Patent Application
20210109101
The invention relates generally to the field of cancer diagnostics and, more specifically to methods for predicting response to a hormone-directed therapy in a prostate cancer (PCa) patient.
Prostate cancer (PCa) remains the most common non-cutaneous cancer in the US. In 2014 alone, the projected incidence of prostate cancer is 233,000 cases with deaths occurring in 29,480 men, making metastatic prostate cancer therapy truly an unmet medical need. Siegel et al., 2014. CA Cancer J Clin. 2014; 64(1):9-29. Epidemiological studies from Europe show comparable data with an estimated incidence of 416,700 new cases in 2012, representing 22.8% of cancer diagnoses in men. In total, 92,200 PCa-specific deaths are expected, making it one of the three cancers men are most likely to die from, with a mortality rate of 9.5%.
The androgen-androgen receptor (AR) signaling pathway is essential for the development and progression of prostate cancer and is a key target of many therapeutic agents. In metastatic prostate cancer (PCa), androgen deprivation therapy (ADT), constitutes the gold standard treatment to induce tumor regression by suppressing AR activation. Despite initial response to ADT, patients often develop resistance and progress to castration resistant prostate cancer (CRPC), an incurable disease with poor prognosis. These patients are often treated with salvage hormone-directed therapies, including agents such as non-steroidal anti-androgens and androgen-synthesis inhibitors. In managing these treatments, predicting therapeutic response and identifying early indicators of therapy resistance are major challenges. The levels of prostate specific antigen (PSA), an androgen regulated protein measured in the serum, is used to monitor therapeutic response in CRPC patients, however its predictive capability for this patient group is limited. In addition, while many studies have identified molecular events that may contribute to therapeutic resistance to androgen-targeting agents, it is difficult to apply these findings due to the limited supply of sequentially acquired tissue and the expected heterogeneity across multiple metastatic deposits present in any individual patient. As such, methods that would allow for non-invasive sequential monitoring through the clinical course of therapy would be of tremendous value to clinicians.
Circulating tumor cells (CTCs) have the potential to provide a non-invasive means of assessing progressive cancers in real time during therapy, and further, to help direct therapy by monitoring phenotypic physiological and genetic changes that occur in response to therapy. In most CRPC patients, the primary tumor has been removed, and CTCs are expected to consist of cells shed from metastases, providing a ‘fluid biopsy’. Currently, the only method approved for CTC enumeration (CellSearch, Veridex) is based on an immune enrichment approach that pre-selects for cells that express Epithelial Cell Adhesion Molecule (EpCAM), an epithelial cell surface marker. Although, numeric quantification of CTCs using CellSearch has yielded some prognostic information in certain cancers, this methodology has limitations such as low sensitivity (cells with low or absent EpCAM expression won't be captured) and the regular presence/contamination of genomically normal leukocytes in the sample preparation that hampers further molecular characterization and data interpretation. Recently, genomic changes based on array CGH and limited sequencing has been reported on CTCs isolated with the CellSearch system. Detailed analysis in paired tumors and metastasis (n=2) and CTCs (n=8) suggested that most mutations detected in CTCs were present at a low-level in the primary tumor. However, because a single timepoint during the clinical course of the disease was investigated this study does not address how a tumor may respond and evolve to therapeutic pressure.
A need exists for diagnostic methods that provide a more comprehensive portrait of the molecular changes occurring, at the single cell level, in a CRPC patient under the treatment pressure in both ADT and chemotherapy settings to allow association of the emergence of distinct CTC subpopulations with the clinical course of the disease. The present invention addresses this need by enabling to trace over time the molecular changes in a patient's CTC population by correlating morphometric and protein expression data with genome wide CNV alterations for individual CTCs isolated at clinically significant timepoints. Related advantages are provided as well.
The present invention provides methods for predicting response to a hormone-directed therapy in a prostate cancer (PCa) patient comprising (a) performing a direct analysis comprising immunofluorescent staining and morphological characteristization of nucleated cells in a blood sample obtained from the patient to identify and enumerate circulating tumor cells (CTC); (b) individually characterizing genotypic, morphometric and protein expression parameters to generate a profile for each of the CTCs, and (c) predicting response to hormone-directed therapy in the prostate cancer PCa patient based on said profile.
The present invention provides methods for predicting response to chemotherapy in a prostate cancer (PCa) patient comprising (a) performing a direct analysis comprising immunofluorescent staining and morphological characteristization of nucleated cells in a blood sample obtained from the patient to identify and enumerate circulating tumor cells (CTC); (b) individually characterizing genotypic, morphometric and protein expression parameters to generate a profile for each of the CTCs, and (c) predicting response to chemotherapy therapy in the prostate cancer PCa patient based on said profile.
In particular embodiments, the methods further comprise isolating the CTCs subsequent to the characterization of the morphometric and protein expression parameters and prior to the characterization of the genotypic parameters.
In some embodiments, the methods comprise repeating steps (a) through (c) at one or more timepoints after initial diagnosis of prostate cancer to sequentially monitor said genotypic, morphometric and protein expression parameters. In some embodiments the timepoints are at intervals that coincide with expected decision points in the standard care of CRPC. In some embodiments the timepoints coincide with clinical progression of the PCa.
In some embodiments, the method further comprises identifying clonal lineages of each CTC by genomic analysis. In additional embodiments, the cancer is metastatic castration resistant PCa (mCRPC). In some embodiments, the hormone directed therapy comprises Androgen Deprivation Therapy (ADT), which can be the first line or second line hormonal therapy. In some embodiments, the second line hormonal therapy blocks synthesis of androgen and is selected from the group consisting of abiraterone acetate, ketoconazole and aminoglutethimide.
In some embodiments the methods for predicting response to a hormone-directed therapy in a prostate cancer (PCa) patient, the immunofluorescent staining of nucleated cells comprises pan cytokeratin, cluster of differentiation (CD) 45, diamidino-2-phenylindole (DAPI) and androgen receptor (AR).
In some embodiments of the disclosed methods for predicting response to a hormone-directed therapy or chemotherapy in a prostate cancer (PCa) patient, the genotypic parameters comprise genomic variations including, for example, structural variations (SVs) and copy number variations (CNVs), simple nucleotide variations (SNVs), including single-nucleotide polymorphisms (SNPs) and small insertions and deletions (INDELs). In particular embodiments of the methods for predicting response to a hormone-directed therapy or chemotherapy in a prostate cancer (PCa) patient, the genotypic parameters comprise copy number variation (CNV) signatures. In some embodiments, CNVs are gene amplifications or deletions. In further embodiments, the gene amplifications comprise genes associated with androgen independent cell growth, for example, AR or v-myc avian myelocytomatosis viral oncogene homolog (MYC). In some embodiments, the genotypic parameters are detected by next generation sequencing (NGS).
In some embodiments the methods for predicting response to a hormone-directed therapy or chemotherapy in a prostate cancer (PCa) patient, the protein expression parameters comprise quantifying protein expression level or subcellular localization of protein expression. In further embodiments, the protein expression level is quantified by measuring strength of immunofluorescent signal using high resolution immunofluorescence imaging. In particular embodiments, the protein expression is AR expression. In some embodiments the methods for predicting response to a hormone-directed therapy in a prostate cancer (PCa) patient, the morphometric parameters comprise cell shape.
In some embodiments of the disclosed methods for predicting response to a hormone-directed therapy or chemotherapy in a prostate cancer (PCa) patient, the response is predicted based on comparison of the profiles between the timepoints. In some embodiments of the disclosed methods for predicting response to a hormone-directed therapy or chemotherapy in a prostate cancer (PCa) patient, the predicted response is emergence of resistant disease. In certain embodiments, the identification of a resistant CTC correlates with the emergence of resistant disease and the resistant CTC represents a clonal lineage that predominates resistant disease. In some embodiments, the re-emergence of AR positive CTCs predicts emergence of resistant disease. In further embodiments, the re-emergence of the AR positive CTCs is accompanied by genomic alterations such as AR or MYC amplification. In some embodiments, the re-emergence of AR positive CTCs is accompanied by morphometric change such as a decrease in cell roundness. In particular embodiments, the methods include determining whether a resistant CTC is AR independent, AR ligand independent or both. In some embodiments, the determination of whether a resistant CTC is AR independent, AR ligand independent or both informs a subsequent therapy selection or therapy change, for example, if the resistant cell is AR positive, the patient is a candidate for AR targeted treatment despite being AR ligand independent.
In some embodiments of the disclosed methods for predicting response to a hormone-directed therapy or chemotherapy in a prostate cancer (PCa) patient, the isolation of the CTCs involves relocation from initial image acquisition, re-imaging of the CTCs and physical extraction of the CTCs.
Other features and advantages of the invention will be apparent from the detailed description, and from the claims.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The present disclosure is based, in part, on the achievement of correlating genomic events with complex phenotypic alterations at single cell level with time resolution in CTCs of a cancer patient. Significantly, the methods disclosed herein enable detection of the emergence of distinct CTC subpopulations endowed with specific molecular alterations along the clinical course of the disease. Based on the detection of these distinct CTC subpopulations characterized by genotypic, morphometric and protein expression alterations, the methods disclosed herein enable the prediction of resistance and clinical escape in a prostate cancer patient undergoing targeted hormone therapy and allow for clinical intervention.
The present disclosure is based on the ability to capture the molecular changes in the CTC population by correlating morphometric and protein expression data with genome wide CNV alterations for individual CTCs isolated at clinically significant timepoints.
As disclosed herein, the invention provides novel methods to achieve a comprehensive portrait of the molecular changes occurring, at the single cell level, in a CRPC patient under the treatment pressure in both ADT and chemotherapy settings. The High Definition-CTC (HD-CTC) method was used for the longitudinal identification and enumeration of CTCs (Marrinucci et al., Phys Biol 9: 016003 (2012)) and to assess for the expression of the AR. Lazar et al., Phys Biol 9: 016002 (2012). The methods employ an unbiased protocol to examine and distinguish CTCs among the surrounding leukocytes based on their cytokeratin positive (CK+) phenotype by using a high resolution immunofluorescence imaging. In addition, the HD-CTC technology preserves the cell morphology in such a way that enables the morphometric and the indirect quantification of AR and CK protein expression levels for all the CTCs identified in the blood sample. To further characterize each CTC, a protocol was developed for extracting individual cells under conditions suitable for subsequent genomic analysis by a modification of the single nucleus sequencing method described by Navin et al., Nature 472: 90-94 (2011), and Baslan et al., Nat Protoc 7: 1024-1041 (2012).
A fundamental and enabling aspect of the present disclosure is the unparalleled robustness of the disclosed methods with regard to the detection of CTCs. The rare event detection disclosed herein with regard to CTCs is based on a direct analysis, i.e. non-enriched, of a population that encompasses the identification of rare events in the context of the surrounding non-rare events. Identification of the rare events according to the disclosed methods inherently identifies the surrounding events as non-rare events. Taking into account the surrounding non-rare events and determining the averages for non-rare events, for example, average cell size of non-rare events, allows for calibration of the detection method by removing noise. The result is a robustness of the disclosed methods that cannot be achieved with methods that are not based on direct analysis, but that instead compare enriched populations with inherently distorted contextual comparisons of rare events. The robustness of the direct analysis methods disclosed herein enables identification, enumeration and characterization of HD-CTCs, including subtypes of CTCs described herein, that enables the morphometric and the indirect quantification of AR and CK protein expression levels for all the CTCs identified in the blood sample that cannot be achieved with other CTC detection methods and that enables the analysis of correlation of genotypic and phenotypic changes in the context of the claimed methods.
The rapid evolution of drug therapies in prostate cancer has vastly improved upon the use of docetaxel since its pivotal US Food and Drug Administration (FDA) approval in 2004 and has brought about a new era where progress has been made beyond the use of androgen deprivation therapy (ADT) with the addition of novel hormonal agents, immunotherapy, second-line chemotherapy as well as radiopharmaceuticals. The choice of sequencing currently relies on patient profiles, whether symptoms of metastatic disease exist or not. While survival outcomes are undeniably improved with the use of these therapies, disease will ultimately progress on each regimen.
Androgens in the form of testosterone or the more potent dihydrotestosterone (DHT) have been well-defined drivers of progression of prostate cancer and differentiation of the prostate gland. As such, the backbone of treatment for advanced prostate cancers was established decades ago when castration in the form of surgical orchiectomy achieved significant prostate tumor regression. Since then, substitution to chemical castration has been employed mostly due to patient preference. ADT has therefore become the standard systemic treatment for locally advanced or metastatic prostate cancer. While ADT is almost always effective in most patients, disease progression to castration resistance inevitably occurs. It is now increasingly recognized that the androgen receptor (AR) remains overexpressed despite seemingly castrate levels of testosterone, since alternative receptors may activate the AR or other target genes may help perpetuate the castrate-resistant phenotype, hence the term “castration-resistance” has become widely adopted in the literature. The enhanced understanding of the role of these androgens in stimulating the growth of prostate cancer has led to the development and approval of both abiraterone and enzalutamide.
Chemotherapy treatment uses drugs to attack cancerous cells directly or indirectly, with the aim of destroying cancer cells or slow their growth. Chemotherapy for prostate cancer can be recommended if a patient is not responding to hormonal therapy and the cancer has spread outside the prostate. Chemotherapy is the use of drugs to destroy cancer cells, usually by stopping their ability to grow and divide. Systemic chemotherapy is delivered through the bloodstream to reach cancer cells throughout the body. Chemotherapy for prostate cancer can help patients with advanced or castration-resistant prostate cancer.
It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a CTC” includes a mixture of two or more CTCs, and the like.
The term “about,” particularly in reference to a given quantity, is meant to encompass deviations of plus or minus five percent.
As used in this application, including the appended claims, the singular forms “a,” “an,” and “the” include plural references, unless the content clearly dictates otherwise, and are used interchangeably with “at least one” and “one or more.”
As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “contains,” “containing,” and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, product-by-process, or composition of matter that comprises, includes, or contains an element or list of elements does not include only those elements but can include other elements not expressly listed or inherent to such process, method, product-by-process, or composition of matter.
The term “patient,” as used herein preferably refers to a human, but also encompasses other mammals. It is noted that, as used herein, the terms “organism,” “individual,” “subject,” or “patient” are used as synonyms and interchangeably.
As used herein, the term “circulating tumor cell” or “CTC” is meant to encompass any rare cell that is present in a biological sample and that is related to prostate cancer. CTCs, which can be present as single cells or in clusters of CTCs, are often epithelial cells shed from solid tumors found in very low concentrations in the circulation of patients.
As used herein, a “HD-CTC” refers to a single CTC that is cytokeratin positive, CD45 negative, contains a DAPI nucleus, and is morphologically distinct from surrounding white blood cells.
As used herein, “HD-CTC analysis” or “HD-SCA” (high definition single cell analysis) refers to analysis of any CTC based on genotypic, morphometric and protein expression parameters to generate a profile for each of the CTCs. High definition in the context of CTC and SC analysis therefore refers to high content analysis of all CTCs or rare cells present in a sample and is not limited to analysis of HD-CTCs.
In one embodiment, the disclosure provides a method of predicting response to a hormone-directed therapy in a prostate cancer (PCa) patient comprising (a) performing a direct analysis comprising immunofluorescent staining and morphological characteristization of nucleated cells in a blood sample obtained from the patient to identify and enumerate circulating tumor cells (CTC); (b) individually characterizing genotypic, morphometric and protein expression parameters to generate a profile for each of the CTCs, and (c) predicting response to hormone-directed therapy in the prostate cancer PCa patient based on the profile.
In one embodiment, the disclosure provides a method of predicting response to chemotherapy in a prostate cancer (PCa) patient comprising (a) performing a direct analysis comprising immunofluorescent staining and morphological characteristization of nucleated cells in a blood sample obtained from the patient to identify and enumerate circulating tumor cells (CTC); (b) individually characterizing genotypic, morphometric and protein expression parameters to generate a profile for each of the CTCs, and (c) predicting response to chemotherapy in the prostate cancer PCa patient based on the profile.
In a further embodiment, the disclosure provides a method of predicting response to a hormone-directed therapy in a prostate cancer (PCa) patient comprising (a) performing a direct analysis comprising immunofluorescent staining and morphological characteristization of nucleated cells in a blood sample obtained from the patient to identify and enumerate circulating tumor cells (CTC); (b) individually characterizing genotypic, morphometric and protein expression parameters to generate a profile for each of the CTCs; (c) identifying clonal lineages of each CTC based on genomic analysis, (d) assigning each CTC to a clonal lineage, and (e) predicting response to hormone-directed therapy in the prostate cancer PCa patient based on a combination the profile and the clonal lineage.
In a further embodiment, the disclosure provides a method of predicting response to chemotherapy in a prostate cancer (PCa) patient comprising (a) performing a direct analysis comprising immunofluorescent staining and morphological characteristization of nucleated cells in a blood sample obtained from the patient to identify and enumerate circulating tumor cells (CTC); (b) individually characterizing genotypic, morphometric and protein expression parameters to generate a profile for each of the CTCs; (c) identifying clonal lineages of each CTC based on genomic analysis, (d) assigning each CTC to a clonal lineage, and (e) predicting response to chemotherapy in the prostate cancer PCa patient based on a combination the profile and the clonal lineage.
In some embodiments, the methods further comprise isolating the CTCs subsequent to the characterization of the morphometric and protein expression parameters and prior to the characterization of said genotypic parameters. In some embodiments, the methods of the invention include an initial step of providing or obtaining a blood sample from the patient.
In metastatic prostate cancer (PCa), androgen deprivation therapy (ADT), constitutes the gold standard treatment to induce tumor regression by suppressing AR activation. ADT can include luteinizing hormone-releasing hormone (LHRH) agonists approved to treat prostate cancer including, for example, leuprolide, goserelin, and buserelin. Despite initial response to ADT, patients often develop resistance and progress to castration resistant prostate cancer (CRPC), an incurable disease with poor prognosis. These patients are often treated with salvage hormone-directed therapies, including agents such as non-steroidal anti-androgens and androgen-synthesis inhibitors. In some embodiments, the cancer is metastatic castration resistant PCa (mCRPC). In additional embodiments, the hormone directed therapy comprises Androgen Deprivation Therapy (ADT). In a further embodiment, the ADT is a second line hormonal therapy. In further embodiments, the second line hormonal therapy blocks synthesis of androgen and is selected from the group consisting of abiraterone acetate, ketoconazole and aminoglutethimide. Abiraterone acetate (Zytiga; Janssen Biotech, Inc. Horsham, Pa., USA) is an FDA-approved inhibitor of androgen biosynthesis, which blocks cytochrome P450-c17 (CYP17), leading to suppression of androgens derived from the adrenal glands, the prostate tumor and the tumor microenvironment.
The method of predicting response to a hormone-directed therapy in a prostate cancer (PCa) patient disclosed herein comprise performing a direct analysis comprising immunofluorescent staining and morphological characteristization of nucleated cells in a blood sample obtained from the patient to identify and enumerate circulating tumor cells (CTC). As used herein in the context of generating CTC data, the term “direct analysis” means that the CTCs are detected in the context of all surrounding nucleated cells present in the sample as opposed to after enrichment of the sample for CTCs prior to detection. In some embodiments, the methods comprise microscopy providing a field of view that includes both CTCs and at least 200 surrounding white blood cells (WBCs). The lack of enrichment of the disclosed methods enables an unbiased approach to examine and distinguish CTCs among the surrounding leukocytes based on their cytokeratin positive (CK+) phenotype by using a high resolution immunofluorescence imaging. In addition, the HD-CTC technology described herein preserves the cell morphology in such a way that enables the morphometric and the indirect quantification of AR and CK protein expression levels for all the CTCs identified in the blood sample. Further enabling the present methods is the ability to extract individual cells under conditions suitable for subsequent genomic analysis as disclosed herein. As described further below, the immunofluorescent staining of nucleated cells comprises pan cytokeratin, cluster of differentiation (CD) 45, diamidino-2-phenylindole (DAPI) and androgen receptor (AR).
In some embodiments, CTCs are individually characterized based on genotypic, morphometric and protein expression parameters to generate a profile for each of the CTCs. In some embodiments of the disclosed methods for predicting response to a hormone-directed therapy or chemotherapy in a prostate cancer (PCa) patient, the genotypic parameters comprise genomic variations including, for example, structural variations (SVs) and copy number variations (CNVs), simple nucleotide variations (SNVs), including single-nucleotide polymorphisms (SNPs) and small insertions and deletions (INDELs). In particular embodiments, genotypic parameters used include detection of copy number variation (CNV) signatures, including genomic amplifications and deletions. In further embodiments, the genotypic parameters measured comprise the number of genomic variations detected and/or the speed of occurrence of new genomic alterations. In some embodiments, the genomic amplifications and deletions affect regions containing oncogenes or genes implicated in the AR signaling axis. In further embodiments, gene amplifications include genes associated with androgen independent cell growth, for example, AR and v-myc avian myelocytomatosis viral oncogene homolog (MYC). In some embodiments, the genotypic parameters are detected by next generation sequencing (NGS). It will be understood by those skilled in the art, that the sequence analysis used in the methods of the invention can employ any useful sequencing technology, including without limitation amplification, polymerase chain reaction (PCR), real-time PCR (qPCR; RT-PCR), Sanger sequencing, next generation sequencing, restriction fragment length polymorphism (RFLP), pyrosequencing, DNA methylation analysis, or a combination thereof.
In some embodiments, protein expression parameters useful in practicing the methods disclosed herein include quantifying protein expression level and subcellular localization of protein expression. In some embodiments, the protein expression level is indirectly quantified by measuring strength of immunofluorescent signal using high resolution immunofluorescence imaging.
In some embodiments, the morphometric parameters useful in practicing the methods disclosed herein include cell shape, in particular, cell roundness. The cell shape (cell roundness) can be analyzed by tracing the cell cytoplasm contour in the composite image of each CTC. The traced cell image can imported into R, and an ellipsis was fitted to the shape using a least squares fitting algorithm described by Halir and Flusser, Proceeding of International Conference in Central Europe on Computer Graphics, Visualization and Interactive Digital Media: 125-132 (1998). The algorithm outputs the cell's major axis, which is the largest radius of the fitted ellipsis as described in the examples below.
In certain embodiments, the disclosure provides a method of predicting response to a hormone-directed therapy or chemotherapy in a prostate cancer (PCa) patient comprising (a) performing a direct analysis comprising immunofluorescent staining and morphological characteristization of nucleated cells in a blood sample obtained from the patient to identify and enumerate circulating tumor cells (CTC); (b) individually characterizing genotypic, morphometric and protein expression parameters to generate a profile for each of the CTCs; (c) repeating steps (a) and (b) at one or more timepoints after initial diagnosis of prostate cancer, and (d) predicting response to hormone-directed therapy in the prostate cancer patient based on sequentially monitoring of the profile at different timepoints after initial diagnosis of the prostate cancer. In particular embodiments, the timepoints are selected to correspond to clinical progression or therapy including, for example, systemic chemotherapy, hormone-directed therapy or radiation. In some embodiments, the blood samples (draws) are taken at timepoints at intervals representing decision points in the standard care of CRPC. For example, in addition to a sample taken at the time of initial diagnosis, the timepoints can be prior to initiation of docetaxel based chemotherapy, prior to initiation of a second line hormone therapy, for example, abiraterone acetate or another highly-selective androgen synthesis inhibitor, as well as at intervals after initiation of second line therapy, for example, after 2, 3, 4, 5, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or more weeks of continuous abiraterone treatment. In some embodiments, a blood draw is taken during ongoing/continuous treatment if a change in clinical symptoms is detected.
In certain embodiments, the method of predicting response to a hormone-directed therapy or chemotherapy in a prostate cancer (PCa) patient encompasses a comparison of the CTC profiles between the timepoints. In some embodiments, the predicted response is emergence of resistant disease. In certain embodiments, a predicted response of emergence of resistant disease is based on identification of a resistant CTC in a blood draw taken at any timepoint. In further embodiments, the resistant CTC is assigned to a clonal lineage that predominates resistant disease.
In some embodiments, re-emergence of AR positive CTCs that had been depleted at a prior timepoint during the course of the disease predicts emergence of resistant disease. In some embodiments, the re-emergence of the AR positive CTCs is accompanied by genomic alterations that were not dominant in CTCs extracted a prior timepoint. In further embodiments, the genomic alterations comprise AR or MYC amplification. In additional embodiments, the re-emergence of the AR positive cells is further accompanied by a morphometric change, in particular, a decrease in cell roundness.
The ability to identify the presence, emergence or re-emergence of a CTC that is representative of a resistant clonal population prior to clinical escape and emergence of resistant disease underlies the predictive power of the claimed methods with regard to response to a hormone-directed therapy or chemotherapy in a prostate cancer (PCa) patient. The ability to predict response to a hormone-directed therapy in a prostate cancer (PCa) patient can inform treatment decisions during a critical period preceding clinical escape and provide a clinician with actionable information as to what treatment course to follow. In some embodiments, a determination of whether a resistant CTC is AR independent, AR ligand independent or both can inform subsequent treatment decisions, for example, if the resistant cell is AR positive, the patient is a candidate for AR targeted treatment despite being AR ligand independent.
In its broadest sense, a biological sample can be any sample that contains CTCs. A sample can comprise a bodily fluid such as blood; the soluble fraction of a cell preparation, or an aliquot of media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA in solution or bound to a substrate; a cell; a tissue; a tissue print; a fingerprint; cells; skin, and the like. A biological sample obtained from a subject can be any sample that contains cells and encompasses any material in which CTCs can be detected. A sample can be, for example, whole blood, plasma, saliva or other bodily fluid or tissue that contains cells.
In particular embodiments, the biological sample is a blood sample. As described herein, a sample can be whole blood, more preferably peripheral blood or a peripheral blood cell fraction. As will be appreciated by those skilled in the art, a blood sample can include any fraction or component of blood, without limitation, T-cells, monocytes, neutrophiles, erythrocytes, platelets and microvesicles such as exosomes and exosome-like vesicles. In the context of this disclosure, blood cells included in a blood sample encompass any nucleated cells and are not limited to components of whole blood. As such, blood cells include, for example, both white blood cells (WBCs) as well as rare cells, including CTCs.
The samples of this disclosure can each contain a plurality of cell populations and cell subpopulation that are distinguishable by methods well known in the art (e.g., FACS, immunohistochemistry). For example, a blood sample can contain populations of non-nucleated cells, such as erythrocytes (e.g., 4-5 million/μl) or platelets (150,000-400,000 cells/μl), and populations of nucleated cells such as WBCs (e.g., 4,500-10,000 cells/μl), CECs or CTCs (circulating tumor cells; e.g., 2-800 cells/μl). WBCs may contain cellular subpopulations of, e.g., neutrophils (2,500-8,000 cells/μl), lymphocytes (1,000-4,000 cells/μl), monocytes (100-700 cells/μl), eosinophils (50-500 cells/μl), basophils (25-100 cells/μl) and the like. The samples of this disclosure are non-enriched samples, i.e., they are not enriched for any specific population or subpopulation of nucleated cells. For example, non-enriched blood samples are not enriched for CTCs, WBC, B-cells, T-cells, NK-cells, monocytes, or the like.
In some embodiments the sample is a blood sample obtained from a healthy subject or a subject deemed to be at high risk for prostate cancer or metastasis of existing prostate cancer based on art known clinically established criteria including, for example, age, race, family and history. In some embodiments the blood sample is from a subject who has been diagnosed with prostate cancer and/or mCRPC based on tissue or liquid biopsy and/or surgery or clinical grounds. In some embodiments, the blood sample is obtained from a subject showing a clinical manifestation of prostate cancer and/or mCRPC well known in the art or who presents with any of the known risk factors for prostate cancer and/or mCRPC.
In some embodiments, the methods of predicting response to a hormone-directed therapy or chemotherapy in a prostate cancer (PCa) patient can further encompass individual patient risk factors and imaging data, which includes any form of imaging modality known and used in the art, for example and without limitation, by X-ray computed tomography (CT), ultrasound, positron emission tomography (PET), electrical impedance tomography and magnetic resonance (MRI). It is understood that one skilled in the art can select an imaging modality based on a variety of art known criteria. As described herein, the methods of the invention can encompass one or more pieces of imaging data. In the methods disclosed herein, one or more individual risk factors can be selected from the group consisting of age, race, family history. It is understood that one skilled in the art can select additional individual risk factors based on a variety of art known criteria. As described herein, the methods of the invention can encompass one or more individual risk factors. Accordingly, parameters can include imaging data, individual risk factors and CTC data. As described herein, parameters also can include, but are not limited to, biological molecules comprising nucleotides, nucleic acids, nucleosides, amino acids, sugars, fatty acids, steroids, metabolites, peptides, polypeptides, proteins, carbohydrates, lipids, hormones, antibodies, regions of interest that serve as surrogates for biological macromolecules and combinations thereof (e.g., glycoproteins, ribonucleoproteins, lipoproteins) as well as portions or fragments of a biological molecule.
CTC data can include both morphological features and immunofluorescent features. As will be understood by those skilled in the art, additional parameters can be biomarker that can include a biological molecule, or a fragment of a biological molecule, the change and/or the detection of which can be correlated, individually or combined with other measurable features, with prostate cancer and/or mCRPC. CTCs, which can be present a single cells or in clusters of CTCs, are often epithelial cells shed from solid tumors and are present in very low concentrations in the circulation of subjects. Accordingly, detection of CTCs in a blood sample can be referred to as rare event detection. CTCs have an abundance of less than 1:1,000 in a blood cell population, e.g., an abundance of less than 1:5,000, 1:10,000, 1:30,000, 1:50:000, 1:100,000, 1:300,000, 1:500,000, or 1:1,000,000. In some embodiments, the a CTC has an abundance of 1:50:000 to 1:100,000 in the cell population.
The samples of this disclosure may be obtained by any means, including, e.g., by means of solid tissue biopsy or fluid biopsy (see, e.g., Marrinucci D. et al., 2012, Phys. Biol. 9 016003). Briefly, in particular embodiments, the process can encompass lysis and removal of the red blood cells in a 7.5 mL blood sample, deposition of the remaining nucleated cells on specialized microscope slides, each of which accommodates the equivalent of roughly 0.5 mL of whole blood. A blood sample may be extracted from any source known to include blood cells or components thereof, such as venous, arterial, peripheral, tissue, cord, and the like. The samples may be processed using well known and routine clinical methods (e.g., procedures for drawing and processing whole blood). In some embodiments, a blood sample is drawn into anti-coagulant blood collection tubes (BCT), which may contain EDTA or Streck Cell-Free DNA™. In other embodiments, a blood sample is drawn into CellSave® tubes (Veridex). A blood sample may further be stored for up to 12 hours, 24 hours, 36 hours, 48 hours, or 60 hours before further processing.
In some embodiments, the methods of this disclosure comprise an initial step of obtaining a white blood cell (WBC) count for the blood sample. In certain embodiments, the WBC count may be obtained by using a HemoCue® WBC device (Hemocue, Ängelholm, Sweden). In some embodiments, the WBC count is used to determine the amount of blood required to plate a consistent loading volume of nucleated cells per slide and to calculate back the equivalent of CTCs per blood volume.
In some embodiments, the methods of this disclosure comprise an initial step of lysing erythrocytes in the blood sample. In some embodiments, the erythrocytes are lysed, e.g., by adding an ammonium chloride solution to the blood sample. In certain embodiments, a blood sample is subjected to centrifugation following erythrocyte lysis and nucleated cells are resuspended, e.g., in a PBS solution.
In some embodiments, nucleated cells from a sample, such as a blood sample, are deposited as a monolayer on a planar support. The planar support may be of any material, e.g., any fluorescently clear material, any material conducive to cell attachment, any material conducive to the easy removal of cell debris, any material having a thickness of <100 μm. In some embodiments, the material is a film. In some embodiments the material is a glass slide. In certain embodiments, the method encompasses an initial step of depositing nucleated cells from the blood sample as a monolayer on a glass slide. The glass slide can be coated to allow maximal retention of live cells (See, e.g., Marrinucci D. et al., 2012, Phys. Biol. 9: 016003). In some embodiments, about 0.5 million, 1 million, 1.5 million, 2 million, 2.5 million, 3 million, 3.5 million, 4 million, 4.5 million, or 5 million nucleated cells are deposited onto the glass slide. In some embodiments, the methods of this disclosure comprise depositing about 3 million cells onto a glass slide. In additional embodiments, the methods of this disclosure comprise depositing between about 2 million and about 3 million cells onto the glass slide. In some embodiments, the glass slide and immobilized cellular samples are available for further processing or experimentation after the methods of this disclosure have been completed.
In some embodiments, the methods of this disclosure comprise an initial step of identifying nucleated cells in the non-enriched blood sample. In some embodiments, the nucleated cells are identified with a fluorescent stain. In certain embodiments, the fluorescent stain comprises a nucleic acid specific stain. In certain embodiments, the fluorescent stain is diamidino-2-phenylindole (DAPI). In some embodiments, immunofluorescent staining of nucleated cells comprises pan cytokeratin (CK), cluster of differentiation (CD) 45 and DAPI. In some embodiments, the immunofluorescent staining of nucleated cells comprises pan cytokeratin, cluster of differentiation (CD) 45, diamidino-2-phenylindole (DAPI) and androgen receptor (AR). In some embodiments further described herein, CTCs comprise distinct immunofluorescent staining from surrounding nucleated cells. In some embodiments, the distinct immunofluorescent staining of CTCs comprises DAPI (+), CK (+) and CD 45 (−). In some embodiments, the identification of CTCs further comprises comparing the intensity of pan cytokeratin fluorescent staining to surrounding nucleated cells. In some embodiments, the CTC data is generated by fluorescent scanning microscopy to detect immunofluorescent staining of nucleated cells in a blood sample. Marrinucci D. et al., 2012, Phys. Biol. 9 016003).
In particular embodiments, all nucleated cells are retained and immunofluorescently stained with monoclonal antibodies targeting cytokeratin (CK), an intermediate filament found exclusively in epithelial cells, a pan leukocyte specific antibody targeting the common leukocyte antigen CD45, and a nuclear stain, DAPI. The nucleated blood cells can be imaged in multiple fluorescent channels to produce high quality and high resolution digital images that retain fine cytologic details of nuclear contour and cytoplasmic distribution. While the surrounding WBCs can be identified with the pan leukocyte specific antibody targeting CD45, CTCs can be identified as DAPI (+), CK (+) and CD 45 (−). In the methods described herein, the CTCs comprise distinct immunofluorescent staining from surrounding nucleated cells.
In further embodiments, the CTC are high definition CTCs (HD-CTCs). HD-CTCs are CK positive, CD45 negative, contain an intact DAPI positive nucleus without identifiable apoptotic changes or a disrupted appearance, and are morphologically distinct from surrounding white blood cells (WBCs). DAPI (+), CK (+) and CD45 (−) intensities can be categorized as measurable features during HD-CTC enumeration as previously described (
While CTCs can be identified as comprises DAPI (+), CK (+) and CD 45 (−) cells, the methods of the invention can be practiced with any other parameters that one of skill in the art selects for generating CTC data and/or identifying CTCs and CTC clusters. One skilled in the art knows how to select a morphological feature, biological molecule, or a fragment of a biological molecule, the change and/or the detection of which can be correlated with a CTC. Molecule parameters include, but are not limited to, biological molecules comprising nucleotides, nucleic acids, nucleosides, amino acids, sugars, fatty acids, steroids, metabolites, peptides, polypeptides, proteins, carbohydrates, lipids, hormones, antibodies, regions of interest that serve as surrogates for biological macromolecules and combinations thereof (e.g., glycoproteins, ribonucleoproteins, lipoproteins). The term also encompasses portions or fragments of a biological molecule, for example, peptide fragment of a protein or polypeptide
In some embodiments, the disclosed method of predicting response to a hormone-directed therapy in a prostate cancer (PCa) patient, which include a step of isolation of the CTCs from the sample, further comprise relocation from the initial fluorescent image acquisition and subsequent re-imaging of the CTCs followed by physical extraction of the CTCs. Included in some embodiments of the claimed methods is a method for HD-CTC fluid phase capture that can be divided into three discrete sequential steps: (1) CTC relocation, (2) cell extraction and (3) physical isolation and manipulation of single CTCs for downstream molecular analyses, as described in the examples provided herewith.
A person skilled in the art will appreciate that a number of methods can be used to generate CTC data, including microscopy based approaches, including fluorescence scanning microscopy (see, e.g., Marrinucci D. et al., 2012, Phys. Biol. 9:016003), mass spectrometry approaches, such as MS/MS, LC-MS/MS, multiple reaction monitoring (MRM) or SRM and product-ion monitoring (PIM) and also including antibody based methods such as immunofluorescence, immunohistochemistry, immunoassays such as Western blots, enzyme-linked immunosorbant assay (ELISA), immunoprecipitation, radioimmunoassay, dot blotting, and FACS. Immunoassay techniques and protocols are generally known to those skilled in the art (Price and Newman, Principles and Practice of Immunoassay, 2nd Edition, Grove's Dictionaries, 1997; and Gosling, Immunoassays: A Practical Approach, Oxford University Press, 2000.) A variety of immunoassay techniques, including competitive and non-competitive immunoassays, can be used (Self et al., Curr. Opin. Biotechnol., 7:60-65 (1996), see also John R. Crowther, The ELISA Guidebook, 1st ed., Humana Press 2000, ISBN 0896037282 and, An Introduction to Radioimmunoassay and Related Techniques, by Chard T, ed., Elsevier Science 1995, ISBN 0444821198).
A person of skill in the art will further appreciate that the presence or absence of parameters may be detected using any class of marker-specific binding reagents known in the art, including, e.g., antibodies, aptamers, fusion proteins, such as fusion proteins including protein receptor or protein ligand components, or parameter-specific small molecule binders. In some embodiments, the presence or absence of CK or CD45 is determined by an antibody.
The antibodies of this disclosure bind specifically to a parameter. The antibody can be prepared using any suitable methods known in the art. See, e.g., Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies: A Laboratory Manual (1988); Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986). The antibody can be any immunoglobulin or derivative thereof, whether natural or wholly or partially synthetically produced. All derivatives thereof which maintain specific binding ability are also included in the term. The antibody has a binding domain that is homologous or largely homologous to an immunoglobulin binding domain and can be derived from natural sources, or partly or wholly synthetically produced. The antibody can be a monoclonal or polyclonal antibody. In some embodiments, an antibody is a single chain antibody. Those of ordinary skill in the art will appreciate that antibody can be provided in any of a variety of forms including, for example, humanized, partially humanized, chimeric, chimeric humanized, etc. The antibody can be an antibody fragment including, but not limited to, Fab, Fab′, F(ab′)2, scFv, Fv, dsFv diabody, and Fd fragments. The antibody can be produced by any means. For example, the antibody can be enzymatically or chemically produced by fragmentation of an intact antibody and/or it can be recombinantly produced from a gene encoding the partial antibody sequence. The antibody can comprise a single chain antibody fragment. Alternatively or additionally, the antibody can comprise multiple chains which are linked together, for example, by disulfide linkages, and any functional fragments obtained from such molecules, wherein such fragments retain specific-binding properties of the parent antibody molecule. Because of their smaller size as functional components of the whole molecule, antibody fragments can offer advantages over intact antibodies for use in certain immunochemical techniques and experimental applications.
A detectable label can be used in the methods described herein for direct or indirect detection of the parameters when generating CTC data in the methods of the invention. A wide variety of detectable labels can be used, with the choice of label depending on the sensitivity required, ease of conjugation with the antibody, stability requirements, and available instrumentation and disposal provisions. Those skilled in the art are familiar with selection of a suitable detectable label based on the assay detection of the parameters in the methods of the invention. Suitable detectable labels include, but are not limited to, fluorescent dyes (e.g., fluorescein, fluorescein isothiocyanate (FITC), Oregon Green™, rhodamine, Texas red, tetra-rhodamine isothiocyanate (TRITC), Cy3, Cy5, Alexa Fluor® 647, Alexa Fluor® 555, Alexa Fluor® 488), fluorescent markers (e.g., green fluorescent protein (GFP), phycoerythrin, etc.), enzymes (e.g., luciferase, horseradish peroxidase, alkaline phosphatase, etc.), nanoparticles, biotin, digoxigenin, metals, and the like.
For mass-spectrometry based analysis, differential tagging with isotopic reagents, e.g., isotope-coded affinity tags (ICAT) or the more recent variation that uses isobaric tagging reagents, iTRAQ (Applied Biosystems, Foster City, Calif.), followed by multidimensional liquid chromatography (LC) and tandem mass spectrometry (MS/MS) analysis can provide a further methodology in practicing the methods of this disclosure.
A chemiluminescence assay using a chemiluminescent antibody can be used for sensitive, non-radioactive detection of proteins. An antibody labeled with fluorochrome also can be suitable. Examples of fluorochromes include, without limitation, DAPI, fluorescein, Hoechst 33258, R-phycocyanin, B-phycoerythrin, R-phycoerythrin, rhodamine, Texas red, and lissamine. Indirect labels include various enzymes well known in the art, such as horseradish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase, urease, and the like. Detection systems using suitable substrates for horseradish-peroxidase, alkaline phosphatase, beta.-galactosidase are well known in the art.
A signal from the direct or indirect label can be analyzed, for example, using a microscope, such as a fluorescence microscope or a fluorescence scanning microscope. Alternatively, a spectrophotometer can be used to detect color from a chromogenic substrate; a radiation counter to detect radiation such as a gamma counter for detection of 125I; or a fluorometer to detect fluorescence in the presence of light of a certain wavelength. If desired, assays used to practice the methods of this disclosure can be automated or performed robotically, and the signal from multiple samples can be detected simultaneously.
In some embodiments, the parameters are immunofluorescent markers. In some embodiments, the immunofluorescent makers comprise a marker specific for epithelial cells In some embodiments, the immunofluorescent makers comprise a marker specific for white blood cells (WBCs). In some embodiments, one or more of the immunofluorescent markers comprise CD45 and CK.
In some embodiments, the presence or absence of immunofluorescent markers in nucleated cells, such as CTCs or WBCs, results in distinct immunofluorescent staining patterns. Immunofluorescent staining patterns for CTCs and WBCs may differ based on which epithelial or WBC markers are detected in the respective cells. In some embodiments, determining presence or absence of one or more immunofluorescent markers comprises comparing the distinct immunofluorescent staining of CTCs with the distinct immunofluorescent staining of WBCs using, for example, immunofluorescent staining of CD45, which distinctly identifies WBCs. There are other detectable markers or combinations of detectable markers that bind to the various subpopulations of WBCs. These may be used in various combinations, including in combination with or as an alternative to immunofluorescent staining of CD45.
In some embodiments, CTCs comprise distinct morphological characteristics compared to surrounding nucleated cells. In some embodiments, the morphological characteristics comprise nucleus size, nucleus shape, cell size, cell shape, and/or nuclear to cytoplasmic ratio. In some embodiments, the method further comprises analyzing the nucleated cells by nuclear detail, nuclear contour, presence or absence of nucleoli, quality of cytoplasm, quantity of cytoplasm, intensity of immunofluorescent staining patterns. A person of ordinary skill in the art understands that the morphological characteristics of this disclosure may include any feature, property, characteristic, or aspect of a cell that can be determined and correlated with the detection of a CTC.
CTC data can be generated with any microscopic method known in the art. In some embodiments, the method is performed by fluorescent scanning microscopy. In certain embodiments the microscopic method provides high-resolution images of CTCs and their surrounding WBCs (see, e.g., Marrinucci D. et al., 2012, Phys. Biol. 9:016003)). In some embodiments, a slide coated with a monolayer of nucleated cells from a sample, such as a non-enriched blood sample, is scanned by a fluorescent scanning microscope and the fluorescence intensities from immunofluorescent markers and nuclear stains are recorded to allow for the determination of the presence or absence of each immunofluorescent marker and the assessment of the morphology of the nucleated cells. In some embodiments, microscopic data collection and analysis is conducted in an automated manner.
In some embodiments, a CTC data includes detecting one or more parameters, for example, CK and CD 45. A parameter is considered “present” in a cell if it is detectable above the background noise of the respective detection method used (e.g., 2-fold, 3-fold, 5-fold, or 10-fold higher than the background; e.g., 2σ or 3σ over background). In some embodiments, a parameter is considered “absent” if it is not detectable above the background noise of the detection method used (e.g., <1.5-fold or <2.0-fold higher than the background signal; e.g., <1.5σ or <2.0σ over background).
In some embodiments, the presence or absence of immunofluorescent markers in nucleated cells is determined by selecting the exposure times during the fluorescence scanning process such that all immunofluorescent markers achieve a pre-set level of fluorescence on the WBCs in the field of view. Under these conditions, CTC-specific immunofluorescent markers, even though absent on WBCs are visible in the WBCs as background signals with fixed heights. Moreover, WBC-specific immunofluorescent markers that are absent on CTCs are visible in the CTCs as background signals with fixed heights. A cell is considered positive for an immunofluorescent marker (i.e., the marker is considered present) if its fluorescent signal for the respective marker is significantly higher than the fixed background signal (e.g., 2-fold, 3-fold, 5-fold, or 10-fold higher than the background; e.g., 2σ or 3σ over background). For example, a nucleated cell is considered CD 45 positive (CD 45+) if its fluorescent signal for CD 45 is significantly higher than the background signal. A cell is considered negative for an immunofluorescent marker (i.e., the marker is considered absent) if the cell's fluorescence signal for the respective marker is not significantly above the background signal (e.g., <1.5-fold or <2.0-fold higher than the background signal; e.g., <1.5σ or <2.0σ over background).
Typically, each microscopic field contains both CTCs and WBCs. In certain embodiments, the microscopic field shows at least 1, 5, 10, 20, 50, or 100 CTCs. In certain embodiments, the microscopic field shows at least 10, 25, 50, 100, 250, 500, or 1,000 fold more WBCs than CTCs. In certain embodiments, the microscopic field comprises one or more CTCs or CTC clusters surrounded by at least 10, 50, 100, 150, 200, 250, 500, 1,000 or more WBCs.
In some embodiments of the methods described herein, generation of the CTC data comprises enumeration of CTCs that are present in the blood sample. In some embodiments, the methods described herein encompass detection of at least 1.0 CTC/mL of blood, 1.5 CTCs/mL of blood, 2.0 CTCs/mL of blood, 2.5 CTCs/mL of blood, 3.0 CTCs/mL of blood, 3.5 CTCs/mL of blood, 4.0 CTCs/mL of blood, 4.5 CTCs/mL of blood, 5.0 CTCs/mL of blood, 5.5 CTCs/mL of blood, 6.0 CTCs/mL of blood, 6.5 CTCs/mL of blood, 7.0 CTCs/mL of blood, 7.5 CTCs/mL of blood, 8.0 CTCs/mL of blood, 8.5 CTCs/mL of blood, 9.0 CTCs/mL of blood, 9.5 CTCs/mL of blood, 10 CTCs/mL of blood, or more.
In some embodiments of methods described herein, generation of the CTC data comprises detecting distinct subtypes of CTCs, including non-traditional CTCs. In some embodiments, the methods described herein encompass detection of at least 0.1 CTC cluster/mL of blood, 0.2 CTC clusters/mL of blood, 0.3 CTC clusters/mL of blood, 0.4 CTC clusters/mL of blood, 0.5 CTC clusters/mL of blood, 0.6 CTC clusters/mL of blood, 0.7 CTC clusters/mL of blood, 0.8 CTC clusters/mL of blood, 0.9 CTC clusters/mL of blood, 1 CTC cluster/mL of blood, 2 CTC clusters/mL of blood, 3 CTC clusters/mL of blood, 4 CTC clusters/mL of blood, 5 CTC clusters/mL of blood, 6 CTC clusters/mL of blood, 7 CTC clusters/mL of blood, 8 CTC clusters/mL of blood, 9 CTC clusters/mL of blood, 10 clusters/mL or more. In a particular embodiment, the methods described herein encompass detection of at least 1 CTC cluster/mL of blood.
In some embodiments, the methods of predicting response to a hormone-directed therapy or chemotherapy in a prostate cancer (PCa) patient can further encompass the use of a predictive model. In further embodiments, the methods of predicting response to a hormone-directed therapy in a prostate cancer (PCa) patient can further encompass comparing a measurable feature with a reference feature. As those skilled in the art can appreciate, such comparison can be a direct comparison to the reference feature or an indirect comparison where the reference feature has been incorporated into the predictive model. In further embodiments, analyzing a measurable feature to prospectively identify resistance to hormone directed therapies in a PCa patient encompasses one or more of a linear discriminant analysis model, a support vector machine classification algorithm, a recursive feature elimination model, a prediction analysis of microarray model, a logistic regression model, a CART algorithm, a flex tree algorithm, a LART algorithm, a random forest algorithm, a MART algorithm, a machine learning algorithm, a penalized regression method, or a combination thereof. In particular embodiments, the analysis comprises logistic regression. In additional embodiments, the prediction of resistance to hormone directed therapies in a PCa patient is expressed as a risk score.
An analytic classification process can use any one of a variety of statistical analytic methods to manipulate the quantitative data and provide for classification of the sample. Examples of useful methods include linear discriminant analysis, recursive feature elimination, a prediction analysis of microarray, a logistic regression, a CART algorithm, a FlexTree algorithm, a LART algorithm, a random forest algorithm, a MART algorithm, machine learning algorithms and other methods known to those skilled in the art.
Classification can be made according to predictive modeling methods that set a threshold for determining the probability that a sample belongs to a given class. The probability preferably is at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% or higher. Classifications also can be made by determining whether a comparison between an obtained dataset and a reference dataset yields a statistically significant difference. If so, then the sample from which the dataset was obtained is classified as not belonging to the reference dataset class. Conversely, if such a comparison is not statistically significantly different from the reference dataset, then the sample from which the dataset was obtained is classified as belonging to the reference dataset class.
The predictive ability of a model can be evaluated according to its ability to provide a quality metric, e.g. AUROC (area under the ROC curve) or accuracy, of a particular value, or range of values. Area under the curve measures are useful for comparing the accuracy of a classifier across the complete data range. Classifiers with a greater AUC have a greater capacity to classify unknowns correctly between two groups of interest. ROC analysis can be used to select the optimal threshold under a variety of clinical circumstances, balancing the inherent tradeoffs that exist between specificity and sensitivity. In some embodiments, a desired quality threshold is a predictive model that will classify a sample with an accuracy of at least about 0.7, at least about 0.75, at least about 0.8, at least about 0.85, at least about 0.9, at least about 0.95, or higher. As an alternative measure, a desired quality threshold can refer to a predictive model that will classify a sample with an AUC of at least about 0.7, at least about 0.75, at least about 0.8, at least about 0.85, at least about 0.9, or higher.
As is known in the art, the relative sensitivity and specificity of a predictive model can be adjusted to favor either the specificity metric or the sensitivity metric, where the two metrics have an inverse relationship. The limits in a model as described above can be adjusted to provide a selected sensitivity or specificity level, depending on the particular requirements of the test being performed. One or both of sensitivity and specificity can be at least about 0.7, at least about 0.75, at least about 0.8, at least about 0.85, at least about 0.9, or higher.
The raw data can be initially analyzed by measuring the values for each measurable feature or parameter, usually in triplicate or in multiple triplicates. The data can be manipulated, for example, raw data can be transformed using standard curves, and the average of triplicate measurements used to calculate the average and standard deviation for each patient. These values can be transformed before being used in the models, e.g. log-transformed, Box-Cox transformed (Box and Cox, Royal Stat. Soc., Series B, 26:211-246 (1964). The data are then input into a predictive model, which will classify the sample according to the state. The resulting information can be communicated to a patient or health care provider.
In some embodiments, the methods of predicting response to a hormone-directed therapy in a prostate cancer (PCa) patient can have a specificity of >60%, >70%, >80%, >90% or higher. In additional embodiments, the methods of predicting response to a hormone-directed therapy in a prostate cancer (PCa) patient can have a specificity >90% at a classification threshold of 7.5 CTCs/mL of blood.
As will be understood by those skilled in the art, an analytic classification process can use any one of a variety of statistical analytic methods to manipulate the quantitative data and provide for classification of the sample. Examples of useful methods include, without limitation, linear discriminant analysis, recursive feature elimination, a prediction analysis of microarray, a logistic regression, a CART algorithm, a FlexTree algorithm, a LART algorithm, a random forest algorithm, a MART algorithm, and machine learning algorithms.
The following examples are provided by way of illustration, not limitation.
This example shows monitoring of treatment response by longitudinal CTC molecular analysis and demonstrates that phenotypic and genotypic changes in circulating cell populations represent sequential steps of genetic evolution in response to a multi-step therapeutic regime culminating in treatment with abiraterone acetate
Patient Clinical History and Blood Draws Collected During Treatment. The study was approved by the institutional review board (IRB) of University of Southern California Comprehensive Cancer Center. The patient provided written informed consent. The patient presented with PCa metastatic to a lumbar vertebrae at diagnosis for which the primary biopsy represents the first specimen in this study. Initial treatment consisted of androgen deprivation therapy (leuprolide acetate). After 5 months, there was clinical progression to CRPC and the patient was enrolled in a clinical trial of docetaxel combined with bevacizumab and everolimus (clinicaltrials.gov identifier: NCT00574769). Before chemotherapy was initiated, a baseline blood draw was taken (Draw 1) according to the sample collection protocol. Clinical progression was noted after 4 months of protocol-specified chemotherapy. Over the next 3 months, additional doses of docetaxel as well as external-beam radiotherapy and samarium (153Sm) lexidronam (a bone-targeting radiopharmaceutical) were employed with limited palliative benefit. At 12 months after diagnosis, treatment with abiraterone acetate, a highly-selective androgen synthesis inhibitor, was initiated. Blood was drawn prior to starting abiraterone (Draw 2), at 3 weeks of continuous treatment coinciding with a clinical response represented by decreased pain and PSA level (Draw 3), and at 9 weeks coinciding with clinical progression represented by increasing pain and PSA levels (Draw 4). Following abiraterone, treatment was changed to cabazitaxel without clinical response followed by a rapid clinical deterioration. The patient died of widely metastatic prostate cancer 4 months following Draw 4 (17 months after diagnosis).
Blood Sample Collection and Processing for CTC Detection. Patient peripheral blood samples were collected according to an IRB approved protocol. Samples were shipped to our laboratory and processed within 24 hours after the time of draw. Sample preparation was previously described in Marrinucci et al., Phys Biol 9: 016003 (2012). In brief, it consists of a red blood cell lysis followed by plating of the nucleated cells as a monolayer on custom made cell-adhesion glass slide followed by storage in a biorepository. Each sample produced at least 14 independent slides for CTC identification and characterization.
Immunofluorescence Staining and CTC Enumeration. For this study, we used a protocol based on the published HD-CTC assay coupled with evaluation of androgen receptor (AR) status within the cytokeratin (CK) positive CTC population. Lazar et al., Phys Biol 9: 016002 (2012). Briefly, the cells were labeled using mouse monoclonal cytokeratin 19 (1:100; Dako) and panCK (1:100; Sigma) primary antibodies to identify cytokeratin (CK) positive cells. AR positive HD-CTCs were identified using a rabbit anti-AR monoclonal antibody (1:250, Cell Signaling Technology). Both the CK and AR antigens were visualized using AlexaFluor secondary antibodies; the CK primary antibodies were recognized with Alexa Fluor 555 IgG1 secondary antibody (1:500, Invitrogen) and the rabbit AR antibody was recognized with Alexa Fluor 488 IgG (H+L) secondary antibody (1:1000, Invitrogen). Alexa Fluor 647 conjugated anti-CD45 (1:125; AbD Serotec) primary antibody was used to identify leukocytes as an exclusion marker. To confirm that the cells are nucleated and to enable the analysis of nuclear morphology all cells were stained with a 4′,6-diamidino-2-phenylindole (DAPI).
The slides were imaged and putative CTCs were recorded using a computerized high-throughput fluorescence microscope at 10× magnification. CTCs were identified by a hematology technician using the previously published criteria of having a DAPI+ nucleus plus cytokeratin positivity and CD45 negativity. Marrinucci et al., Phys Biol 9: 016003 (2012). Androgen receptor protein expression and localization were evaluated using two criteria (1) presence (AR+) or absence (AR−) of AR staining, and (2) AR subcellular localization (nuclear AR versus cytoplasmic staining or both). The threshold for AR positivity was defined as a signal more than 6 standard deviations over the mean signal intensity (SDOM) observed in the surroundings leukocytes (background). Subcellular localization was measured using the relative pixel density of AR staining over the nucleus and cytoplasm.
HD-CTC Assay Reproducibility. The HD-CTC assay was technically validated with cell line spiking experiments to reach an R2=0.9997 on linearity testing as previously reported. These experiments were performed using SK-BR-3 cell lines and 0 to 3×102 cells per mL of normal donor control blood. The coefficient of variation is 16% and inter-processor correlation is R2=0.979. Sample preparation process adhered to standard operating procedures for patient samples through a bar coded system for all consumables and instrumentation. All off-the-shelf instrumentation was calibrated according to the technical validation protocols established during the commissioning. Nair et al., PLoS One 8: e67733 (2013)
Extraction of Single Cells. As a standard procedure, aimed at minimizing DNA fragmentation cells were picked within 5 days of the initial staining procedure. The experimental protocol for HD-CTC fluid phase capture was divided into three discrete sequential steps: (1) CTC relocation, (2) cell extraction and (3) isolation and manipulation of single CTCs for downstream molecular analyses.
HD-CTCs were relocated (step 1) using a transformation matrix from the initial data acquisition for HD-CTC identification. After calibration and relocation, each candidate cell was re-imaged at 40× resolution for the detailed morphometric analysis. For the cell extraction (step 2) an Eppendorf Transfer Man NK2 micromanipulator was used to capture the cell of interest inside a 25° jagged micropipette (Piezo Drill Tip ES, Eppendorf) by applying fluid suction. Once the cell of interest was captured inside the micropipette (step 3), the cell was rinsed with PBS and deposited inside a 0.2 mL PCR tube containing 2 μL of lysis buffer (200 mM KOH; 50 mM DTT). The sample was then and immediately frozen and stored at −80° C. until further processing. All instruments and consumables were decontaminated using a DNAase solution and exposure to UV light for 30 min prior to the experiment.
Single Cell Next Generation Sequencing and Bioinformatic Analysis. The cell containing vials were transferred in dry ice to the sequencing laboratory. Briefly, the lysed cell mixture was thawed and subjected to WGA and sequencing library construction as previously reported by Baslan et al, Nat Protoc 7: 1024-1041 (2012).
WGA was carried out manually in a 96-well plate format using the WGA4 Genomeplex Single Cell Whole Genome Amplification Kit (Sigma-Aldrich), followed by purification using a QIAquick 96 PCR Purification Kit (Qiagen). Concentration of eluted DNA was measured using a Nanodrop 8000 (Thermo Scientific). For each well, amplification was considered successful if the resulting DNA concentration was ≥70 ng/μl (elution volume of 50 μl), followed by further Quality Control (QC) to confirm the appropriate sample size distribution using the Agilent 2100 Bioanalyzer (High-Sensitivity DNA Assay and Kit, Agilent Technologies).
In addition, detailed methods used to analyze sequencing data were published recently by our group in Baslan et al, Nat Protoc 7: 1024-1041 (2012). Briefly, the informatics methods involves three steps: first, deconvoluting the sequence reads based on barcodes; second, mapping the reads to the human genome (hg19, Genome Reference Consortium GRCh37, UCSC Genome Browser database) (Meyer et al., Nucleic Acids Res 41: D64-69 (2013)), and removing PCR duplicates; and third, normalizing for guanine-cytosine (GC) content and estimating copy number using the CBS segmentation algorithm. The copy number profiles in this report are based on 20,000 variable length genome bins, averaging a length of ˜150 kilo-base pairs each, and were calculated as ratio compared to normal (hg 19). The data reported here had a median count of 1.78 million uniquely mapping reads, with a range from 244,190 (minimum cut off 200,000) to 5.33 million.
Cluster Analysis. The hierarchical clustering was performed in R (Team RC (2012) R: A Language and Environment for Statistical Computing) using the heatmap. 2 function in the gplots package. Ward's method with Euclidean distance metric was used for the clustering. The heatmap is colored according to the cutoffs described above and the clustering was performed using median centered data.
Frequency Analysis to Define Genomic Alterations. Using median centered CNV profiles, cutoff ratios versus the median of 0.8 and 1.25 were used to define deletions and amplifications, respectively. These cutoffs were used both to color the heatmap and to do the frequency analysis.
Statistics and Cell Morphology Analysis. The cell shape (cell roundness) was analyzed by tracing the cell cytoplasm contour in the composite image of each CTC. The traced cell image was imported into R, and an ellipsis was fitted to the shape using a least squares fitting algorithm described by Halir and Flusser, Proceeding of International Conference in Central Europe on Computer Graphics, Visualization and Interactive Digital Media: 125-132 (1998). The algorithm outputs the cell's major axis, which is the largest radius of the fitted ellipsis (Refer to supporting information). The cell roundness (c) is estimated as the fraction of the de facto cell area (A) and the area of a circle with the radius (r) set to the cell's major axis.
C=A/πr
2
The p-value used in the comparison of the roundness between the CTCs in Draw 3 and 4 was calculated using the Wilcoxon sum-rank test.
In order to assess the patient's response to treatment high content single cell analysis including: (1) AR protein expression phenotype, (2) AR subcellular localization and (3) CNV genomic profiling were performed in the CTCs identified in the blood samples collected across four different intervals representing decision points in the standard care of CRPC including: (Draw 1) immediately prior to initiation of docetaxel based chemotherapy, (Draw 2) immediately prior to abiraterone acetate (a highly-selective androgen synthesis inhibitor), (Draw 3) after three weeks, and (Draw 4) after nine weeks of continuous abiraterone treatment. The specific data for all profiled cells is presented in the supporting information. In addition, a similar sequencing based method was used to obtain the CNV profile of one metastatic site from the patient using a bone biopsy taken at the time of diagnosis (5 months prior to draw 1) prior to receiving any cancer-specific therapy. As shown in
The genomic CNV profiles of CK+ cells from Draws 1 and 2 were of two types (
At Draw 3, after three weeks of abiraterone acetate treatment, the patient displayed a clear clinical response as defined by decrease in PSA and pain (
The apparent effect of treatment was also evident in the genomic analysis of Draw 3 where the altered phenotypic states correlated with distinct genomic profiles. The majority (10/12) of phenotypically AR− cells from Draw 3 were not amplified for AR and exhibited apparently normal or near normal (pseudodiploid) profiles (
Draw 4 was collected at the point of clinical progression, when PSA levels increased after 9 weeks on abiraterone (
Alterations in response to therapy were again apparent at the genomic level, as (6/10) cells formed the majority of a new, apparently clonal, subpopulation (Cluster C in
In addition, morphometric analysis of AR subcellular localization showed that AR was generally localized in the nucleus of cells from Draws 1 and 2, but was identified as significantly less localized to the nucleus in the CTCs isolated in Draw 4 collected at progression (p=0.00017 Wilcoxon rank-sum test) (
Although our study is based on longitudinal study of a single patient, our findings are consistent with previous studies involving genomic analysis from either CTCs or circulating cell-free DNA isolated from patients with metastatic prostate cancer. Magbanua et al., BMC Cancer 12: 78 (2012), Heitzer et al., Genome Med 5: 30 (2013). However, these prior studies were generally limited to the characterization of pooled samples from a single timepoint, and therefore do not shed light into the temporal and dynamic evolution of cancer under therapeutic selective pressure. Regardless, consistent with these prior reports we observed copy number alterations in chromosome 8 (particularly gain in 8q and loss in 8p), which is one of the most frequent somatic mutations described in prostate cancer. Taylor et al., Cancer Cell 18: 11-22 (2010). In addition, our finding that AR amplification was not found in sample obtained before initial androgen-deprivation therapy, but occurred at high frequency in later samples representing CRPC is consistent with multiple prior studies linking AR amplification with androgen-independent prostate cancer growth.
Clonal evolution of cancer is a well-established principle that has been validated in multiple published studies (Navin et al., Genome Res 20: 68-80 (2010), Gerlinger et al., N Engl J Med 366: 883-892 (2012), Almendro et al., Cancer Res 74: 1338-1348 (2014)), as well as, the appearance of somatic mutations in tumors in response to therapeutic selective pressure Sequist et al., Sci Transl Med 3 (2011), Shi et al. Cancer Discov 4: 80-93 (2014). We interpret the phenotypic and genotypic changes in circulating cell populations presented here as representing sequential steps of genetic evolution in response to a multi-step therapeutic regime culminating in treatment with abiraterone acetate.
The bulk metastatic biopsy taken prior to initiation of therapy provides the root CNV profile to from which the subsequent time course CTC profiles have evolved. It exhibits a backbone of CNV elements that defines a lineage, based on CNV breakpoints, that is carried forward in the circulating cells from blood draws taken during later treatment. The first two of these draws were taken after an initial course of androgen deprivation therapy (ADT) (leuprolide acetate). One population in Draws 1 and 2 (Clone A) was a clearly a direct descendant of the met biopsy profile with the exception that all cells showed high-copy AR amplification and strong AR protein expression. We interpret these cells to be the products of metastatic deposits that had evolved to amplify the AR gene locus and overexpress androgen receptor protein as a result of genetic selection for resistance to the initial round of ADT. It is noteworthy that in addition to the AR amplified cells, both draws contained a significant fraction of cytokeratin positive, AR negative cells with near-normal (pseudodiploid) genomes forming a separate CNV cluster (
In contrast to the similarity of cell phenotype and genotype in Draws 1 and 2, the selective effects of abiraterone acetate were very evident in Draws 3 and 4. After three weeks of treatment the androgen dependent, AR positive cells in Draws 1 and 2 were nearly absent and the CK+ population consisted almost entirely of AR negative pseudodiploid cells. The clone (Clone C) that would become dominant at the nine-week timepoint (Draw 4) was first seen as a single incidence in Draw 3. By Draw 4, AR+ cells had once again become a substantial fraction of the population, albeit with a significantly altered CNV profile (
Through this selective process, the population of AR negative, pseudodiploid cells remained a significant fraction of cytokeratin positive cells. The presence of these phenotypically (
In an era of clinical oncology that is progressively moving towards targeted cancer therapy, approaches that allow for non-invasive monitoring of therapeutic response at both phenotypic and genetic levels are essential. We have chosen to approach this goal through a combined phenotypic and genetic analysis of non-leukocyte circulating nucleated cells, without a pre-selection step that may bias the CTC population. Our method allows us to correlate genomic events with complex phenotypes based on protein expression and cell morphology. Alternative methods, such as sequencing of free DNA from plasma (ctDNA) are also powerful tools and can yield both mutation and copy number information, but only for an admixture of the various cellular components. Murtaza et al., Nature 497: 108-112 (2013). In this case study, we show the remarkable extent and speed of the genomic reorganization as putative-resistant clones emerge at the time of treatment failure. Although, we cannot establish a mechanistic relationship between the large CNV changes and the eventual resistance to abiraterone acetate based on a single patient, it appeared that after 9 weeks of targeted therapy the original CTC population was not completely eliminated and an apparently drug-resistant clone was present. Finally, the integration of data across multiple subjects will open the door for a deeper understanding of the mechanisms and timing of resistance and allow for rationally-designed, personalized treatments based on sequential, combined, or intermittent application of therapeutic agents.
The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.
From the foregoing description, it will be apparent that variations and modifications can be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
This patent application is a continuation of U.S. patent application Ser. No. 15/024,483 (filed Mar. 24, 2016; now pending), which is a § 371 U.S. national phase filing of PCT International Patent Application No. PCT/US2014/058304 (filed Sep. 30, 2014; now expired), which claims the benefit of priority to U.S. Provisional Patent Application No. 61/884,835 (filed Sep. 30, 2013; now expired). The full disclosures of the priority applications are incorporated herein by reference in their entirety and for all purposes.
This invention was made with government support under grant number CA143906 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61884835 | Sep 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15024483 | Mar 2016 | US |
| Child | 16884737 | US |
