD4, SMARCB1 and STK11.
  • 41. The method of any of claims 38-40, wherein the sequence analysis comprises Next Generation Sequencing.
  • 42. The method of any of claims 38-41, wherein the sequence analysis of BRAF comprises PCR.
  • 43. A method of identifying one or more candidate treatment for a uveal melanoma cancer in a subject in need thereof, comprising: (a) determining a molecular profile for a sample from the subject by assessing a panel of gene or gene products, wherein the panel of gene or gene products are assessed as indicated in Table 13, FIG. 33G or FIG. 33H; and(b) identifying one or more treatment that is beneficially associated with the molecular profile of the subject, and optionally one or more treatment associated with lack of benefit, according to the determining in (a) and one or more rules in Table 14, thereby identifying the one or more candidate treatment.
  • 44. The method of claim 43, wherein the panel of gene or gene products comprises ABL1, AKT1, ALK, APC, AR, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ER, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HER2, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MGMT, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PGP, PIK3CA, PR, PTEN, PTPN11, RB1, RET, RRM1, SMAD4, SMARCB1, SMO, SPARC, STK11, TLE3, TOP2A, TOPO1, TP53, TS, TUBB3, VHL.
  • 45. The method of claim 43, wherein assessing the panel of gene or gene products comprises using ISH to assess cMET, HER2.
  • 46. The method of claim 43, wherein assessing the panel of gene or gene products comprises using IHC to assess AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3.
  • 47. The method of claim 43, wherein assessing the panel of gene or gene products comprises using sequence analysis to assess ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL.
  • 48. The method of claim 43, wherein assessing the panel of gene or gene products comprises using ISH to assess cMET, HER2; using IHC to assess AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3; and/or using sequence analysis to assess ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL.
  • 49. The method of claim 47 or 48, wherein assessing the panel of gene or gene products comprises using sequence analysis to assess CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1, PTPN11, RB1, SMAD4, SMARCB1 and STK11.
  • 50. The method of any of claims 47-49, wherein the sequence analysis comprises Next Generation Sequencing.
  • 51. The method of any of claims 47-50, wherein the sequence analysis of BRAF comprises PCR.
  • 52. A method of identifying one or more candidate treatment for a colorectal cancer in a subject in need thereof, comprising: (a) determining a molecular profile for a sample from the subject by assessing a panel of gene or gene products, wherein the panel of gene or gene products are assessed as indicated in Table 15, FIG. 33M or FIG. 33N; and(b) identifying one or more treatment that is beneficially associated with the molecular profile of the subject, and optionally one or more treatment associated with lack of benefit, according to the determining in (a) and one or more rules in Table 16, thereby identifying the one or more candidate treatment.
  • 53. The method of claim 52, wherein the panel of gene or gene products comprises ABL1, AKT1, ALK, APC, AR, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ER, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HER2, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MGMT, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PGP, PIK3CA, PR, PTEN, PTPN11, RB1, RET, RRM1, SMAD4, SMARCB1, SMO, SPARC, STK11, TLE3, TOP2A, TOPO1, TP53, TS, TUBB3, VHL.
  • 54. The method of claim 52, wherein assessing the panel of gene or gene products comprises using ISH to assess cMET, HER2.
  • 55. The method of claim 52, wherein assessing the panel of gene or gene products comprises using IHC to assess AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3.
  • 56. The method of claim 52, wherein assessing the panel of gene or gene products comprises using sequence analysis to assess ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL.
  • 57. The method of claim 52, wherein assessing the panel of gene or gene products comprises using ISH to assess cMET, HER2; using IHC to assess AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3; and/or using sequence analysis to assess ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL.
  • 58. The method of claim 56 or 57, wherein assessing the panel of gene or gene products comprises using sequence analysis to assess CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1, PTPN11, RB1, SMAD4, SMARCB1 and STK11.
  • 59. The method of any of claims 56-58, wherein the sequence analysis comprises Next Generation Sequencing.
  • 60. A method of identifying one or more candidate treatment for a lung cancer in a subject in need thereof, comprising: (a) determining a molecular profile for a sample from the subject by assessing a panel of gene or gene products, wherein the panel of gene or gene products are assessed as indicated in Table 17, FIG. 33I or FIG. 33J; and(b) identifying one or more treatment that is beneficially associated with the molecular profile of the subject, and optionally one or more treatment associated with lack of benefit, according to the determining in (a) and one or more rules in Table 18, thereby identifying the one or more candidate treatment.
  • 61. The method of claim 60, wherein the panel of gene or gene products comprises ABL1, AKT1, ALK, APC, AR, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ER, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HER2, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MGMT, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PGP, PIK3CA, PR, PTEN, PTPN11, RB1, RET, ROS1, RRM1, SMAD4, SMARCB1, SMO, SPARC, STK11, TLE3, TOP2A, TOPO1, TP53, TS, TUBB3, VHL.
  • 62. The method of claim 60, wherein assessing the panel of gene or gene products comprises using ISH to assess ALK, cMET, HER2, ROS1.
  • 63. The method of claim 60, wherein assessing the panel of gene or gene products comprises using IHC to assess AR, cMET, EGFR (H-score), ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3.
  • 64. The method of claim 60, wherein assessing the panel of gene or gene products comprises using sequence analysis to assess ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL.
  • 65. The method of claim 60, wherein assessing the panel of gene or gene products comprises using ISH to assess ALK, cMET, HER2, ROS1; using IHC to assess AR, cMET, EGFR (H-score), ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3; and/or using sequence analysis to assess ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL.
  • 66. The method of claim 64 or 65, wherein assessing the panel of gene or gene products comprises using sequence analysis to assess CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1, PTPN11, RB1, SMAD4, SMARCB1 and STK11.
  • 67. The method of any of claims 64-66, wherein the sequence analysis comprises Next Generation Sequencing.
  • 68. The method of any of claims 60-67, wherein the lung cancer comprises non-small cell lung cancer (NSCLC) or bronchioloalveolar cancer (BAC).
  • 69. A method of identifying one or more candidate treatment for a glioma brain cancer in a subject in need thereof, comprising: (a) determining a molecular profile for a sample from the subject by assessing a panel of gene or gene products, wherein the panel of gene or gene products are assessed as indicated in Table 21, FIG. 33O or FIG. 33P; and(b) identifying one or more treatment that is beneficially associated with the molecular profile of the subject, and optionally one or more treatment associated with lack of benefit, according to the determining in (a) and one or more rules in Table 19, thereby identifying the one or more candidate treatment.
  • 70. The method of claim 69, wherein the panel of gene or gene products comprises ABL1, AKT1, ALK, APC, AR, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, EGFRvIII, ER, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HER2, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR (VEGFR2), KRAS, MGMT-Me, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PGP, PIK3CA, PR, PTEN, PTPN11, RB1, RET, RRM1, SMAD4, SMARCB1, SMO, SPARCm, SPARCp, STK11, TLE3, TOP2A, TOPO1, TP53, TS, TUBB3, VHL.
  • 71. The method of claim 69, wherein assessing the panel of gene or gene products comprises using ISH to assess cMET, HER2.
  • 72. The method of claim 69, wherein assessing the panel of gene or gene products comprises using IHC to assess AR, cMET, ER, HER2, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3.
  • 73. The method of claim 69, wherein assessing the panel of gene or gene products comprises assessing methylation of the MGMT promoter region.
  • 74. The method of claim 73, wherein assessing methylation of the MGMT promoter region comprises pyrosequencing.
  • 75. The method of claim 69, wherein assessing the panel of gene or gene products comprises sequence analysis of IDH2.
  • 76. The method of claim 75, wherein sequence analysis of IDH2 comprises Sanger sequencing or Next Generation Sequencing.
  • 77. The method of claim 69, wherein assessing the panel of gene or gene products comprises detection of the EGFRvIII variant.
  • 78. The method of claim 77, wherein the EGFRvIII variant is detected by fragment analysis.
  • 79. The method of claim 69, wherein assessing the panel of gene or gene products comprises using sequence analysis to assess ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL.
  • 80. The method of claim 69, wherein assessing the panel of gene or gene products comprises using ISH to assess cMET, HER2; using IHC to assess AR, cMET, ER, HER2, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3; using pyrosequencing to detect methylation of the MGMT promoter; using Sanger sequencing to assess the sequence of IDH2; using fragment analysis to detect the EGFRvIII variant; and/or using sequence analysis to assess ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL.
  • 81. The method of claim 79 or 80, wherein assessing the panel of gene or gene products comprises using sequence analysis to assess CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1, PTPN11, RB1, SMAD4, SMARCB1 and STK11.
  • 82. The method of any of claims 79-81, wherein the sequence analysis comprises Next Generation Sequencing.
  • 83. A method of identifying one or more candidate treatment for a gastrointestinal stromal tumor (GIST) cancer in a subject in need thereof, comprising: (a) determining a molecular profile for a sample from the subject by assessing a panel of gene or gene products, wherein the panel of gene or gene products are assessed as indicated in Table 21; and(b) identifying one or more treatment that is beneficially associated with the molecular profile of the subject, and optionally one or more treatment associated with lack of benefit, according to the determining in (a) and one or more rules in Table 20, thereby identifying the one or more candidate treatment.
  • 84. The method of claim 83, wherein the panel of gene or gene products comprises ABL1, AKT1, ALK, APC, AR, ATM, BRAF, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ER, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HER2, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KRAS, MGMT, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PGP, PIK3CA, PR, PTEN, PTPN11, RB1, RET, RRM1, SMAD4, SMARCB1, SMO, SPARC, STK11, TLE3, TOP2A, TOPO1, TP53, TS, TUBB3, VHL.
  • 85. The method of claim 83, wherein assessing the panel of gene or gene products comprises using ISH to assess cMET, HER2.
  • 86. The method of claim 83, wherein assessing the panel of gene or gene products comprises using IHC to assess AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3.
  • 87. The method of claim 83, wherein assessing the panel of gene or gene products comprises using sequence analysis to assess ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL.
  • 88. The method of claim 83, wherein assessing the panel of gene or gene products comprises using ISH to assess cMET, HER2; using IHC to assess AR, cMET, ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOP2A, TOPO1, TS, TUBB3; and/or using sequence analysis to assess ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL.
  • 89. The method of claim 87 or 88, wherein assessing the panel of gene or gene products comprises using sequence analysis to assess CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1, PTPN11, RB1, SMAD4, SMARCB1 and STK11.
  • 90. The method of any of claims 87-89, wherein the sequence analysis comprises Next Generation Sequencing.
  • 91. A method of identifying one or more candidate treatment for a cancer in a subject in need thereof, comprising: (a) determining a molecular profile for a sample from the subject by assessing a panel of gene or gene products, wherein the panel of gene or gene products are assessed using IHC for AR, cMET, EGFR (including H-score for NSCLC), ER, HER2, MGMT, PGP, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOPO1, TOP2A, TS, TUBB3; FISH or CISH for ALK, cMET, HER2, ROS1, TOP2A; Mutational Analysis of BRAF (e.g., Cobas® PCR), IDH2 (e.g., Sanger Sequencing), MGMT promoter methylation (e.g., by PyroSequencing), EGFR (e.g., fragment analysis to detect EGFRvIII); and/or Mutational Analysis (e.g., by Next-Generation Sequencing) of ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CSF1R, CTNNB1, EGFR, ERBB2 (HER2), ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, STK11, TP53, VHL; and(b) identifying one or more treatment that is beneficially associated with the molecular profile of the subject, and optionally one or more treatment associated with lack of benefit, according to the determining in (a) and one or more rules in any of Tables 7-22, thereby identifying the one or more candidate treatment.
  • 92. The method of any preceding claim, further comprising additional molecular profiling according to FIG. 33Q.
  • 93. A method of identifying one or more candidate treatment for a prostate cancer in a subject in need thereof, comprising: (a) determining a molecular profile for a sample from the subject on a panel of gene or gene products, wherein the panel of gene or gene products comprises immunohistochemistry (IHC) of AR, MRP1, TOPO1, TLE3, EGFR, TS, PGP, TUBB3, RRM1, PTEN and/or MGMT; in situ hybridization (ISH) of EGFR and/or cMYC; and/or sequencing of TP53, PTEN, CTNNB1, PIK3CA, RB11, ATM, cMET, K/HRAS, ERBB4, ALK, BRAF and/or cKIT; and(b) identifying one or more treatment that is beneficially associated with the molecular profile of the subject, and optionally one or more treatment associated with lack of benefit, according to the determining in (a) and one or more rules in Table 22, thereby identifying the one or more candidate treatment.
  • 94. The method of claim 93, where the rules include one or more of: (a) imatinib for patients with high cKIT or PDGFRA;(b) cetuximab for patients with EGFR positivity;(c) cabozantinib for patients with cMET aberrations;(d) PAM pathway inhibitors (e.g., BEZ234, everolimus) for patients with PIK3CA pathway activation;(e) HDAC inhibitors for patients with cMYC amplification;(f) 5-FU for patients with low TS;(g) gemcitabine for patients with low RRM1;(h) temozolomide for patients with low MGMT;(i) cabazitaxel for patients with low TUBB3 or PGP, or high TLE3; and(j) anti-androgen agents (e.g., enzalutamide) for patients with high AR.
  • 95. A method of identifying one or more candidate treatment for a cancer in a subject in need thereof, comprising: (a) determining a molecular profile for a sample from the subject by sequencing a panel of gene or gene products, wherein the panel of gene or gene products comprises one or more gene in Table 24; and(b) identifying one or more treatment that is beneficially associated with the molecular profile of the subject, and optionally one or more treatment associated with lack of benefit, according to the determining in (a) and one or more rules in Table 25 or any of Tables 7-22, thereby identifying the one or more candidate treatment.
  • 96. The method of claim 95, wherein assessing the panel of gene or gene products comprises using sequence analysis to assess ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CSF1R, CTNNB1, EGFR, ERBB2 (HER2), ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, STK11, TP53, VHL.
  • 97. The method of claim 95, wherein assessing the panel of gene or gene products comprises using sequence analysis to assess ABL1, AKT1, ALK, APC, ATM, BRAF, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO, TP53, VHL.
  • 98. The method of claim 95, wherein assessing the panel of gene or gene products comprises using sequence analysis to assess ABL1, APC, BRAF, EGFR, FLT3, GNAQ, IDH1, JAK2, cKIT, KRAS, MPL, NPM1, NRAS, PDGFRA, VHL.
  • 99. The method of claim 95, wherein assessing the panel of gene or gene products comprises using sequence analysis to assess ABL1, APC, BRAF, EGFR, FLT3, GNAQ, IDH1, JAK2, cKIT, KRAS, MPL, NRAS, PDGFRA, VHL.
  • 100. The method of any preceding claim, wherein identifying the one or more treatment that is beneficially associated with the molecular profile of the subject, and optionally the one or more treatment associated with lack of benefit, comprises: (a) correlating the molecular profile with the one or more rules, wherein the one or more rules comprise a mapping of treatments whose efficacy has been previously determined in individuals having cancers that have different levels of, overexpress, underexpress, and/or have mutations in one or more members of the panel of gene or gene products; and(b) identifying one or more treatment that is associated with treatment benefit based on the correlating in (a); and optionally (c) identifying one or more treatment that is associated with lack of treatment benefit based on the correlating in (a).
  • 101. The method of claim 100, wherein the mapping of treatments is shown in any of Tables 3-5, 7-23, FIGS. 33A-Q, FIGS. 35A-I, or FIGS. 36A-F.
  • 102. The method of any preceding claim, further comprising identifying one or more candidate clinical trial for the subject based on the molecular profiling.
  • 103. A method of identifying one or more candidate clinical trial for a subject having a cancer, comprising: (a) determining a molecular profile for a sample from the subject on a panel of gene or gene products; and(b) identifying one or more clinical trial associated with the molecular profile of the subject according to the determining in (a) and one or more biomarker-clinical trial association rules, thereby identifying the one or more candidate clinical trial.
  • 104. The method of claim 103, wherein the molecular profile comprises IHC for AR, cMET, EGFR (including H-score for NSCLC), ER, HER2, MGMT, Pgp, PR, PTEN, RRM1, SPARCm, SPARCp, TLE3, TOPO1, TOP2A, TS, TUBB3; FISH or CISH for ALK, cMET, HER2, ROS1, TOP2A; Mutational Analysis of BRAF (e.g., Cobas® PCR), IDH2 (e.g., Sanger Sequencing), MGMT promoter methylation (e.g., by PyroSequencing), EGFR (e.g., fragment analysis to detect EGFRvIII); and/or Mutational Analysis (e.g., by Next-Generation Sequencing) of ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CSF1R, CTNNB1, EGFR, ERBB2 (HER2), ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, STK11, TP53, VHL.
  • 105. The method of any of claims 102-104, wherein identifying the one or more clinical trial associated with the molecular profile of the subject according to the determining in (a) comprises: 1) matching to clinical trials for non-standard of care treatments for the patient's cancer (e.g., off NCCN compendium treatments) indicated as potentially beneficial according to the biomarker—drug association rules herein; 2) matching to clinical trials based on biomarker eligibility requirements of the trial; and/or 3) matching to clinical trials based on the molecular profile of the patient, biology of the disease and/or associated signaling pathways.
  • 106. The method of claim 105, wherein matching to clinical trials based on the molecular profile of the patient, biology of the disease and/or associated signaling pathways comprises: 1) matching trials with therapeutic agents directly targeting a gene and/or gene product in the molecular profile; 2) matching trials with therapeutic agents that target another gene or gene product in a biological pathway that directly target a gene and/or gene product in the molecular profile; 3) matching trials with therapeutic agents that target another gene or gene product in a biological pathway that indirectly target a gene and/or gene product in the molecular profile.
  • 107. The method of any of claims 102-106, wherein identifying the one or more candidate clinical trial is according to one or more biomarker-clinical trial association rules in Tables 28-29.
  • 108. The method of any preceding claim, wherein the sample comprises formalin-fixed paraffin-embedded (FFPE) tissue, fixed tissue, core needle biopsy, fine needle aspirate, unstained slides, fresh frozen (FF) tissue, formalin samples, tissue comprised in a solution that preserves nucleic acid or protein molecules, and/or a bodily fluid sample.
  • 109. The method of any preceding claim, wherein the molecular profile comprises one or more additional gene or gene product listed in Table 2, Table 6 or Table 25.
  • 110. The method of claim 109, wherein the one or more additional gene or gene product listed in Table 2, Table 6 or Table 25 is assessed by next generation sequencing.
  • 111. The method of any preceding claim, wherein the sample comprises cells from a solid tumor.
  • 112. The method of any of claims 1-110, wherein the sample comprises a bodily fluid.
  • 113. The method of claim 112, wherein the bodily fluid comprises a malignant fluid.
  • 114. The method of claim 112, wherein the bodily fluid comprises a pleural or peritoneal fluid.
  • 115. The method of claim 112, wherein the bodily fluid comprises peripheral blood, sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, or umbilical cord blood.
  • 116. The method of any preceding claim, wherein the sample comprises a microvesicle population.
  • 117. The method of claim 116, wherein one or more members of the panel of gene or gene products is associated with the microvesicle population.
  • 118. The method of any preceding claim, wherein a prioritized list of the one or more candidate treatment is identified.
  • 119. The method of any preceding claim, wherein the one or more candidate treatment is selected from those listed in any of Tables 3-5, 7-22, 28, 29, 33, 36 or 37.
  • 120. The method of any preceding claim, wherein the subject has not previously been treated with the one or more candidate treatment that is associated with treatment benefit.
  • 121. The method of any preceding claim, wherein the cancer comprises a metastatic cancer.
  • 122. The method of any preceding claim, wherein the cancer comprises a recurrent cancer.
  • 123. The method of any preceding claim, wherein the cancer is refractory to a prior treatment.
  • 124. The method of claim 123, wherein the prior treatment comprises the standard of care for the cancer.
  • 125. The method of claim 123, wherein the cancer is refractory to all known standard of care treatments.
  • 126. The method of any of claims 1-122, wherein the subject has not previously been treated for the cancer.
  • 127. The method of any preceding claim, wherein progression free survival (PFS) or disease free survival (DFS) for the subject is extended by administration of the one or more candidate treatment to the subject.
  • 128. The method of any preceding claim, wherein the subject's lifespan is extended by administration of the one or more candidate treatment to the subject.
  • 129. The method of any preceding claim, wherein the molecular profile is compared to the one or more rules using a computer.
  • 130. The method of claim 129, wherein the one or more rules are comprised within a computer database.
  • 131. A method of generating a molecular profiling report comprising preparing a report comprising results of the molecular profile determined by any preceding claim.
  • 132. The method of claim 131, wherein the report further comprises a list of the one or more candidate treatment that is associated with benefit for treating the cancer.
  • 133. The method of claim 132, wherein the report further comprises a list of one or more treatment that is associated with lack of benefit for treating the cancer.
  • 134. The method of claim 132, wherein the report further comprises a list of one or more treatment that is associated with indeterminate benefit for treating the cancer.
  • 135. The method of claim 132, wherein the report further comprises identification of the one or more candidate treatment as standard of care or not for the cancer lineage.
  • 136. The method of claim 131, wherein the report further comprises a listing of members of the panel of genes or gene products assessed with description of each.
  • 137. The method of claim 131, wherein the report further comprises a listing of members of the panel of genes or gene products assessed by one or more of ISH, IHC, Next Generation sequencing, Sanger sequencing, PCR, pyrosequencing and fragment analysis.
  • 138. The method of claim 131, wherein the report further comprises a list of clinical trials for which the subject is eligible based on the molecular profile.
  • 139. The method of claim 131, wherein the report further comprises a list of evidence supporting the identification of certain treatments as likely to benefit the patient, not benefit the patient, or having indeterminate benefit.
  • 140. The method of claim 131, wherein the report further comprises: 1) a list of the genes and/or gene products in the molecular profile; 2) a description of the molecular profile of the genes and/or gene products as determined for the subject; 3) a treatment associated with one or more of the genes and/or gene products in the molecular profile; and 4) and an indication whether each treatment is likely to benefit the patient, not benefit the patient, or has indeterminate benefit.
  • 141. The method of claim 140, wherein the description of the molecular profile of the genes and/or gene products as determined for the subject comprises the technique used to assess the gene and/or gene products and the results of the assessment.
  • 142. A method of generating a molecular profiling report comprising preparing a report comprising results of the molecular profile determined by any of claims 103 or 104-141 as depends from claim 103.
  • 143. The method of claim 142, wherein the report further comprises a list of the one or more identified candidate clinical trial.
  • 144. The method of any of claims 131-143, wherein the molecular profile report is computer generated.
  • 145. The method of claim 144, wherein the molecular profile report is a printed report or a computer file.
  • 146. The method of claim 144, wherein the molecular profile report is accessible via a web portal.
  • 147. Use of a reagent in carrying out the method of any previous claim.
  • 148. Use of a reagent in the manufacture of a reagent or kit for carrying out the method of any of claims 1-146.
  • 149. A kit comprising a reagent for carrying out the method of any of claims 1-146.
  • 150. The use of claim 147-148 or kit of claim 149, wherein the reagent comprises one or more of a reagent for extracting nucleic acid from a sample, a reagent for performing ISH, a reagent for performing IHC, a reagent for performing PCR, a reagent for performing Sanger sequencing, a reagent for performing next generation sequencing, a reagent for a DNA microarray, a reagent for performing pyrosequencing, a nucleic acid probe, a nucleic acid primer, an antibody, a reagent for performing bisulfite treatment of nucleic acid.
  • 151. A report generated by the method of any of claims 131-146.
  • 152. A computer system for generating the report of claim 151.
  • 153. A system for identifying one or more candidate treatment for a cancer comprising: (a) a host server; (b) a user interface for accessing the host server to access and input data;(c) a processor for processing the inputted data;(d) a memory coupled to the processor for storing the processed data and instructions for: i. accessing a molecular profile generated by the method of any of claims 1-130;ii. identifying one or more candidate treatment that is associated with likely treatment benefit by comparing the molecular profiling results to the one or more rules;iii. optionally identifying one or more treatment that is associated with likely lack of treatment benefit by comparing the molecular profiling results to the one or more rules; andiv. optionally identifying one or more treatment that is associated with indeterminate treatment benefit by comparing the molecular profiling results to the one or more rules; and(e) a display for displaying the identified one or more candidate treatment that is associated with likely treatment benefit and the optional one or more treatment that is associated with likely lack of treatment benefit and one or more treatment that is associated with indeterminate treatment benefit.
  • 154. The system of claim 153, wherein the display comprises a report of claim 151.
  • 155. The system of claim 153, further comprising instructions for identifying one or more clinical trial that is associated with likely treatment benefit by comparing the molecular profiling results to one or more biomarker-clinical trial association rules.
  • 156. A system for identifying one or more candidate clinical trial for a cancer comprising: (a) a host server;(b) a user interface for accessing the host server to access and input data;(c) a processor for processing the inputted data;(d) a memory coupled to the processor for storing the processed data and instructions for: i. accessing a molecular profile generated by the method of any of claims 103 or 104-141 as depends from claim 103; andii. identifying one or more candidate candidate clinical trial by comparing the molecular profiling results to the one or more rules; and(e) a display for displaying the identified one or more candidate candidate clinical trial.
  • 157. The system of claim 156, wherein the display comprises a report of claim 151 as depends from claim 142.
  • 158. A computer medium comprising one or more rules from any of Tables 7, 9, 11, 13, 15, 17, 21 and Table 28.
  • 159. The computer medium of claim 158, comprising one or more rules selected from: (a) performing IHC on RRM1 to determine likely benefit or lack of benefit from an antimetabolite and/or gemcitabine;(b) performing IHC on TS to determine likely benefit or lack of benefit from a TOPO1 inhibitor, irinotecan and/or topotecan;(c) performing IHC on TS to determine likely benefit or lack of benefit from an antimetabolite, fluorouracil, capecitabine, and/or pemetrexed;(d) performing IHC on MGMT to determine likely benefit or lack of benefit from an alkylating agent, temozolomide, and/or dacarbazine;(e) performing IHC on AR to determine likely benefit or lack of benefit from an anti-androgen, bicalutamide, flutamide, and/or abiraterone;(f) performing IHC on ER to determine likely benefit or lack of benefit from a hormonal agent, tamoxifen, fulvestrant, letrozole, and/or anastrozole;(g) performing IHC on one or more of ER and PR to determine likely benefit or lack of benefit from a hormonal agent, tamoxifen, toremifene, fulvestrant, letrozole, anastrozole, exemestane, megestrol acetate, leuprolide, and/or goserelin;(h) performing one or more of IHC on HER2 and ISH on HER2 to determine likely benefit or lack of benefit from a tyrosine kinase inhibitor and/or lapatinib;(i) performing one or more of IHC on HER2 and ISH on HER2 to determine likely benefit or lack of benefit from an antibody therapy, trastuzumab, pertuzumab, and/or ado-trastuzumab emtansine (T-DM1);(j) performing one or more of ISH on TOP2A, ISH on HER2, IHC on TOP2A and IHC on PGP to determine likely benefit or lack of benefit from an anthracyclines, doxorubicin, liposomal-doxorubicin, and/or epirubicin;(k) performing sequencing on one or more of cKIT and PDGFRA to determine likely benefit or lack of benefit from a tyrosine kinase inhibitor and/or imatinib;(l) performing one or more of ISH on ALK and ISH on ROS1 to determine likely benefit or lack of benefit from a tyrosine kinase inhibitor and/or crizotinib;(m) performing sequencing on PIK3CA to determine likely benefit or lack of benefit from an mTOR inhibitor, everolimus, and/or temsirolimus;(n) performing sequencing on RET to determine likely benefit or lack of benefit from a tyrosine kinase inhibitor, and/or vandetanib;(o) performing IHC on one or more of SPARC, TUBB3 and PGP to determine likely benefit or lack of benefit from a taxane, paclitaxel, docetaxel, nab-paclitaxel;(p) performing IHC on one or more of SPARC, TLE3, TUBB3 and PGP to determine likely benefit or lack of benefit from a taxane, paclitaxel, docetaxel, nab-paclitaxel;(q) performing one or more of PCR and sequencing on BRAF to determine likely benefit or lack of benefit from a tyrosine kinase inhibitor, vemurafenib, dabrafenib, and/or trametinib;(r) performing one or more of sequencing on KRAS, sequencing on BRAF, sequencing on NRAS, sequencing on PIK3CA and IHC on PTEN to determine likely benefit or lack of benefit from an EGFR-targeted antibody, cetuximab, and/or panitumumab;(s) performing one or more of sequencing on EGFR, sequencing on KRAS, ISH on cMET, sequencing on PIK3CA and IHC onn PTEN to determine likely benefit or lack of benefit from a tyrosine kinase inhibitor, erlotinib, and/or gefitinib;(t) performing sequencing on EGFR to determine likely benefit or lack of benefit from a tyrosine kinase inhibitor, and/or afatinib; and(u) performing sequencing on cKIT to determine likely benefit or lack of benefit from a tyrosine kinase inhibitor, and/or sunitinib.
  • 160. The computer medium of claim 158, comprising one or more rules selected from Table 28.
CROSS REFERENCE

This application claims the benefit of U.S. Provisional Patent Application Nos. 61/733,396, filed Dec. 4, 2012; 61/757,701, filed Jan. 28, 2013; 61/759,986, filed Feb. 1, 2013; 61/830,018, filed May 31, 2013; 61/847,057, filed Jul. 16, 2013; 61/865,957, filed Aug. 14, 2013; 61/878,536, filed Sep. 16, 2013; 61/879,498, filed Sep. 18, 2013; 61/885,456, filed Oct. 1, 2013; 61/887,971, filed Oct. 7, 2013; 61/904,398, filed Nov. 14, 2013; all of which applications are incorporated herein by reference in their entirety.

PCT Information
Filing Document Filing Date Country Kind
PCT/US13/73184 12/4/2013 WO 00
Provisional Applications (11)
Number Date Country
61757701 Jan 2013 US
61830018 May 2013 US
61879498 Sep 2013 US
61759986 Feb 2013 US
61904398 Nov 2013 US
61878536 Sep 2013 US
61887971 Oct 2013 US
61733396 Dec 2012 US
61885456 Oct 2013 US
61847057 Jul 2013 US
61865957 Aug 2013 US