Biomarkers and methods for determining sensitivity to epidermal growth factor receptor modulators

Abstract
EGFR biomarkers useful in a method for predicting the likelihood that a mammal that will respond therapeutically to a method of treating cancer comprising administering an EGFR modulator, wherein the method comprises (a) measuring in the mammal the level of at least one biomarker selected from epiregulin and amphiregulin, (b) exposing a biological sample from the mammal to the EGFR modulator, and (c) following the exposing of step (b), measuring in the biological sample the level of the at least one biomarker, wherein an increase in the level of the at least one biomarker measured in step (c) compared to the level of the at least one biomarker measured in step (a) indicates an increased likelihood that the mammal will respond therapeutically to the method of treating cancer.
Description
SEQUENCE LISTING

A compact disc labeled “Copy 1” contains the Sequence Listing as 10646 PCT.ST25.txt. The Sequence Listing is 1241 KB in size and was recorded Aug. 24, 2006. The compact disk is 1 of 2 compact disks. A duplicate copy of the compact disc is labeled “Copy 2” and is 2 of 2 compact discs.


The compact disc and duplicate copy are identical and are hereby incorporated by reference into the present application.


FIELD OF THE INVENTION

The present invention relates generally to the field of pharmacogenomics, and more specifically to methods and procedures to determine drug sensitivity in patients to allow the identification of individualized genetic profiles which will aid in treating diseases and disorders.


BACKGROUND OF THE INVENTION

Cancer is a disease with extensive histoclinical heterogeneity. Although conventional histological and clinical features have been correlated to prognosis, the same apparent prognostic type of tumors varies widely in its responsiveness to therapy and consequent survival of the patient.


New prognostic and predictive markers, which would facilitate an individualization of therapy for each patient, are needed to accurately predict patient response to treatments, such as small molecule or biological molecule drugs, in the clinic. The problem may be solved by the identification of new parameters that could better predict the patient's sensitivity to treatment. The classification of patient samples is a crucial aspect of cancer diagnosis and treatment. The association of a patient's response to a treatment with molecular and genetic markers can open up new opportunities for treatment development in non-responding patients, or distinguish a treatment's indication among other treatment choices because of higher confidence in the efficacy. Further, the pre-selection of patients who are likely to respond well to a medicine, drug, or combination therapy may reduce the number of patients needed in a clinical study or accelerate the time needed to complete a clinical development program (Cockett et al., Current Opinion in Biotechnology, 11:602-609 (2000)).


The ability to predict drug sensitivity in patients is particularly challenging because drug responses reflect not only properties intrinsic to the target cells, but also a host's metabolic properties. Efforts to use genetic information to predict drug sensitivity have primarily focused on individual genes that have broad effects, such as the multidrug resistance genes, mdr1 and mrp1 (Sonneveld, J. Intern. Med., 247:521-534 (2000)).


The development of microarray technologies for large scale characterization of gene mRNA expression pattern has made it possible to systematically search for molecular markers and to categorize cancers into distinct subgroups not evident by traditional histopathological methods (Khan et al., Cancer Res., 58:5009-5013 (1998); Alizadeh et al., Nature, 403:503-511 (2000); Bittner et al., Nature, 406:536-540 (2000); Khan et al., Nature Medicine, 7(6):673-679 (2001); and Golub et al., Science, 286:531-537 (1999); Alon et al., P. N. A. S. USA, 96:6745-6750 (1999)). Such technologies and molecular tools have made it possible to monitor the expression level of a large number of transcripts within a cell population at any given time (see, e.g., Schena et al., Science, 270:467-470 (1995); Lockhart et al., Nature Biotechnology, 14:1675-1680 (1996); Blanchard et al., Nature Biotechnology, 14:1649 (1996); U.S. Pat. No. 5,569,588).


Recent studies demonstrate that gene expression information generated by microarray analysis of human tumors can predict clinical outcome (van't Veer et al., Nature, 415:530-536 (2002); Sorlie et al., P. N. A. S. USA, 98:10869-10874 (2001); M. Shipp et al., Nature Medicine, 8(1):68-74 (2002): Glinsky et al., The Journal of Clin. Invest., 113(6):913-923 (2004)). These findings bring hope that cancer treatment will be vastly improved by better predicting the response of individual tumors to therapy.


The epidermal growth factor receptor (EGFR) and its downstream signaling effectors, notably members of the Ras/Raf/MAP kinase pathway, play an important role in both normal and malignant epithelial cell biology (Normanno et al., Gene 366, 2-16 (2006)) and have therefore become established targets for therapeutic development. Whereas the anti-EGFR antibody cetuximab and the EGFR small molecular tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib have demonstrated activity in a subset of patients (Baselga and Arteaga, J. Clin. Oncol. 23, 2445-2459 (2005)), their initial clinical development has not benefited from an accompanying strategy for identifying the patient populations that would most likely derive benefit. The hypothesis that only a relatively small number of tumors are “EGFR-pathway dependent” and therefore likely to respond to EGFR inhibitors might explain the limited clinical activity that is observed with this class of therapeutics. For example, in patients with refractory metastatic colorectal cancer clinical response rates with cetuximab consistently range from 11% in a monotherapy setting to 23% in a combination setting with chemotherapy (Cunningham et al., N. Engl. J. Med 351, 337-345 (2004)). To date, significant efforts have been focused on elucidating the mechanisms of sensitivity or resistance to EGFR inhibition, particularly through evaluation of EGFR protein expression, kinase domain mutations, and gene copy number.


While relative protein expression of the EGFR as measured by immunohistochemistry (IHC) has been demonstrated in many solid tumors (Ciardiello and Tortora, Eur. J. Cancer 39, 1348-1354 (2003)), no consistent association between EGFR expression and response to EGFR inhibitors has been established. Clinical studies of cetuximab in a monotherapy setting and in combination with irinotecan in patients with mCRC failed to reveal an association between radiographic response and EGFR protein expression as measured by IHC (Cunningham et al., N. Engl. J. Med 351, 337-345 (2004); Saltz et al., J. Clin. Oncol. 22, 1201-1208 (2004)). Furthermore, clinical responses have been demonstrated in patients with undetectable EGFR protein expression (Chung et al., J. Clin. Oncol., 23, 1803-1810 (2005); Lenz et al., Activity of cetuximab in patients with colorectal cancer refractory to both irinotecan and oxaliplatin. Paper presented at: 2004 ASCO Annual Meeting Proceedings; Saltz, Clin Colorectal Cancer, 5 Suppl. 2, S98-100 (2005)). In comparison, clinical studies of erlotinib in NSCLC patients and gefitinib in ovarian cancer did demonstrate an association between EGFR expression, response, and survival (Schilder et al., Clin. Cancer Res., 11, 5539-5548 (2005); Tsao et al., N. Engl. J. Med., 353, 133-144 (2005)). The presence of somatic mutations in the tyrosine kinase domain, particularly in NSCLC has been extensively described (Janne et al., J. Clin. Oncol., 23, 3227-3234 (2005)). In both preclinical and clinical settings, these mutations are found to correlate with sensitivity to gefitinib and erlotinib but not to cetuximab (Janne et al., J. Clin. Oncol., 23, 3227-3234 (2005); Tsuchihashi et al., N. Engl. J. Med., 353, 208-209 (2005)). In addition, the lack of EGFR kinase domain mutations in CRC patients suggests that such mutations do not underlie the response to cetuximab. EGFR gene copy number has also been evaluated as a potential predictor of response to EGFR inhibitors. Clinical studies of gefitinib demonstrated an association between increased EGFR copy number, mutational status, and clinical response (Cappuzzo et al., J. Natl. Cancer Inst., 97, 643-655 (2005)). A similar association was identified in a small number of patients treated with the anti-EGFR monoclonal antibodies cetuximab and panitumumab (Moroni et al., Lancet Oncol., 6, 279-286 (2005)). Additional potential predictive biomarkers have also been evaluated. For example, in glioblastoma patients, a significant association between co-expression of EGFRvIII and PTEN and response to EGFR small molecule inhibitors was found (Mellinghoff et al., N. Engl. J. Med., 353, 2012-2024 (2005)).


The anti-tumor activity of cetuximab has been attributed to its ability to block EGFR ligand binding and ligand-dependent EGFR activation. Clinical activity of cetuximab has been shown in multiple epithelial tumor types (Bonner et al., N. Engl. J. Med., 354, 567-578 (2006); Cunningham et al., N. Engl. J. Med., 351, 337-345 (2004)), however responses continue to be seen in only a fraction of patients. Previous attempts to identify predictors of sensitivity or resistance as described above have focused on specific biomarkers rather than using genomic discovery approaches. In addition, RNA-, DNA- and protein-based markers have rarely been examined in the same patient population in a single study, making comparisons challenging.


Biomarkers useful for determining sensitivity to EGFR modulators have been described in published PCT applications WO2004/063709, WO2005/067667, and WO2005/094332.


Needed are new and alternative methods and procedures to determine drug sensitivity in patients to allow the development of individualized genetic profiles which are necessary to treat diseases and disorders based on patient response at a molecular level.


SUMMARY OF THE INVENTION

The invention provides methods and procedures for determining patient sensitivity to one or more Epidermal Growth Factor Receptor (EGFR) modulators. The invention also provides methods of determining or predicting whether an individual requiring therapy for a disease state such as cancer will or will not respond to treatment, prior to administration of the treatment, wherein the treatment comprises administration of one or more EGFR modulators. The one or more EGFR modulators are compounds that can be selected from, for example, one or more EGFR-specific ligands, one or more small molecule EGFR inhibitors, or one or more EGFR binding monoclonal antibodies.


In one aspect, the invention provides a method for predicting the likelihood a mammal will respond therapeutically to a method of treating cancer comprising administering an EGFR modulator, wherein the method comprises: (a) measuring in the mammal the level of at least one biomarker selected from epiregulin and amphiregulin; (b) exposing a biological sample from the mammal to the EGFR modulator; (c) following the exposing of step (b), measuring in the biological sample the level of the at least one biomarker, wherein an increase in the level of the at least one biomarker measured in step (c) compared to the level of the at least one biomarker measured in step (a) indicates an increased likelihood that the mammal will respond therapeutically to the method of treating cancer. In one aspect, the at least one biomarker comprises epiregulin and amphiregulin. In yet another aspect, the at least one biomarker further comprises at least one additional biomarker selected from Table 1. In another aspect, the biological sample is a tissue sample comprising cancer cells and the method further comprises the step of determining whether the cancer cells have the presence of a mutated K-RAS, wherein detection of a mutated K-RAS indicates a decreased likelihood that that the mammal will respond therapeutically to the method of treating cancer.


The biological sample can be, for example, a tissue sample comprising cancer cells and the tissue is fixed, paraffin-embedded, fresh, or frozen.


In another aspect, the EGFR modulator is cetuximab and the cancer is colorectal cancer.


In another aspect, the invention is a method for predicting the likelihood a mammal will respond therapeutically to a method of treating cancer comprising administering an EGFR modulator, wherein the method comprises: (a) measuring in the mammal the level of at least one biomarker that comprises CD73; (b) exposing a biological sample from the mammal to the EGFR modulator; (c) following the exposing of step (b), measuring in the biological sample the level of the at least one biomarker, wherein an increase in the level of the at least one biomarker measured in step (c) compared to the level of the at least one biomarker measured in step (a) indicates a decreased likelihood that the mammal will respond therapeutically to the method of treating cancer. In another aspect, the at least one biomarker further comprises at least one additional biomarker selected from Table 1. In another aspect, the method further comprises the step of determining whether the cancer cells have the presence of a mutated K-RAS, wherein detection of a mutated K-RAS indicates a decreased likelihood that that the mammal will respond therapeutically to the method of treating cancer.


A difference in the level of the biomarker that is sufficient to predict the likelihood that the mammal will or will not respond therapeutically to the method of treating cancer can be readily determined by one of skill in the art using known techniques. The increase or decrease in the level of the biomarker can be correlated to determine whether the difference is sufficient to predict the likelihood that a mammal will respond therapeutically. The difference in the level of the biomarker that is sufficient can, in one aspect, be predetermined prior to predicting the likelihood that the mammal will respond therapeutically to the treatment. In one aspect, the difference in the level of the biomarker is a difference in the mRNA level (measured, for example, by RT-PCR or a microarray), such as at least a two-fold difference, at least a three-fold difference, or at least a four-fold difference in the level of expression. In another aspect, the difference in the level of the biomarker is determined by IHC. In another aspect, the difference in the level of the biomarker refers to a p-value of <0.05 in Anova (t test) analysis. In yet another aspect, the difference is determined in an ELISA assay.


As used herein, respond therapeutically refers to the alleviation or abrogation of the cancer. This means that the life expectancy of an individual affected with the cancer will be increased or that one or more of the symptoms of the cancer will be reduced or ameliorated. The term encompasses a reduction in cancerous cell growth or tumor volume. Whether a mammal responds therapeutically can be measured by many methods well known in the art, such as PET imaging.


The mammal can be, for example, a human, rat, mouse, dog, rabbit, pig sheep, cow, horse, cat, primate, or monkey.


The method of the invention can be, for example, an in vitro method wherein the step of measuring in the mammal the level of at least one biomarker comprises taking a biological sample from the mammal and then measuring the level of the biomarker(s) in the biological sample. The biological sample can comprise, for example, at least one of serum, whole fresh blood, peripheral blood mononuclear cells, frozen whole blood, fresh plasma, frozen plasma, urine, saliva, skin, hair follicle, bone marrow, or tumor tissue.


The level of the at least one biomarker can be, for example, the level of protein and/or mRNA transcript of the biomarker. The level of the biomarker can be determined, for example, by RT-PCR or another PCR-based method, immunohistochemistry, proteomics techniques, or any other methods known in the art, or their combination.


In another aspect, the invention provides a method for identifying a mammal that will respond therapeutically to a method of treating cancer comprising administering of an EGFR modulator, wherein the method comprises: (a) measuring in the mammal the level of at least one biomarker selected from the biomarkers of Table 1; (b) exposing a biological sample from the mammal to the EGFR modulator; (c) following the exposing in step (b), measuring in said biological sample the level of the at least one biomarker, wherein a difference in the level of the at least one biomarker measured in step (c) compared to the level of the at least one biomarker measured in step (a) indicates that the mammal will respond therapeutically to the said method of treating cancer.


In another aspect, the invention provides a method for identifying a mammal that will respond therapeutically to a method of treating cancer comprising administering an EGFR modulator, wherein the method comprises: (a) exposing a biological sample from the mammal to the EGFR modulator; (b) following the exposing of step (a), measuring in said biological sample the level of at least one biomarker selected from the biomarkers of Table 1, wherein a difference in the level of the at least one biomarker measured in step (b), compared to the level of the at least one biomarker in a mammal that has not been exposed to said EGFR modulator, indicates that the mammal will respond therapeutically to said method of treating cancer.


In yet another aspect, the invention provides a method for testing or predicting whether a mammal will respond therapeutically to a method of treating cancer comprising administering an EGFR modulator, wherein the method comprises: (a) measuring in the mammal the level of at least one biomarker selected from the biomarkers of Table 1; (b) exposing the mammal to the EGFR modulator; (c) following the exposing of step (b), measuring in the mammal the level of the at least one biomarker, wherein a difference in the level of the at least one biomarker measured in step (c) compared to the level of the at least one biomarker measured in step (a) indicates that the mammal will respond therapeutically to said method of treating cancer.


In another aspect, the invention provides a method for determining whether a compound inhibits EGFR activity in a mammal, comprising: (a) exposing the mammal to the compound; and (b) following the exposing of step (a), measuring in the mammal the level of at least one biomarker selected from the biomarkers of Table 1, wherein a difference in the level of said biomarker measured in step (b), compared to the level of the biomarker in a mammal that has not been exposed to said compound, indicates that the compound inhibits EGFR activity in the mammal.


In yet another aspect, the invention provides a method for determining whether a mammal has been exposed to a compound that inhibits EGFR activity, comprising (a) exposing the mammal to the compound; and (b) following the exposing of step (a), measuring in the mammal the level of at least one biomarker selected from the biomarkers of Table 1, wherein a difference in the level of said biomarker measured in step (b), compared to the level of the biomarker in a mammal that has not been exposed to said compound, indicates that the mammal has been exposed to a compound that inhibits EGFR activity.


In another aspect, the invention provides a method for determining whether a mammal is responding to a compound that inhibits EGFR activity, comprising (a) exposing the mammal to the compound; and (b) following the exposing of step (a), measuring in the mammal the level of at least one biomarker selected from the biomarkers of Table 1, wherein a difference in the level of the at least one biomarker measured in step (b), compared to the level of the at least one biomarker in a mammal that has not been exposed to said compound, indicates that the mammal is responding to the compound that inhibits EGFR activity.


As used herein, “responding” encompasses responding by way of a biological and cellular response, as well as a clinical response (such as improved symptoms, a therapeutic effect, or an adverse event), in a mammal.


The invention also provides an isolated biomarker selected from the biomarkers of Table 1. The biomarkers of the invention comprise sequences selected from the nucleotide and amino acid sequences provided in Table 1 and the Sequence Listing, as well as fragments and variants thereof.


The invention also provides a biomarker set comprising two or more biomarkers selected from the biomarkers of Table 1.


The invention also provides kits for determining or predicting whether a patient would be susceptible or resistant to a treatment that comprises one or more EGFR modulators. The patient may have a cancer or tumor such as, for example, colorectal cancer, NSCLC, or head and neck cancer.


In one aspect, the kit comprises a suitable container that comprises one or more specialized microarrays of the invention, one or more EGFR modulators for use in testing cells from patient tissue specimens or patient samples, and instructions for use. The kit may further comprise reagents or materials for monitoring the expression of a biomarker set at the level of mRNA or protein.


In another aspect, the invention provides a kit comprising two or more biomarkers selected from the biomarkers of Table 1.


In yet another aspect, the invention provides a kit comprising at least one of an antibody and a nucleic acid for detecting the presence of at least one of the biomarkers selected from the biomarkers of Table 1. In one aspect, the kit further comprises instructions for determining whether or not a mammal will respond therapeutically to a method of treating cancer comprising administering a compound that inhibits EGFR activity. In another aspect, the instructions comprise the steps of (a) measuring in the mammal the level of at least one biomarker selected from the biomarkers of Table 1, (b) exposing the mammal to the compound, (c) following the exposing of step (b), measuring in the mammal the level of the at least one biomarker, wherein a difference in the level of the at least one biomarker measured in step (c) compared to the level of the at least one biomarker measured in step (a) indicates that the mammal will respond therapeutically to said method of treating cancer.


The invention also provides screening assays for determining if a patient will be susceptible or resistant to treatment with one or more EGFR modulators.


The invention also provides a method of monitoring the treatment of a patient having a disease, wherein said disease is treated by a method comprising administering one or more EGFR modulators.


The invention also provides individualized genetic profiles which are necessary to treat diseases and disorders based on patient response at a molecular level.


The invention also provides specialized microarrays, e.g., oligonucleotide microarrays or cDNA microarrays, comprising one or more biomarkers having expression profiles that correlate with either sensitivity or resistance to one or more EGFR modulators.


The invention also provides antibodies, including polyclonal or monoclonal, directed against one or more biomarkers of the invention.


The invention will be better understood upon a reading of the detailed description of the invention when considered in connection with the accompanying figures.





BRIEF DESCRIPTION OF THE FIGURES


FIG. 1 illustrates a scheme used for identifying the biomarkers described herein.



FIG. 2 illustrates the expression profiling of the biomarkers described herein.



FIG. 3 (FIGS. 3A and 3B) illustrates the mRNA expression profiles of epiregulin and amphiregulin in 30 patients.



FIG. 4 illustrates the biological relationship of biomarkers described herein using Ingenuity Pathway Analysis.



FIG. 5 illustrates a comparison of a single biomarker model to multiple biomarker models.



FIG. 6 illustrates the filtering of candidate markers for cetuximab response. Expression data on 640 probe sets from 164 primary colorectal tumors was subjected to an unsupervised hierarchical clustering. The 164 tumors were divided into 3 major classes (Class 1, 2 and 3). The 640 probe sets were divided into 5 clusters (labeled A through E). Cluster A, which contains cancer antigens such as CEACAM 6 and CD24, also contains EREG and AREG. Cluster A is most highly expressed in Class 1a, which represents approximately 25% of the 164 colorectal tumor specimens.



FIG. 7 (FIGS. 7A and 7B) illustrates the mRNA levels of epiregulin and amphiregulin in 80 patients. Affymetrix mRNA levels of epiregulin (EREG, 205767_at) and amphiregulin (AREG, 205239_at) are plotted on the y axis. Subjects are ordered by best clinical response. There is a statistically significant difference in gene expression levels between the disease control group (CR, PR and SD) and the non-responder group (EREG p=1.474e−05, AREG p=2.489e−05).



FIG. 8 (FIGS. 8A and 8B) illustrates receiver operating characteristic (ROC) curves for prediction of patient response. FIG. 8A provides ROC using EREG to predict on test samples. EREG was the top single gene predictor using the discriminant function analysis, and has an area under the ROC curve (AUC) of 0.845 on the test set, indicating a high performance for prediction. FIG. 8B provides ROC using AREG to predict on the test set. The AREG gene, which was found to be coordinately regulated with the EREG gene, has an AUC of 0.815 on the test set, indicating that it too has a good prediction power as a single gene predictor.



FIG. 9 illustrates the results obtained from validation of AREG and EREG Affymetrix expression by qRT-PCR. A good correlation between the two methods (Pearson>0.85, R2>0.7) was seen. High expression on Affymetrix arrays (y axis) corresponds to low ΔCt values from TaqMan qPCR assays for both AREG and EREG (x axis).





DETAILED DESCRIPTION OF THE INVENTION

Identification of biomarkers that provide rapid and accessible readouts of efficacy, drug exposure, or clinical response is increasingly important in the clinical development of drug candidates. Embodiments of the invention include measuring changes in the levels of secreted proteins, or plasma biomarkers, which represent one category of biomarker. In one aspect, plasma samples, which represent a readily accessible source of material, serve as surrogate tissue for biomarker analysis.


The invention provides biomarkers that respond to the modulation of a specific signal transduction pathway and also correlate with EGFR modulator sensitivity or resistance. These biomarkers can be employed for predicting response to one or more EGFR modulators. In one aspect, the biomarkers of the invention are those provided in Table 1 and the Sequence Listing, including both polynucleotide and polypeptide sequences. The invention also includes nucleotide sequences that hybridize to the polynucleotides provided in Table 1.









TABLE 1







Biomarkers











Affymetrix


Unigene title and SEQ ID NO:
Affymetrix Description
Probe Set





NT5E: 5′-nucleotidase, ecto
gb: NM_002526.1 /DEF = Homo sapiens
203939_at


(CD73) (LOC4907)
5 nucleotidase (CD73) (NT5), mRNA.


SEQ ID NOS: 1 (DNA) and 129
/FEA = mRNA /GEN = NT5 /PROD = 5


(amino acid)
nucleotidase /DB_XREF = gi: 4505466



/UG = Hs.153952 5 nucleotidase (CD73)



/FL = gb: NM_002526.1


EREG: epiregulin (LOC2069)
gb: NM_001432.1 /DEF = Homo sapiens.
205767_at


SEQ ID NOS: 2 (DNA) and 130
epiregulin (EREG), mRNA.


(amino acid)
/FEA = mRNA /GEN = EREG



/PROD = epiregulin precursor



/DB_XREF = gi: 4557566



/UG = Hs.115263 epiregulin



/FL = gb: D30783.1 gb: NM_001432.1


AREG: amphiregulin
gb: NM_001657.1 /DEF = Homo sapiens
205239_at


(schwannoma-derived growth
amphiregulin (schwannoma-derived


factor) (LOC374)
growth factor) (AREG), mRNA.


SEQ ID NOS: 3 (DNA) and 131
/FEA = mRNA /GEN = AREG


(amino acid)
/PROD = amphiregulin (schwannoma-



derived growth factor)



/DB_XREF = gi: 4502198



/UG = Hs.270833 amphiregulin



(schwannoma-derived growth factor)



/FL = gb: M30704.1 gb: NM_001657.1


LYZ: lysozyme (renal
Consensus includes gb: AV711904
213975_s_at


amyloidosis) (LOC4069)
/FEA = EST /DB_XREF = gi: 10731210


SEQ ID NOS: 4 (DNA) and 132
/DB_XREF = est: AV711904


(amino acid)
/CLONE = DCAAIE08 /UG = Hs.277431




Homo sapiens cDNA: FLJ23356 fis,




clone HEP14919


BST2: bone marrow stromal cell
gb: NM_004335.2 /DEF = Homo sapiens
201641_at


antigen 2 (LOC684)
bone marrow stromal cell antigen 2


SEQ ID NOS: 5 (DNA) and 133
(BST2), mRNA. /FEA = mRNA


(amino acid)
/GEN = BST2 /PROD = bone marrow



stromal cell antigen 2



/DB_XREF = gi: 7262372



/UG = Hs.118110 bone marrow stromal



cell antigen 2 /FL = gb: D28137.1



gb: NM_004335.2


DUSP6: dual specificity
gb: BC005047.1 /DEF = Homo sapiens,
208893_s_at


phosphatase 6 (LOC1848)
clone MGC: 12852, mRNA, complete


SEQ ID NOS: 6 (DNA) and 134
cds. /FEA = mRNA /PROD = Unknown


(amino acid)
(protein for MGC: 12852)



/DB_XREF = gi: 13477170



/UG = Hs.180383 dual specificity



phosphatase 6 /FL = gb: BC003562.1



gb: BC003143.1 gb: BC005047.1



gb: AB013382.1 gb: NM_001946.1


VAV3: vav 3 oncogene
gb: NM_006113.2 /DEF = Homo sapiens
218807_at


(LOC10451)
vav 3 oncogene (VAV3), mRNA.


SEQ ID NOS: 7 (DNA) and 135
/FEA = mRNA /GEN = VAV3


(amino acid)
/PROD = vav 3 oncogene



/DB_XREF = gi: 7262390



/UG = Hs.267659 vav 3 oncogene



/FL = gb: AF067817.1 gb: AF118887.1



gb: NM_006113.2


VAV3: vav 3 oncogene
gb: AF118887.1 /DEF = Homo sapiens
218806_s_at


(LOC10451)
VAV-3 protein (VAV-3) mRNA,


SEQ ID NOS: 8 (DNA) and 136
alternatively spliced, complete cds.


(amino acid)
/FEA = mRNA /GEN = VAV-3



/PROD = VAV-3 protein



/DB_XREF = gi: 4416407



/UG = Hs.267659 vav 3 oncogene



/FL = gb: AF067817.1 gb: AF118887.1



gb: NM_006113.2


CCL2: chemokine (C-C motif)
Consensus includes gb: S69738.1
216598_s_at


ligand 2 (LOC6347)
/DEF = MCP-1 = monocyte chemotactic


SEQ ID NOS: 9 (DNA) and 137
protein human, aortic endothelial cells,


(amino acid)
mRNA, 661 nt. /FEA = mRNA



/GEN = MCP-1 /PROD = MCP-1



/DB_XREF = gi: 545464



/UG = Hs.303649 small inducible



cytokine A2 (monocyte chemotactic



protein 1, homologous to mouse Sig-je)


SATB2: SATB family member 2
Consensus includes gb: AB028957.1
213435_at


(LOC23314)
/DEF = Homo sapiens mRNA for


SEQ ID NOS: 10 (DNA) and
KIAA1034 protein, partial ccls.


138 (amino acid)
/FEA = mRNA /GEN = KIAA1034



/PROD = KIAA1034 protein



/DB_XREF = gi: 5689404



/UG = Hs.12896 KIAA1034 protein


AKAP12: A kinase (PRKA)
gb: AB003476.1 /DEF = Homo sapiens
210517_s_at


anchor protein (gravin) 12
mRNA for gravin, complete cds.


(LOC9590)
/FEA = mRNA /PROD = gravin


SEQ ID NOS: 11 (DNA) and
/DB_XREF = gi: 2081606 /UG = Hs.788


139 (amino acid)
A kinase (PRKA) anchor protein



(gravin) 12 /FL = gb: AB003476.1


GCNT3: glucosaminyl (N-
gb: NM_004751.1 /DEF = Homo sapiens
219508_at


acetyl) transferase 3, mucin type
glucosaminyl (N-acetyl) transferase 3,


(LOC9245)
mucin type (GCNT3), mRNA.


SEQ ID NOS: 12 (DNA) and
/FEA = mRNA /GEN = GCNT3


140 (amino acid)
/PROD = glucosaminyl (N-acetyl)



transferase 3, mucintype



/DB_XREF = gi: 4758421



/UG = Hs.194710 glucosaminyl (N-



acetyl) transferase 3, mucin type



/FL = gb: AF102542.1 gb: AF038650.1



gb: NM_004751.1


SCRN1: secernin 1 (LOC9805)
gb: NM_014766.1 /DEF = Homo sapiens
201462_at


SEQ ID NOS: 13 (DNA) and
KIAA0193 gene product (KIAA0193),


141 (amino acid)
mRNA. /FEA = mRNA



/GEN = KIAA0193 /PROD = KIAA0193



gene product /DB_XREF = gi: 7661983



/UG = Hs.75137 KIAA0193 gene



product /FL = gb: D83777.1



gb: NM_014766.1


FGFR3: fibroblast growth factor
gb: NM_000142.2 /DEF = Homo sapiens
204379_s_at


receptor 3 (achondroplasia,
fibroblast growth factor receptor 3


thanatophoric dwarfism)
(achondroplasia, thanatophoric


(LOC2261)
dwarfism) (FGFR3), transcript variant


SEQ ID NOS: 14 (DNA) and
1, mRNA. /FEA = mRNA


142 (amino acid)
/GEN = FGFR3 /PROD = Fibroblast



growth factor receptor 3, isoform



1 precursor /DB_XREF = gi: 13112046



/UG = Hs.1420 fibroblast growth factor



receptor 3 (achondroplasia,



thanatophoric dwarfism)



/FL = gb: NM_000142.2 gb: M58051.1


LY96: lymphocyte antigen 96
gb: NM_015364.1 /DEF = Homo sapiens
206584_at


(LOC23643)
MD-2 protein (MD-2), mRNA.


SEQ ID NOS: 15 (DNA) and
/FEA = mRNA /GEN = MD-2


143 (amino acid)
/PROD = MD-2 protein



/DB_XREF = gi: 7662503



/UG = Hs.69328 MD-2 protein



/FL = gb: AB018549.1 gb: NM_015364.1



gb: AF168121.1


CKB: creatine kinase, brain
gb: NM_001823.1 /DEF = Homo sapiens
200884_at


(LOC1152)
creatine kinase, brain (CKB), mRNA.


SEQ ID NOS: 16 (DNA) and
/FEA = mRNA /GEN = CKB


144 (amino acid)
/PROD = creatine kinase, brain



/DB_XREF = gi: 4502850



/UG = Hs.173724 creatine kinase, brain



/FL = gb: L47647.1 gb: BC001190.1



gb: BC004914.1 gb: M16364.1



gb: M16451.1 gb: NM_001823.1


IFI16: interferon, gamma-
gb: NM_005531.1 /DEF = Homo sapiens
206332_s_at


inducible protein 16 (LOC3428)
interferon, gamma-inducible protein 16


SEQ ID NOS: 17 (DNA) and
(IFI16), mRNA. /FEA = mRNA


145 (amino acid)
/GEN = IFI16 /PROD = interferon,



gamma-inducible protein 16



/DB_XREF = gi: 5031778



/UG = Hs.155530 interferon, gamma-



inducible protein 16 /FL = gb: M63838.1



gb: NM_005531.1


PRSS8: protease, serine, 8
gb: NM_002773.1 /DEF = Homo sapiens
202525_at


(prostasin) (LOC5652)
protease, serine, 8 (prostasin) (PRSS8),


SEQ ID NOS: 18 (DNA) and
mRNA. /FEA = mRNA /GEN = PRSS8


146 (amino acid)
/PROD = protease, serine, 8 (prostasin)



/DB_XREF = gi: 4506152



/UG = Hs.75799 protease, serine, 8



(prostasin) /FL = gb: BC001462.1



gb: NM_002773.1 gb: L41351.1


IL1R2: interleukin 1 receptor,
gb: NM_004633.1 /DEF = Homo sapiens
205403_at


type II (LOC7850)
interleukin 1 receptor, type II (IL1R2),


SEQ ID NOS: 19 (DNA) and
mRNA. /FEA = mRNA /GEN = IL1R2


147 (amino acid)
/PROD = interleukin 1 receptor, type II



/DB_XREF = gi: 4758597



/UG = Hs.25333 interleukin 1 receptor,



type II /FL = gb: U74649.1



gb: NM_004633.1


BHLHB3: basic helix-loop-helix
Consensus includes gb: BE857425
221530_s_at


domain containing, class B, 3
/FEA = EST /DB_XREF = gi: 10371439


(LOC79365)
/DB_XREF = est: 7f97a11.x1


SEQ ID NOS: 20 (DNA) and
/CLONE = IMAGE: 3304892


148 (amino acid)
/UG = Hs.33829 bHLH protein DEC2



/FL = gb: AB044088.1


HLA-DRB4: major
gb: BC005312.1 /DEF = Homo sapiens,
209728_at


histocompatibility complex,
clone MGC: 12387, mRNA, complete


class II, DR beta 4 (LOC3126)
cds. /FEA = mRNA /PROD = Unknown


SEQ ID NOS: 21 (DNA) and
(protein for MGC: 12387)


149 (amino acid)
/DB_XREF = gi: 13529055



/UG = Hs.318720 Homo sapiens, clone



MGC: 12387, mRNA, complete cds



/FL = gb: BC005312.1 gb: M16942.1


CD163: CD163 antigen
Consensus includes gb: Z22969.1
215049_x_at


(LOC9332)
/DEF = H. sapiens mRNA for M130


SEQ ID NOS: 22 (DNA) and
antigen cytoplasmic variant 1.


150 (amino acid)
/FEA = mRNA /PROD = M130 antigen



cytoplasmic variant 1



/DB_XREF = gi: 312143 /UG = Hs.74076



CD163 antigen


CD163: CD163 antigen
gb: NM_004244.1 /DEF = Homo sapiens
203645_s_at


(LOC9332)
CD163 antigen (CD163), mRNA.


SEQ ID NOS: 23 (DNA) and
/FEA: = mRNA /GEN = CD163


151 (amino acid)
/PROD = CD163 antigen



/DB_XREF = gi: 4758721



/UG = Hs.74076 CD163 antigen



/FL = gb: NM_004244.1


C13orf18: chromosome 13 open
gb: NM_025113.1 /DEF = Homo sapiens
219471_at


reading frame 18 (LOC80183)
hypothetical protein FLJ21562


SEQ ID NOS: 24 (DNA) and
(FLJ21562), mRNA. /FEA = mRNA


152 (amino acid)
/GEN = FLJ21562 /PROD = hypothetical



protein FLJ21562



/DB_XREF = gi: 13376686



/UG = Hs.288708 hypothetical protein



FLJ21562 /FL = gb: NM_025113.1


CCL11: chemokine (C-C motif)
gb: D49372.1 /DEF = Human mRNA for
210133_at


ligand 11 (LOC6356)
eotaxin, complete cds. /FEA = mRNA


SEQ ID NOS: 25 (DNA) and
/PROD = eotaxin


153 (amino acid)
/DB_XREF = gi: 1552240



/UG = Hs.54460 small inducible



cytokine subfamily A (Cys-Cys),



member 11 (eotaxin) /FL = gb: U46573.1



gb: D49372.1 gb: NM_002986.1


SLC26A2: solute carrier family
Consensus includes gb: AI025519
205097_at


26 (sulfate transporter), member
/FEA = EST /DB_XREF = gi: 3241132


2 (LOC1836)
/DB_XREF = est: ov75c04.x1


SEQ ID NOS: 26 (DNA) and
/CLONE = IMAGE: 1643142


154 (amino acid)
/UG = Hs.29981 solute carrier family 26



(sulfate transporter), member 2



/FL = gb: NM_000112.1 gb: U14528.1


HLA-DQB1: major
gb: M32577.1 /DEF = Human MHC
211656_x_at


histocompatibility complex,
HLA-DQ beta mRNA, complete cds.


class II, DQ beta 1 (LOC3119)
/FEA = mRNA /GEN = HLA-DQB1


SEQ ID NOS: 27 (DNA) and
/DB _XREF = gi: 188194


155 (amino acid)
/FL = gb: M32577.1


ENPP2: ectonucleotide
gb: L35594.1 /DEF = Human autotaxin
209392_at


pyrophosphatase/phosphodiesterase
mRNA, complete cds. /FEA = mRNA


2 (autotaxin) (LOC5168)
/PROD = autotaxin


SEQ ID NOS: 28 (DNA) and
/DB_XREF = gi: 537905


156 (amino acid)
/UG = Hs.174185 ectonucleotide



pyrophosphatasephosphodiesterase 2



(autotaxin) /FL = gb: L35594.1


PRSS3: protease, serine, 3
gb: NM_002770.1 /DEF = Homo sapiens
205402_x_at


(mesotrypsin) (LOC5646)
protease, serine, 2 (trypsin 2) (PRSS2),


SEQ ID NOS: 29 (DNA) and
mRNA. /FEA = mRNA /GEN = PRSS2


157 (amino acid)
/PROD = protease, serine, 2 (trypsin 2)



/DB_XREF = gi: 4506146



/UG = Hs.241561 protease, serine, 2



(trypsin 2) /FL = gb: NM_002770.1



gb: M27602.1


CXCR4: chemokine (C—X—C
Consensus includes gb: AJ224869
217028_at


motif) receptor 4 (LOC7852)
/DEF = Homo sapiens CXCR4 gene


SEQ ID NOS: 30 (DNA) and
encoding receptor CXCR4


158 (amino acid)
/FEA = mRNA /DB_XREF = gi: 3059119



/UG = Hs.89414 chemokine (C—X—C



motif), receptor 4 (fusin)


SERPINB5: serine (or cysteine)
gb: NM_002639.1 /DEF = Homo sapiens
204855_at


proteinase inhibitor, clade B
serine (or cysteine) proteinase inhibitor,


(ovalbumin), member 5
clade B (ovalbumin), member 5


(LOC5268)
(SERPINB5), mRNA. /FEA = mRNA


SEQ ID NOS: 31 (DNA) and
/GEN = SERPINB5 /PROD = serine (or


159 (amino acid)
cysteine) proteinase inhibitor, cladeB



(ovalbumin), member 5



/DB_XREF = gi: 4505788



/UG = Hs.55279 serine (or cysteine)



proteinase inhibitor, clade B



(ovalbumin), member 5



/FL = gb: NM_002639.1 gb: U04313.1


HLA-DPB1: major
gb: NM_002121.1 /DEF = Homo sapiens
201137_s_at


histocompatibility complex,
major histocompatibility complex, class


class II, DP beta 1 (LOC3115)
II, DP beta 1 (HLA-DPB1), mRNA.


SEQ ID NOS: 32 (DNA) and
/FEA = mRNA /GEN = HLA-DPB1


160 (amino acid)
/PROD = major histocompatibility



complex, class II, DPbeta 1



/DB_XREF = gi: 4504404 /UG = Hs.814



major histocompatibility complex, class



II, DP beta 1 /FL = gb: J03041.1



gb: M57466.1 gb: M83664.1



gb: NM_002121.1 gb: M28200.1



gb: M28202.1


AIF1: allograft inflammatory
Consensus includes gb: BF213829
215051_x_at


factor 1 (LOC199)
/FEA = EST /DB_XREF = gi: 11107415


SEQ ID NOS: 33 (DNA) and
/DB_XREF = est: 601848003F1


161 (amino acid)
/CLONE = IMAGE: 4078849



/UG = Hs.76364 allograft inflammatory



factor 1


IL8: interleukin 8 (LOC3576)
gb: NM_000584.1 /DEF = Homo sapiens
202859_x_at


SEQ ID NOS: 34 (DNA) and
interleukin 8 (IL8), mRNA.


162 (amino acid)
/FEA = mRNA /GEN = IL8



/PROD = interleukin 8



/DB_XREF = gi: 10834977 /UG = Hs.624



interleukin 8 /FL = gb: NM_000584.1



gb: M17017.1 gb: M26383.1


IL8: interleukin 8 (LOC3576)
gb: AF043337.1 /DEF = Homo sapiens
211506_s_at


SEQ ID NOS: 35 (DNA) and
interleukin 8 C-terminal variant (IL8)


163 (amino acid)
mRNA, complete cds. /FEA = mRNA



/GEN = IL8 /PROD = interleukin 8 C-



terminal variant



/DB_XREF = gi: 12641914 /UG = Hs.624



interleukin 8 /FL = gb: AF043337.1


LY6G6D: lymphocyte antigen 6
gb: NM_021246.1 /DEF = Homo sapiens
207457_s_at


complex, locus G6D
megakaryocyte-enhanced gene


(LOC58530)
transcript 1 protein (MEGT1), mRNA.


SEQ ID NOS: 36 (DNA) and
/FEA = mRNA /GEN = MEGT1


164 (amino acid)
/PROD = megakaryocyte-enhanced gene



transcript 1protein



/DB_XREF = gi: 10864054



/UG = Hs.241587 megakaryocyte-



enhanced gene transcript 1 protein



/FL = gb: NM_021246.1 gb: AF195764.1


CYP3A5: cytochrome P450,
gb: NM_000777.1 /DEF = Homo sapiens
205765_at


family 3, subfamily A,
cytochrome P450, subfamily IIIA


polypeptide 5 (LOC1577)
(niphedipine oxidase), polypeptide 5


SEQ ID NOS: 37 (DNA) and
(CYP3A5), mRNA. /FEA = mRNA


165 (amino acid)
/GEN = CYP3A5 /PROD = cytochrome



P450, subfamily IIIA, polypeptide 5



/DB_XREF = gi: 4503230



/UG = Hs.104117 cytochrome P450,



subfamily IIIA (niphedipine oxidase),



polypeptide 5 /FL gb: J04813.1



gb: NM_000777.1


CSPG2: chondroitin sulfate
Consensus includes gb: BF590263
204619_s_at


proteoglycan 2 (versican)
/FEA = EST /DB_XREF = gi: 11682587


(LOC1462)
/DB_XREF = est: nab22b12.x1


SEQ ID NOS: 38 (DNA) and
/CLONE = IMAGE: 3266638


166 (amino acid)
/UG = Hs.81800 chondroitin sulfate



proteoglycan 2 (versican)



/FL = gb: NM_004385.1


CA9: carbonic anhydrase IX
gb: NM_001216.1 /DEF = Homo sapiens
205199_at


(LOC768)
carbonic anhydrase IX (CA9), mRNA.


SEQ ID NOS: 39 (DNA) and
/FEA = mRNA /GEN = CA9


167 (amino acid)
/PROD = carbonic anhydrase IX



precursor /DB_XREF = gi: 9955947



/UG = Hs.63287 carbonic anhydrase IX



/FL = gb: NM_001216.1


ACE2: angiotensin I converting
gb: NM_021804.1 /DEF = Homo sapiens
219962_at


enzyme (peptidyl-dipeptidase A)
angiotensin I converting enzyme


2 (LOC59272)
(peptidyl-dipeptidase A) 2 (ACE2),


SEQ ID NOS: 40 (DNA) and
mRNA. /FEA = mRNA /GEN = ACE2


168 (amino acid)
/PROD = angiotensin I converting



enzyme(peptidyl-dipeptidase A) 2



/DB_XREF = gi: 11225608



/UG = Hs.178098 angiotensin I



converting enzyme (peptidyl-



dipeptidase A) 2



/FL = gb: NM_021804.1 gb: AB046569.1



gb: AF241254.1 gb: AF291820.1


CXCL13: chemokine (C—X—C
gb: NM_006419.1 /DEF = Homo sapiens
205242_at


motif) ligand 13 (B-cell
small inducible cytokine B subfamily


chemoattractant) (LOC10563)
(Cys-X-Cys motif), member 13 (B-cell


SEQ ID NOS: 41 (DNA) and
chemoattractant) (SCYB13), mRNA.


169 (amino acid)
/FEA = mRNA /GEN = SCYB13



/PROD = small inducible cytokine B



subfamily (Cys-X-Cysmotif), member



13 (B-cell chemoattractant)



/DB_XREF = gi: 5453576



/UG = Hs.100431 small inducible



cytokine B subfamily (Cys-X-Cys



motif), member 13 (B-cell



chemoattractant) /FL = gb: AF044197.1



gb: AF029894.1 gb: NM_006419.1


COL10A1: collagen, type X,
Consensus includes gb: X98568
217428_s_at


alpha 1(Schmid metaphyseal
/DEF = H. sapiens type X collagen gene


chondrodysplasia) (LOC1300)
/FEA = mRNA /DB_XREF = gi: 1405722


SEQ ID NOS: 42 (DNA) and
/UG = Hs.179729 collagen, type X,


170 (amino acid)
alpha 1 (Schmid metaphyseal



chondrodysplasia)


CPNE1: copine I (LOC8904)
gb: NM_003915.1 /DEF = Homo sapiens
206918_s_at


SEQ ID NOS: 43 (DNA) and
copine I (CPNE1), mRNA.


171 (amino acid)
/FEA = mRNA /GEN = CPNE1



/PROD = copine I



/DB_XREF = gi: 4503012



/UG = Hs.166887 copine I



/FL = gb: U83246.1 gb: NM_003915.1


C13orf18: chromosome 13 open
Cluster Incl. AI129310: qc48a05.x1
44790_s_at


reading frame 18 (LOC80183)

Homo sapiens cDNA, 3 end



SEQ ID NOS: 44 (DNA) and
/clone = IMAGE-1712816 /clone_end = 3′


172 (amino acid)
/gb = AI129310 /gi = 3597824



/ug = Hs.234923 /len = 811


GREM1: gremlin 1 homolog,
gb: NM_013372.1 /DEF = Homo sapiens
218469_at


cysteine knot superfamily
cysteine knot superfamily 1, BMP


(Xenopus laevis) (LOC26585)
antagonist 1 (CKTSF1B1), mRNA.


SEQ ID NOS: 45 (DNA) and
/FEA = mRNA /GEN = CKTSF1B1


173 (amino acid)
/PROD = cysteine knot superfamily 1,



BMP antagonist 1



/DB_XREF = gi: 7019348



/UG = Hs.40098 cysteine knot



superfamily 1, BMP antagonist 1



/FL = gb: AF154054.1 gb: AF045800.1



gb: AF110137.2 gb: NM_013372.1


HLA-DQB1: major
gb: M17955.1 /DEF = Human MHC
209823_x_at


histocompatibility complex,
class II HLA-DQ-beta mRNA,


class II, DQ beta 1 (LOC3119)
complete cds. /FEA = mRNA


SEQ ID NOS: 46 (DNA) and
/DB_XREF = gi: 188178 /UG = Hs.73931


174 (amino acid)
major histocompatibility complex, class



II, DQ beta 1 /FL = gb: M33907.1



gb: M17955.1 gb: M17563.1



gb: M26042.1 gb: M20432.1



gb: M16996.1


TCN1: transcobalamin I
gb: NM_001062.1 /DEF = Homo sapiens
205513_at


(vitamin B12 binding protein, R
transcobalamin I (vitamin B12 binding


binder family) (LOC6947)
protein, R binder family) (TCN1),


SEQ ID NOS: 47 (DNA) and
mRNA. /FEA = mRNA /GEN = TCN1


175 (amino acid)
/PROD = transcobalamin I (vitamin B12



binding protein, Rbinder family)



/DB_XREF = gi: 4507406 /UG = Hs.2012



transcobalamin I (vitamin B12 binding



protein, R binder family)



/FL = gb: J05068.1 gb: NM_001062.1


PIGR: polymeric
gb: NM_002644.1 /DEF = Homo sapiens
204213_at


immunoglobulin receptor
polymeric immunoglobulin receptor


(LOC5284)
(PIGR), mRNA. /FEA = mRNA


SEQ ID NOS: 48 (DNA) and
/GEN = PIGR /PROD = polymeric


176 (amino acid)
immunoglobulin receptor



/DB_XREF = gi: 11342673



/UG = Hs.288579 polymeric



immunoglobulin receptor



/FL = gb: NM_002644.1


COL10A1: collagen, type X,
Consensus includes gb: AI376003
205941_s_at


alpha 1(Schmid metaphyseal
/FEA = EST /DB_XREF = gi: 4175993


chondrodysplasia) (LOC1300)
/DB_XREF = est: tc30d11.x1


SEQ ID NOS: 49 (DNA) and
/CLONE = IMAGE: 2066133


177 (amino acid)
/UG = Hs.179729 collagen, type X,



alpha 1 (Schmid metaphyseal



chondrodysplasia)



/FL: = gb: NM_000493.1


KCTD12: potassium channel
Consensus includes gb: AI718937
212192_at


tetramerisation domain
/FEA = EST /DB_XREF = gi: 5036193


containing 12 (LOC115207)
/DB_XREF = est: as50b04.x1


SEQ ID NOS: 50 (DNA) and
/CLONE = IMAGE: 2320591


178 (amino acid)
/UG = Hs.109438 Homo sapiens clone



24775 mRNA sequence


LCK: lymphocyte-specific
gb: NM_005356.1 /DEF = Homo sapiens
204891_s_at


protein tyrosine kinase
lymphocyte-specific protein tyrosine


(LOC3932)
kinase (LCK), mRNA. /FEA = mRNA


SEQ ID NOS: 51 (DNA) and
/GEN = LCK /PROD = lymphocyte-


179 (amino acid)
specific protein tyrosine kinase



/DB_XREF = gi: 4885448 /UG = Hs.1765



lymphocyte-specific protein tyrosine



kinase /FL = gb: M36881.1 gb: U07236.1



gb: NM_005356.1


LAPTM4B: lysosomal
gb: NM_018407.1 /DEF = Homo sapiens
208029_s_at


associated protein
putative integral membrane transporter


transmembrane 4 beta
(LC27), mRNA. /FEA = mRNA


(LOC55353)
/GEN = LC27 /PROD = putative integral


SEQ ID NOS: 52 (DNA) and
membrane transporter


180 (amino acid)
/DB_XREF = gi: 8923827



/FL = gb: NM_018407.1


CEACAM5: carcinoembryonic
gb: NM_004363.1 /DEF = Homo sapiens
201884_at


antigen-related cell adhesion
carcinoembryonic antigen-related cell


molecule 5 (LOC1048)
adhesion molecule 5 (CEACAM5),


SEQ ID NOS: 53 (DNA) and
mRNA. /FEA = mRNA


181 (amino acid)
/GEN = CEACAM5



/PROD = carcinoembryonic antigen-



related cell adhesionmolecule 5



/DB_XREF = gi: 11386170



/UG = Hs.220529 carcinoembryonic



antigen-related cell adhesion molecule



5 /FL = gb: NM_004363.1 gb: M29540.1


LDHB: lactate dehydrogenase B
gb: NM_002300.1 /DEF = Homo sapiens
201030_x_at


(LOC3945)
lactate dehydrogenase B (LDHB),


SEQ ID NOS: 54 (DNA) and
mRNA. /FEA = mRNA /GEN = LDHB


182 (amino acid)
/PROD = lactate dehydrogenase B



/DB_XREF = gi: 4557031



/UG = Hs.234489 lactate dehydrogenase



B /FL = gb: BC002362.1



gb: NM_002300.1


IFI27: interferon, alpha-
gb: NM_005532.1 /DEF = Homo sapiens
202411_at


inducible protein 27 (LOC3429)
interferon, alpha-inducible protein 27


SEQ ID NOS: 55 (DNA) and
(IFI27), mRNA. /FEA = mRNA


183 (amino acid)
/GEN = IFI27 /PROD = interferon, alpha-



inducible protein 27



/DB_XREF = gi: 5031780



/UG = Hs.278613 interferon, alpha-



inducible protein 27



/FL = gb: NM_005532.1


EPHB2: EphB2 (LOC2048)
gb: D31661.1 /DEF = Human mRNA for
211165_x_at


SEQ ID NOS: 56 (DNA) and
tyrosine kinase, complete cds.


184 (amino acid)
/FEA = mRNA /GEN = ERK



/PROD = tyrosine kinase precursor



/DB_XREF = gi: 495677



/UG = Hs.125124 EphB2



/FL = gb: D31661.1


ACACA: acetyl-Coenzyme A
Consensus includes gb: BE855983
212186_at


carboxylase alpha (LOC31)
/FEA = EST /DB_XREF = gi: 10368561


SEQ ID NOS: 57 (DNA) and
/DB_XREF = est: 7f85g11.x1


185 (amino acid)
/CLONE = IMAGE: 3303812



/UG = Hs.7232 acetyl-Coenzyme A



carboxylase alpha



/FL = gb: NM_000664.1 gb: U19822.1


CD14: CD14 antigen (LOC929)
gb: NM_000591.1 /DEF = Homo sapiens
201743_at


SEQ ID NOS: 58 (DNA) and
CD14 antigen (CD14), mRNA.


186 (amino acid)
/FEA = mRNA /GEN = CD14



/PROD = CD14 antigen precursor



/DB_XREF = gi: 4557416



/UG = Hs.75627 CD14 antigen



/FL = gb: M86511.1 gb: AF097942.1



gb: NM_000591.1


ABHD2: abhydrolase domain
Cluster Incl. AI832249: td14g10.x1
87100_at


containing 2 (LOC11057)

Homo sapiens cDNA, 3 end



SEQ ID NOS: 59 (DNA) and
/clone = IMAGE-2075682 /clone_end = 3′


187 (amino acid)
/gb = AI832249 /gi = 5452920



/ug = Hs.211522 /len = 545


TNFRSF6B: tumor necrosis
gb: NM_003823.1 /DEF = Homo sapiens
206467_x_at


factor receptor superfamily,
tumor necrosis factor receptor


member 6b, decoy (LOC8771)
superfamily, member 6b, decoy


SEQ ID NOS: 60 (DNA) and
(TNFRSF6B), mRNA. /FEA = mRNA


188 (amino acid)
/GEN = TNFRSF6B /PROD = decoy



receptor 3 /DB_XREF = gi: 4507584



/UG = Hs.278556 tumor necrosis factor



receptor superfamily, member 6b,



decoy /FL = gb: AF104419.1



gb: NM_003823.1 gb: AF134240.1



gb: AF217794.1


GREM1: gremlin 1 homolog,
gb: AF154054.1 /DEF = Homo sapiens
218468_s_at


cysteine knot superfamily
DRM (DRM) mRNA, complete cds.


(Xenopus laevis) (LOC26585)
/FEA = mRNA /GEN = DRM


SEQ ID NOS: 61 (DNA) and
/PROD = DRM


189 (amino acid)
/DB_XREF = gi: 10863087



/UG = Hs.40098 cysteine knot



superfamily 1, BMP antagonist 1



/FL = gb: AF154054.1 gb: AF045800.1



gb: AF110137.2 gb: NM_013372.1


ACE2: angiotensin I converting
Consensus includes gb: AK026461.1
222257_s_at


enzyme (peptidyl-dipeptidase A)
/DEF = Homo sapiens cDNA: FLJ22808


2 (LOC59272)
fis, clone KAIA2925. /FEA = mRNA


SEQ ID NOS: 62 (DNA) and
/DB_XREF = gi: 10439331


190 (amino acid)
/UG = Hs.178098 angiotensin I



converting enzyme (peptidyl-



dipeptidase A) 2


COL5A2: collagen, type V,
Consensus includes gb: NM_000393.1
221730_at


alpha 2 (LOC1290)
/DEF = Homo sapiens collagen, type V,


SEQ ID NOS: 63 (DNA) and
alpha 2 (COL5A2), mRNA.


191 (amino acid)
/FEA = CDS /GEN = COL5A2



/PROD = collagen, type V, alpha 2



/DB_XREF = gi: 4502958



/UG = Hs.82985 collagen, type V, alpha



2 /FL = gb: NM_000393.1


CXCL9: chemokine (C—X—C
gb: NM_002416.1 /DEF = Homo sapiens
203915_at


motif) ligand 9 (LOC4283)
monokine induced by gamma


SEQ ID NOS: 64 (DNA) and
interferon (MIG), mRNA.


192 (amino acid)
/FEA = mRNA /GEN = MIG



/PROD = monokine induced by gamma



interferon /DB_XREF = gi: 4505186



/UG = Hs.77367 monokine induced by



gamma interferon



/FL = gb: NM_002416.1


HOXC6: homeo box C6
gb: NM_004503.1 /DEF = Homo sapiens
206858_s_at


(LOC3223)
homeo box C6 (HOXC6), mRNA.


SEQ ID NOS: 65 (DNA) and
/FEA = mRNA /GEN = HOXC6


193 (amino acid)
/PROD = homeo box C6



/DB_XREF = gi: 4758553 /UG = Hs.820



homeo box C6 /FL = gb: NM_004503.1


KCNMA1: potassium large
gb: U11058.2 /DEF = Homo sapiens
221584_s_at


conductance calcium-activated
large conductance calcium- and


channel, subfamily M, alpha
voltage-dependent potassium channel


member 1 (LOC3778)
alpha subunit (MaxiK) mRNA,


SEQ ID NOS: 66 (DNA) and
complete cds. /FEA = mRNA


194 (amino acid)
/GEN = MaxiK /PROD = large



conductance calcium- and voltage-



dependentpotassium channel alpha



subunit /DB_XREF = gi: 7914977



/UG = Hs.89463 potassium large



conductance calcium-activated channel,



subfamily M, alpha member 1



/FL = gb: AF025999.1 gb: NM_002247.1



gb: AF118141.1 gb: U13913.1



gb: U11717.1 gb: U23767.1



gb: U11058.2


MMP1: matrix metalloproteinase
gb: NM_002421.2 /DEF = Homo sapiens
204475_at


1 (interstitial collagenase)
matrix metalloproteinase 1 (interstitial


(LOC4312)
collagenase) (MMP1), mRNA.


SEQ ID NOS: 67 (DNA) and
/FEA = mRNA /GEN = MMP1


195 (amino acid)
/PROD = matrix metalloproteinase 1



preproprotein



/DB_XREF = gi: 13027798



/UG = Hs.83169 matrix



metalloproteinase 1 (interstitial



collagenase) /FL = gb: NM_002421.2



gb: M13509.1


PLCB4: phospholipase C, beta 4
Consensus includes gb: AL535113
203895_at


(LOC5332)
/FEA = EST /DB_XREF = gi: 12798606


SEQ ID NOS: 68 (DNA) and
/DB_XREF = est: AL535113


196 (amino acid)
/CLONE = CS0DF008YC23 (3 prime)



/UG = Hs.283006 phospholipase C, beta



4 /FL = gb: NM_000933.1 gb: L41349.1


PTPRD: protein tyrosine
Consensus includes gb: BF062299
214043_at


phosphatase, receptor type, D
/FEA = EST /DB_XREF = gi: 10821197


(LOC5789)
/DB_XREF = est: 7k76c03.x1


SEQ ID NOS: 69 (DNA) and
/CLONE = IMAGE: 3481325


197 (amino acid)
/UG = Hs.323079 Homo sapiens



mRNA; cDNA DKFZp564P116 (from



clone DKFZp564P116)


KCNK1: potassium channel,
gb: U90065.1 /DEF = Human potassium
204678_s_at


subfamily K, member 1
channel KCNO1 mRNA, complete cds.


(LOC3775)
/FEA = mRNA /PROD = potassium


SEQ ID NOS: 70 (DNA) and
channel KCNO1


198 (amino acid)
/DB_XREF = gi: 1916294



/UG = Hs.79351 potassium channel,



subfamily K, member 1 (TWIK-1)



/FL = gb: U33632.1 gb: U90065.1



gb: U76996.1 gb: NM_002245.1


ALOX5: arachidonate 5-
gb: NM_000698.1 /DEF = Homo sapiens
204446_s_at


lipoxygenase (LOC240)
arachidonate 5-lipoxygenase (ALOX5),


SEQ ID NOS: 71 (DNA) and
mRNA. /FEA = mRNA /GEN = ALOX5


199 (amino acid)
/PROD = arachidonate 5-lipoxygenase



/DB_XREF = gi: 4502056



/UG = Hs.89499 arachidonate 5-



/ipoxygenase /FL = gb: J03600.1



gb: J03571.1 gb: NM_000698.1


CXCL10: chemokine (C—X—C
gb: NM_001565.1 /DEF = Homo sapiens
204533_at


motif) ligand 10 (LOC3627)
small inducible cytokine subfamily B


SEQ ID NOS: 72 (DNA) and
(Cys-X-Cys), member 10 (SCYB10),


200 (amino acid)
mRNA. /FEA = mRNA



/GEN = SCYB10 /PROD = interferon



gamma-induced precursor



/DB_XREF = gi: 4504700 /UG = Hs.2248



small inducible cytokine subfamily B



(Cys-X-Cys), member 10



/FL = gb: NM_001565.1


TMPRSS2: transmembrane
gb: AF270487.1 /DEF = Homo sapiens
211689_s_at


protease, serine 2 (LOC7113)
androgen-regulated serine protease


SEQ ID NOS: 73 (DNA) and
TMPRSS2 precursor (TMPRSS2)


201 (amino acid)
mRNA, complete cds. /FEA = mRNA



/GEN = TMPRSS2 /PROD = androgen-



regulated serine protease



TMPRSS2precursor



/DB_XREF = gi: 13540003



/FL = gb: AF270487.1


PRG1: proteoglycan 1, secretory
gb: J03223.1 /DEF = Human secretory
201858_s_at


granule (LOC5552)
granule proteoglycan peptide core


SEQ ID NOS: 74 (DNA) and
mRNA, complete cds. /FEA = mRNA


202 (amino acid)
/GEN = PRG1 /DB_XREF = gi: 190419



/UG = Hs.1908 proteoglycan 1,



secretory granule /FL = gb: J03223.1



gb: NM_002727.1


HLA-DQA1: major
Consensus includes gb: BG397856
212671_s_at


histocompatibility complex,
/FEA = EST /DB_XREF = gi: 13291304


class II, DQ alpha 1 (LOC3117)
/DB_XREF = est: 602438950F1


SEQ ID NOS: 75 (DNA) and
/CLONE = IMAGE: 4564956


203 (amino acid)
/UG = Hs.198253 major



histocompatibility complex, class II,



DQ alpha 1


NR4A2: nuclear receptor
Consensus includes gb: S77154.1
216248_s_at


subfamily 4, group A, member 2
/DEF = TINUR = NGFI-Bnur77 beta-


(LOC4929)
type transcription factor homolog


SEQ ID NOS: 76 (DNA) and
human, T lymphoid cell line, PEER,


204 (amino acid)
mRNA, 2469 nt. /FEA = mRNA



/GEN = TINUR /DB_XREF = gi: 913966



/UG = Hs.82120 nuclear receptor



subfamily 4, group A, member 2


KCTD12: potassium channel
Consensus includes gb: AA551075
212188_at


tetramerisation domain
/FEA = EST /DB_XREF = gi: 2321327


containing 12 (LOC115207)
/DB_XREF = est: nk74h06.s1


SEQ ID NOS: 77 (DNA) and
/CLONE = IMAGE: 1019291


205 (amino acid)
/UG = Hs.109438 Homo sapiens clone



24775 mRNA sequence


RARRES3: retinoic acid
gb: NM_004585.2 /DEF = Homo sapiens
204070_at


receptor responder (tazarotene
retinoic acid receptor responder


induced) 3 (LOC5920)
(tazarotene induced) 3 (RARRES3),


SEQ ID NOS: 78 (DNA) and
mRNA. /FEA = mRNA


206 (amino acid)
/GEN = RARRES3 /PROD = retinoic



acid receptor responder



(tazaroteneinduced) 3



/DB_XREF = gi: 8051633



/UG = Hs.17466 retinoic acid receptor



responder (tazarotene induced) 3



/FL = gb: AF060228.1 gb: AF092922.1



gb: NM_004585.2 gb: AB030815.1


LDHB: lactate dehydrogenase B
Consensus includes gb: BE042354
213564_x_at


(LOC3945)
/FEA = EST /DB_XREF = gi: 8359407


SEQ ID NOS: 79 (DNA) and
/DB_XREF = est: ho19b09.x1


207 (amino acid)
/CLONE = IMAGE: 3037817



/UG = Hs.234489 lactate dehydrogenase B


CLECSF2: C-type (calcium
gb: BC005254.1 /DEF = Homo sapiens,
209732_at


dependent, carbohydrate-
Similar to C-type (calcium dependent,


recognition domain) lectin,
carbohydrate-recognition domain)


superfamily member 2
lectin, superfamily member 2


(activation-induced) (LOC9976)
(activation-induced), clone


SEQ ID NOS: 80 (DNA) and
MGC: 12289, mRNA, complete cds.


208 (amino acid)
/FEA = mRNA /PROD = Siniilar to C-



type (calcium dependent, carbohydrate-



recognition domain) lectin,



superfamilymember 2 (activation-



induced) /DB_XREF = gi: 13528920



/UG = Hs.85201 C-type (calcium



dependent, carbohydrate-recognition



domain) lectin, superfamily member 2



(activation-induced)



/FL = gb: BC005254.1 gb: AB015628.1



gb: NM_005127.1


FLNA: filamin A, alpha (actin
Consensus includes gb: AW051856
213746_s_at


binding protein 280) (LOC2316)
/FEA = EST /DB_XREF = gi: 5914215


SEQ ID NOS: 81 (DNA) and
/DB_XREF = est: wz04a05.x1


209 (amino acid)
/CLONE = IMAGE: 2557040



/UG = Hs.195464 filamin A, alpha



(actin-binding protein-280)


CXCL5: chemokine (C—X—C
Consensus includes gb: AK026546.1
214974_x_at


motif) ligand 5 (LOC6374)
/DEF = Homo sapiens cDNA: FLJ22893


SEQ ID NOS: 82 (DNA) and
fis, clone KAT04792. /FEA = mRNA


210 (amino acid)
/DB_XREF = gi: 10439427



/UG = Hs.287716 Homo sapiens cDNA:



FLJ22893 fis, clone KAT04792


AEBP1: AE binding protein 1
gb: NM_001129.2 /DEF = Homo sapiens
201792_at


(LOC165)
AE-binding protein 1 (AEBP1),


SEQ ID NOS: 83 (DNA) and
mRNA. /FEA = mRNA /GEN = AEBP1


211 (amino acid)
/PROD = adipocyte enhancer binding



protein 1 precursor



/DB_XREF = gi: 4755145



/UG = Hs.118397 AE-binding protein 1



/FL = gb: D86479.1 gb: AF053944. 1



gb: NM_001129.2


BGN: biglycan (LOC633)
Consensus includes gb: AA845258
213905_x_at


SEQ ID NOS: 84 (DNA) and
/FEA = EST /DB_XREF = gi: 2931709


212 (amino acid)
/DB_XREF = est: ak84a11.s1



/CLONE = IMAGE: 1414556



/UG = Hs.821 biglycan


SULF1: sulfatase 1 (LOC23213)
Consensus includes gb: AI479175
212353_at


SEQ ID NOS: 85 (DNA) and
/FEA = EST /DB_XREF = gi: 4372343


213 (amino acid)
/DB_XREF = est: tm55c05.x1



/CLONE = IMAGE: 2162024



/UG = Hs.70823 KIAA1077 protein


COL6A2: collagen, type VI,
gb: AY029208.1 /DEF = Homo sapiens
209156_s_at


alpha 2 (LOC1292)
type VI collagen alpha 2 chain


SEQ ID NOS: 86 (DNA) and
precursor (COL6A2) mRNA, complete


214 (amino acid)
cds, alternatively spliced.



/FEA = mRNA /GEN = COL6A2



/PROD = type VI collagen alpha 2 chain



precursor /DB_XREF = gi: 13603393



/UG = Hs.159263 collagen, type VI,



alpha 2 /FL = gb: AY029208.1


THBS2: thrombospondin 2
gb: NM_003247.1 /DEF = Homo sapiens
203083_at


(LOC7058)
thrombospondin 2 (THBS2), mRNA.


SEQ ID NOS: 87 (DNA) and
/FEA = mRNA /GEN = THBS2


215 (amino acid)
/PROD = thrombospondin 2



/DB_XREF = gi: 4507486



/UG = Hs.108623 thrombospondin 2



/FL = gb: L12350.1 gb: NM_003247.1


PLCB4: phospholipase C, beta 4
gb: NM_000933.1 /DEF = Homo sapiens
203896_s_at


(LOC5332)
phospholipase C, beta 4 (PLCB4),


SEQ ID NOS: 88 (DNA) and
mRNA. /FEA = mRNA /GEN = PLCB4


216 (amino acid)
/PROD = phospholipase C, beta 4



/DB_XREF = gi: 4505866



/UG = Hs.283006 phospholipase C, beta



4 /FL = gb: NM_000933.1 gb: L41349.1


CALD1: caldesmon 1 (LOC800)
gb: NM_004342.2 /DEF = Homo sapiens
201617_x_at


SEQ ID NOS: 89 (DNA) and
caldesmon 1 (CALD1), mRNA.


217 (amino acid)
/FEA = mRNA /GEN = CALD1



/PROD = caldesmon 1



/DB_XREF = gi: 11091984



/UG = Hs.325474 caldesmon 1



/FL = gb: NM_004342.2 gb: M64110.1


NGFRAP1: nerve growth factor
gb: NM_014380.1 /DEF = Homo sapiens
217963_s_at


receptor (TNFRSF16) associated
p75NTR-associated cell death


protein 1 (LOC27018)
executor; ovarian granulosa cell protein


SEQ ID NOS: 90 (DNA) and
(13 kD) (DXS6984E), mRNA.


218 (amino acid)
/FEA = mRNA /GEN = DXS6984E



/PROD = p75NTR-associated cell death



executor; ovariangranulosa cell protein



(13 kD) /DB_XREF = gi: 7657043



/UG = Hs.17775 p75NTR-associated



cell death executor; ovarian granulosa



cell protein (13 kD)



/FL = gb: NM_014380.1 gb: AF187064.1


IFI16: interferon, gamma-
Consensus includes gb: BG256677
208965_s_at


inducible protein 16 (LOC3428)
/FEA = EST /DB_XREF = gi: 12766493


SEQ ID NOS: 91 (DNA) and
/DB_XREF = est: 602370865F1


219 (amino acid)
/CLONE = IMAGE: 4478872



/UG = Hs.155530 interferon, gamma-



inducible protein 16



/FL = gb: AF208043.1


RAB31: RAB31, member RAS
gb: NM_006868.1 /DEF = Homo sapiens
217763_s_at


oncogene family (LOC11031)
RAB31, member RAS oncogene family


SEQ ID NOS: 92 (DNA) and
(RAB31), mRNA. /FEA = mRNA


220 (amino acid)
/GEN = RAB31 /PROD = RAB31,



member RAS oncogene family



/DB_XREF = gi: 5803130



/UG = Hs.223025 RAB31, member RAS



oncogene family /FL = gb: AF234995.1



gb: BC001148.1 gb: U59877.1



gb: U57091.1 gb: NM_006868.1



gb: AF183421.1


COL5A1: collagen, type V,
Consensus includes gb: AI130969
203325_s_at


alpha 1 (LOC1289)
/FEA = EST /DB_XREF = gi: 3600985


SEQ ID NOS: 93 (DNA) and
/DB_XREF = est: qc15e01.x1


221 (amino acid)
/CLONE = IMAGE: 1709688



/UG = Hs.146428 collagen, type V,



alpha 1 /FL = gb: M76729.1



gb: D90279.1 gb: NM_000093.1


KLK10: kallikrein 10
gb: BC002710.1 /DEF = Homo sapiens,
209792_s_at


(LOC5655)
kallikrein 10, clone MGC: 3667,


SEQ ID NOS: 94 (DNA) and
mRNA, complete cds. /FEA = mRNA


222 (amino acid)
/PROD = kallikrein 10



/DB_XREF = gi: 12803744



/UG = Hs.69423 kallikrein 10



/FL = gb: BC002710.1


PCP4: Purkinje cell protein 4
gb: NM_006198.1 /DEF = Homo sapiens
205549_at


(LOC5121)
Purkinje cell protein 4 (PCP4), mRNA.


SEQ ID NOS: 95 (DNA) and
/FEA = mRNA /GEN = PCP4


223 (amino acid)
/PROD = Purkinje cell protein 4



/DB_XREF = gi: 5453857



/UG = Hs.80296 Purkinje cell protein 4



/FL = gb: U52969.1 gb: NM_006198.1


NR4A2: nuclear receptor
gb: NM_006186.1 /DEF = Homo sapiens
204622_x_at


subfamily 4, group A, member 2
nuclear receptor subfamily 4, group A,


(LOC4929)
member 2 (NR4A2), mRNA.


SEQ ID NOS: 96 (DNA) and
/FEA = mRNA /GEN = NR4A2


224 (amino acid)
/PROD = nuclear receptor subfamily 4,



group A, member 2



/DB_XREF = gi: 5453821



/UG = Hs.82120 nuclear receptor



subfamily 4, group A, member 2



/FL = gb: NM_006186.1


IGFBP3: insulin-like growth
gb: M31159.1 /DEF = Human growth
210095_s_at


factor binding protein 3
hormone-dependent insulin-like growth


(LOC3486)
factor-binding protein mRNA,


SEQ ID NOS: 97 (DNA) and
complete cds. /FEA = mRNA


225 (amino acid)
/GEN = IGFBP1 /DB_XREF = gi: 183115



/UG = Hs.77326 insulin-like growth



factor binding protein 3



/FL = gb: BC000013.1 gb: M31159.1


STAT1: signal transducer and
gb: BC002704.1 /DEF = Homo sapiens,
209969_s_at


activator of transcription 1,
Similar to signal transducer and


91 kDa (LOC6772)
activator of transcription 1, 91 kD,


SEQ ID NOS: 98 (DNA) and
clone MGC: 3493, mRNA, complete


226 (amino acid)
cds. /FEA = mRNA /PROD = Similar to



signal transducer and activator



oftranscription 1, 91 kD



/DB_XREF = gi: 12803734



/UG = Hs.21486 signal transducer and



activator of transcription 1, 91 kD



/FL = gb: BC002704.1


CYP1B1: cytochrome P450,
Consensus includes gb: AU144855
202436_s_at


family 1, subfamily B,
/FEA = EST /DB_XREF = gi: 11006376


polypeptide 1 (LOC1545)
/DB_XREF = est: AU144855


SEQ ID NOS: 99 (DNA) and
/CLONE = HEMBA1003161


227 (amino acid)
/UG = Hs.154654 cytochrome P450,



subfamily I (dioxin-inducible),



polypeptide 1 (glaucoma 3, primary



infantile) /FL = gb: NM_000104.2



gb: U03688.1


COL1A1: collagen, type I, alpha
Consensus includes gb: AI743621
202311_s_at


1 (LOC1277)
/FEA = EST /DB_XREF = gi: 5111909


SEQ ID NOS: 100 (DNA) and
/DB_XREF = est: wg51h09.x1


228 (amino acid)
/CLONE = IMAGE: 2368673



/UG = Hs.172928 collagen, type I, alpha



1 /FL = gb: NM_000088.1


DKFZP434F0318: hypothetical
gb: NM_030817.1 /DEF = Homo sapiens
221031_s_at


protein DKFZp434F0318
hypothetical protein DKFZp434F0318


(LOC81575)
(DKFZP434F0318), mRNA.


SEQ ID NOS: 101 (DNA) and
/FEA = mRNA /GEN = DKFZP434F0318


229 (amino acid)
/PROD = hypothetical protein



DKFZp434F0318



/DB_XREF = gi: 13540611



/FL = gb: NM_030817.1


TUBA3: tubulin, alpha 3
gb: AF141347.1 /DEF = Homo sapiens
209118_s_at


(LOC7846)
hum-a-tub2 alpha-tubulin mRNA,


SEQ ID NOS: 102 (DNA) and
complete cds. /FEA = mRNA


230 (amino acid)
/PROD = alpha-tubulin



/DB_XREF = gi: 4929133



/UG = Hs.272897 Tubulin, alpha, brain-



specific /FL = gb: AF141347.1



gb: NM_006009.1


GZMB: granzyme B (granzyme
gb: J03189.1 /DEF = Human proteolytic
210164_at


2, cytotoxic T-lymphocyte-
serine esterase-like protein (SECT)


associated serine esterase 1)
gene, complete cds. /FEA = mRNA


(LOC3002)
/DB_XREF = gi: 338010 /UG = Hs.1051


SEQ ID NOS: 103 (DNA) and
granzyme B (granzyme 2, cytotoxic T-


231 (amino acid)
lymphocyte-associated serine esterase



1) /FL = gb: J04071.1 gb: J03189.1



gb: M17016.1 gb: NM_004131.2


ROBO1: roundabout, axon
Consensus includes gb: BF059159
213194_at


guidance receptor, homolog 1
/FEA = EST /DB_XREF = gi: 10813055


(Drosophila) (LOC6091)
/DB_XREF = est: 7k66g04.x1


SEQ ID NOS: 104 (DNA) and
/CLONE = IMAGE: 3480391


232 (amino acid)
/UG = Hs.301198 roundabout (axon



guidance receptor, Drosophila)



homolog 1 /FL = gb: AF040990.1



gb: NM_002941.1


CHGA: chromogranin A
gb: NM_001275.2 /DEF = Homo sapiens
204697_s_at


(parathyroid secretory protein 1)
chromogranin A (parathyroid secretory


(LOC1113)
protein 1) (CHGA), mRNA.


SEQ ID NOS: 105 (DNA) and
/FEA = mRNA /GEN = CHGA


233 (amino acid)
/PROD = chromogranin A



/DB_XREF = gi: 10800418



/UG = Hs.172216 chromogranin A



(parathyroid secretory protein 1)



/FL = gb: NM_001275.2 gb: BC001059.1



gb: J03483.1 gb: J03915.1


SLC7A8: solute carrier family 7
gb: NM_012244.1 /DEF = Homo sapiens
202752_x_at


(cationic amino acid transporter,
solute carrier family 7 (cationic amino


y+ system), member 8
acid transporter, y+ system), member 8


(LOC23428)
(SLC7A8), mRNA. /FEA = mRNA


SEQ ID NOS: 106 (DNA) and
/GEN = SLC7A8 /PROD = solute carrier


234 (amino acid)
family 7 (cationic amino



acidtransporter, y+ system), member 8



/DB_XREF = gi: 6912669



/UG = Hs.22891 solute carrier family 7



(cationic amino acid transporter, y+



system), member 8



/FL = gb: AB037669.1 gb: AF171669.1



gb: NM_012244.1


GPA33: glycoprotein A33
gb: NM_005814.1 /DEF = Homo sapiens
205929_at


(transmembrane) (LOC10223)
glycoprotein A33 (transmembrane)


SEQ ID NOS: 107 (DNA) and
(GPA33), mRNA. /FEA = mRNA


235 (amino acid)
/GEN = GPA33 /PROD = transmembrane



glycoprotein A33 precursor



/DB_XREF = gi: 5031560



/UG = Hs.143131 glycoprotein A33



(transmembrane) /FL = gb: U79725.1



gb: NM_005814.1


QPRT: quinolinate
gb: NM_014298.2 /DEF = Homo sapiens
204044_at


phosphoribosyltransferase
quinolinate phosphoribosyltransferase


(nicotinate-nucleotide
(nicotinate-nucleotide


pyrophosphorylase
pyrophosphorylase (carboxylating))


(carboxylating)) (LOC23475)
(QPRT), mRNA. /FEA = mRNA


SEQ ID NOS: 108 (DNA) and
/GEN = QPRT /PROD = quinolinate


236 (amino acid)
phosphoribosyltransferase



/DB_XREF = gi: 9257236 /UG = Hs.8935



quinolinate phosphoribosyltransferase



(nicotinate-nucleotide



pyrophosphorylase (carboxylating))



/FL = gb: D78177.1 gb: BC005060.1



gb: NM_014298.2


DDC: dopa decarboxylase
gb: NM_000790.1 /DEF = Homo sapiens
205311_at


(aromatic L-amino acid
dopa decarboxylase (aromatic L-amino


decarboxylase) (LOC1644)
acid decarboxylase) (DDC), mRNA.


SEQ ID NOS: 109 (DNA) and
/FEA = mRNA /GEN = DDC


237 (amino acid)
/PROD = dopa decarboxylase (aromatic



L-amino aciddecarboxylase)



/DB_XREF = gi: 4503280



/UG = Hs.150403 dopa decarboxylase



(aromatic L-amino acid decarboxylase)



/FL = gb: BC000485.1 gb: M76180.1



gb: M88700.1 gb: NM_000790.1


COL11A1: collagen, type XI,
gb: NM_001854.1 /DEF = Homo sapiens
204320_at


alpha 1 (LOC1301)
collagen, type XI, alpha 1 (COL11A1),


SEQ ID NOS: 110 (DNA) and
mRNA. /FEA = mRNA


238 (amino acid)
/GEN = COL11A1 /PROD = collagen,



type XI, alpha 1



/DB_XREF = gi: 4502938



/UG = Hs.82772 collagen, type XI, alpha



1 /FL = gb: J04177.1 gb: NM_001854.1


C2orf23: chromosome 2 open
Consensus includes gb: BE535746
204364_s_at


reading frame 23 (LOC65055)
/FEA = EST /DB_XREF = gi: 9764391


SEQ ID NOS: 111 (DNA) and
/DB_XREF = est: 601060419F1


239 (amino acid)
/CLONE = IMAGE: 3446788



/UG = Hs.7358 hypothetical protein



FLJ13110 /FL = gb: NM_022912.1


SULF1: sulfatase 1 (LOC23213)
Consensus includes gb: BE500977
212354_at


SEQ ID NOS: 112 (DNA) and
/FEA = EST /DB_XREF = gi: 9703385


240 (amino acid)
/DB_XREF = est: 7a33h02.x1



/CLONE = IMAGE: 3220563



/UG = Hs.70823 KIAA1077 protein


PCOLCE: procollagen C-
gb: NM_002593.2 /DEF = Homo sapiens
202465_at


endopeptidase enhancer
procollagen C-endopeptidase enhancer


(LOC5118)
(PCOLCE), mRNA. /FEA = mRNA


SEQ ID NOS: 113 (DNA) and
/GEN = PCOLCE /PROD = procollagen


241 (amino acid)
C-endopeptidase enhancer



/DB_XREF = gi: 7262388



/UG = Hs.202097 procollagen C-



endopeptidase enhancer



/FL = gb: BC000574.1 gb: AB008549.1



gb: L33799.1 gb: NM_002593.2


C14orf78: chromosome 14 open
Consensus includes gb: AI935123
212992_at


reading frame 78 (LOC113146)
/FEA = EST /DB_XREF = gi: 5673993


SEQ ID NOS: 114 (DNA) and
/DB_XREF = est: wp13h09.x1


242 (amino acid)
/CLONE = IMAGE: 2464769



/UG = Hs.57548 ESTs


CXCR4: chemokine (C—X—C
gb: L01639.1 /DEF = Human (clone
209201_x_at


motif) receptor 4 (LOC7852)
HSY3RR) neuropeptide Y receptor


SEQ ID NOS: 115 (DNA) and
(NPYR) mRNA, complete cds.


243 (amino acid)
/FEA = mRNA /GEN = NPYR



/PROD = neuropeptide Y receptor



/DB_XREF = gi: 189313 /UG = Hs.89414



chemokine (C—X—C motif), receptor 4



(fusin) /FL = gb: L01639.1



gb: AF025375.1 gb: M99293.1



gb: L06797.1 gb: NM_003467.1



gb: AF147204.1


CSPG2: chondroitin sulfate
Consensus includes gb: R94644
215646_s_at


proteoglycan 2 (versican)
/FEA = EST /DB_XREF = gi: 970039


(LOC1462)
/DB_XREF = est: yq42a12.r1


SEQ ID NOS: 116 (DNA) and
/CLONE = IMAGE: 198430


244 (amino acid)
/UG = Hs.306542 Homo sapiens



versican Vint isoform, mRNA, partial



cds


SERPINF1: serine (or cysteine)
gb: NM_002615.1 /DEF = Homo sapiens
202283_at


proteinase inhibitor, clade F
serine (or cysteine) proteinase inhibitor,


(alpha-2 antiplasmin, pigment
clade F (alpha-2 antiplasmin, pigment


epithelium derived factor),
epithelium derived factor), member 1


member 1 (LOC5176)
(SERPINF1), mRNA. /FEA = mRNA


SEQ ID NOS: 117 (DNA) and
/GEN = SERPINF1 /PROD = serine (or


245 (amino acid)
cysteine) proteinase inhibitor, cladeF



(alpha-2 antiplasmin, pigment



epithelium derivedfactor), member 1



/DB_XREF = gi: 4505708



/UG = Hs.173594 serine (or cysteine)



proteinase inhibitor, clade F (alpha-2



antiplasmin, pigment epithelium



derived factor), member 1



/FL = gb: M90439.1 gb: BC000522.1



gb: M76979.1 gb: NM_002615.1


SPON1: spondin 1, extracellular
Consensus includes gb: AB018305.1
209436_at


matrix protein (LOC10418)
/DEF = Homo sapiens mRNA for


SEQ ID NOS: 118 (DNA) and
KIAA0762 protein, partial cds.


246 (amino acid)
/FEA = mRNA /GEN = KIAA0762



/PROD = KIAA0762 protein



/DB_XREF = gi: 3882244 /UG = Hs.5378



spondin 1, (f-spondin) extracellular



matrix protein /FL = gb: AB051390.1


COL11A1: collagen, type XI,
Cluster Incl. J04177: Human alpha-1
37892_at


alpha 1 (LOC1301)
type XI collagen (COL11A1) mRNA,


SEQ ID NOS: 119 (DNA) and
complete cds /cds = (161,5581)


247 (amino acid)
/gb = J04177 /gi = 179729 /ug = Hs.82772



/len = 6158


MAFB: v-maf
gb: NM_005461.1 /DEF = Homo sapiens
218559_s_at


musculoaponeurotic
Kreisler (mouse) maf-related leucine


fibrosarcoma oncogene homolog
zipper homolog (KRML), mRNA.


B (avian) (LOC9935)
/FEA = mRNA /GEN = KRML


SEQ ID NOS: 120 (DNA) and
/PROD = Kreisler (mouse) maf-related


248 (amino acid)
leucine zipperhomolog



/DB_XREF = gi: 4885446



/UG = Hs.169487 Kreisler (mouse) maf-



related leucine zipper homolog



/FL = gb: AF134157.1 gb: NM_005461.1


DDX17: DEAD (Asp-Glu-Ala-
Consensus includes gb: AW188131
213998_s_at


Asp) box polypeptide 17
/FEA = EST /DB_XREF = gi: 6462567


(LOC10521)
/DB_XREF = est: xj92f11.x1


SEQ ID NOS: 121 (DNA) and
/CLONE = IMAGE: 2664717


249 (amino acid)
/UG = Hs.6179 DEADH (Asp-Glu-Ala-



AspHis) box polypeptide 17 (72 kD)


PHLDA1: pleckstrin homology-
Consensus includes gb: NM_007350.1
217999_s_at


like domain, family A, member
/DEF = Homo sapiens pleckstrin


1 (LOC22822)
homology-like domain, family A,


SEQ ID NOS: 122 (DNA) and
member 1 (PHLDA1), mRNA.


250 (amino acid)
/FEA = mRNA /GEN = PHLDA1



/PROD = pleckstrin homology-like



domain, family A, member 1



/DB_XREF = gi: 6679302



/UG = Hs.82101 pleckstrin homology-



like domain, family A, member 1



/FL = gb: NM_007350.1


ETV5: ets variant gene 5 (ets-
gb: NM_004454.1 /DEF = Homo sapiens
203349_s_at


related molecule) (LOC2119)
ets variant gene 5 (ets-related


SEQ ID NOS: 123 (DNA) and
molecule) (ETV5), mRNA.


251 (amino acid)
/FEA = mRNA /GEN = ETV5



/PROD = ets variant gene 5 (ets-related



molecule) /DB_XREF = gi: 4758315



/UG = Hs.43697 ets variant gene 5 (ets-



related molecule)



/FL = gb: NM_004454.1


DUSP4: dual specificity
gb: BC002671.1 /DEF = Homo sapiens,
204015_s_at


phosphatase 4 (LOC1846)
dual specificity phosphatase 4, clone


SEQ ID NOS: 124 (DNA) and
MGC: 3713, mRNA, complete cds.


252 (amino acid)
/FEA = mRNA /PROD = dual specificity



phosphatase 4



/DB_XREF = gi: 12803670



/UG = Hs.2359 dual specificity



phosphatase 4 /FL = gb: U48807.1



gb: NM_001394.2 gb: BC002671.1



gb: U21108.1


DUSP4: dual specificity
gb: NM_001394.2 /DEF = Homo sapiens
204014_at


phosphatase 4 (LOC1846)
dual specificity phosphatase 4


SEQ ID NOS: 125 (DNA) and
(DUSP4), mRNA. /FEA = mRNA


253 (amino acid)
/GEN = DUSP4 /PROD = dual specificity



phosphatase 4



/DB_XREF = gi: 12707552



/UG = Hs.2359 dual specificity



phosphatase 4 /FL = gb: U48807.1



gb: NM_001394.2 gb: BC002671.1



gb: U21108.1


POFUT1: protein O-
Consensus includes gb: AL045513
212349_at


fucosyltransferase 1
/FEA = EST /DB_XREF = gi: 5433649


(LOC23509)
/DB_XREF = est: DKFZp434J015_r1


SEQ ID NOS: 126 (DNA) and
/CLONE = DKFZp434J015


254 (amino acid)
/UG = Hs.178292 KIAA0180 protein


TBXAS1: thromboxane A
gb: NM_030984.1 /DEF = Homo sapiens
208130_s_at


synthase 1 (platelet, cytochrome
thromboxane A synthase 1 (platelet,


P450, family 5, subfamily A)
cytochrome P450, subfamily V)


(LOC6916)
(TBXAS1), transcript variant TXS-II,


SEQ ID NOS: 127 (DNA) and
mRNA. /FEA = mRNA


255 (amino acid)
/GEN = TBXAS1 /PROD = thromboxane



A synthase 1 (platelet,



cytochromeP450, subfamily V),



isoform TXS-II



/DB_XREF = gi: 13699839



/FL = gb: NM_030984.1


KCNK5: potassium channel,
gb: NM_003740.1 /DEF = Homo sapiens
219615_s_at


subfamily K, member 5
potassium channel, subfamily K,


(LOC8645)
member 5 (TASK-2) (KCNK5),


SEQ ID NOS: 128 (DNA) and
mRNA. /FEA = mRNA /GEN = KCNK5


256 (amino acid)
/PROD = potassium channel, subfamily



K, member 5(TASK-2)



/DB_XREF = gi: 4504850



/UG = Hs.127007 potassium channel,



subfamily K, member 5 (TASK-2)



/FL = gb: AF084830.1 gb: NM_003740.1









The biomarkers provided in Table 1, which include the nucleotide sequences of SEQ ID NOS:1-128 and the amino acid sequences of SEQ ID NOS:129-256, are referred to herein as a total of 128 biomarkers with reference to the Unigene Title.


The biomarkers have expression levels in cells that may be dependent on the activity of the EGFR signal transduction pathway, and that are also highly correlated with EGFR modulator sensitivity exhibited by the cells. Biomarkers serve as useful molecular tools for predicting the likelihood of a response to EGFR modulators, preferably biological molecules, small molecules, and the like that affect EGFR kinase activity via direct or indirect inhibition or antagonism of EGFR kinase function or activity.


Wild Type K-Ras and Mutated K-Ras

As used herein, wild type K-Ras can be selected from the K-Ras variant a and variant b nucleotide and amino acid sequences. Wild type K-Ras variant a has a nucleotide sequence that is 5436 nucleotides (GenBank Accession No. NM033360.2) and encodes a protein that is 189 amino acids (GenBank Accession No. NP203524.1). Wild type K-Ras variant b has a nucleotide sequence that is 5312 nucleotides (GenBank Accession No. NM004985.3) and encodes a protein that is 188 amino acids (GenBank Accession No. NP004976.2).


A mutated form of K-Ras is a nucleotide or amino acid sequence that differs from wild type K-Ras at least at one position, preferably at least one nucleotide position that encodes an amino acid that differs from wild type K-Ras. In one aspect, the mutated form of K-Ras includes at least one mutation in exon 2. In another aspect, the mutated form of K-RAS includes at least one of the following mutations in exon 2 (base change (amino acid change)): 200G>A (V7M); 216G>C (G12A); 215G>T (G12C); 216G>A (G12D); 215G>C (G12R); 215G>A (G12S); 216G>T (G12V); 218G>T (G13C); 219G>A (G13D).


Methods for detecting K-Ras mutations are well known in the art and include, for example, the methods described in PCT Publication No. Wo2005/118876.


EGFR Modulators

As used herein, the term “EGFR modulator” is intended to mean a compound or drug that is a biological molecule or a small molecule that directly or indirectly modulates EGFR activity or the EGFR signal transduction pathway. Thus, compounds or drugs as used herein is intended to include both small molecules and biological molecules. Direct or indirect modulation includes activation or inhibition of EGFR activity or the EGFR signal transduction pathway. In one aspect, inhibition refers to inhibition of the binding of EGFR to an EGFR ligand such as, for example, EGF. In another aspect, inhibition refers to inhibition of the kinase activity of EGFR.


EGFR modulators include, for example, EGFR-specific ligands, small molecule EGFR inhibitors, and EGFR monoclonal antibodies. In one aspect, the EGFR modulator inhibits EGFR activity and/or inhibits the EGFR signal transduction pathway. In another aspect, the EGFR modulator is an EGFR monoclonal antibody that inhibits EGFR activity and/or inhibits the EGFR signal transduction pathway.


EGFR modulators include biological molecules or small molecules. Biological molecules include all lipids and polymers of monosaccharides, amino acids, and nucleotides having a molecular weight greater than 450. Thus, biological molecules include, for example, oligosaccharides and polysaccharides; oligopeptides, polypeptides, peptides, and proteins; and oligonucleotides and polynucleotides. Oligonucleotides and polynucleotides include, for example, DNA and RNA.


Biological molecules further include derivatives of any of the molecules described above. For example, derivatives of biological molecules include lipid and glycosylation derivatives of oligopeptides, polypeptides, peptides, and proteins.


Derivatives of biological molecules further include lipid derivatives of oligosaccharides and polysaccharides, e.g., lipopolysaccharides. Most typically, biological molecules are antibodies, or functional equivalents of antibodies. Functional equivalents of antibodies have binding characteristics comparable to those of antibodies, and inhibit the growth of cells that express EGFR. Such functional equivalents include, for example, chimerized, humanized, and single chain antibodies as well as fragments thereof.


Functional equivalents of antibodies also include polypeptides with amino acid sequences substantially the same as the amino acid sequence of the variable or hypervariable regions of the antibodies. An amino acid sequence that is substantially the same as another sequence, but that differs from the other sequence by means of one or more substitutions, additions, and/or deletions, is considered to be an equivalent sequence. Preferably, less than 50%, more preferably less than 25%, and still more preferably less than 10%, of the number of amino acid residues in a sequence are substituted for, added to, or deleted from the protein.


The functional equivalent of an antibody is preferably a chimerized or humanized antibody. A chimerized antibody comprises the variable region of a non-human antibody and the constant region of a human antibody. A humanized antibody comprises the hypervariable region (CDRs) of a non-human antibody. The variable region other than the hypervariable region, e.g., the framework variable region, and the constant region of a humanized antibody are those of a human antibody.


Suitable variable and hypervariable regions of non-human antibodies may be derived from antibodies produced by any non-human mammal in which monoclonal antibodies are made. Suitable examples of mammals other than humans include, for example, rabbits, rats, mice, horses, goats, or primates.


Functional equivalents further include fragments of antibodies that have binding characteristics that are the same as, or are comparable to, those of the whole antibody. Suitable fragments of the antibody include any fragment that comprises a sufficient portion of the hypervariable (i.e., complementarity determining) region to bind specifically, and with sufficient affinity, to EGFR tyrosine kinase to inhibit growth of cells that express such receptors.


Such fragments may, for example, contain one or both Fab fragments or the F(ab′)2 fragment. Preferably, the antibody fragments contain all six complementarity determining regions of the whole antibody, although functional fragments containing fewer than all of such regions, such as three, four, or five CDRs, are also included.


In one aspect, the fragments are single chain antibodies, or Fv fragments. Single chain antibodies are polypeptides that comprise at least the variable region of the heavy chain of the antibody linked to the variable region of the light chain, with or without an interconnecting linker. Thus, Fv fragment comprises the entire antibody combining site. These chains may be produced in bacteria or in eukaryotic cells.


The antibodies and functional equivalents may be members of any class of immunoglobulins, such as IgG, IgM, IgA, IgD, or IgE, and the subclasses thereof. In one aspect, the antibodies are members of the IgG1 subclass. The functional equivalents may also be equivalents of combinations of any of the above classes and subclasses.


In one aspect, EGFR antibodies can be selected from chimerized, humanized, fully human, and single chain antibodies derived from the murine antibody 225 described in U.S. Pat. No. 4,943,533.


In another aspect, the EGFR antibody is cetuximab (IMC-C225) which is a chimeric (human/mouse) IgG monoclonal antibody, also known under the tradename ERBITUX. Cetuximab Fab contains the Fab fragment of cetuximab, i.e., the heavy and light chain variable region sequences of murine antibody M225 (U.S. Application No. 2004/0006212, incorporated herein by reference) with human IgG1 CH1 heavy and kappa light chain constant domains. Cetuximab includes all three IgG1 heavy chain constant domains.


In another aspect, the EGFR antibody can be selected from the antibodies described in U.S. Pat. No. 6,235,883, U.S. Pat. No. 5,558,864, and U.S. Pat. No. 5,891,996. The EGFR antibody can be, for example, AGX-EGF (Amgen Inc.) (also known as panitumumab) which is a fully human IgG2 monoclonal antibody. The sequence and characterization of ABX-EGF, which was formerly known as clone E7.6.3, is disclosed in U.S. Pat. No. 6,235,883 at column 28, line 62 through column 29, line 36 and FIGS. 29-34, which is incorporated by reference herein. The EGFR antibody can also be, for example, EMD72000 (Merck KGaA), which is a humanized version of the murine EGFR antibody EMD 55900. The EGFR antibody can also be, for example: h-R3 (TheraCIM), which is a humanized EGFR monoclonal antibody; Y10 which is a murine monoclonal antibody raised against a murine homologue of the human EGFRvIII mutation; or MDX-447 (Medarex Inc.).


In addition to the biological molecules discussed above, the EGFR modulators useful in the invention may also be small molecules. Any molecule that is not a biological molecule is considered herein to be a small molecule. Some examples of small molecules include organic compounds, organometallic compounds, salts of organic and organometallic compounds, saccharides, amino acids, and nucleotides. Small molecules further include molecules that would otherwise be considered biological molecules, except their molecular weight is not greater than 450. Thus, small molecules may be lipids, oligosaccharides, oligopeptides, and oligonucleotides and their derivatives, having a molecular weight of 450 or less.


It is emphasized that small molecules can have any molecular weight. They are merely called small molecules because they typically have molecular weights less than 450. Small molecules include compounds that are found in nature as well as synthetic compounds. In one embodiment, the EGFR modulator is a small molecule that inhibits the growth of tumor cells that express EGFR. In another embodiment, the EGFR modulator is a small molecule that inhibits the growth of refractory tumor cells that express EGFR.


Numerous small molecules have been described as being useful to inhibit EGFR.


One example of a small molecule EGFR antagonist is ERESSA (ZD1939), which is a quinozaline derivative that functions as an ATP-mimetic to inhibit EGFR. See, U.S. Pat. No. 5,616,582; WO 96/33980 at page 4. Another example of a small molecule EGFR antagonist is TARCEVA (OSI-774), which is a 4-(substitutedphenylamino)quinozaline derivative [6,7-Bis(2-methoxy-ethoxy)-quinazolin-4-yl]-(3-ethynyl-1-phenyl)amine hydrochloride] EGFR inhibitor. See WO 96/30347 (Pfizer Inc.) at, for example, page 2, line 12 through page 4, line 34 and page 19, lines 14-17. TARCEVA may function by inhibiting phosphorylation of EGFR and its downstream PI3/Akt and MAP (mitogen activated protein) kinase signal transduction pathways resulting in p27-mediated cell-cycle arrest. See Hidalgo et al., Abstract 281 presented at the 37th Annual Meeting of ASCO, San Francisco, Calif., 12-15 May 2001.


Other small molecules are also reported to inhibit EGFR, many of which are thought to be specific to the tyrosine kinase domain of an EGFR. Some examples of such small molecule EGFR antagonists are described in WO 91/116051, WO96/30347, WO96/33980, WO97/27199. WO97/30034, WO97/42187, WO97/49688, WO98/33798, WO00/18761, and WO00/31048. Examples of specific small molecule EGFR antagonists include C1-1033 (Pfizer Inc.), which is a quinozaline (N-[4-(3-chloro-4-fluoro-phenylamino)-7-(3-mprpholin-4-yl-propoxy)-quinazolin-6-yl]-acrylamide) inhibitor of tyrosine kinases, particularly EGFR and is described in WO00/31048 at page 8, lines 22-6; PKI166 (Novartis), which is a pyrrolopyrimidine inhibitor of EGFR and is described in WO97/27199 at pages 10-12; GW2016 (GlaxoSmithKline), which is an inhibitor of EGFR and HER2; EKB569 (Wyeth), which is reported to inhibit the growth of tumor cells that overexpress EGFR or HER2 in vitro and in vivo; AG-1478 (Tryphostin), which is a quinazoline small molecule that inhibits signaling from both EGFR and erbB-2; AG-1478 (Sugen), which is a bisubstrate inhibitor that also inhibits protein kinase CK2; PD 153035 (Parke-Davis) which is reported to inhibit EGFR kinase activity and tumor growth, induce apoptosis in cells in culture, and enhance the cytotoxicity of cytotoxic chemotherapeutic agents; SPM-924 (Schwarz Pharma), which is a tyrosine kinase inhibitor targeted for treatment of prostrate cancer; CP-546,989 (OSI Pharmaceuticals), which is reportedly an inhibitor of angiogenesis for treatment of solid tumors; ADL-681, which is a EGFR kinase inhibitor targeted for treatment of cancer; PD 158780, which is a pyridopyrimidine that is reported to inhibit the tumor growth rate of A4431 xenografts in mice; CP-358,774, which is a quinzoline that is reported to inhibit autophosphorylation in HN5 xenografts in mice; ZD1839, which is a quinoline that is reported to have antitumor activity in mouse xenograft models including vulvar, NSCLC, prostrate, ovarian, and colorectal cancers; CGP 59326A, which is a pyrrolopyrimidine that is reported to inhibit growth of EGFR-positive xenografts in mice; PD 165557 (Pfizer); CGP54211 and CGP53353 (Novartis), which are dianilnophthalimides. Naturally derived EGFR tyrosine kinase inhibitors include genistein, herbimycin A, quercetin, and erbstatin.


Further small molecules reported to inhibit EGFR and that are therefore within the scope of the present invention are tricyclic compounds such as the compounds described in U.S. Pat. No. 5,679,683; quinazoline derivatives such as the derivatives described in U.S. Pat. No. 5,616,582; and indole compounds such as the compounds described in U.S. Pat. No. 5,196,446.


Further small molecules reported to inhibit EGFR and that are therefore within the scope of the present invention are styryl substituted heteroaryl compounds such as the compounds described in U.S. Pat. No. 5,656,655. The heteroaryl group is a monocyclic ring with one or two heteroatoms, or a bicyclic ring with 1 to about 4 heteroatoms, the compound being optionally substituted or polysubstituted.


Further small molecules reported to inhibit EGFR and that are therefore within the scope of the present invention are bis mono and/or bicyclic aryl heteroaryl, carbocyclic, and heterocarbocyclic compounds described in U.S. Pat. No. 5,646,153.


Further small molecules reported to inhibit EGFR and that are therefore within the scope of the present invention is the compound provided FIG. 1 of Fry et al., Science 265, 1093-1095 (1994) that inhibits EGFR.


Further small molecules reported to inhibit EGFR and that are therefore within the scope of the present invention are tyrphostins that inhibit EGFR/HER1 and HER 2, particularly those in Tables I, II, III, and IV described in Osherov et al., J. Biol. Chem., 25; 268(15):11134-42 (1993).


Further small molecules reported to inhibit EGFR and that are therefore within the scope of the present invention is a compound identified as PD166285 that inhibits the EGFR, PDGFR, and FGFR families of receptors. PD166285 is identified as 6-(2,6-dichlorophenyl)-2-(4-(2-diethylaminoethyoxy)phenylamino)-8-methyl-8H-pyrido(2,3-d)pyrimidin-7-one having the structure shown in FIG. 1 on page 1436 of Panek et al., Journal of Pharmacology and Experimental Therapeutics 283, 1433-1444 (1997).


It should be appreciated that useful small molecule to be used in the invention are inhibitors of EGFR, but need not be completely specific for EGFR.


Biomarkers and Biomarker Sets

The invention includes individual biomarkers and biomarker sets having both diagnostic and prognostic value in disease areas in which signaling through EGFR or the EGFR pathway is of importance, e.g., in cancers or tumors, in immunological disorders, conditions or dysfunctions, or in disease states in which cell signaling and/or cellular proliferation controls are abnormal or aberrant. The biomarker sets comprise a plurality of biomarkers such as, for example, a plurality of the biomarkers provided in Table 1, that highly correlate with resistance or sensitivity to one or more EGFR modulators.


The biomarkers and biomarker sets of the invention enable one to predict or reasonably foretell the likely effect of one or more EGFR modulators in different biological systems or for cellular responses. The biomarkers and biomarker sets can be used in in vitro assays of EGFR modulator response by test cells to predict in vivo outcome. In accordance with the invention, the various biomarkers and biomarker sets described herein, or the combination of these biomarker sets with other biomarkers or markers, can be used, for example, to predict how patients with cancer might respond to therapeutic intervention with one or more EGFR modulators.


A biomarker and biomarker set of cellular gene expression patterns correlating with sensitivity or resistance of cells following exposure of the cells to one or more EGFR modulators provides a useful tool for screening one or more tumor samples before treatment with the EGFR modulator. The screening allows a prediction of cells of a tumor sample exposed to one or more EGFR modulators, based on the expression results of the biomarker and biomarker set, as to whether or not the tumor, and hence a patient harboring the tumor, will or will not respond to treatment with the EGFR modulator.


The biomarker or biomarker set can also be used as described herein for monitoring the progress of disease treatment or therapy in those patients undergoing treatment for a disease involving an EGFR modulator.


The biomarkers also serve as targets for the development of therapies for disease treatment. Such targets may be particularly applicable to treatment of colorectal cancer. Indeed, because these biomarkers are differentially expressed in sensitive and resistant cells, their expression patterns are correlated with relative intrinsic sensitivity of cells to treatment with EGFR modulators. Accordingly, the biomarkers highly expressed in resistant cells may serve as targets for the development of new therapies for the tumors which are resistant to EGFR modulators, particularly EGFR inhibitors.


The level of biomarker protein and/or mRNA can be determined using methods well known to those skilled in the art. For example, quantification of protein can be carried out using methods such as ELISA, 2-dimensional SDS PAGE, Western blot, immunopreciptation, immunohistochemistry, fluorescence activated cell sorting (FACS), or flow cytometry. Quantification of mRNA can be carried out using methods such as PCR, array hybridization, Northern blot, in-situ hybridization, dot-blot, Taqman, or RNAse protection assay.


Microarrays

The invention also includes specialized microarrays, e.g., oligonucleotide microarrays or cDNA microarrays, comprising one or more biomarkers, showing expression profiles that correlate with either sensitivity or resistance to one or more EGFR modulators. Such microarrays can be employed in in vitro assays for assessing the expression level of the biomarkers in the test cells from tumor biopsies, and determining whether these test cells are likely to be resistant or sensitive to EGFR modulators. For example, a specialized microarray can be prepared using all the biomarkers, or subsets thereof, as described herein and shown in Table 1. Cells from a tissue or organ biopsy can be isolated and exposed to one or more of the EGFR modulators. In one aspect, following application of nucleic acids isolated from both untreated and treated cells to one or more of the specialized microarrays, the pattern of gene expression of the tested cells can be determined and compared with that of the biomarker pattern from the control panel of cells used to create the biomarker set on the microarray. Based upon the gene expression pattern results from the cells that underwent testing, it can be determined if the cells show a resistant or a sensitive profile of gene expression. Whether or not the tested cells from a tissue or organ biopsy will respond to one or more of the EGFR modulators and the course of treatment or therapy can then be determined or evaluated based on the information gleaned from the results of the specialized microarray analysis.


Antibodies

The invention also includes antibodies, including polyclonal or monoclonal, directed against one or more of the polypeptide biomarkers. Such antibodies can be used in a variety of ways, for example, to purify, detect, and target the biomarkers of the invention, including both in vitro and in vivo diagnostic, detection, screening, and/or therapeutic methods.


Kits

The invention also includes kits for determining or predicting whether a patient would be susceptible or resistant to a treatment that comprises one or more EGFR modulators. The patient may have a cancer or tumor such as, for example, colorectal cancer. Such kits would be useful in a clinical setting for use in testing a patient's biopsied tumor or other cancer samples, for example, to determine or predict if the patient's tumor or cancer will be resistant or sensitive to a given treatment or therapy with an EGFR modulator. The kit comprises a suitable container that comprises: one or more microarrays, e.g., oligonucleotide microarrays or cDNA microarrays, that comprise those biomarkers that correlate with resistance and sensitivity to EGFR modulators, particularly EGFR inhibitors; one or more EGFR modulators for use in testing cells from patient tissue specimens or patient samples; and instructions for use. In addition, kits contemplated by the invention can further include, for example, reagents or materials for monitoring the expression of biomarkers of the invention at the level of mRNA or protein, using other techniques and systems practiced in the art such as, for example, RT-PCR assays, which employ primers designed on the basis of one or more of the biomarkers described herein, immunoassays, such as enzyme linked immunosorbent assays (ELISAs), immunoblotting, e.g., Western blots, or in situ hybridization, and the like.


Application of Biomarkers and Biomarker Sets

The biomarkers and biomarker sets may be used in different applications. Biomarker sets can be built from any combination of biomarkers listed in Table 1 to make predictions about the effect of an EGFR modulator in different biological systems. The various biomarkers and biomarkers sets described herein can be used, for example, as diagnostic or prognostic indicators in disease management, to predict how patients with cancer might respond to therapeutic intervention with compounds that modulate the EGFR, and to predict how patients might respond to therapeutic intervention that modulates signaling through the entire EGFR regulatory pathway.


The biomarkers have both diagnostic and prognostic value in diseases areas in which signaling through EGFR or the EGFR pathway is of importance, e.g., in immunology, or in cancers or tumors in which cell signaling and/or proliferation controls have gone awry.


In one aspect, cells from a patient tissue sample, e.g., a tumor or cancer biopsy, can be assayed to determine the expression pattern of one or more biomarkers prior to treatment with one or more EGFR modulators. In one aspect, the tumor or cancer is colorectal. Success or failure of a treatment can be determined based on the biomarker expression pattern of the cells from the test tissue (test cells), e.g., tumor or cancer biopsy, as being relatively similar or different from the expression pattern of a control set of the one or more biomarkers. Thus, if the test cells show a biomarker expression profile which corresponds to that of the biomarkers in the control panel of cells which are sensitive to the EGFR modulator, it is highly likely or predicted that the individual's cancer or tumor will respond favorably to treatment with the EGFR modulator. By contrast, if the test cells show a biomarker expression pattern corresponding to that of the biomarkers of the control panel of cells which are resistant to the EGFR modulator, it is highly likely or predicted that the individual's cancer or tumor will not respond to treatment with the EGFR modulator.


The invention also provides a method of monitoring the treatment of a patient having a disease treatable by one or more EGFR modulators. The isolated test cells from the patient's tissue sample, e.g., a tumor biopsy or tumor sample, can be assayed to determine the expression pattern of one or more biomarkers before and after exposure to an EGFR modulator wherein, preferably, the EGFR modulator is an EGFR inhibitor. The resulting biomarker expression profile of the test cells before and after treatment is compared with that of one or more biomarkers as described and shown herein to be highly expressed in the control panel of cells that are either resistant or sensitive to an EGFR modulator. Thus, if a patient's response is sensitive to treatment by an EGFR modulator, based on correlation of the expression profile of the one or biomarkers, the patient's treatment prognosis can be qualified as favorable and treatment can continue. Also, if, after treatment with an EGFR modulator, the test cells don't show a change in the biomarker expression profile corresponding to the control panel of cells that are sensitive to the EGFR modulator, it can serve as an indicator that the current treatment should be modified, changed, or even discontinued. This monitoring process can indicate success or failure of a patient's treatment with an EGFR modulator and such monitoring processes can be repeated as necessary or desired.


EXAMPLES
Example 1
Interim Analysis Identification of Biomarkers

The CA225-045 pharmacogenomics trial is a phase II randomized exploratory study of ERBITUX (cetuximab) monotherapy in patients with refractory metastatic colorectal cancer (mCRC). An interim analysis of data from samples obtained from this trial was performed to examine the preclinically discovered markers in the clinical samples and to identify response prediction markers de novo.


Clinical Samples:

49 RNA patient samples isolated from pre-treatment tumor biopsies of the metastatic site were randomized into five blocks and profiled on U133A v2.0 chips (Affymetrix, Santa Clara, Calif.). Profiling data from 30/49 patients were included in the analysis based on meeting the following criteria: completion of at least two cycles of therapy; availability of sufficient clinical data to evaluate response; presence of tumor cells in biopsy sample; and good quality profiling data from chip.


The 30 patient expression profiles consisted of 24 liver metastases and 6 other tissue types. The Best Clinical Response information from the 30 patients identified 4 partial responders (PR), 5 stable disease (SD) and 21 progressive disease (PD) patients. Assessment of response was performed according to a modified version of the World Health Organization (WHO) criteria (Miller et al., Cancer, 47: 207-214 (1981)). Overall response was determined based on evaluation of target, non-target, and new lesions. Partial response (PR) was defined as at least a 50% decrease in the sum of the product of diameters (SPD) of target lesions, taking as reference the baseline SPD. Progressive disease (PD) was defined as a 25% or greater increase in the SPD of target lesions, taking as reference the smallest SPD recorded since the treatment started or the appearance of new lesions. Stable disease (SD) was defined as neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD.


Gene Expression Profiling:

Pre-treatment biopsies were obtained from the metastatic site for RNA isolation. RNA was isolated from the pre-treatment biopsies using the RNeasy mini kit (Qiagen, Valencia, Calif.). The quality of RNA was checked by measuring the 28S:18S ribosomal RNA ratio using an Agilent 2100 Bioanalyzer (Agilent Technologies, Rockville, Md.). Concentration of total RNA was determined spectrophotometrically. 1 μg of total RNA was used to prepare biotinylated probes according to the Affymetrix Genechip Expression Analysis Technical Manual. Targets were hybridized to human HG-U133A v2.0 gene chips according to the manufacturer's instructions. Data were preprocessed using the MAS 5.0 software (Affymetrix, Santa Clara, Calif.).


Data Analysis:

Of the 22,215 probesets present on the U133A v2.0 chip, 17,261 probesets that had present calls in at least two liver metastatic tissues were included for data analysis. Data was analyzed by performing a two-sided unequal variance t test with Microsoft Excel or Anova analysis using PartekPro Pattern Recognition Software (Partek, St. Charles, Mo.). The statistical analyses were performed using MAS 5.0 quantile normalized values for signal intensity for 17,261 probe sets.


Analysis of Biomarkers Using T Test and ANOVA Analysis:

The first step was to examine 42 probesets that were identified preclinically (FIG. 1) in the transcriptional profiles of 30 metastatic tumors. This was done to examine whether the preclinical markers are differentially expressed between patients who derive clinical benefit (PR and SD) from ERBITUX treatment and those who do not (PD).


A two-sided unequal variance t test was performed between the 9 patients who derive clinical benefit and the 21 patients who have progressive disease. Three probesets out of 42 are differentially expressed between 9 (PR+SD) patients and 21 (PD) patients (p<0.05). These probesets represent the mRNA expression of Annexin A1 (ANXA1 201012_at), serine proteinase inhibitor Glade B member 5 (SERPINB5 204855_at), and fibroblast growth factor receptor 3 (FGFR3 204379_s_at).


Next, a broader list of 640 genes from which the 42 probe set list had been derived (FIG. 1) was examined. 635 out of the 640 probesets were present in the 17,261 probe sets that are included in the analysis. The 635 probesets were identified as being highly variably expressed in transcriptional profiles of 164 primary untreated CRC tumors. They expressed at a moderate to high level in colon tumors (at least one expression value of two times the mean value for the array, i.e., 3000 expression units) and with a population variance value of >0.1.


The 635 probe sets were examined in transcriptional profiles of 30 metastatic tumors from the CA225-045 trial. 39 out of 635 probesets were found to be differentially expressed between 9 (PR+SD) and 21 (PD), p<0.05 and are described in Table 2. 19 of the 39 probe sets are resistance markers for ERBITUX and 20 of these are sensitivity markers for ERBITUX (FIG. 2).









TABLE 2







39 Markers for Response Prediction to ERBITUX












Affymetrix
p





ID
value
Gene name
Symbol















1
205767_at
0.0002
epiregulin
EREG


2
201012_at
0.006
annexin A1
ANXA1


3
205239_at
0.0068
amphiregulin
AREG


4
213435_at
0.0098
SATB family member 2
SATB2


5
209260_at
0.0122
stratifin
SFN


6
204379_s_at
0.0129
fibroblast growth factor receptor 3
FGFR3


7
205295_at
0.0143
creatine kinase, mitochondrial 2
CKMT2


8
204678_s_at
0.0148
potassium channel, subfamily K, memb. 1
KCNK1


9
204044_at
0.0151
quinolinate phosphoribosyltransferase
QPRT


10
203726_s_at
0.0154
laminin, alpha 3
LAMA3


11
219555_s_at
0.0165
uncharacterized bone marrow prtn BM039
BM039


12
216598_s_at
0.0188
chemokine (C-C motif) ligand 2
CCL2


13
209425_at
0.0195
alpha-methylacyl-CoA racemase
AMACR


14
204855_at
0.0207
serine proteinase inhibitor, clade B, memb. 5
SERPINB5


15
218807_at
0.0213
vav 3 oncogene
VAV3


16
210764_s_at
0.0261
cysteine-rich, angiogenic inducer, 61
CYR61


17
210511_s_at
0.0265
inhibin, beta A
INHBA


18
220834_at
0.0266
membrane-spanning 4-domains, subfly A,
MS4A12





12


19
210809_s_at
0.0268
periostin, osteoblast specific factor
POSTN


20
213385_at
0.0304
chimerin 2
CHN2


21
218468_s_at
0.0323
gremlin 1 homolog, cysteine knot
GREM1





superfamily


22
202859_x_at
0.033
interleukin 8
IL8


23
206754_s_at
0.0337
cytochrome P450, 2B6
CYP2B6


24
218806_s_at
0.034
vav 3 oncogene
VAV3


25
218469_at
0.0342
gremlin 1 homolog, cysteine knot
GREM1





superfamily


26
219508_at
0.0347
glucosaminyl (N-acetyl) transferase 3,
GCNT3





mucin type


27
204364_s_at
0.0367
chromosome 2 open reading frame 23
C2orf23


28
219471_at
0.0376
chromosome 13 open reading frame 18
C13orf18


29
219014_at
0.0396
placenta-specific 8
PLAC8


30
203939_at
0.04
5′-nucleotidase, ecto (CD73)
NT5E


31
211506_s_at
0.0401
interleukin 8
IL8


32
206143_at
0.0404
solute carrier family 26, member 3
SLC26A3


33
44790_s_at
0.0425
chromosome 13 open reading frame 18
C13orf18


34
202075_s_at
0.0427
phospholipid transfer protein
PLTP


35
201650_at
0.0436
keratin 19
KRT19


36
205259_at
0.046
nuclear receptor subfamily 3, C2
NR3C2


37
208893_s_at
0.0466
dual specificity phosphatase 6
DUSP6


38
209436_at
0.048
spondin 1, extracellular matrix protein
SPON1


39
218087_s_at
0.0496
sorbin and SH3 domain containing 1
SORBS1









The top 3 markers based on lowest p value were epiregulin (EREG, 205767_at), annexin A1 (ANXA1 201012_at), and amphiregulin (AREG, 205239_at). Interestingly, epiregulin and amphiregulin are ligands for EGFR. Examination of their individual mRNA expression profiles indicates that they appear to be more highly expressed in patients who derive clinical benefit from ERBITUX treatment (FIGS. 3A and 3B). This suggests that patients who have high levels of epiregulin and amphiregulin have tumors that are addicted to the EGFR signaling pathway that is being driven by these two ligands.


The expression levels of epidermal growth factor (EGF, 206254_at), transforming growth factor alpha (TGFα, 205016_at), betacellulin (BTC, 207326_at), and heparin binding-EGF (HB-EGF, 203821_at), which are the other known ligands for EGFR, were also examined. Their expression levels showed no correlation with response to ERBITUX.


Determination of Biological Relationships Between 39 Biomarkers:

The Ingenuity Pathway Analysis web-based application (Ingenuity Systems Inc., Mountain View, Calif.) was used to test the biological relationship between the 39 biomarkers of Table 2. This application makes use of the Ingenuity Knowledge Base, a curated database consisting of millions of individually modeled relationships between proteins, genes, complexes, cells, tissues, drugs, and diseases. The 39 genes were inputted into the Pathway Analysis application. The Ingenuity Knowledge base had information on 25 of the 39 genes. Strikingly, of the 25 “network eligible” genes, 17 mapped to the EGFR network (FIG. 4, 17 genes are shaded) indicating a strong link between the EGFR signaling status in the tumors and response to ERBITUX. No other network emerged from the analysis of the 39 genes. Of the 17 genes, DUSP6 is a member of the ERK/MAPK signaling pathway and SFN is a member of the PI3K/AKT signaling pathway, which are the two key pathways downstream of EGFR signaling.


Multivariate Analysis:

The t test and ANOVA analysis was used to assess the ability of individual biomarkers to separate PR/SD patients from PD patients. Multivariate discriminant analysis was used to assess the prediction power of the 39 markers on patient response, and identify the set of variables/biomarkers that would be the best predictors of response to ERBITUX treatment.


SAS discriminant function analysis (SAS Scientific Discovery Solutions, release 8.02, SAS Institute Inc., Cary, N.C.) was applied to the data set of 39 markers. Discriminant function analysis was broken into a 2-step process: (1) testing the significance of a set of discriminant functions; and (2) using these functions to classify the sample objects to the corresponding response groups. The first step was accomplished by a SAS “stepwise” procedure using the forward variable selection method. The derived discriminant functions were passed on to the second SAS procedure, called the “discrim” procedure, for classification of the given samples.


Given the small sample size of 30 patients, the samples were not partitioned into separate training and test data sets. Instead a single data set was used, and the leave-one-out cross-validation method was applied to test the prediction power of the identified biomarker predictors. A SAS cross-validation protocol was developed, which implemented leave-one-out cross-validation method in a SAS program, and was run on this data set to define the number of predictors that could be used for building the discriminant function models. This method allowed a comparison of a single biomarker model to multiple biomarker models (up to 15 biomarkers) (FIG. 5). The single gene predictor model was found to have 0.7037 prediction power as measured by AUC coverage (area under the Receiver Operating Characteristic (ROC) curve which shows the tradeoff between sensitivity and specificity). An area of 1 represents completely accurate prediction. When the number of predictors included in the model goes up to three biomarkers, the prediction power increases to 0.9. When the number of predictors included in the model exceeds three, there tends to be a decrease in prediction power. These results indicate that the best prediction power is achieved by building a discriminant function model with 3 out of the 39 biomarkers.


Correlation of the 39 Biomarkers:

Ingenuity Pathway analysis suggested that at least 17 of the 39 biomarkers identified belong to a single interaction network. A correlation analysis using SAS “corr” procedure was applied to investigate the correlation of genes identified from the discriminant analysis. Table 3 shows an example of a correlation matrix of some of the top predictors selected by the SAS procedure. Some of the genes show very high correlation coefficient values which suggests they are highly correlated. For example, 205767_at (EREG) and 205239_at (AREG), or 205767_at (EREG) and 218807_at (VAV3), or 206754_s_at (CYP2B6) and 209260_at (SFN) were found to be highly correlated. The highly correlated genes could replace each other to explain a certain proportion of the variation between the groups of patients who derive clinical benefit and those that do not. These results show excellent agreement between the possible biological mechanism as elucidated by Ingenuity Pathway Analysis and literature, and the statistical prediction as determined by the SAS procedure.









TABLE 3







Pearson Correlation Co-Efficients on 7 Most Frequent Probesets That


Were Identified As Top Variables For Discriminant Analysis














Affymetrix









ID
205767_at
201012_at
205239_at
206754_at
209260_at
205259_at
218807_at

















205767_at
1
−0.28587
0.84089
−0.16409
−0.04261
−0.02338
0.64133


201012_at
−0.28587
1
−0.16652
−0.41722
0.31615
−0.45851
0.28141


205239_at
0.84089
−0.16652
1
−0.21894
0.07064
−0.19815
0.60752


206754_s_at
−0.16409
−0.41722
−0.21894
1
−0.47769
0.53511
−0.21663


209260_at
−0.04261
0.31615
0.07064
−0.47769
1
−0.26621
0.26204


205259_at
−0.02338
−0.45851
−0.19815
0.53511
−0.26621
1
−0.02668


218807_at
0.64133
−0.28141
0.60752
−0.21663
0.26204
−0.02668
1









Best Prediction Models:

The best prediction models were determined using the SAS stepwise procedure. 205767_at (EREG) was always picked first. This suggests that the expression of the EGFR ligand epiregulin can explain most of the variation that exists between the group of patients that are PR/SD and the group of patients who are PD. The second predictor aids in picking up the largest proportion of the unexplained variation from the first variable function (predictor) and so on. The misclassification rates of the best SAS selected models were:













Model
Error rate
















205767_at (EREG)
0.2143


205767_at (EREG), 206754_s_at (CYP2B6)
0.127


205767_at (EREG), 206754_s_at (CYP2B6), 201650_at
0.1032


(KRT19)


205767_at (EREG), 206754_s_at (CYP2B6), 201650_at
0.1032


(KRT19), 204678_at (KCNK1)










Biomarkers were also selected based on their biological, functional, and co-regulation information, and the derived prediction functions were used to classify the 30 sample data set using the SAS “discrim” procedure. Using this approach, some optimal combinations of biomarker variables and their corresponding misclassification rates were identified, such as:













Model
Error rate
















205767_at (EREG), 206754_s_at (CYP2B6)
0.1032


201650_at (KRT19)


205767_at (EREG), 209260_at (SFN), 205259_at (NR3C2)
0.079


201012_at (ANXA1), 205239_at (AREG), 209260_at
0.07


(SFN), 205259_at (NR3C2), 218807_at (VAV3)


209260_at (SFN), 218807_at (VAV3)
0.1270









Example 2
Identification of Biomarkers Following Interim Analysis

As mentioned above, the CA225-045 pharmacogenomics trial is a phase II randomized exploratory study of ERBITUX (cetuximab) monotherapy in patients with refractory metastatic colorectal cancer (mCRC). This trial enrolled 111 patients. A standard cetuximab dosing regimen was followed for the first 3 weeks of therapy, thereafter patients were eligible for dose escalation every 3 weeks to a maximum dose of 400 mg/m2 provided they had not experienced a >grade 2 skin rash. During the pre-treatment phase, all patients underwent a tumor biopsy procedure involving three passes with an 18-gauge needle of a single metastatic lesion. Two pre-treatment core needle biopsies were stored in a single tube of RNALater at room temperature and one core needle biopsy was formalin-fixed and embedded in paraffin for subsequent analyses. All subjects also underwent a pre-treatment blood draw. All specimens were obtained from patients with appropriate informed consent and IRB approval.


Tumor response was evaluated every nine weeks (one cycle of therapy) according to the modified World Health Organization criteria (Miller et al., Cancer, 47, 207-214 (1981)). Overall response was determined based on evaluation of target, non-target and new lesions. For this analysis, subjects experiencing a complete (CR) or partial response (PR), or stable disease (SD), were grouped as the disease control group; progressive disease (PD) and select unable to determine (UTD) subjects were grouped as non-responders. The UTD subjects that were included in the non-responder group for analysis were those that died prior to the response assessment. All other UTD subjects were excluded from the analysis.


RNA and DNA Extraction:

For each subject's tumor sample, RNA and DNA were isolated from two pre-treatment core needle biopsies provided in a single tube of RNALater at room temperature within seven days from the date of the biopsy procedure. RNA was isolated using the RNeasy mini kit (Qiagen, Valencia, Calif.). The quality of RNA was checked by measuring the 28S:18S ribosomal RNA ratio using an Agilent 2100 Bioanalyzer (Agilent Technologies, Rockville, Md.). DNA was isolated from the flow-through collected during the RNA isolation procedure using the DNeasy mini kit (Qiagen). Concentration of RNA and DNA was determined spectrophotometrically.


Gene Expression Profiling and Statistical Analysis:

For each sample from which sufficient RNA was available, 1 μg of total RNA was used to prepare biotinylated probes according to the Affymetrix GeneChip Expression Analysis Technical Manual. Targets were hybridized to human HG-U133A v2.0 GeneChips according to the manufacturer's instructions. Data were preprocessed using the MAS 5.0 software (Affymetrix, Santa Clara, Calif.) and statistical analyses were performed using quantile normalized values for signal intensity. Univariate analysis was done by using a two-sided unequal variance t-test. For multivariate analysis samples were randomly partitioned 50-50 into a training set and a test set. Top candidate predictors were selected from the training set using a t-test. This was followed by model construction using stepwise discriminant analysis (v8.2, SAS, Cary, N.C.). Class prediction was assessed using 10-fold cross validation. The models developed from the training set were evaluated using a test set.


In addition to the profiling of RNA from the clinical study, an expression database of 164 primary colorectal tumors (Banerjea et al., Mol. Cancer, 3, 21 (2004)) was examined to identify potential predictive markers. Data from the 640 probe sets that passed the filtering steps described above in the results were then subjected to an unsupervised average linkage hierarchical clustering using CLUSTER and the results were displayed by using TREEVIEW.


RT-qPCR for Gene Expression Analysis:

For each sample from which RNA was available, approximately 100 ng RNA was converted into cDNA by the random priming method using MultiScribe Reverse Transcriptase according to the manufacturer's instructions (TaqMan Reverse Transcription Reagents, Applied Biosystems Inc. ((ABI), Foster City, Calif.). The resulting cDNA was measured on the ABI 7900HT Sequence Detection System using ABI Assay-on-Demand primer/probe sets directed against the amphiregulin (Hs00155832_m1) and epiregulin (Hs00154995_m1) genes. Relative expression levels were calculated using the ΔCt method in which average values of duplicate reactions were compared, with GAPDH (Hs001266705_g1) serving as the internal reference. In this experimental design, low ΔCt values correspond to high levels of expression.


Nucleotide Sequence Analysis:

Mutational analyses of EGFR, K-RAS, and BRAF were performed using available genomic DNAs isolated from tumor specimens. Primers used for EGFR exons 18-21, coding for the TK domain, were published previously (Lynch et al., N. Engl. J. Med., 350, 2129-2139 (2004)). The primers used to evaluate exon 2 of K-RAS and exon 15 of BRAF were as follows: K-RAS F 5′-TAAGGCCTGCTGAAAATGACTG-3′ (SEQ ID NO:257) and K-RAS R 5′-TGGTCCTGCACCAGTAA TATGC-3′ (SEQ ID NO:258); BRAF F 5′-TCATAATGCTTGCTCTGATAGGA-3′ (SEQ ID NO:259) and BRAF R 5′-GGCCAAAAATTTAATCAGTGGA-3′ (SEQ ID NO:260). PCR was performed using conditions as previously described (Chen et al., Hum. Mutat., 27, 427-435 (2006)). PCR fragments were cleaned with QIAquick PCR Purification Kit (Qiagen), sequenced on an ABI 3100A Capillary Genetic Analyzer (Applied Biosystems Inc.) and analyzed in both sense and antisense directions for the presence of heterozygous mutations. Analysis of the DNA sequence was performed using SEQUENCHER v4.2 (Gene Codes, Ann Arbor, Mich.) followed by visual analysis of each electropherogram by two independent reviewers. Appropriate positive and negative controls were included for each of the exons evaluated. Mutational analyses were done without knowledge of clinical outcome including tumor response.


Results
Patients' Characteristics and Clinical Outcome:

The primary objective of this study was to identify predictive markers of response to cetuximab therapy in CRC. Evaluable RNA and/or DNA and/or plasma samples were available for 103 out of 111 subjects. The objective response determination for these 103 subjects were: one complete response (CR), six partial response (PR), twenty-eight stable disease (SD), fifty-six progressive disease (PD), and twelve patients who died prior to their first radiographic assessment and are therefore unable to determine (UTD). Thirty-four percent of the subjects either responded or had disease stabilization whereas the remaining 66% were classified as non-responders.


Genomic Analysis of Tumor-Derived RNAs:

In order to identify genes that were differentially expressed between the disease control and non-responder groups, gene expression profiling was carried out on RNA isolated from 95 pre-treatment biopsies. Seventy percent of the biopsies were taken from the liver metastatic tissue, and 30% of the biopsies were taken from non-hepatic tissue sites. 91 out of the 95 samples yielded >500 ng RNA and were randomized into ten blocks and profiled on U133A v2.0 chips (Affymetrix). High quality transcriptional profiling data were obtained from 87 patients. Seven patients were excluded from further analysis either because they withdrew from the study prior to the first assessment, experienced hypersensitivity or withdrew their consent. Final data analysis was carried out using best clinical response assessments for the remaining 80 patients and expression profiles from these patients were included in the statistical analysis. These 80 patients included 1 CR, 5 PR, 19 SD, 43 PD, and 12 UTD.


An initial candidate set of genes was identified that were variably expressed in an independent set of 164 primary colorectal tumors by filtering transcriptional data from all 22,215 probe sets. This filtering yielded 640 probe sets that were expressed at a moderate to high level in colon tumors (at least one expression value of two times the mean value for the array i.e. 3000 expression units) and with a population variance value of >0.1. It was proposed that these 640 probe sets that had a highly dynamic range of expression across a population of CRC tumors were most likely to yield markers that would be useful for patient selection. Unsupervised hierarchical clustering of the 640 probe sets across the 164 primary colon tumors showed that biologically interesting genes that might be predictive of response to cetuximab were preferentially expressed in a subset of colorectal tumors (FIG. 6). In FIG. 6, the 164 tumors were divided into 3 major classes (Class 1, 2 and 3). The 640 probe sets were divided into 5 clusters (labeled A through E). Cluster A, which contains cancer antigens such as CEACAM 6 and CD24, also contains the EGFR ligands EREG and AREG. Cluster A is most highly expressed iii Class 1a, which represents approximately 25% of the 164 colorectal tumor specimens.


Out of 22,215 probe sets, data analysis was conducted on 17,137 probe sets that were found to be expressed in at least 10% of the liver metastases patient samples. 629 of the previously identified 640 probe sets were present in the 17,137 probe set list. Their gene expression profiles were examined in the data from 80 patients and were correlated with response assessments. 121 out of the 629 probe sets were found to be differentially expressed between 25 patients with disease control and 55 non-responders, p<0.05 (t test of the disease group (CR, PR, SD) vs. non-responders) as shown in Table 4.









TABLE 4







121 Probe Sets Differentially Expressed Between 25 patients


with disease control and 55 non-responders, p < 0.05










Affymetrix





ID
p value
Gene name
Symbol













203939_at
3.787E−07
5′-nucleotidase, ecto (CD73)
NT5E


205767_at
1.474E−05
epiregulin
EREG


205239_at
2.489E−05
amphiregulin (schwannoma-
AREG




derived growth factor)


213975_s_at
3.617E−05
lysozyme (renal amyloidosis) ///
LYZ /// LILRB1




leukocyte immunoglobulin-like




receptor, subfamily B (with TM




and ITIM domains), member 1


201641_at
9.146E−05
bone marrow stromal cell antigen 2
BST2


208893_s_at
0.000257
dual specificity phosphatase 6
DUSP6


218807_at
0.000507
vav 3 oncogene
VAV3


218806_s_at
0.000513
vav 3 oncogene
VAV3


216598_s_at
0.000680
chemokine (C-C motif) ligand 2
CCL2


213435_at
0.000909
SATB family member 2
SATB2


210517_s_at
0.001636
A kinase (PRKA) anchor protein
AKAP12




(gravin) 12


219508_at
0.001935
glucosaminyl (N-acetyl) transferase
GCNT3




3, mucin type


201462_at
0.001937
secernin 1
SCRN1


204379_s_at
0.002008
fibroblast growth factor receptor 3
FGFR3




(achondroplasia, thanatophoric




dwarfism)


206584_at
0.002018
lymphocyte antigen 96
LY96


200884_at
0.002042
creatine kinase, brain
CKB


206332_s_at
0.002612
interferon, gamma-inducible
IFI16




protein 16


202525_at
0.002630
protease, serine, 8 (prostasin)
PRSS8


205403_at
0.002869
interleukin 1 receptor, type II
IL1R2


221530_s_at
0.002881
basic helix-loop-helix domain
BHLHB3




containing, class B, 3


209728_at
0.003260
major histocompatibility complex,
HLA-DRB4




class II, DR beta 4 /// major




histocompatibility complex, class




II, DR beta 4


215049_x_at
0.004039
CD163 antigen
CD163


203645_s_at
0.004182
CD163 antigen
CD163


219471_at
0.004627
chromosome 13 open reading
C13orf18




frame 18


210133_at
0.004790
chemokine (C-C motif) ligand 11
CCL11


205097_at
0.005553
solute carrier family 26 (sulfate
SLC26A2




transporter), member 2


211656_x_at
0.006050
major histocompatibility complex,
HLA-DQB1




class II, DQ beta 1 /// major




histocompatibility complex, class




II, DQ beta 1


209392_at
0.006150
ectonucleotide
ENPP2




pyrophosphatase/phosphodiesterase




2 (autotaxin)


205402_x_at
0.006181
protease, serine, 2 (trypsin 2)
PRSS2


217028_at
0.006582
chemokine (C—X—C motif) receptor 4
CXCR4


204855_at
0.006615
serpin peptidase inhibitor, clade B
SERPINB5




(ovalbumin), member 5


201137_s_at
0.007369
major histocompatibility complex,
HLA-DPB1




class II, DP beta 1


215051_x_at
0.007563
allograft inflammatory factor 1
AIF1


202859_x_at
0.007872
interleukin 8
IL8


211506_s_at
0.008119
interleukin 8
IL8


207457_s_at
0.008600
lymphocyte antigen 6 complex,
LY6G6D




locus G6D


205765_at
0.009101
cytochrome P450, family 3,
CYP3A5




subfamily A, polypeptide 5


204619_s_at
0.009733
chondroitin sulfate proteoglycan 2
CSPG2




(versican)


205199_at
0.010621
carbonic anhydrase IX
CA9


219962_at
0.010751
angiotensin I converting enzyme
ACE2




(peptidyl-dipeptidase A) 2


205242_at
0.011022
chemokine (C—X—C motif) ligand
CXCL13




13 (B-cell chemoattractant)


217428_s_at
0.011274
collagen, type X, alpha 1(Schmid
COL10A1




metaphyseal chondrodysplasia)


206918_s_at
0.011540
copine I
CPNE1


44790_s_at
0.011645
chromosome 13 open reading
C13orf18




frame 18


218469_at
0.011704
gremlin 1, cysteine knot
GREM1




superfamily, homolog (Xenopus





laevis)



209823_x_at
0.011862
major histocompatibility complex,
HLA-DQB1




class II, DQ beta 1


205513_at
0.011867
transcobalamin I (vitamin B12
TCN1




binding protein, R binder family)


204213_at
0.012198
polymeric immunoglobulin
PIGR




receptor


205941_s_at
0.012335
collagen, type X, alpha 1(Schmid
COL10A1




metaphyseal chondrodysplasia)


212192_at
0.012522
potassium channel tetramerisation
KCTD12




domain containing 12


204891_s_at
0.012755
lymphocyte-specific protein
LCK




tyrosine kinase


208029_s_at
0.012800
lysosomal associated protein
LAPTM4B




transmembrane 4 beta /// lysosomal




associated protein transmembrane




4 beta


201884_at
0.013032
carcinoembryonic antigen-related
CEACAM5




cell adhesion molecule 5


201030_x_at
0.013074
lactate dehydrogenase B
LDHB


202411_at
0.013302
interferon, alpha-inducible protein
IFI27




27


211165_x_at
0.013671
EPH receptor B2
EPHB2


212186_at
0.014902
acetyl-Coenzyme A carboxylase
ACACA




alpha


201743_at
0.015156
CD14 antigen /// CD14 antigen
CD14


87100_at
0.015861




206467_x_at
0.015975
tumor necrosis factor receptor
TNFRSF6B ///




superfamily, member 6b, decoy ///
RTEL1




regulator of telomere elongation




helicase 1


218468_s_at
0.016329
gremlin 1, cysteine knot
GREM1




superfamily, homolog (Xenopus





laevis)



222257_s_at
0.016397
angiotensin I converting enzyme
ACE2




(peptidyl-dipeptidase A) 2


221730_at
0.016992
collagen, type V, alpha 2
COL5A2


203915_at
0.017412
chemokine (C—X—C motif) ligand 9
CXCL9


206858_s_at
0.017492
homeo box C6
HOXC6


221584_s_at
0.017554
potassium large conductance
KCNMA1




calcium-activated channel,




subfamily M, alpha member 1


204475_at
0.018085
matrix metallopeptidase 1
MMP1




(interstitial collagenase)


203895_at
0.018353
phospholipase C, beta 4
PLCB4


214043_at
0.018926
Protein tyrosine phosphatase,
PTPRD




receptor type, D


204678_s_at
0.019645
potassium channel, subfamily K,
KCNK1




member 1


204446_s_at
0.019912
arachidonate 5-lipoxygenase
ALOX5


204533_at
0.020226
chemokine (C—X—C motif) ligand
CXCL10




10


211689_s_at
0.020262
transmembrane protease, serine 2
TMPRSS2




/// transmembrane protease, serine 2


201858_s_at
0.020471
proteoglycan 1, secretory granule
PRG1


212671_s_at
0.020852
major bistocompatibility complex,
HLA-DQA1 ///




class II, DQ alpha 1 /// major
HLA-DQA2




histocompatibility complex, class




II, DQ alpha 2


216248_s_at
0.021062
nuclear receptor subfamily 4, group
NR4A2




A, member 2


212188_at
0.021225
potassium channel tetramerisation
KCTD12




domain containing 12 /// potassium




channel tetramerisation domain




containing 12


204070_at
0.021833
retinoic acid receptor responder
RARRES3




(tazarotene induced) 3


213564_x_at
0.022061
lactate dehydrogenase B
LDHB


209732_at
0.022699
C-type lectin domain family 2,
CLEC2B




member B


213746_s_at
0.023141
filamin A, alpha (actin binding
FLNA




protein 280)


214974_x_at
0.023351
chemokine (C—X—C motif) ligand 5
CXCL5


201792_at
0.023592
AE binding protein 1
AEBP1


213905_x_at
0.023638
biglycan /// serologically defined
BGN ///




colon cancer antigen 33
SDCCAG33


212353_at
0.024175
sulfatase 1
SULF1


209156_s_at
0.024926
collagen, type VI, alpha 2
COL6A2


203083_at
0.025140
thrombospondin 2
THBS2


203896_s_at
0.025311
phospholipase C, beta 4
PLCB4


201617_x_at
0.025316
caldesmon 1
CALD1


217963_s_at
0.025667
nerve growth factor receptor
NGFRAP1




(TNFRSF16) associated protein 1


208965_s_at
0.025706
interferon, gamma-inducible
IFI16




protein 16


217763_s_at
0.026315
RAB31, member RAS oncogene
RAB31




family


203325_s_at
0.026698
collagen, type V, alpha 1
COL5A1


209792_s_at
0.026893
kallikrein 10
KLK10


205549_at
0.027028
Purkinje cell protein 4
PCP4


204622_x_at
0.028026
nuclear receptor subfamily 4, group
NR4A2




A, member 2


210095_s_at
0.030712
insulin-like growth factor binding
IGFBP3




protein 3


209969_s_at
0.031010
signal transducer and activator of
STAT1




transcription 1, 91 kDa


202436_s_at
0.031792
cytochrome P450, family 1,
CYP1B1




subfamily B, polypeptide 1


202311_s_at
0.032306
collagen, type I, alpha 1
COL1A1


221031_s_at
0.032415
hypothetical protein
DKFZP434F0318




DKFZp434F0318 /// hypothetical




protein DKFZp434F0318


209118_s_at
0.032949
tubulin, alpha 3
TUBA3


210164_at
0.033266
granzyme B (granzyme 2,
GZMB




cytotoxic T-lymphocyte-associated




serine esterase 1) /// granzyme B




(granzyme 2, cytotoxic T-




lymphocyte-associated serine




esterase 1)


213194_at
0.034686
roundabout, axon guidance
ROBO1




receptor, homolog 1 (Drosophila)


204697_s_at
0.034934
chromogranin A (parathyroid
CHGA




secretory protein 1)


202752_x_at
0.035921
solute carrier family 7 (cationic
SLC7A8




amino acid transporter, y+ system),




member 8


205929_at
0.037216
glycoprotein A33 (transmembrane)
GPA33


204044_at
0.037293
quinolinate
QPRT




phosphoribosyltransferase




(nicotinate-nucleotide




pyrophosphorylase




(carboxylating))


205311_at
0.037673
dopa decarboxylase (aromatic L-
DDC




amino acid decarboxylase)


204320_at
0.038710
collagen, type XI, alpha 1
COL11A1


204364_s_at
0.040104
chromosome 2 open reading frame
C2orf23




23


212354_at
0.040347
sulfatase 1
SULF1


202465_at
0.040639
procollagen C-endopeptidase
PCOLCE




enhancer


212992_at
0.041178
chromosome 14 open reading
C14orf78




frame 78


209201_x_at
0.042126
chemokine (C—X—C motif) receptor 4
CXCR4


215646_s_at
0.043050
chondroitin sulfate proteoglycan 2
CSPG2




(versican) /// chondroitin sulfate




proteoglycan 2 (versican)


202283_at
0.045795
serpin peptidase inhibitor, clade F
SERPINF1




(alpha-2 antiplasmin, pigment




epithelium derived factor), member 1


209436_at
0.046099
spondin 1, extracellular matrix
SPON1




protein


37892_at
0.048675
collagen, type XI, alpha 1
COL11A1


218559_s_at
0.048679
v-maf musculoaponeurotic
MAFB




fibrosarcoma oncogene homolog B




(avian)


213998_s_at
0.049742
DEAD (Asp-Glu-Ala-Asp) box
DDX17




polypeptide 17









The top three candidate markers based on lowest p value were 5′ nucleotidase ecto (CD73, 203939_at), epiregulin (EREG, 205767_at) and amphiregulin (AREG, 205239_at). CD73 is a purine metabolizing enzyme that may have prognostic value in colorectal and pancreatic cancer (Eroglu et al., Med. Oncol., 17, 319-324 (2000); Giovannetti et al., Cancer Res., 66, 3928-3935 (2006)). Examination of its mRNA profile showed that it is expressed at higher levels in the non-responder group. Epiregulin and amphiregulin are ligands for EGFR (Singh and Harris, Cell Signal, 17, 1183-1193 (2005)). Examination of their individual mRNA expression profiles revealed that they were more highly expressed in patients in the disease control group (FIGS. 7A and 7B). FIGS. 7A and 7B provide mRNA levels of EGFR ligands epiregulin and amphiregulin. Affymetrix mRNA levels of Epiregulin (EREG, 205767_at) and Amphiregulin (AREG, 205239_at) are plotted on the y axis. There is a statistically significant difference in gene expression levels between the disease control group (CR, PR and SD) and the non-responder group (EREG p=1.474e−05, AREG p=2.489e−05). These results suggest that patients who have high levels of EREG and AREG have tumors that are addicted to the EGFR signaling pathway and are therefore most likely to experience disease control on treatment with cetuximab.


In addition to the gene filtering approach described above, a de novo analysis was performed on the transcriptional profiles of the same 80 patients. A two-sided unequal-variance t-test was done on all 17,137 probe sets. The top 10 genes are provided in Table 5.









TABLE 5







Top 10 Genes from De Novo Analysis










Affymetrix





ID
p value
Gene name
Symbol













203939_at
3.787E−07
5′-nucleotidase, ecto (CD73)
NT5E


217999_s_at
7.056E−06
Pleckstrin homology-like domain, family A,
PHLDA1




member 1


205767_at
1.474E−05
epiregulin
EREG


203349_s_at
1.704E−05
ets variant gene 5 (ets-related molecule)
ETV5


204015_s_at
1.812E−05
dual specificity phosphatase 4
DUSP4


204014_at
1.856E−05
dual specificity phosphatase 4
DUSP4


212349_at
2.395E−05
protein O-fucosyltransferase 1
POFUT1


205239_at
2.489E−05
amphiregulin (schwannoma-derived growth
AREG




factor)


208130_s_at
2.646E−05
thromboxane A synthase 1 (platelet,
TBXAS1




cytochrome P450, family 5, subfamily A) ///




thromboxane A synthase 1 (platelet,




cytochrome P450, family 5, subfamily A)


219615_s_at
3.153E−05
potassium channel, subfamily K, member 5
KCNK5










Examination of the top 10 genes with the lowest p value revealed that EREG and AREG were once again found to be top sensitivity markers. CD73, dual specificity phosphatase 4 (DUSP4, 204015_s_at and 204014_at), and pleckstrin homology like domain A1 (PHLDA1, 217999_s_at) were found to be top resistance markers. The mRNA expression levels of epidermal growth factor (EGF, 206254_at), transforming growth factor alpha (TGFα, 205016_at), betacellulin (BTC, 207326_at) and heparin binding-EGF (HB-EGF, 203821_at), some of the other known ligands for EGFR, were also reviewed. Their expression levels showed no correlation with response to cetuximab. It is also worth noting that no correlation was seen between EGFR (201983_s_at) mRNA levels and response to cetuximab. These results suggest that a de novo analysis using only the transcriptional profiling data gathered from this clinical study could find the candidate markers EREG and AREG. However, given the issue of multiple test comparisons, the identification of EREG and AREG using an independent filtering approach described above lends additional support to their being candidates for predicting cetuximab response.


From the t-test analyses, the ability of individual biomarkers to separate the disease control group from the non-responders could be assessed. Using discriminant function analysis, the prediction power of a set of the 100 top candidate markers for patient response was assessed in order to identify the set of variables that would be the best predictors of disease control with cetuximab treatment. The AUC (area under the receiver operating characteristic curve) values of the different multi-gene models showed that as the number of genes in the model increased from one to fifteen the predictive power of the model did not improve. The AUC value of a single gene model was >0.8. An independent test was done to assess the performance of the most frequently identified gene, EREG, and also of AREG, as individual predictors. EREG has an AUC value of 0.845, and AREG has an AUC value of 0.815, indicating that they are both highly powerful predictive markers for patient selection (FIGS. 8A and 8B).


Analysis of Candidate Markers Epiregulin and Amphiregulin:

In order to independently verify gene expression with a different technology platform that may ultimately be more easily transferable into a diagnostic test, AREG and EREG transcript levels were measured using quantitative RT-PCR TaqMan assays. Expression levels of these genes were obtained for tumor samples from 73 of the subjects using both array-based and qRT-PCR methods (Table 6).









TABLE 6







Expression Levels of Amphiregulin and Epiregulin by quantitative RT-


PCR TaqMan Assays



















KRAS
KRAS
Order


Best


qRT-
qRT-
Mutation
Mutation
of


Clinical
AffyQ
AffyQ
PCR
PCR
codon
amino
sample


Response
AREG
EREG
AREG
EREG
base
acid
on


Assessment
expression
expression
dCt
dCt
change
change
FIG. 7

















CR
2573.74
1659.91
5.80
5.32


1


PD
949.81
450.25
7.79
7.20
WT

36


SD
3353.93
2336.8
9.58
8.89
c.35G > T
G12V
7


SD
105.82
89.23
9.35
9.31
WT

8


UTD
1581.54
603.27
6.48
6.20
c.35G > A
G12D
73


SD


PD
1626.87
668.84
5.40
5.48
c.35G > T
G12V
32


PD
122.3
46.36




58


UTD
321.51
56.59
9.20
9.31
c.35G > A
G12D
69


SD


SD


PD
177.95
128.85
9.01
8.76
c.35G > A
G12D
67


PD
2550.49
655.04
4.57
5.64
WT

30


PR
3974.98
1108.91
3.23
4.38
WT

2


PD
1084.91
622.01
5.35
5.46
WT

26


PD
611.84
573.66
6.17
5.60
WT

47


SD
955.24
292.33
6.22
7.30
WT

11


PR
5083.12
1166.18


WT

5


PD


SD
2481.22
1154.9
4.56
4.99
WT

12


SD
2527.86
1395.95
5.37
4.35
WT

13


SD




WT


PD




c.35G > A
G12D


PD
402.53
419.27
9.34
6.14
c.35G > A
G12D
62


PR
3395.09
1447.49
3.76
4.14
WT

3


PD
2134.23
906.03
7.11
6.45
c.35G > T
G12V
37


PD
1163.17
100.48
6.39
9.52
c.35G > T
G12V
27


UTD
1086.48
113.14
UTD
UTD
WT

70


UTD
301.36
241.05
8.82
8.30
WT

74


SD
4414.67
1331.61
3.77
4.67
WT

14


SD
609.57
62.96


c.35G > A
G12D
15


PD




WT


PD
901.86
459.6
8.30
7.43
WT

68


PD




WT


PR
3332.21
2042.92
5.17
3.47
WT

6


PD
42.03
78.71
11.81
9.19
WT

48


SD




WT


PD




c.35G > C
G12A


PR
1418.75
2411.15
4.91
3.40
WT

4


UTD
872.72
469.76
6.32
5.55
c.35G > A
G12D
71


SD
1384.71
632.61
5.75
5.60
na

9


PD
503.53
206.2
6.83
7.10
na

59


PD
75.64
50.98
10.33
9.52


61


PD
1879.09
587.4
7.50
7.25
na

41


PD
471.68
36.46
5.60
4.77


34


PD
39.27
8.15
12.33
13.18
WT

55


PD
111.94
107.83
10.02
8.30
WT

43


PD




na


PR




na


PD
1464.45
298.7
5.94
7.16
WT

51


SD
5533.18
2232.8


na

10


PD
236.8
42.59
8.96
UTD


54


SD
1416.68
819.85


WT

16


PD
719.16
550.72
6.38
5.90
c.35G > A
G12D
42


PD


UTD
127.95
12.85
9.86
10.64
c.35G > A
G12D
72


PD
331.54
307.55
8.22
6.83
WT

33


PD
936.71
64.49
8.28
10.95
WT

65


PD
132.01
28.72
10.55
12.04
c.35G > A
G12D
35


UTD
760.08
221.16
6.27
8.55


75


PD
162.74
71.16
10.21
11.17
WT

28


UTD
865.02
258.5
7.95
8.94
c.34G > A
G12S
76


PD
489.57
224.81
8.17
7.70
c.35G > T
G12V
46


PD
813.24
529.95
7.16
6.79
c.35G > A
G12D
38


PD


PD


PD
1556.84
703.23
5.70
5.40
c.35G > C
G12A
60


SD


PD
1646.55
1127.43
6.44
5.39
WT

57


PD


PD
27.71
1.05
13.23
UTD
WT

56


PD
1182.47
76.66
7.48
10.91
c.34G > A
G12S
50


PD


PD
532.55
171.22
8.87
8.79
c.35G > C
G12A
45


PD
12.43
13.62
UTD
13.67
WT

63


SD
2809.16
804.93
6.13
5.20
WT

17


UTD
1656.76
665.01
6.14
5.07
c.38G > A
G13D
77


SD
18.88
2.2
10.67
12.31
WT

18


SD
1479.28
799.93
5.74
6.28
WT

19


PD
1034.32
384.07
6.64
7.29
WT

53


UTD
24.18
15.47
UTD
UTD
WT

78


UTD
54.13
11.49
9.44
11.32
WT

79


SD
1554.57
646.2
5.23
5.86
WT

20


SD
3536.88
1764.91
5.82
3.45
WT

21


SD




WT


SD
6390.33
3078.94
3.47
4.02
WT

22


PD


PD
801.39
486.2
6.81
7.14
WT

40


SD




c.35G > A
G12D


UTD
1945.99
240.5
8.21
10.16
c.38G > A
G13D
80


PD
1984.72
897.89
4.21
4.31
c.35G > T
G12V
64


SD
5830.27
1980.37
2.58
3.11
WT

23


PD
2321
784.77
5.41
5.21
c.35G > T
G12V
29


PD




WT


PD
1095.66
468.77
9.03
7.75
c.38G > A
G13D
66


PD
442.29
77.8
9.84
10.39
c.35G > A
G12D
49


SD
1610.75
442.09
5.25
6.21
WT

24


SD
2615.62
1113.89
5.67
7.03
WT

25


PD
1737.75
694.22
6.05
7.01
WT

44


SD




WT


PD
2271.37
634.05
5.32
5.61
c.35G > A
G12D
39


PD
1858.06
870.14
6.27
6.34
c.35G > A
G12D
52


PD
1018.25
859.41
8.08
5.91
WT

31










There was good correlation between the two methods (for log2-transformed array data, Pearson >0.85, R2>0.7), with high expression on Affymetrix arrays corresponding to low ΔCt values from TaqMan assays for both amphiregulin and epiregulin (FIG. 9).


Genetic Analysis of DNA Isolated from Tumor Biopsies and Whole Blood:


Somatic mutations in the EGFR tyrosine kinase domain are found to be strongly associated with sensitivity to gefitinib and erlotinib in NSCLC (Janne et al., J. Clin. Oncol., 23, 3227-3234 (2005)). It has been reported that somatic mutations in the EGFR TK domain are not required for response to cetuximab, nor do they appear to be predictive of response to cetuximab (Tsuchihashi et al., N. Engl. J. Med., 353, 208-209 (2005)). Somatic mutations in K-RAS are associated with a lack of sensitivity to gefitinib and erlotinib in NSCLC but their role in cetuximab sensitivity in CRC is unclear (Moroni et al., Lancet Oncol., 6, 279-286 (2005); Pao et al., PLoS Med., 2, e17 (2005)). DNA from 80 tumor biopsies was evaluated for mutations in EGFR, K-RAS and BRAF. Not a single heterozygous mutation was detected in either the EGFR kinase domain or in exon 15 of the BRAF gene. K-RAS exon 2 mutations affecting codon 12 and 13 were detected in 30 out of 80 (38%) analyzed samples (Table 6). K-RAS mutations were detected in only 3 Stable Disease patients out of the 27 Disease Control Group (5 PR and 22 SD) patients tested (11%). On the other hand, K-RAS mutations were detected in 27 out of 53 non-responders (51%). The data clearly show that the presence of a K-RAS mutation correlates with a lack of response to cetuximab therapy.


Discussion:

The key findings from the analysis of pre-treatment biopsies are that patients whose tumors express high levels of the EGFR ligands epiregulin and amphiregulin are most likely to benefit from cetuximab therapy. In addition, it was found that patients whose tumors do not have K-RAS mutations have a significantly higher disease control rate than those with K-RAS mutations.


The genes for the EGFR ligands epiregulin and amphiregulin are co-localized on chromosome 4q13.3 (Conti et al., Mol. Endocrinol., 20, 715-723 (2006)). It was observed that the expression of epiregulin and amphiregulin was coordinately regulated (Pearson correlation=0.85). Epiregulin is known to bind more weakly to EGFR and ERBB4 than the EGF ligand, but is a much more potent mitogen than EGF and leads to a prolonged state of receptor activation (Shelly et al., J. Biol. Chem., 273, 10496-10505 (1998)). Elevated expression of epiregulin and/or amphiregulin may play an important role in tumor growth and survival by stimulating an autocrine loop through EGFR. This may characterize a tumor that is “EGFR-dependent” and therefore sensitive to the ability of cetuximab to block ligand-receptor interaction. The observations that constitutive epiregulin and amphiregulin expression in L2987 cells is decreased upon EGFR inhibitor treatment, is stimulated by EGF treatment, and that cetuximab treatment blocks L2987 cell growth, support the hypothesis that these EGFR ligands are beacons of an activated EGFR pathway and perhaps autocrine stimulators. This hypothesis is also supported by results in a lung cancer mouse model in which high expression of epiregulin and amphiregulin, as well as ERBB3, was dependent on EGFR activation (Fujimoto et al., Cancer Res., 65, 11478-11485 (2005)).


It is not surprising that the findings of epiregulin and amphiregulin RNA expression was not translated into protein-based assays. The mRNA transcripts may code for the membrane-anchored precursor forms that are eventually cleaved to generate soluble forms. In the case of amphiregulin, it has been shown that the membrane-anchored isoform, as well as the soluble form, are biologically active and may induce juxtacrine, autocrine or paracrine signaling (Singh and Harris, Cell Signal, 17, 1183-1193 (2005)). It is interesting to note that in contrast to these findings, elevated serum levels of amphiregulin and TGFα have been reported to predict poor response to gefitinib in patients with advanced NSCLC. (Ishikawa et al., Cancer Res., 65, 9176-9184 (2005)). It remains to be determined whether the tumors of the patients with high serum levels of amphiregulin and TGFα described in that study may have other genetic aberrations such as K-RAS mutation that may allow by-pass of their dependence on EGFR signaling for growth and survival.


Epiregulin and amphiregulin can be used to identify other tumor types that might be sensitive to cetuximab. Epiregulin and amphiregulin expression is increased in androgen-independent prostate cancer cells and after castration in an androgen-sensitive prostate cancer xenograft (Torring et al., Prostate, 64, 1-8 (2005); Torring et al., Anticancer Res., 20, 91-95 (2000)). Epiregulin expression is higher in pancreatic cancer where it stimulates cell growth (Zhu et al., Biochem. Biophys. Res. Commun., 273, 1019-1024 (2000)) and in bladder cancer patients where it is correlated with survival (Thogersen et al., Cancer Res., 61, 6227-6233 (2001)). The enhanced expression of amphiregulin is found to be significantly correlated with overall survival in non-small cell lung cancer (NSCLC) (Fontanini et al., Clin. Cancer Res., 4, 241-249 (1998)). Amphiregulin expression is higher in multiple myeloma cells expressing ERBB receptors and promotes their growth (Mahtouk et al., Oncogene, 24, 3512-3524 (2005)). Recently, it has been found that high levels of lutenizing hormone may elevate the risk of ovarian and breast cancers through the stimulation of epiregulin and amphiregulin which in turn could stimulate mitogenic EGFR signaling (Freimann et al., Biochem. Pharmacol., 68, 989-996 (2004)). Finally, the observation that EGFR and estrogen receptor (ERα) mediate expression of amphiregulin (Britton et al., Breast Cancer Res. Treat., 96, 131-146 (2006)) suggests that a subset of breast cancer patients (EGFR+, ER+, amphiregulin+) may benefit from cetuximab therapy. It is notable that among metastatic breast cancer patients treated with the EGFR inhibitor gefitinib in combination with taxotere, significantly better response rates were seen in ER positive than in ER negative tumors (Ciardiello et al., Br. J. Cancer, 94, 1604-1609 (2006)).


In addition to the observation that the two EGFR ligands are predictive of response to cetuximab, it was found that patients without K-RAS mutations have a higher disease control rate (48%) than those with K-RAS mutations (10%). This result confirms findings from a recently reported study that shows that patients without K-RAS mutations have a higher disease control rate (76%) than those with K-RAS mutations (31%) (Lievre et al., Cancer Res., 66, 3992-3995 (2006)). Interestingly, a majority of the patients described in the previous study were treated with a combination of cetuximab and chemotherapy, suggesting that the K-RAS mutations are predictive of disease progression in both the monotherapy and combination therapy settings. K-RAS plays a crucial role in the RAS/MAPK pathway, which is located downstream of EGFR and other growth factor receptors, and is involved in cell proliferation. The presence of activating mutations in K-RAS might be expected to circumvent the inhibitory activity of cetuximab. K-RAS mutations have also been found to be associated with resistance to gefitinib and erlotinib in NSCLC (Pao et al., PLoS Med., 2, e17 (2005)). These data consistently support the role of K-RAS mutations in predicting response to cetuximab and/or other EGFR inhibitors, and should continue to be evaluated in cancers where RAS mutations are prevalent such as CRC, NSCLC and pancreatic cancer (Minamoto et al., Cancer Detect. Prev., 24, 1-12 (2000)).


In contrast to what has been observed in patients with NSCLC (Janne et al., J. Clin. Oncol., 23, 3227-3234 (2005)), mutations in the EGFR gene (exons 18-21) in the patients enrolled in this CRC study were not detected, confirming the paucity of mutations in patients with CRC (Tsuchihashi et al., N. Engl. J. Med., 353, 208-209 (2005)). Mutations in BRAF (exon 15) were not detected, though such mutations have been observed at a low frequency (<5%) in other studies (Moroni et al., Lancet Oncol., 6, 279-286 (2005)). An increase in EGFR gene copy number was observed in less than 10% of the patients evaluated in this study and while there was a trend towards higher copy number in the patients with disease control, the result was more in line with that of Lievre et al (10% of patients had amplification) than with Moroni et al (31% of patients had amplification). Assessment of the performance of a model using the combination of K-RAS mutation status and epiregulin mRNA expression levels showed excellent prediction power (AUC value of 0.89).


Example 3
Production of Antibodies Against the Biomarkers

Antibodies against the biomarkers can be prepared by a variety of methods. For example, cells expressing a biomarker polypeptide can be administered to an animal to induce the production of sera containing polyclonal antibodies directed to the expressed polypeptides. In one aspect, the biomarker protein is prepared and isolated or otherwise purified to render it substantially free of natural contaminants, using techniques commonly practiced in the art. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity for the expressed and isolated polypeptide.


In one aspect, the antibodies of the invention are monoclonal antibodies (or protein binding fragments thereof). Cells expressing the biomarker polypeptide can be cultured in any suitable tissue culture medium, however, it is preferable to culture cells in Earle's modified Eagle's medium supplemented to contain 10% fetal bovine serum (inactivated at about 56° C.), and supplemented to contain about 10 g/l nonessential amino acids, about 1.00 U/ml penicillin, and about 100 μg/ml streptomycin.


The splenocytes of immunized (and boosted) mice can be extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line can be employed in accordance with the invention, however, it is preferable to employ the parent myeloma cell line (SP2/0), available from the ATCC (Manassas, Va.). After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (1981, Gastroenterology, 80:225-232). The hybridoma cells obtained through such a selection are then assayed to identify those cell clones that secrete antibodies capable of binding to the polypeptide immunogen, or a portion thereof.


Alternatively, additional antibodies capable of binding to the biomarker polypeptide can be produced in a two-step procedure using anti-idiotypic antibodies. Such a method makes use of the fact that antibodies are themselves antigens and, therefore, it is possible to obtain an antibody that binds to a second antibody. In accordance with this method, protein specific antibodies can be used to immunize an animal, preferably a mouse. The splenocytes of such an immunized animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones that produce an antibody whose ability to bind to the protein-specific antibody can be blocked by the polypeptide. Such antibodies comprise anti-idiotypic antibodies to the protein-specific antibody and can be used to immunize an animal to induce the formation of further protein-specific antibodies.


Example 4
Immunofluorescence Assays

The following immunofluorescence protocol may be used, for example, to verify EGFR biomarker protein expression on cells or, for example, to check for the presence of one or more antibodies that bind EGFR biomarkers expressed on the surface of cells. Briefly, Lab-Tek II chamber slides are coated overnight at 4° C. with 10 micrograms/milliliter (μg/ml) of bovine collagen Type II in DPBS containing calcium and magnesium (DPBS++). The slides are then washed twice with cold DPBS++ and seeded with 8000 CHO-CCR5 or CHO pC4 transfected cells in a total volume of 125 μl and incubated at 37° C. in the presence of 95% oxygen/5% carbon dioxide.


The culture medium is gently removed by aspiration and the adherent cells are washed twice with DPBS-++ at ambient temperature. The slides are blocked with DPBS-++ containing 0.2% BSA (blocker) at 0-4° C. for one hour. The blocking solution is gently removed by aspiration, and 125 μl of antibody containing solution (an antibody containing solution may be, for example, a hybridoma culture supernatant which is usually used undiluted, or serum/plasma which is usually diluted, e.g., a dilution of about 1/100 dilution). The slides are incubated for 1 hour at 0-4° C. Antibody solutions are then gently removed by aspiration and the cells are washed five times with 400 μl of ice cold blocking solution. Next, 125 μl of 1 μg/ml rhodamine labeled secondary antibody (e.g., anti-human IgG) in blocker solution is added to the cells. Again, cells are incubated for 1 hour at 0-4° C.


The secondary antibody solution is then gently removed by aspiration and the cells are washed three times with 400 μl of ice cold blocking solution, and five times with cold DPBS++. The cells are then fixed with 125 μl of 3.7% formaldehyde in DPBS++ for 15 minutes at ambient temperature. Thereafter, the cells are washed five times with 400 μl of DPBS++ at ambient temperature. Finally, the cells are mounted in 50% aqueous glycerol and viewed in a fluorescence microscope using rhodamine filters.

Claims
  • 1. A method for predicting the likelihood a mammal will respond therapeutically to a method of treating cancer comprising administering an EGFR modulator, wherein the method comprises: (a) measuring in the mammal the level of at least one biomarker selected from epiregulin and amphiregulin;(b) exposing a biological sample from said mammal to the EGFR modulator;(c) following the exposing of step (b), measuring in said biological sample the level of the at least one biomarker,wherein an increase in the level of the at least one biomarker measured in step (c) compared to the level of the at least one biomarker measured in step (a) indicates an increased likelihood that the mammal will respond therapeutically to said method of treating cancer.
  • 2. The method of claim 1 wherein said at least one biomarker comprises epiregulin and amphiregulin.
  • 3. The method of claim 1 wherein said at least one biomarker further comprises at least one additional biomarker selected from Table 1.
  • 4. The method of claim 1 wherein said biological sample is a tissue sample comprising cancer cells and said tissue is fixed, paraffin-embedded, fresh, or frozen.
  • 5. The method of claim 4 that further comprises the step of determining whether said cancer cells have the presence of a mutated K-RAS, wherein detection of a mutated K-RAS indicates a decreased likelihood that that the mammal will respond therapeutically to said method of treating cancer.
  • 6. The method of claim 4 wherein said EGFR modulator is cetuximab and said cancer is colorectal cancer.
  • 7. A method for predicting the likelihood a mammal will respond therapeutically to a method of treating cancer comprising administering an EGFR modulator, wherein the method comprises: (a) measuring in the mammal the level of at least one biomarker that comprises CD73;(b) exposing a biological sample from said mammal to the EGFR modulator;(c) following the exposing of step (b), measuring in said biological sample the level of the at least one biomarker,wherein an increase in the level of the at least one biomarker measured in step (c) compared to the level of the at least one biomarker measured in step (a) indicates a decreased likelihood that the mammal will respond therapeutically to said method of treating cancer.
  • 8. The method of claim 7 wherein said at least one biomarker further comprises at least one additional biomarker selected from Table 1.
  • 9. The method of claim 8 wherein said biological sample is a tissue sample comprising cancer cells and said tissue is fixed, paraffin-embedded, fresh, or frozen.
  • 10. The method of claim 9 that further comprises the step of determining whether said cancer cells have the presence of a mutated K-RAS, wherein detection of a mutated K-RAS indicates a decreased likelihood that that the mammal will respond therapeutically to said method of treating cancer.
PCT Information
Filing Document Filing Date Country Kind 371c Date
PCT/US2006/033073 8/24/2006 WO 00 2/20/2008
Provisional Applications (1)
Number Date Country
60711054 Aug 2005 US