# Patent

## Title
Gene expression profiling of EGFR positive cancer

## Abstract

The present invention concerns prognostic markers associated with EGFR positive cancer. In particular, the invention concerns prognostic methods based on the molecular characterization of gene expression in paraffin-embedded, fixed tissue samples of EGFR-expressing cancer, which allow a physician to predict whether a patient is likely to respond well to treatment with an EGFR inhibitor.

## Background
This application is a divisional application of U.S. application Ser. No. 10/714,195, filed Nov. 14, 2003, which claims priority under 35 U.S.C. §119(e) to provisional application No. 60/427,090 filed on Nov. 15, 2002, the entire disclosures of which are hereby expressly incorporated by reference.


BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention concerns gene expression profiling of tissue samples obtained from EGFR-positive cancer. More specifically, the invention provides diagnostic, prognostic and predictive methods based on the molecular characterization of gene expression in paraffin-embedded, fixed tissue samples of EGFR-expressing cancer, which allow a physician to predict whether a patient is likely to respond well to treatment with an EGFR inhibitor. In addition, the present invention provides treatment methods based on such findings.
2. Description of the Related Art
Oncologists have a number of treatment options available to them, including different combinations of chemotherapeutic drugs that are characterized as “standard of care,” and a number of drugs that do not carry a label claim for particular cancer, but for which there is evidence of efficacy in that cancer. Best likelihood of good treatment outcome requires that patients be assigned to optimal available cancer treatment, and that this assignment be made as quickly as possible following diagnosis.
Currently, diagnostic tests used in clinical practice are single analyte, and therefore do not capture the potential value of knowing relationships between dozens of different markers. Moreover, diagnostic tests are frequently not quantitative, relying on immunohistochemistry. This method often yields different results in different laboratories, in part because the reagents are not standardized, and in part because the interpretations are subjective and cannot be easily quantified. RNA-based tests have not often been used because of the problem of RNA degradation over time and the fact that it is difficult to obtain fresh tissue samples from patients for analysis. Fixed paraffin-embedded tissue is more readily available and methods have been established to detect RNA in fixed tissue. However, these methods typically do not allow for the study of large numbers of genes (DNA or RNA) from small amounts of material. Thus, traditionally fixed tissue has been rarely used other than for immunohistochemistry detection of proteins.
Recently, several groups have published studies concerning the classification of various cancer types by microarray gene expression analysis (see, e.g. Golub et al., Science 286:531-537 (1999); Bhattacharjae et al., Proc. Natl. Acad. Sci. USA 98:13790-13795 (2001); Chen-Hsiang et al., Bioinformatics 17 (Suppl. 1):S316-S322 (2001); Ramaswamy et al., Proc. Natl. Acad. Sci. USA 98:15149-15154 (2001)). Certain classifications of human breast cancers based on gene expression patterns have also been reported (Martin et al., Cancer Res. 60:2232-2238 (2000); West et al., Proc. Natl. Acad. Sci. USA 98:11462-11467 (2001); Sorlie et al., Proc. Natl. Acad. Sci. USA 98:10869-10874 (2001); Yan et al., Cancer Res. 61:8375-8380 (2001)). However, these studies mostly focus on improving and refining the already established classification of various types of cancer, including breast cancer, and generally do not link the findings to treatment strategies in order to improve the clinical outcome of cancer therapy.
Although modern molecular biology and biochemistry have revealed more than 100 genes whose activities influence the behavior of tumor cells, state of their differentiation, and their sensitivity or resistance to certain therapeutic drugs, with a few exceptions, the status of these genes has not been exploited for the purpose of routinely making clinical decisions about drug treatments. One notable exception is the use of estrogen receptor (ER) protein expression in breast carcinomas to select patients to treatment with anti-estrogen drugs, such as tamoxifen. Another exceptional example is the use of ErbB2 (Her2) protein expression in breast carcinomas to select patients with the Her2 antagonist drug Herceptin® (Genentech, Inc., South San Francisco, Calif.).
Despite recent advances, the challenge of cancer treatment remains to target specific treatment regimens to pathogenically distinct tumor types, and ultimately personalize tumor treatment in order to optimize outcome. Hence, a need exists for tests that simultaneously provide predictive information about patient responses to the variety of treatment options.
SUMMARY OF THE INVENTION
The present invention is based on findings of Phase II clinical studies of gene expression in tissue samples obtained from EGFR-expressing head and neck cancer or colon cancer of human patients who responded well or did not respond to (showed resistance to) treatment with EGFR inhibitors.
Based upon such findings, in one aspect the present invention concerns a method for predicting the likelihood that a patient diagnosed with an EGFR-expressing cancer will respond to treatment with an EGFR inhibitor, comprising determining the expression level of one or more prognostic RNA transcripts or their products in a sample comprising EGFR-expressing cancer cells obtained from the patient, wherein the prognostic transcript is the transcript of one or more genes selected from the group consisting of: Bak; Bclx; BRAF; BRK; Cad17; CCND3; CD105; CD44s; CD82; CD9; CGA; CTSL; EGFRd27; ErbB3; EREG; GPC3; GUS; HGF; ID1; IGFBP3; ITGB3; ITGB3; p27; P53; PTPD1; RB1; RPLPO; STK15; SURV; TERC; TGFBR2; TIMP2; TITF1; XIAP; YB-1; A-Catenin; AKT1; AKT2; APC; Bax; B-Catenin; BTC; CA9; CCNA2; CCNE1; CCNE2; CD134; CD44E; CD44v3; CD44v6; CD68; CDC25B; CEACAM6; Chk2; cMet; COX2; cripto; DCR3; DIABLO; DPYD; DR5; EDN1 endothelin; EGFR; EIF4E; ERBB4; ERK1; fas; FRP1; GRO1; HB-EGF; HER2; IGF1R; IRS1; ITGA3; KRT17; LAMC2; MTA1; NMYC; P14ARF; PAI1; PDGFA; PDGFB; PGK1; PLAUR; PPARG; RANBP2; RASSF1; RIZ1; SPRY2; Src; TFRC; TP53BP1; UPA; and VEGFC, wherein (a) the patient is unlikely to benefit from treatment with an EGFR inhibitor if the normalized levels of any of the following genes A-Catenin; AKT1; AKT2; APC; Bax; B-Catenin; BTC; CA9; CCNA2; CCNE1; CCNE2; CD134; CD44E; CD44v3; CD44v6; CD68; CDC25B; CEACAM6; Chk2; cMet; COX2; cripto; DCR3; DIABLO; DPYD; DR5; EDN1 endothelin; EGFR; EIF4E; ERBB4; ERK1; fas; FRP1; GRO1; HB-EGF; HER2; IGF1R; IRS1; ITGA3; KRT17; LAMC2; MTA1; NMYC; P14ARF; PAI1; PDGFA; PDGFB; PGK1; PLAUR; PPARG; RANBP2; RASSF1; RIZ1; SPRY2; Src; TFRC; TP53BP1; upa; VEGFC, or their products are elevated above defined expression thresholds, and (b) the patient is likely to benefit from treatment with an EGFR inhibitor if the normalized levels of any of the following genes Bak; Bclx; BRAF; BRK; Cad17; CCND3; CD105; CD44s; CD82; CD9; CGA; CTSL; EGFRd27; ErbB3; EREG; GPC3; GUS; HGF; ID1; IGFBP3; ITGB3; ITGB3; p27; P53; PTPD1; RB1; RPLPO; STK15; SURV; TERC; TGFBR2; TIMP2; TITF1; XIAP; and YB-1, or their products are elevated above defined expression thresholds.
In another aspect, the present invention concerns a prognostic method comprising

    
    
        (a) subjecting a sample comprising EGFR-expressing cancer cells obtained from a patient to quantitative analysis of the expression level of at least one gene selected from the group consisting of CD44v3; CD44v6; DR5; GRO1; KRT17; and LAMC2 gene or their products, and
        (b) identifying the patient as likely to show resistance to treatment with an EGFR-inhibitor if the expression levels of such gene or genes, or their products, are elevated above a defined threshold. In a particular embodiment, the gene is LAMC2.
    
    


In yet another aspect, the invention concerns a method for predicting the likelihood that a patient diagnosed with an EGFR-expressing head or neck cancer will respond to treatment with an EGFR inhibitor, comprising determining the expression level of one or more prognostic RNA transcripts or their products in a sample comprising EGFR-expressing cancer cells obtained from such patient, wherein the prognostic transcript is the transcript of one or more genes selected from the group consisting of: CD44s; CD82; CGA; CTSL; EGFRd27; IGFBP3; p27; P53; RB1; TIMP2; YB-1; A-Catenin; AKT1; AKT2; APC; Bax; B-Catenin; BTC; CCNA2; CCNE1; CCNE2; CD105; CD44v3; CD44v6; CD68; CEACAM6; Chk2; cMet; COX2; cripto; DCR3; DIABLO; DPYD; DR5; EDN1 endothelin; EGFR; EIF4E; ERBB4; ERK1; fas; FRP1; GRO1; HB-EGF; HER2; IGF1R; IRS1; ITGA3; KRT17; LAMC2; MTA1; NMYC; PAI1; PDGFA; PGK1; PTPD1; RANBP2; SPRY2; TP53BP1; and VEGFC, wherein (a) normalized expression of one or more of A-Catenin; AKT1; AKT2; APC; Bax; B-Catenin; BTC; CCNA2; CCNE1; CCNE2; CD105; CD44v3; CD44v6; CD68; CEACAM6; Chk2; cMet; COX2; cripto; DCR3; DIABLO; DPYD; DR5; EDN1 endothelin; EGFR; EIF4E; ERBB4; ERK1; fas; FRP1; GRO1; HB-EGF; HER2; IGF1R; IRS1; ITGA3; KRT17; LAMC2; MTA1; NMYC; PAI1; PDGFA; PGK1; PTPD1; RANBP2; SPRY2; TP53BP1; VEGFC, or the corresponding gene product, above determined expression thresholds indicates that the patient is likely to show resistance to treatment with an EGFR inhibitor, and (b) normalized expression of one or more of CD44s; CD82; CGA; CTSL; EGFRd27; IGFBP3; p27; P53; RB1; TIMP2; YB-1, or the corresponding gene product, above defined expression thresholds indicates that the patient is likely to respond well to treatment with an EGFR inhibitor.
In a further aspect, the invention concerns a method for predicting the likelihood that a patient diagnosed with an EGFR-expressing colon cancer will respond to treatment with an EGFR inhibitor, comprising determining the expression level of one or more prognostic RNA transcripts or their products in a sample comprising EGFR-expressing cancer cells obtained from the patient, wherein the prognostic transcript is the transcript of one or more genes selected from the group consisting of Bak; Bclx; BRAF; BRK; Cad17; CCND3; CCNE1; CCNE2; CD105; CD9; COX2; DIABLO; ErbB3; EREG; FRP1; GPC3; GUS; HER2; HGF; ID1; ITGB3; PTPD1; RPLPO; STK15; SURV; TERC; TGFBR2; TITF1; XIAP; CA9; CD134; CD44E; CD44v3; CD44v6; CDC25B; CGA; DR5; GRO1; KRT17; LAMC2; P14ARF; PDGFB; PLAUR; PPARG; RASSF1; RIZ1; Src; TFRC; and UPA, wherein (a) elevated expression of one or more of CA9; CD134; CD44E; CD44v3; CD44v6; CDC25B; CGA; DR5; GRO1; KRT17; LAMC2; P14ARF; PDGFB; PLAUR; PPARG; RASSF1; RIZ1; Src; TFRC; and UPA, or the corresponding gene product, above defined expression thresholds indicates that the patient is likely to show resistance to treatment with an EGFR inhibitor, and normalized expression of one or more of Bak; Bclx; BRAF; BRK; Cad17; CCND3; CCNE1; CCNE2; CD105; CD9; COX2; DIABLO; ErbB3; EREG; FRP1; GPC3; GUS; HER2; HGF; ID1; ITGB3; PTPD1; RPLPO; STK15; SURV; TERC; TGFBR2; TITF1; XIAP, or the corresponding gene product, above certain expression thresholds indicates that the patient is likely to respond well to treatment with an EGFR inhibitor.
In another aspect, the invention concerns a method comprising treating a patient diagnosed with an EGFR-expressing cancer and determined to have elevated normalized levels of one or more of the RNA transcripts of Bak; Bclx; BRAF; BRK; Cad17; CCND3; CD105; CD44s; CD82; CD9; CGA; CTSL; EGFRd27; ErbB3; EREG; GPC3; GUS; HGF; ID1; IGFBP3; ITGB3; ITGB3; p27; P53; PTPD1; RB1; RPLPO; STK15; SURV; TERC; TGFBR2; TIMP2; TITF1; XIAP; YB-1; A-Catenin; AKT1; AKT2; APC; Bax; B-Catenin; BTC; CA9; CCNA2; CCNE1; CCNE2; CD134; CD44E; CD44v3; CD44v6; CD68; CDC25B; CEACAM6; Chk2; cMet; COX2; cripto; DCR3; DIABLO; DPYD; DR5; EDN1 endothelin; EGFR; EIF4E; ERBB4; ERK1; fas; FRP1; GRO1; HB-EGF; HER2; IGF1R; IRS1; ITGA3; KRT17; LAMC2; MTA1; NMYC; P14ARF; PAI1; PDGFA; PDGFB; PGK1; PLAUR; PPARG; RANBP2; RASSF1; RIZ1; SPRY2; Src; TFRC; TP53BP1; UPA; and VEGFC genes, or the corresponding gene products in the cancer, with an effective amount of an EGFR-inhibitor, wherein elevated RNA transcript level is defined by a defined expression threshold.
In yet another aspect, the invention concerns a method comprising treating a patient diagnosed with an EGFR-expressing head or neck cancer and determined to have elevated normalized expression of one or more of the RNA transcripts of CD44s; CD82; CGA; CTSL; EGFRd27; IGFBP3; p27; P53; RB1; TIMP2; YB-1; A-Catenin; AKT1; AKT2; APC; Bax; B-Catenin; BTC; CCNA2; CCNE1; CCNE2; CD105; CD44v3; CD44v6; CD68; CEACAM6; Chk2; cMet; COX2; cripto; DCR3; DIABLO; DPYD; DR5; EDN1 endothelin; EGFR; EIF4E; ERBB4; ERK1; fas; FRP1; GRO1; HB-EGF; HER2; IGF1R; IRS1; ITGA3; KRT17; LAMC2; MTA1; NMYC; PAI1; PDGFA; PGK1; PTPD1; RANBP2; SPRY2; TP53BP1; VEGFC genes, or the corresponding gene products in said cancer, with an effective amount of an EGFR-inhibitor, wherein elevated normalized RNA transcript level is defined by a defined expression threshold.
In a further aspect, the invention concerns a method comprising treating a patient diagnosed with an EGFR-expressing colon cancer and determined to have elevated normalized expression of one or more of the RNA transcripts of Bak; Bclx; BRAF; BRK; Cad17; CCND3; CCNE1; CCNE2; CD105; CD9; COX2; DIABLO; ErbB3; EREG; FRP1; GPC3; GUS; HER2; HGF; ID1; ITGB3; PTPD1; RPLPO; STK15; SURV; TERC; TGFBR2; TITF1; XIAP; CA9; CD134; CD44E; CD44v3; CD44v6; CDC25B; CGA; DR5; GRO1; KRT17; LAMC2; P14ARF; PDGFB; PLAUR; PPARG; RASSF1; RIZ1; Src; TFRC; UPA genes, or the corresponding gene products in such cancer, with an effective amount of an EGFR-inhibitor, wherein elevated normalized RNA transcript level is defined by a defined expression threshold.
The invention further concerns an array comprising (a) polynucleotides hybridizing to the following genes: Bak; Bclx; BRAF; BRK; Cad17; CCND3; CD105; CD44s; CD82; CD9; CGA; CTSL; EGFRd27; ErbB3; EREG; GPC3; GUS; HGF; ID1; IGFBP3; ITGB3; ITGB3; p27; P53; PTPD1; RB1; RPLPO; STK15; SURV; TERC; TGFBR2; TIMP2; TITF1; XIAP; YB-1; A-Catenin; AKT1; AKT2; APC; Bax; B-Catenin; BTC; CA9; CCNA2; CCNE1; CCNE2; CD134; CD44E; CD44v3; CD44v6; CD68; CDC25B; CEACAM6; Chk2; cMet; COX2; cripto; DCR3; DIABLO; DPYD; DR5; EDN1 endothelin; EGFR; EIF4E; ERBB4; ERK1; fas; FRP1; GRO1; HB-EGF; HER2; IGF1R; IRS1; ITGA3; KRT17; LAMC2; MTA1; NMYC; P14ARF; PAI1; PDGFA; PDGFB; PGK1; PLAUR; PPARG; RANBP2; RASSF1; RIZ1; SPRY2; Src; TFRC; TP53BP1; UPA; VEGFC; or (b) an array comprising polynucleotides hybridizing to the following genes: CD44v3; CD44v6; DR5; GRO1; KRT17; and LAMC2, immobilized on a solid surface; or (c) an array comprising polynucleotides hybridizing to the following genes: CD44s; CD82; CGA; CTSL; EGFRd27; IGFBP3; p27; P53; RB1; TIMP2; YB-1; A-Catenin; AKT1; AKT2; APC; Bax; B-Catenin; BTC; CCNA2; CCNE1; CCNE2; CD105; CD44v3; CD44v6; CD68; CEACAM6; Chk2; cMet; COX2; cripto; DCR3; DIABLO; DPYD; DR5; EDN1 endothelin; EGFR; EIF4E; ERBB4; ERK1; fas; FRP1; GRO1; HB-EGF; HER2; IGF1R; IRS1; ITGA3; KRT17; LAMC2; MTA1; NMYC; PAI1; PDGFA; PGK1; PTPD1; RANBP2; SPRY2; TP53BP1; and VEGFC, immobilized on a solid surface, or (d) an array comprising polynucleotides hybridizing to the following genes: Bak; Bclx; BRAF; BRK; Cad17; CCND3; CCNE1; CCNE2; CD105; CD9; COX2; DIABLO; ErbB3; EREG; FRP1; GPC3; GUS; HER2; HGF; ID1; ITGB3; PTPD1; RPLPO; STK15; SURV; TERC; TGFBR2; TITF1; XIAP; CA9; CD134; CD44E; CD44v3; CD44v6; CDC25B; CGA; DR5; GRO1; KRT17; LAMC2; P14ARF; PDGFB; PLAUR; PPARG; RASSF1; RIZ1; Src; TFRC; and UPA, immobilized on a solid surface.
In a further aspect, the invention concerns a method in which RNA is isolated from a fixed, paraffin-embedded tissue specimen by a procedure comprising:

    
    
        (a) incubating a section of the fixed, paraffin-embedded tissue specimen at a temperature of about 56° C. to 70° C. in a lysis buffer, in the presence of a protease, without prior dewaxing, to form a lysis solution;
        (b) cooling the lysis solution to a temperature where the wax solidifies; and
        (c) isolating the nucleic acid from the lysis solution.
    
    


In a different aspect, the invention concerns a kit comprising one or more of (1) extraction buffer/reagents and protocol; (2) reverse transcription buffer/reagents and protocol; and (3) qPCR buffer/reagents and protocol suitable for performing the gene expression analysis methods of the invention.
In a further aspect, the invention concerns a method for measuring levels of mRNA products of genes listed in Tables 5A and 5B by quantitative RT-PCR (qRT-PCR) reaction, by using an amplicon listed in Tables 5A and 5B and a corresponding primer-probe set listed in Tables 6A-6F.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a chart illustrating the overall workflow of the process of the invention for measurement of gene expression. In the Figure, FPET stands for “fixed paraffin-embedded tissue,” and “RT-PCR” stands for “reverse transcriptase-PCR.” RNA concentration is determined by using the commercial RiboGreenTM RNA Quantitation Reagent and Protocol.
FIG. 2 is a flow chart showing the steps of an RNA extraction method according to the invention alongside a flow chart of a representative commercial method.



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
A. Definitions
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992), provide one skilled in the art with a general guide to many of the terms used in the present application.
One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention; the following terms are defined below.
The term “microarray” refers to an ordered arrangement of hybridizable array elements, preferably polynucleotide probes, on a substrate.
The term “polynucleotide,” when used in singular or plural, generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Thus, for instance, polynucleotides as defined herein include, without limitation, single- and double-stranded DNA, DNA including single- and double-stranded regions, single- and double-stranded RNA, and RNA including single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or include single- and double-stranded regions. In addition, the term “polynucleotide” as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or front different molecules. The regions May include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. The term “polynucleotide” specifically includes cDNAs. The term includes DNAs (including cDNAs) and RNAs that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are “polynucleotides” as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritiated bases, are included within the term “polynucleotides” as defined herein. In general, the term “polynucleotide” embraces all chemically, enzymatically and/or metabolically modified forms of unmodified polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells.
The term “oligonucleotide” refers to a relatively short polynucleotide, including, without limitation, single-stranded deoxyribonucleotides, single- or double-stranded ribonucleotides, RNA:DNA hybrids and double-stranded DNAs. Oligonucleotides, such as single-stranded DNA probe oligonucleotides, are often synthesized by chemical methods, for example using automated oligonucleotide synthesizers that are commercially available. However, oligonucleotides can be made by a variety of other methods, including in vitro recombinant DNA-mediated techniques and by expression of DNAs in cells and organisms.
The terms “differentially expressed gene,” “differential gene expression” and their synonyms, which are used interchangeably, refer to a gene whose expression is activated to a higher or lower level in a subject suffering from a disease, specifically cancer, such as breast cancer, relative to its expression in a normal or control subject. The terms also include genes whose expression is activated to a higher or lower level at different stages of the same disease. It is also understood that a differentially expressed gene may be either activated or inhibited at the nucleic acid level or protein level, or may be subject to alternative splicing to result in a different polypeptide product. Such differences may be evidenced by a change in mRNA levels, surface expression, secretion or other partitioning of a polypeptide, for example. Differential gene expression may include a comparison of expression between two or more genes or their gene products, or a comparison of the ratios of the expression between two or more genes or their gene products, or even a comparison of two differently processed products of the same gene, which differ between normal subjects and subjects suffering from a disease, specifically cancer, or between various stages of the same disease. Differential expression includes both quantitative, as well as qualitative, differences in the temporal or cellular expression pattern in a gene or its expression products among, for example, normal and diseased cells, or among cells which have undergone different disease events or disease stages. For the purpose of this invention, “differential gene expression” is considered to be present when there is at least an about two-fold, preferably at least about four-fold, more preferably at least about six-fold, most preferably at least about ten-fold difference between the expression of a given gene in normal and diseased subjects, or in various stages of disease development in a diseased subject.
The term “normalized” with regard to a gene transcript or a gene expression product refers to the level of the transcript or gene expression product relative to the mean levels of transcripts/products of a set of reference genes, wherein the reference genes are either selected based on their minimal variation across, patients, tissues or treatments (“housekeeping genes”), or the reference genes are the totality of tested genes. In the latter case, which is commonly referred to as “global normalization”, it is important that the total number of tested genes be relatively large, preferably greater than 50. Specifically, the term ‘normalized’ with respect to an RNA transcript refers to the transcript level relative to the mean of transcript levels of a set of reference genes. More specifically, the mean level of an RNA transcript as measured by TaqMan® RT-PCR refers to the Ct value minus the mean Ct values of a set of reference gene transcripts.
The terms “expression threshold,” and “defined expression threshold” are used interchangeably and refer to the level of a gene or gene product in question above which the gene or gene product serves as a predictive marker for patient response or resistance to a drug, in the present case an EGFR inhibitor drug. The threshold is defined experimentally from clinical studies such as those described in examples 1 and 2, below. The expression threshold can be selected either for maximum sensitivity (for example, to detect all responders to a drug), or for maximum selectivity (for example to detect only responders to a drug), or for minimum error.
The phrase “gene amplification” refers to a process by which multiple copies of a gene or gene fragment are formed in a particular cell or cell line. The duplicated region (a stretch of amplified DNA) is often referred to as “amplicon.” Usually, the amount of the messenger RNA (mRNA) produced, i.e., the level of gene expression, also increases in the proportion of the number of copies made of the particular gene expressed.
The term “diagnosis” is used herein to refer to the identification of a molecular or pathological state, disease or condition, such as the identification of a molecular subtype of head and neck cancer, colon cancer, or other type of cancer. The term “prognosis” is used herein to refer to the prediction of the likelihood of cancer-attributable death or progression, including recurrence, metastatic spread, and drug resistance, of a neoplastic disease, such as breast cancer, or head and neck cancer. The term “prediction” is used herein to refer to the likelihood that a patient will respond either favorably or unfavorably to a drug or set of drugs, and also the extent of those responses, or that a patient will survive, following surgical removal or the primary tumor and/or chemotherapy for a certain period of time without cancer recurrence. The predictive methods of the present invention can be used clinically to make treatment decisions by choosing the most appropriate treatment modalities for any particular patient. The predictive methods of the present invention are valuable tools in predicting if a patient is likely to respond favorably to a treatment regimen, such as surgical intervention, chemotherapy with a given drug or drug combination, and/or radiation therapy, or whether long-term survival of the patient, following surgery and/or termination of chemotherapy or other treatment modalities is likely.
The term “long-term” survival is used herein to refer to survival for at least 5 years, more preferably for at least 8 years, most preferably for at least 10 years following surgery or other treatment.
The term “increased resistance” to a particular drug or treatment option, when used in accordance with the present invention, means decreased response to a standard dose of the drug or to a standard treatment protocol.
The term “decreased sensitivity” to a particular drug or treatment option, when used in accordance with the present invention, means decreased response to a standard dose of the drug or to a standard treatment protocol, where decreased response can be compensated for (at least partially) by increasing the dose of drug, or the intensity of treatment.
“Patient response” can be assessed using any endpoint indicating a benefit to the patient, including, without limitation, (1) inhibition, to some extent, of tumor growth, including slowing down and complete growth arrest; (2) reduction in the number of tumor cells; (3) reduction in tumor size; (4) inhibition (i.e., reduction, slowing down or complete stopping) of tumor cell infiltration into adjacent peripheral organs and/or tissues; (5) inhibition (i.e. reduction, slowing down or complete stopping) of metastasis; (6) enhancement of anti-tumor immune response, which may, but does not have to, result in the regression or rejection of the tumor; (7) relief, to some extent, of one or more symptoms associated with the tumor; (8) increase in the length of survival following treatment; and/or (9) decreased mortality at a given point of time following treatment.
The term “treatment” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. In tumor (e.g., cancer) treatment, a therapeutic agent may directly decrease the pathology of tumor cells, or render the tumor cells more susceptible to treatment by other therapeutic agents, e.g., radiation and/or chemotherapy.
The term “tumor,” as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include but are not limited to, breast cancer, colon cancer, lung cancer, prostate cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, head and neck cancer, and brain cancer.
The “pathology” of cancer includes all phenomena that compromise the well-being of the patient. This includes, without limitation, abnormal or uncontrollable cell growth, metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels, suppression or aggravation of inflammatory or immunological response, neoplasia, premalignancy, malignancy, invasion of surrounding or distant tissues or organs, such as lymph nodes, etc.
The term “EGFR inhibitor” as used herein refers to a molecule having the ability to inhibit a biological function of a native epidermal growth factor receptor (EGFR). Accordingly, the term “inhibitor” is defined in the context of the biological role of EGFR. While preferred inhibitors herein specifically interact with (e.g. bind to) an EGFR, molecules that inhibit an EGFR biological activity by interacting with other members of the EGFR signal transduction pathway are also specifically included within this definition. A preferred EGFR biological activity inhibited by an EGFR inhibitor is associated with the development, growth, or spread of a tumor.
The term “housekeeping gene” refers to a group of genes that codes for proteins whose activities are essential for the maintenance of cell function. These genes are typically similarly expressed in all cell types. Housekeeping genes include, without limitation, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Cyp1, albumin, actins, e.g. β-actin, tubulins, cyclophilin, hypoxantine phosphoribosyltransferase (HRPT), L32. 28S, and 18S.
B. Detailed Description
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, 2nd edition (Sambrook et al., 1989); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal Cell Culture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (Academic Press, Inc.); “Handbook of Experimental Immunology”, 4th edition (D. M. Weir & C. C. Blackwell, eds., Blackwell Science Inc., 1987); “Gene Transfer Vectors for Mammalian Cells” (J. M. Miller & M. P. Calos, eds., 1987); “Current Protocols in Molecular Biology” (F. M. Ausubel et al., eds., 1987); and “PCR: The Polymerase Chain Reaction”, (Mullis et al., eds., 1994).
1. Gene Expression Profiling
In general, methods of gene expression profiling can be divided into two large groups: methods based on hybridization analysis of polynucleotides, and methods based on sequencing of polynucleotides. The most commonly used methods known in the art for the quantification of mRNA expression in a sample include northern blotting and in situ hybridization (Parker & Barnes, Methods in Molecular Biology 106:247-283 (1999)); RNAse protection assays (Hod, Biotechniques 13:852-854 (1992)); and reverse transcription polymerase chain reaction (RT-PCR) (Weis et al., Trends in Genetics 8:263-264 (1992)). Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Representative methods for sequencing-based gene expression analysis include Serial Analysis of Gene Expression (SAGE), and gene expression analysis by massively parallel signature sequencing (MPSS).
2. Reverse Transcriptase PCR (RT-PCR)
Of the techniques listed above, the most sensitive and most flexible quantitative method is RT-PCR, which can be used to compare mRNA levels in different sample populations, in normal and tumor tissues, with or without drug treatment, to characterize patterns of gene expression, to discriminate between closely related mRNAs, and to analyze RNA structure.
The first step is the isolation of mRNA from a target sample. The starting material is typically total RNA isolated from human tumors or tumor cell lines, and corresponding normal tissues or cell lines, respectively. Thus RNA can be isolated from a variety of primary tumors, including breast, lung, colon, prostate, brain, liver, kidney, pancreas, spleen, thymus, testis, ovary, uterus, head and neck, etc., tumor, or tumor cell lines, with pooled DNA from healthy donors. If the source of mRNA is a primary tumor, mRNA can be extracted, for example, from frozen or archived paraffin-embedded and fixed (e.g. formalin-fixed) tissue samples.
General methods for mRNA extraction are well known in the art and are disclosed in standard textbooks of molecular biology, including Ausubel et al., Current Protocols of Molecular Biology, John Wiley and Sons (1997). Methods for RNA extraction from paraffin embedded tissues are disclosed, for example, in Rupp and Locker, Lab Invest. 56:A67 (1987), and De Andrés et al., BioTechniques 18:42044 (1995). In particular, RNA isolation can be performed using purification kit, buffer set and protease from commercial manufacturers, such as Qiagen, according to the manufacturer's instructions. For example, total RNA from cells in culture can be isolated using Qiagen RNeasy mini-columns. Other commercially available RNA isolation kits include MasterPureTM Complete DNA and RNA Purification Kit (EPICENTRE®, Madison, Wis.), and Paraffin Block RNA Isolation Kit (Ambion, Inc.). Total RNA from tissue samples can be isolated using RNA Stat-60 (Tel-Test). RNA prepared from tumor can be isolated, for example, by cesium chloride density gradient centrifugation.
As RNA cannot serve as a template for PCR, the first step in gene expression profiling by RT-PCR is the reverse transcription of the RNA template into cDNA, followed by its exponential amplification in a PCR reaction. The two most commonly used reverse transcriptases are avilo myeloblastosis virus reverse transcriptase (AMV-RT) and Moloney murine leukemia virus reverse transcriptase (MMLV-RT). The reverse transcription step is typically primed using specific primers, random hexamers, or oligo-dT primers, depending on the circumstances and the goal of expression profiling. For example, extracted RNA can be reverse-transcribed using a GeneAmp RNA PCR kit (Perkin Elmer, CA, USA), following the manufacturer's instructions. The derived cDNA can then be used as a template in the subsequent PCR reaction.
Although the PCR step can use a variety of thermostable DNA-dependent DNA polymerases, it typically employs the Taq DNA polymerase, which has a 5′-3′ nuclease activity but lacks a 3′-5′ proofreading endonuclease activity. Thus, TaqMan® PCR typically utilizes the 5′-nuclease activity of Taq or Tth polymerase to hydrolyze a hybridization probe bound to its target amplicon, but any enzyme with equivalent 5′ nuclease activity can be used. Two oligonucleotide primers are used to generate an amplicon typical of a PCR reaction. A third oligonucleotide, or probe, is designed to detect nucleotide sequence located between the two PCR primers. The probe is non-extendible by Taq DNA polymerase enzyme, and is labeled with a reporter fluorescent dye and a quencher fluorescent dye. Any laser-induced emission from the reporter dye is quenched by the quenching dye when the two dyes are located close together as they are on the probe. During the amplification reaction, the Taq DNA polymerase enzyme cleaves the probe in a template-dependent manner. The resultant probe fragments disassociate in solution, and signal from the released reporter dye is free from the quenching effect of the second fluorophore. One molecule of reporter dye is liberated for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data.
TaqMan® RT-PCR can be performed using commercially available equipment, such as, for example, ABI PRISM 7700TM Sequence Detection SystemTM (Perkin-Elmer-Applied Biosystems, Foster City, Calif., USA), or Lightcycler (Roche Molecular Biochemicals, Mannheim, Germany). In a preferred embodiment, the 5′ nuclease procedure is run on a real-time quantitative PCR device such as the ABI PRISM 7700TM Sequence Detection SystemTM. The system consists of a thermocycler, laser, charge-coupled device (CCD), camera and computer. The system amplifies samples in a 96-well format on a thermocycler. During amplification, laser-induced fluorescent signal is collected in real-time through fiber optics cables for all 96 wells, and detected at the CCD. The system includes software for running the instrument and for analyzing the data.
5′-Nuclease assay data are initially expressed as Ct, or the threshold cycle. As discussed above, fluorescence values are recorded during every cycle and represent the amount of product amplified to that point in the amplification reaction. The point when the fluorescent signal is first recorded as statistically significant is the threshold cycle (Ct).
To minimize errors and the effect of sample-to-sample variation, RT-PCR is usually performed using an internal standard. The ideal internal standard is expressed at a constant level among different tissues, and is unaffected by the experimental treatment. RNAs most frequently used to normalize patterns of gene expression are mRNAs for the housekeeping genes glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and β-actin.
A more recent variation of the RT-PCR technique is the real time quantitative PCR, which measures PCR product accumulation through a dual-labeled fluorigenic probe (i.e., TaqMan® probe). Real time PCR is compatible both with quantitative competitive PCR, where internal competitor for each target sequence is used for normalization, and with quantitative comparative PCR using a normalization gene contained within the sample, or a housekeeping gene for RT-PCR. For further details see, e.g. Held et al., Genome Research 6:986-994 (1996).
According to one aspect of the present invention, PCR primers and probes are designed based upon intron sequences present in the gene to be amplified. In this embodiment, the first step in the primer/probe design is the delineation of intron sequences within the genes. This can be done by publicly available software, such as the DNA BLAT software developed by Kent, W. J., Genome Res. 12(4):656-64 (2002), or by the BLAST software including its variations. Subsequent steps follow well established methods of PCR primer and probe design.
In order to avoid non-specific signals, it is important to mask repetitive sequences within the introns when designing the primers and probes. This can be easily accomplished by using the Repeat Masker program available on-line through the Baylor College of Medicine, which screens DNA sequences against a library of repetitive elements and returns a query sequence in which the repetitive elements are masked. The masked intron sequences can then be used to design primer and probe sequences using any commercially or otherwise publicly available primer/probe design packages, such as Primer Express (Applied Biosystems); MGB assay-by-design (Applied Biosystems); Primer3 (Steve Rozen and Helen J. Skaletsky (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology, Humana Press, Totowa, N.J., pp 365-386)
The most important factors considered in PCR primer design include primer length, melting temperature (Tm), and G/C content, specificity, complementary primer sequences, and 3′-end sequence. In general, optimal PCR primers are generally 17-30 bases in length, and contain about 20-80%, such as, for example, about 50-60% G+C bases. Tm's between 50 and 80° C., e.g. about 50 to 70° C. are typically preferred.
For further guidelines for PCR primer and probe design see, e.g. Dieffenbach, C. W. et al., “General Concepts for PCR Primer Design” in: PCR Primer, A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1995, pp. 133-155; Innis and Gelfand, “Optimization of PCRs” in: PCR Protocols, A Guide to Methods and Applications, CRC Press, London, 1994, pp. 5-11; and Plasterer, T. N. Primer select: Primer and probe design. Methods Mol. Biol. 70:520-527 (1997), the entire disclosures of which are hereby expressly incorporated by reference.
3. Microarrays
Differential gene expression can also be identified, or confirmed using the microarray technique. Thus, the expression profile of breast cancer-associated genes can be measured in either fresh or paraffin-embedded tumor tissue, using microarray technology. In this method, polynucleotide sequences of interest (including cDNAs and oligonucleotides) are plated, or arrayed, on a microchip substrate. The arrayed sequences are then hybridized with specific DNA probes from cells or tissues of interest. Just as in the RT-PCR method, the source of mRNA typically is total RNA isolated from human tumors or tumor cell lines, and corresponding normal tissues or cell lines. Thus RNA can be isolated from a variety of primary tumors or tumor cell lines. If the source of mRNA is a primary tumor, mRNA can be extracted, for example, from frozen or archived paraffin-embedded and fixed (e.g. formalin-fixed) tissue samples, which are routinely prepared and preserved in everyday clinical practice.
In a specific embodiment of the microarray technique, PCR amplified inserts of cDNA clones are applied to a substrate in a dense array. Preferably at least 10,000 nucleotide sequences are applied to the substrate. The microarrayed genes, immobilized on the microchip at 10,000 elements each, are suitable for hybridization under stringent conditions. Fluorescently labeled cDNA probes may be generated through incorporation of fluorescent nucleotides by reverse transcription of RNA extracted from tissues of interest. Labeled cDNA probes applied to the chip hybridize with specificity to each spot of DNA on the array. After stringent washing to remove non-specifically bound probes, the chip is scanned by confocal laser microscopy or by another detection method, such as a CCD camera. Quantitation of hybridization of each arrayed element allows for assessment of corresponding mRNA abundance. With dual color fluorescence, separately labeled cDNA probes generated from two sources of RNA are hybridized pairwise to the array. The relative abundance of the transcripts from the two sources corresponding to each specified gene is thus determined simultaneously. The miniaturized scale of the hybridization affords a convenient and rapid evaluation of the expression pattern for large numbers of genes. Such methods have been shown to have the sensitivity required to detect rare transcripts, which are expressed at a few copies per cell, and to reproducibly detect at least approximately two-fold differences in the expression levels (Schena et al., Proc. Natl. Acad. Sci. USA 93(2):106-149 (1996)). Microarray analysis can be performed by commercially available equipment, following manufacturer's protocols, such as by using the Affymetrix GenChip technology, or Incyte's microarray technology.
The development of microarray methods for large-scale analysis of gene expression makes it possible to search systematically for molecular markers of cancer classification and outcome prediction in a variety of tumor types.
4. Serial Analysis of Gene Expression (SAGE)
Serial analysis of gene expression (SAGE) is a method that allows the simultaneous and quantitative analysis of a large number of gene transcripts, without the need of providing an individual hybridization probe for each transcript. First, a short sequence tag (about 10-14 bp) is generated that contains sufficient information to uniquely identify a transcript, provided that the tag is obtained from a unique position within each transcript. Then, many transcripts are linked together to form long serial molecules, that can be sequenced, revealing the identity of the multiple tags simultaneously. The expression pattern of any population of transcripts can be quantitatively evaluated by determining the abundance of individual tags, and identifying the gene corresponding to each tag. For more details see, e.g. Velculescu et al., Science 270:484-487 (1995); and Velculescu et al., Cell 88:243-51 (1997).
5. MassARRAY Technology
The MassARRAY (Sequenom, San Diego, Calif.) technology is an automated, high-throughput method of gene expression analysis using mass spectrometry (MS) for detection. According to this method, following the isolation of RNA, reverse transcription and PCR amplification, the cDNAs are subjected to primer extension. The cDNA-derived primer extension products are purified, and dipensed on a chip array that is pre-loaded with the components needed for MALTI-TOF MS sample preparation. The various cDNAs present in the reaction are quantitated by analyzing the peak areas in the mass spectrum obtained.
6. Gene Expression Analysis by Massively Parallel Signature Sequencing (MPSS
This method, described by Brenner et al., Nature Biotechnology 18:630-634 (2000), is a sequencing approach that combines non-gel-based signature sequencing with in vitro cloning of millions of templates on separate 5 μm diameter microbeads. First, a microbead library of DNA templates is constructed by in vitro cloning. This is followed by the assembly of a planar array of the template-containing microbeads in a flow cell at a high density (typically greater than 3×106 microbeads/cm2). The free ends of the cloned templates on each microbead are analyzed simultaneously, using a fluorescence-based signature sequencing method that does not require DNA fragment separation. This method has been shown to simultaneously and accurately provide, in a single operation, hundreds of thousands of gene signature sequences from a yeast cDNA library.
7. Immunohistochemistry
Immunohistochemistry methods are also suitable for detecting the expression levels of the prognostic markers of the present invention. Thus, antibodies or antisera, preferably polyclonal antisera, and most preferably monoclonal antibodies specific for each marker are used to detect expression. The antibodies can be detected by direct labeling of the antibodies themselves, for example, with radioactive labels, fluorescent labels, hapten labels such as, biotin, or an enzyme such as horse radish peroxidase or alkaline phosphatase. Alternatively, unlabeled primary antibody is used in conjunction with a labeled secondary antibody, comprising antisera, polyclonal antisera or a monoclonal antibody specific for the primary antibody. Immunohistochemistry protocols and kits are well known in the art and are commercially available.
8. Proteomics
The term “proteome” is defined as the totality of the proteins present in a sample (e.g. tissue, organism, or cell culture) at a certain point of time. Proteomics includes, among other things, study of the global changes of protein expression in a sample (also referred to as “expression proteomics”). Proteomics typically includes the following steps: (1) separation of individual proteins in a sample by 2-D gel electrophoresis (2-D PAGE); (2) identification of the individual proteins recovered from the gel, e.g. my mass spectrometry or N-terminal sequencing, and (3) analysis of the data using bioinformatics. Proteomics methods are valuable supplements to other methods of gene expression profiling, and can be used, alone or in combination with other methods, to detect the products of the prognostic markers of the present invention.
9. Improved Method for Isolation of Nucleic Acid from Archived Tissue Specimens
In the first step of the method of the invention, total RNA is extracted from the source material of interest, including fixed, paraffin-embedded tissue specimens, and purified sufficiently to act as a substrate in an enzyme assay. While extration of total RNA can be performed by any method known in the art, in a particular embodiment, the invention relies on an improved method for the isolation of nucleic acid from archived, e.g. fixed, paraffin-embedded tissue specimens (FPET).
Measured levels of mRNA species are useful for defining the physiological or pathological status of cells and tissues. RT-PCR (which is discussed above) is one of the most sensitive, reproducible and quantitative methods for this “gene expression profiling”. Paraffin-embedded, formalin-fixed tissue is the most widely available material for such studies. Several laboratories have demonstrated that it is possible to successfully use fixed-paraffin-embedded tissue (FPET) as a source of RNA for RT-PCR (Stanta et al., Biotechniques 11:304-308 (1991); Stanta et al., Methods Mol. Biol. 86:23-26 (1998); Jackson et al., Lancet 1:1391 (1989); Jackson et al., J. Clin. Pathol. 43:499-504 (1999); Finke et al., Biotechniques 14:448-453 (1993); Goldsworthy et al., Mol. Carcinog. 25:86-91 (1999); Stanta and Bonin, Biotechniques 24:271-276 (1998); Godfrey et al., J. Mol. Diagnostics 2:84 (2000); Specht et al., J. Mol. Med. 78:B27 (2000); Specht et al., Am. J. Pathol. 158:419-429 (2001)). This allows gene expression profiling to be carried out on the most commonly available source of human biopsy specimens, and therefore potentially to create new valuable diagnostic and therapeutic information.
The most widely used protocols utilize hazardous organic solvents, such as xylene, or octane (Finke et al., supra) to dewax the tissue in the paraffin blocks before nucleic acid (RNA and/or DNA) extraction. Obligatory organic solvent removal (e.g. with ethanol) and rehydration steps follow, which necessitate multiple manipulations, and addition of substantial total time to the protocol, which can take up to several days. Commercial kits and protocols for RNA extraction from FPET [MasterPureTM Complete DNA and RNA Purification Kit (EPICENTRE®, Madison, Wis.); Paraffin Block RNA Isolation Kit (Ambion, Inc.) and RNeasyTM Mini kit (Qiagen, Chatsworth, Calif.)] use xylene for deparaffinization, in procedures which typically require multiple centrifugations and ethanol buffer changes, and incubations following incubation with xylene.
The method that can be used in the present invention provides an improved nucleic acid extraction protocol that produces nucleic acid, in particular RNA, sufficiently intact for gene expression measurements. The key step in this improved nucleic acid extraction protocol is the performance of dewaxing without the use of any organic solvent, thereby eliminating the need for multiple manipulations associated with the removal of the organic solvent, and substantially reducing the total time to the protocol. According to the improved method, wax, e.g. paraffin is removed from wax-embedded tissue samples by incubation at 65-75° C. in a lysis buffer that solubilizes the tissue and hydrolyzes the protein, following by cooling to solidify the wax.
FIG. 2 shows a flow chart of the improved RNA extraction protocol used herein in comparison with a representative commercial method, using xylene to remove wax. The times required for individual steps in the processes and for the overall processes are shown in the chart. As shown, the commercial process requires approximately 50% more time than the improved process used in performing the methods of the invention.
The lysis buffer can be any buffer known for cell lysis. It is, however, preferred that oligo-dT-based methods of selectively purifying polyadenylated mRNA not be used to isolate RNA for the present invention, since the bulk of the mRNA molecules are expected to be fragmented and therefore will not have an intact polyadenylated tail, and will not be recovered or available for subsequent analytical assays. Otherwise, any number of standard nucleic acid purification schemes can be used. These include chaotrope and organic solvent extractions, extraction using glass beads or filters, salting out and precipitation based methods, or any of the purification methods known in the art to recover total RNA or total nucleic acids from a biological source.
Lysis buffers are commercially available, such as, for example, from Qiagen, Epicentre, or Ambion. A preferred group of lysis buffers typically contains urea, and Proteinase K or other protease. Proteinase K is very useful in the isolation of high quality, undamaged DNA or RNA, since most mammalian DNases and RNases are rapidly inactivated by this enzyme, especially in the presence of 0.5-1% sodium dodecyl sulfate (SDS). This is particularly important in the case of RNA, which is more susceptible to degradation than DNA. While DNases require metal ions for activity, and can therefore be easily inactivated by chelating agents, such as EDTA, there is no similar co-factor requirement for RNases.
Cooling and resultant solidification of the wax permits easy separation of the wax from the total nucleic acid, which can be conveniently precipitated, e.g. by isopropanol. Further processing depends on the intended purpose. If the proposed method of RNA analysis is subject to bias by contaminating DNA in an extract, the RNA extract can be further treated, e.g. by DNase, post purification to specifically remove DNA while preserving RNA. For example, if the goal is to isolate high quality RNA for subsequent RT-PCR amplification, nucleic acid precipitation is followed by the removal of DNA, usually by DNase treatment. However, DNA can be removed at various stages of nucleic acid isolation, by DNase or other techniques well known in the art.
While the advantages of the improved nucleic acid extraction discussed above are most apparent for the isolation of RNA from archived, paraffin embedded tissue samples, the wax removal step of the present invention, which does not involve the use of an organic solvent, can also be included in any conventional protocol for the extraction of total nucleic acid (RNA and DNA) or DNA only.
By using heat followed by cooling to remove paraffin, the improved process saves valuable processing time, and eliminates a series of manipulations, thereby potentially increasing the yield of nucleic acid.
10. 5′-Multiplexed Gene Specific Priming of Reverse Transcription
RT-PCR requires reverse transcription of the test RNA population as a first step. The most commonly used primer for reverse transcription is oligo-dT, which works well when RNA is intact. However, this primer will not be effective when RNA is highly fragmented as is the case in FPE tissues.
The present invention includes the use of gene specific primers, which are roughly 20 bases in length with a Tm optimum between about 58° C. and 60° C. These primers will also serve as the reverse primers that drive PCR DNA amplification.
An alternative approach is based on the use of random hexamers as primers for cDNA synthesis. However, we have experimentally demonstrated that the method of using a multiplicity of gene-specific primers is superior over the known approach using random hexamers.
11. Normalization Strategy
An important aspect of the present invention is to use the measured expression of certain genes by EGFR-expressing cancer tissue to provide information about the patient's likely response to treatment with an EGFR-inhibitor. For this purpose it is necessary to correct for (normalize away) both differences in the amount of RNA assayed and variability in the quality of the RNA used. Therefore, the assay typically measures and incorporates the expression of certain normalizing genes, including well known housekeeping genes, such as GAPDH and Cyp1. Alternatively or in addition, normalization can be based on the mean or median signal (Ct in the case of RT-PCR) of all of the assayed genes or a large subset thereof (global normalization approach). On a gene-by-gene basis, measured normalized amount of a patient tumor mRNA is compared to the amount found in a reference set of cancer tissue of the same type (e.g. head and neck cancer, colon cancer, etc.). The number (N) of cancer tissues in this reference set should be sufficiently high to ensure that different reference sets (as a whole) behave essentially the same way. If this condition is met, the identity of the individual cancer tissues present in a particular set will have no significant impact on the relative amounts of the genes assayed. Usually, the cancer tissue reference set consists of at least about 30, preferably at least about 40 different FPE cancer tissue specimens. Unless noted otherwise, normalized expression levels for each mRNA/tested tumor/patient will be expressed as a percentage of the expression level measured in the reference set. More specifically, the reference set of a sufficiently high number (e.g. 40) of tumors yields a distribution of normalized levels of each mRNA species. The level measured in a particular tumor sample to be analyzed falls at some percentile within this range, which can be determined by methods well known in the art. Below, unless noted otherwise, reference to expression levels of a gene assume normalized expression relative to the reference set although this is not always explicitly stated.
12. EGFR Inhibitors
The epidermal growth factor receptor (EGFR) family (which includes EGFR, erb-B2, erb-B3, and erb-B4) is a family of growth factor receptors that are frequently activated in epithelial malignancies. Thus, the epidermal growth factor receptor (EGFR) is known to be active in several tumor types, including, for example, ovarian cancer, pancreatic cancer, non-small cell lung cancer, breast cancer, colon cancer and head and neck cancer. Several EGFR inhibitors, such as ZD1839 (also known as gefitinib or Iressa); and OS1774 (Erlotinib, TarcevaTM), are promising drug candidates for the treatment of EGFR-expressing cancer.
Iressa, a small synthetic quinazoline, competitively inhibits the ATP binding site of EGFR, a growth-promoting receptor tyrosine kinase, and has been in Phase III clinical trials for the treatment of non-small-cell lung carcinoma. Another EGFR inhibitor, [agr]cyano-[bgr]methyl-N-[(trifluoromethoxy)phenyl]-propenamide (LFM-A12), has been shown to inhibit the proliferation and invasiveness of EGFR positive human breast cancer cells.
Cetuximab is a monoclonal antibody that blocks the EGFR and EGFR-dependent cell growth. It is currently being tested in phase III clinical trials.
TarcevaTM has shown promising indications of anti-cancer activity in patients with advanced ovarian cancer, and non-small cell lung and head and neck carcinomas.
The present invention provides valuable tools to predict whether an EGFR-positive tumor is likely to respond to treatment with an EGFR-inhibitor.
Recent publications further confirm the involvement of EGFR in gastrointestinal (e.g. colon) cancer, and associate its expression with poor survival. See, e.g. Khorana et al., Proc. Am. Soc. Clin. Oncol 22:317 (2003).
While the listed examples of EGFR inhibitors a small organic molecules, the findings of the present invention are equally applicable to other EGFR inhibitors, including, without limitation, anti-EGFR antibodies, antisense molecules, small peptides, etc.
Further details of the invention will be apparent from the following non-limiting Examples.
EXAMPLE 1
A Phase II Study of Gene Expression in Head and Neck Tumors
A gene expression study was designed and conducted with the primary goal to molecularly characterize gene expression in paraffin-embedded, fixed tissue samples of head and neck cancer patients who responded or did not respond to treatment with an EGFR inhibitor. The results are based on the use of five different EGFR inhibitor drugs.
Study Design
Molecular assays were performed on paraffin-embedded, formalin-fixed head and neck tumor tissues obtained from 14 individual patients diagnosed with head and neck cancer. Patients were included in the study only if histopathologic assessment, performed as described in the Materials and Methods section, indicated adequate amounts of tumor tissue.
Materials and Methods
Each representative tumor block was characterized by standard histopathology for diagnosis, semi-quantitative assessment of amount of tumor, and tumor grade. A total of 6 sections (10 microns in thickness each) were prepared and placed in two Costar Brand Microcentrifuge Tubes (Polypropylene, 1.7 mL tubes, clear; 3 sections in each tube). If the tumor constituted less than 30% of the total specimen area, the sample may have been crudely dissected by the pathologist, using gross microdissection, putting the tumor tissue directly into the Costar tube.
If more than one tumor block was obtained as part of the surgical procedure, all tumor blocks were subjected to the same characterization, as described above, and the block most representative of the pathology was used for analysis.
Gene Expression Analysis
mRNA was extracted and purified from fixed, paraffin-embedded tissue samples, and prepared for gene expression analysis as described above.
Molecular assays of quantitative gene expression were performed by RT-PCR, using the ABI PRISM 7900TM Sequence Detection SystemTM (Perkin-Elmer-Applied Biosystems, Foster City, Calif., USA). ABI PRISM 7900TM consists of a thermocycler, laser, charge-coupled device (CCD), camera and computer. The system amplifies samples in a 384-well format on a thermocycler. During amplification, laser-induced fluorescent signal is collected in real-time through fiber optics cables for all 384 wells, and detected at the CCD. The system includes software for running the instrument and for analyzing the data.
Analysis and Results
Tumor tissue was analyzed for 185 cancer-related genes and 7 reference genes. The threshold cycle (CT) values for each patient were normalized based on the mean of all genes for that particular patient. Clinical outcome data were available for all patients.
Outcomes were classified as either response or no response. The results were analyzed in two different ways using two different criteria for response: partial response, or clinical benefit. The latter criterion combines partial or complete response with stable disease (minimum 3 months). In this study, there were no complete responses, four cases of partial response and two cases of disease stabilization.
We evaluated the relationship between gene expression and partial response by logistic regression and have identified the following genes as significant (p<0.15), as indicated in the attached Table 1. The logistic model provides a means of predicting the probability (Pr) of a subject as being either a partial responder or not. The following equation defined the expression threshold for response.




  
    
      
        
          Pr
          ⁡
          
            (
            Response
            )
          
        
        =
        
        ⁢
        
          1
          
            1
            +
            
              e
              
                Intercept
                +
                
                  Slope
                  ×
                  Reference
                  ⁢
                  
                    
                  
                  ⁢
                  Normalized
                  ⁢
                  
                    
                  
                  ⁢
                  CT
                
              
            
          
        
      
    
  
  
    
      
        
        ⁢
        
          and
          ⁢
          
            
          
          ⁢
          
            Pr
            ⁡
            
              (
              
                No
                ⁢
                
                  
                
                ⁢
                Response
              
              )
            
          
        
      
    
  
  
    
      
        =
        
        ⁢
        
          1
          -
          
            Pr
            ⁡
            
              (
              Response
              )
            
          
        
      
    
  




In Table 1, the term “negative” indicates that greater expression of the gene decreased likelihood of response to treatment with EGFR inhibitor, and “positive” indicates that increased expression of the gene increased likelihood of response to EGFR inhibitor. Results from analysis of head and neck cancer patient data using clinical benefit criteria are shown in Table 2.
Overall increased expression of the following genes correlated with resistance of head and neck cancer to EGFR inhibitor treatment: A-Catenin; AKT1; AKT2; APC; Bax; B-Catenin; BTC; CCNA2; CCNE1; CCNE2; CD105; CD44v3; CD44v6; CD68; CEACAM6; Chk2; cMet; COX2; cripto; DCR3; DIABLO; DPYD; DR5; EDN1 endothelin; EGFR; EIF4E; ERBB4; ERK1; fas; FRP1; GRO1; HB-EGF; HER2; IGF1R; IRS1; ITGA3; KRT17; LAMC2; MTA1; NMYC; PAI1; PDGFA; PGK1; PTPD1; RANBP2; SPRY2; TP53BP1; and VEGFC; and increased expression of the following genes correlated with response of head and neck cancer to EGFR inhibitor treatment: CD44s; CD82; CGA; CTSL; EGFRd27; IGFBP3; p27; P53; RB1; TIMP2; and YB-1.
EXAMPLE 2
A Phase II Study of Gene Expression in Colon Cancer
In a study analogous to the study of head and neck cancer patients described in Example 1, gene expression markers were sought that correlate with increased or decreased likelihood of colon cancer response to EGFR inhibitors. Sample preparation and handling and gene expression and data analysis were performed as in Example 1.
Twenty-three colon adenocarcinoma patients in all were studied, using a 192 gene assay. 188 of the 192 genes were expressed above the limit of detection. Both pathological and clinical responses were evaluated. Following treatment with EGFR inhibitor, three patients were determined to have had a partial response, five to have stable disease and fifteen to have progressive disease.
Table 3 shows the results obtained using the partial response criterion.
Results from analysis of colon cancer patient data using clinical benefit criteria are shown in Table 4.
Overall, increased expression of the following genes correlated with resistance of colon cancer to EGFR inhibitor treatment: CA9; CD134; CD44E; CD44v3; CD44v6; CDC25B; CGA; DR5; GRO1; KRT17; LAMC2; P14ARF; PDGFB; PLAUR; PPARG; RASSF1; RIZ1; Src; TFRC; and UPA, and increased expression of the following genes correlated with sensitivity of colon cancer to EGFR inhibitor treatment: CD44s; CD82; CGA; CTSL; EGFRd27; IGFBP3; p27; P53; RB1; TIMP2; and YB-1.
Finally, it is noteworthy that increased expression of the following genes correlated with resistance to EGFR inhibitor treatment in both head and neck and colon cancer: CD44v3; CD44v6; DR5; GRO1; KRT17; LAMC2.
In similar experiments; the elevated expression of LAMC2, B-Catenin, Bax, GRO1, Fas, or ITGA3 in EGFR-positive head and neck cancer was determined to be an indication that the patient is not likely to respond well to treatment with an EGFR inhibitor. On the other hand, elevated expression of YB-1, PTEN, CTSL, P53, STAT3, ITGB3, IGFBP3, RPLPO or p27 in EGFR-positive head and neck cancer was found to be an indication that the patient is likely to respond to EGFR inhibitor treatment.
In another set of similar experiments, elevated expression of the following genes in EGFR-expressing colon cancer correlated with positive response to treatment: BAK; BCL2; BRAF; BRK; CCND3; CD9; ER2; ERBB4; EREG; ERK1; FRP1. Elevated expression of the following genes in EGFR-expressing colon cancer correlated with resistance to treatment APN; CA9; CCND1; CDC25B; CD134; LAMC2; PDGFB; CD44v6; CYP1; DR5; GAPDH; IGFBP2; PLAUR; RASSF1; UPA.
All references cited throughout the specification are hereby expressly incorporated by reference.
Although the present invention is illustrated with reference to certain embodiments, it is not so limited. Modifications and variations are possible without diverting from the spirit of the invention. All such modifications and variations, which will be apparent to those skilled in the art, are specifically within the scope of the present invention. While the specific examples disclosed herein concern head and neck cancer and colon cancer, the methods of the present invention are generally applicable and can be extended to all EGFR-expressing cancers, and such general methods are specifically intended to be within the scope herein.







TABLE 1







Partial Response Genes for Head and Neck Study













Logistic Discriminat

Likelihood


Gene

Function

Ratio Test












Name
Response
Intercept
Slope
R2
P Value















cMet
Negative
26.5168713
4.57143179
0.6662
0.0011


LAMC2
Negative
5.29706425
1.28137295
0.6155
0.0017


ITGA3
Negative
22.6008544
3.17707499
0.5063
0.0044


CD44v6
Negative
6.92255059
4.3069909
0.492
0.005


B-Catenin
Negative
7.85913706
2.52965454
0.4805
0.0055


PDGFA
Negative
6.0016358
1.10386463
0.4318
0.0085


GRO1
Negative
8.37646635
1.74815793
0.4146
0.0099


ERK1
Negative
6.14712633
1.64819007
0.4024
0.0111


CD44v3
Negative
5.95094528
3.36594473
0.3451
0.0186


Bax
Negative
5.34006632
1.19383253
0.3361
0.0202


CGA
Positive
−78.121148
−10.503757
0.3266
0.0221


fas
Negative
7.27491015
1.38464586
0.3251
0.0224


IGFBP3
Positive
−2.1529531
−2.7937517
0.3097
0.0258


MTA1
Negative
6.07167277
1.23786874
0.3072
0.0264


YB-1
Positive
1.73598983
−4.0859174
0.2814
0.0336


DR5
Negative
9.0550349
1.46349944
0.2703
0.0373


APC
Negative
5.775003
1.88324269
0.2512
0.0447


ERBB4
Negative
11.9466285
1.58606697
0.2357
0.0518


CD68
Negative
3.60605487
1.0645631
0.2319
0.0537


cripto
Negative
19.5004373
2.64909385
0.2251
0.0574


P53
Positive
−4.1976158
−1.5541169
0.2208
0.0598


VEGFC
Negative
6.33634489
0.90613473
0.2208
0.0598


A-Catenin
Negative
4.41215235
1.7591194
0.2199
0.0603


COX2
Negative
8.00968996
1.27597736
0.202
0.0718


CD82
Positive
−1.8999985
−1.171157
0.1946
0.0772


PAI1
Negative
2.94777884
0.97480364
0.1944
0.0774


AKT2
Negative
2.45598587
1.64608189
0.1889
0.0817


HER2
Negative
4.25059223
0.97748483
0.1845
6.0853


DIABLO
Negative
17.035069
2.93939741
0.1809
0.0884


p27
Positive
−1.9798519
−1.9041142
0.1792
0.09


RANBP2
Negative
2.85994976
0.41878666
0.1757
0.0931


EIF4E
Negative
2.91202768
0.56099402
0.1722
0.0965


EDN1 endothelin
Negative
6.06858911
0.87185553
0.1688
0.0998


IGF1R
Negative
6.14387144
1.68865744
0.1674
0.1012


AKT1
Negative
5.02676228
1.50585593
0.1659
0.1028


CCNA2
Negative
3.95684559
0.63089954
0.184
0.1033


HB-EGF
Negative
5.1019713
0.70368632
0.1627
0.1061


TIMP2
Positive
2.58975885
−1.0832648
0.1625
0.1064


EGFRd27
Positive
−38.789016
−5.2513587
0.1607
0.1083


Chk2
Negative
6.8797175
1.21671205
0.1581
0.1112


IRS1
Negative
12.0545078
1.59632708
0.1578
0.1115


FRP1
Negative
3.38233862
0.49053452
0.1569
0.1126


CCNE2
Negative
5.78828731
1.11609099
0.1566
0.1129


SPRY2
Negative
4.68447069
0.86747803
0.1552
0.1145


KRT17
Negative
0.34280253
0.412313
0.151
0.1195


DPYD
Negative
2.78071456
0.78918833
0.1504
0.1202


CD105
Negative
3.13613733
0.51406689
0.1391
0.1351


TP53BP1
Negative
3.18676588
0.58622276
0.1361
0.1395


PTPD1
Negative
5.85217342
1.08545385
0.1357
0.1401


CTSL
Positive
−2.2283797
−1.4833372
0.1354
0.1405
















TABLE 2







Clinical Benefit Genes for Head and Neck Study













Logistic Discriminat

Likelihood


Gene

Function

Ratio Test












Name
Response
Intercept
Slope
R2
P Value















cMet.2
Negative
23.583252
4.4082875
0.6444
0.0007


GRO1.2
Negative
10.10717
2.46904056
0.5388
0.0019


A-Catenin.2
Negative
5.13298651
2.60834812
0.3628
0.0107


AKT1.3
Negative
7.7652606
2.83068092
0.3044
0.0194


DCR3.3
Negative
10.2957141
1.85012996
0.293
0.0219


B-Catenin.3
Negative
4.21267279
1.5417788
0.2791
0.0252


EDN1 endothelin.1
Negative
6.83022814
1.14550062
0.2758
0.0261


CCNE1.1
Negative
7.43731399
1.21270723
0.2661
0.0289


LAMC2.2
Negative
1.79659862
0.56623898
0.2498
0.0342


CD44v6.1
Negative
2.55050577
1.87838162
0.2071
0.0539


DIABLO.1
Negative
16.5051841
2.99910512
0.2066
0.0542


CD44v3.2
Negative
3.02492619
2.05469571
0.2002
0.058


NMYC.2
Negative
23.2010327
3.20767305
0.1955
0.061


CD82.3
Positive
−2.7521937
−1.1692268
0.188
0.0662


RANBP2.3
Negative
2.02076788
0.42173233
0.1807
0.0718


RB1.1
Positive
−5.7352964
−1.7540651
0.1761
0.0754


HER2.3
Negative
3.87564158
1.11486016
0.1732
0.0779


MTA1.1
Negative
3.9020256
0.92255645
0.1628
0.0874


CGA.3
Positive
−41.909839
−5.5686182
0.1619
0.0883


CEACAM6.1
Negative
1.66596967
0.59307792
0.1602
0.0899


PTPD1.2
Negative
5.51242763
1.18616068
0.1601
0.0901


ERK1.3
Negative
2.4144706
0.72072834
0.154
0.0964


Bax.1
Negative
2.91338256
0.76334619
0.152
0.0987


STMY3.3
Positive
−0.9946728
−0.6053981
0.1483
0.1028


COX2.1
Negative
5.79279616
1.0312018
0.1478
0.1034


EIF4E.1
Negative
2.08005397
0.55985052
0.1468
0.1045


YB-1.2
Positive
0.45158771
−2.2935538
0.1426
0.1096


fas.1
Negative
4.05538424
0.8686042
0.1397
0.1134


PDGFA.3
Negative
2.43388275
0.53168307
0.1371
0.1168


FRP1.3
Negative
2.17320245
0.41529609
0.137
0.1169


PGK1.1
Negative
1.86416703
1.92395917
0.1338
0.1212


AKT2.3
Negative
1.45131206
1.43341036
0.1281
0.1294


BTC.3
Negative
12.1153734
1.67411928
0.1281
0.1294


APC.4
Negative
2.50791938
0.92506412
0.128
0.1296


CCNE2.2
Negative
3.98727145
0.89372321
0.1267
0.1315


OPN, osteopontin.3
Positive
−0.522697
−0.5069258
0.1225
0.1382


ITGA3.2
Negative
2.23381763
0.3800099
0.1203
0.1417


KRT17.2
Negative
−0.4861169
0.43917211
0.1184
0.1449


CD44s.1
Positive
−0.9768133
−0.8896223
0.118
0.1456


EGFR.2
Negative
0.43258354
0.46719029
0.1162
0.1487
















TABLE 3







Partial Response Genes for Colon Study













Logistic Discriminat

Likelihood


Gene

Function

Ratio Test












Name
Response
Intercept
Slope
R2
P Value















Bclx_2
Positive
2.04896151
−2.1025144
0.172
0.0801


BRAF_2
Positive
−2.5305788
−3.0987684
0.2532
0.0337


BRK 2
Positive
−2.6096501
−1.577388
0.2998
0.0209


CA9_3
Negative
2.65287578
0.83720397
0.2758
0.0267


Cad17_1
Positive
−0.0419396
−1.8773242
0.2096
0.0533


CCND3_1
Positive
−1.014844
−5.1111617
0.348
0.0128


CCNE1_1
Positive
−6.5821701
−0.8939912
0.1914
0.0648


CCNE2_2
Positive
26.1675642
−1.0709109
0.1707
0.0812


CD105_1
Positive
5.85359096
−1.2349006
0.1302
0.1278


CD134_2
Negative
−5.9286576
1.51119518
0.1212
0.1418


CD44v3_2
Negative
−1.8184898
1.12771829
0.2064
0.0552


CDC25B_1
Negative
10.4351019
1.59196005
0.2455
0.0365


DR5_2
Negative
−1.7399226
1.60177588
0.1759
0.0767


ErbB3_1
Positive
3.65681435
−0.760436
0.1222
0.1401


EREG_1
Positive
−2.3409861
−1.1217612
0.2542
0.0333


GPC3_1
Positive
4.03889935
−1.9097648
0.3752
0.0097


GRO1.2
Negative
2.77545378
0.74734483
0.124
0.1359


GUS_1
Positive
8.29578416
−1.9015759
0.2105
0.0529


HGF_4
Positive
5.10609383
−1.1947949
0.2361
0.0403


ID1_1
Positive
10.6703203
−1.654146
0.216
0.0498


ITGB3_1
Positive
0.79232612
−0.827508
0.3321
0.015


KRT17_2
Negative
5.93738146
0.93514633
0.2133
0.0513


LAMC2_2
Negative
−0.3325052
1.41542034
0.2475
0.0357


P14ARF_1
Negative
4.36456658
4.10859002
0.2946
0.022


PDGFB_3
Negative
−4.7055966
1.96517114
0.3299
0.0154


PLAUR_3
Negative
7.51817646
0.6862142
0.1534
0.0983


PTPD1_2
Positive
−11.659761
−1.2559081
0.1247
0.1362


RASSF1_3
Negative
6.60631474
0.9862129
0.1708
0.0811


RIZ1_2
Negative
2.83817546
0.86281199
0.1255
0.1349


Src_2
Negative
4.91364145
1.96089745
0.1324
0.1247


TFRC_3
Negative
−4.0754666
3.03617052
0.19
0.0658


TITF1_1
Positive
−1.8849815
−2.1890987
0.1349
0.1211


upa_3
Negative
4.1059421
1.14053848
0.1491
0.1032


XIAP_1
Positive
−16.296951
−2.9502191
0.2661
0.0295
















TABLE 4







Clinical Benefit Genes for Colon Study













Logistic Discriminat

Likelihood


Gene

Function

Ratio Test












Name
Response
Intercept
Slope
R2
P Value















Bak
Positive
−1.347937
−0.993212
0.1189
0.0602


BRK
Positive
−3.237705
−1.1479379
0.2567
0.0057


CD134
Negative
9.9358537
1.68440149
0.1927
0.0167


CD44E
Negative
3.188991
0.59091622
0.0958
0.0916


CD44v6
Negative
5.7352464
1.77571293
0.2685
0.0047


CDC25B
Negative
2.0664209
0.67140598
0.0783
0.1272


CGA
Negative
2.7903424
0.43834476
0.1035
0.0794


COX2
Positive
−1.262804
−0.4741852
0.0733
0.1398


DIABLO
Positive
−2.514199
−1.0753148
0.1028
0.0805


FRP1
Positive
−0.401936
−0.3555899
0.0937
0.0952


GPC3
Positive
−7.875276
−1.7437079
0.3085
0.0025


HER2
Positive
0.1228609
−0.5549133
0.073
0.1408


ITGB3
Positive
−1.593092
−0.5249778
0.1352
0.045


PPARG
Negative
8.6479233
1.36115361
0.1049
0.0774


PTPD1
Positive
−3.203607
−1.2049773
0.1356
0.0447


RPLPO
Positive
3.5110353
−1.030518
0.0752
0.135


STK15
Positive
−0.664989
−0.5936475
0.0873
0.1072


SURV
Positive
−1.409619
−0.6214924
0.074
0.1381


TERC
Positive
1.7755749
−0.5180083
0.1073
0.0742


TGFBR2
Positive
1.5172396
−0.9288498
0.0934
0.0957



















TABLES 5A-5B





Gene
Accessin
Sequence
Seq. ID


















A-Catenin
NM_00190
CGTTCCGATCCTCTATACTGCATCCCAGGCATGCCTACAGCACCCTGATGTCGCAGCCTATAAGGCCAACA 
1




GGGACCT



 


AKT1
NM_00516
CGCTTCTATGGCGCTGAGATTGTGTCAGCCCTGGACTACCTGCACTCGGAGAAGAACGTGGTGTACCGGGA
2


 


AKT2
NM_00162
TCCTGCCACCCTTCAAACCTCAGGTCACGTCCGAGGTCGACACAAGGTACTTCGATGATGAATTTACCGCC
3


 


APC
NM_00003
GGACAGCAGGAATGTGTTTCTCCATACAGGTCACGGGGAGCCAATGGTTCAGAAACAAATCGAGTGGGT
4


 


B-Catenin
NM_00190
GGCTCTTGTGCGTACTGTCCTTCGGGCTGGTGACAGGGAAGACATCACTGAGCCTGCCATCTGTGCTCTTC
5




GTCATCTGA



 


Bak
NM_00118
CCATTCCCACCATTCTACCTGAGGCCAGGACGTCTGGGGTGTGGGGATTGGTGGGTCTATGTTCCC
6


 


Bax
NM_00432
CCGCCGTGGACACAGACTCCCCCCGAGAGGTCTTTTTCCGAGTGGCAGCTGACATGTTTTCTGACGGCAA
7


 


Bclx
NM_00119
CTTTTGTGGAACTCTATGGGAACAATGCAGCAGCCGAGAGCCGAAAGGGCCAGGAACGCTTCAACCGCTG
8


 


BRAF
NM_00433
CCTTCCGACCAGCAGATGAAGATCATCGAAATCAATTTGGGCAACGAGACCGATCCTCATCAGCTCCCAAT
9




GTGCATATAAA



 


BRK
NM_00597
GTGCAGGAAAGGTTCACAAATGTGGAGTGTCTGCGTCCAATACACGCGTGTGCTCCTCTCCTTACTCCATC
10




GTGTGTGC



 


BTC
NM_00172
AGGGAGATGCCGCTTCGTGGTGGCCGAGCAGACGCCCTCCTGTGTCTGTGATGAAGGCTACATTGGAGCAA
11




GGTGTGAGAG



 


CA9
NM_00121
ATCCTAGCCCTGGTTTTTGGCCTCCTTTTTGCTGTCACCAGCGTCGCGTTCCTTGTGCAGATGAGAAGGCA
12




G



 


Cad17
NM_100406
GAAGGCCAAGAACCGAGTCAAATTATATTCCAGTTTAAGGCCAATCCTCCTGCTGTGACTTTTGAACTAAC
13




TGGGGA



 


CCNA2
NM_00123
CCATACCTCAAGTATTTGCCATCAGTTATTGCTGGAGCTGCCTTTCATTTAGCACTCTACACAGTCACGGG
14




ACAAAGCT



 


CCND3
NM_00176
CCTCTGTGCTACAGATTATACCTTTGCCATGTACCCGCCATCCATGATCGCCACGGGCAGCATTGGGGCTG
15




CAGTG



 


CCNE1
NM_00123
AAAGAAGATGATGACCGGGTTTACCCAAACTCAACGTGCAAGCCTCGGATTATTGCACCATCCAGAGGCTC
16


 


CCNE2
NM_05774
ATGCTGTGGCTCGTTCCTAACTGGGGCTTTCTTGACATGTAGGTTGCTTGGTAATAACCTTTTTGTATACA
17




CAATTTGGGT



 


CD105
NM_00011
GCAGGTGTCAGCAAGTATGATCAGCAATGAGGCGGTGGTCAATATCCTGTCGAGCTCATCACCACAGCGGA
18




AAAA



 


CD134
NM_00332
GCCCAGTGCGGAGAACAGOTCCAGCTTGATTCTCGTCTCTGCACTTAAGCTGTTCTCCAGGTGCGTGTGAT
19




T



 


CD44E
X55150
ATCACCGACAGCACAGACAGAATCCCTGCTACCAATATGGACTCCAGTCATAGTACAACGCTTCAGCCTAC
20




TGCAAATCCAAACACAGGT



 


CD44s
M59040
GACGAAGACAGTCCCTGGATCACCGACAGCACAGACAGAATCCCTGCTACCAGAGACCAAGACACATTCCA
21




CCCCAGT



 


CD44v3
AJ251595t
CACACAAAACAGAACCAGGACTGGACCCAGTGGAACCCAAGCCATTCAAATCCGGAAGTGCTACTTCAG
22


 


CD44v6
AJ251595s
CTCATACCAGCCATCCAATGCAAGGAAGGACAACACCAAGCCCAGAGGACAGTTCCTGGACTGATTTCTTC
23




AACCCAA



 


CD68
NM_00125
TGGTTCCCAGCCCTGTGTCCACCTCCAAGCCCAGATTCAGATTCGAGTCATGTACACAACCCAGGGTGGAG
24




GAG



 


CD82
NM_00223
GTGCAGGCTCAGGTGAAGTGCTGCGGCTGGGTCAGCTTCTACAACTGGACAGACAACGCTGAGCTCATGAA
25




TCGCCCTGAGGTC



 


CD9
NM_00176
GGGCGTGGAACAGITTATCTCAGACATCTGCCCCAAGAAGGACGTACTCGAAACCTTCACCGTG
26


 


CDC25B
NM_02187
AAACGAGCAGTTTGCCATCAGACGCTTCCAGTCTATGCCGGTGAGGCTGCTGGGCCACAGCCCCGTGCTTC
27




GGAACATCACCAAC



 


CEACAM6
NM_00248
CACAGCCTCACTTCTAACCTTTCTGGAACCCACCCACCACTGCCAAGCTCACTATTGAATCCACGCCATTC
28




AA



 


CGA
NM_00127
CTGAAGGAGCTCCAAGACCTCGCTCTCCAAGGCGCCAAGGAGAGGGCACATCAGCAGAAGAAACACAGCGG
29




TTTTG



 


Chk2
NM_00719
ATGTGGAACCCCCACCTACTTGGCGCCTGAAGTTCTTGTTTCTGTTGGGACTGCTGGGTATAACCGTGCTG
30




TGGACTG



 


cMet
NM_00024
GACATTTCCAGTCCTGCAGTCAATGCCTCTCTGCCCCACCCTTTGTTCAGTGTGGCTGGTGCCACGACAAA
31




TGTGTGCGATCGGAG



 


COX2
NM_00096
TCTGCAGAGTTGGAAGCACTCTATGGTGACATCGATGCTGTGGAGCTGTATCCTGOCCTTCTGGTAGAAAA
32




GCCTCGGC



 


cripto
NM_00321
GGGTCTGTGCCCCATGACACCTGGCTGCCCAAGAAGTGTTCCCTGTGTAAATGCTGGCACGGTCA
33


 


CTSL
NM_00191
GGGAGGCTTATCTCACTGAGTGAGCAGAATCTGGTAGACTGCTCTGGGCCTCAAGGCAATGAAGGCTGCAA
34




TGG



 


DCR3
NM_01643
GACCAAGGTCCTGGAATGTCTGCAGCAGAAGGTGAATGGCATCCTGGAGAGCCCTACGGGTACAGGGAAGA
35




C



 


DIABLO
NM_01988
CACAATGGCGGCTCTGAAGAGTTGGCTGTCGCGCAGCGTAACTTCATTCTTCAGGTACAGACAGTGTTTGT
36




GT



 


DPYD
NM_00011
AGGACGCAAGGAGGGTTTGTCACTGGCAGACTCGAGACTGTAGGCACTGCCATGGCCCCTGTGCTCAGTAA
37




GGACTCGGCGGACATC



 


DR5
NM_00384
CTCTGAGACAGTGCTTCGATGACTTTGCAGACTTGGTGCCCTTTGACTCCTGGGAGCCGCTCATGAGGAAG
38




TTGGGCCTCATGG



 


EDN1 
NM_00195
TGCCACCTGGACATCATTTGGGTCAACACTCCCGAGCACGTTGTTCCGTATGGACTTGGAAGCCCTAGGTC
39


endo

CA



 


EGFR
NM_00522
TGTCGATGGACTTCCAGAACCACCTGGGCAGCTGCCAAAAGTGTGATCCAAGCTGTCCCAAT
40


 


EGFRd27
EGRd27
GAGTCGGGCTCTGGAGGAAAAGAAAGGTAATTATGTGGTGACAGATCACGGCTCGTGCGTCCGAGCCTGTG
41




G



 


EIF4E
NM_00196
GATCTAAGATGGCGACTGTCGAACCGGAAACCACCCCTACTCCTAATCCCCCGACTACAGAAGAGGAGAAA
42




ACGGAATCTAA



 


ErbB3
NM_00198
CGGTTATGTCATGCCAGATACACACCTCAAAGGTACTCCCTCCTCCCGGGAAGGCACCCTTTCTTCAGTGG
43




GTCTCAGTTC



 


ERBB4
NM_00523
TGGCTCTTAATCAGTTTCGTTACCTGCCTCTGGAGAATTTACGCATTATTCGTGGGACAAAACTTTATGAG
44




GATCGATATGCCTTG



 


EREG
NM_00143
ATAACAAAGTGTAGCTCTGACATGAATGGCTATTGTTTGCATGGACAGTGCATCTATCTGGTGGACATGAG
45




TCAAAACTACTGCAGGTGTG



 


ERK1
Z11696
ACGGATCACAGTGGAGGAAGCGCTGGCTCACCCCTACCTGGAGCAGTACTATGACCCGACGGATGAG
46


 


fas
NM_00004
GGATTGCTCAACAACCATGCTGGGCATCTGGACCCTCCTACCTCTGGTTCTTACGTCTGTTGCTAGATTAT
47




CGTCCAAAAGTGTTAATGCC



 


FRP1
NM_00301
TTGGTACCTGTGaGTTAGCATCAAGTTCTCCCCAGGGTAAATTCAATCAGAGCTCCAGTTTGCATTTGGAT
48




GTG



 


GPC3
NM_00448
TGATGCGCCTGGAAACAGTCAGCAGGCAACTCCGAAGGACAACGAGATAAGCACCITTCACAACCTCG
49


 


GRO1
NM_00151
CGAAAAGATGCTGAACAGTGACAAATCCAACTGACCAGAAGGGAGGAGGAAGCTCACTGGTGGCTGTTCCT
50




GA



 


GUS
NM_00018
CCCACTCAGTAGCCAAGTCACAATGTTTGGAAAACAGCCCGTTTACTTGAGCAAGACTGATACCACCTGCG
51




TG



 


HB-EGF
NM_00194
GACTCCTTCGTCCCCAGTTGCCGTCTAGGATTGGGCCTCCCATAATTGCTTTGCCAAAATACCAGAGCCTT
52




CAAGTGCCA



 


HER2
NM_00444
CGGTGTGAGAAGTGCAGCAAGCCCTGTGCCCGAGTGTGCTATGGTCTGGGCATGGAGCACTTGCGAGAGG
53


 


HGF
M2145
CCGAAATCCAGATGATGATGCTCATGGACCCTGGTGCTACACGGGAAATCCACTCATTCCTTGGG
54


 


ID1
NM_00216
AGAACCGCAAGGTGAGCAAGGTGGAGATTCTCCAGCACGTCATCGACTACATCAGGGACCTTCAGTTGGA
55


 


1GF1R
NM_00087
GCATGGTAGCCGAAGATTTCACAGTCAAAATCGGAGATTTTGGTATGACGCGAGATATCTATGAGACAGAC
56




TATTACCGGAAA



 


IGFBP3
NM_00059
ACGCACCGGGTGTCTGATCCCAAGTTCCACCCCCTCCATTCAAAGATAATCATCATCAAGAAAGGGCA
57


 


IRSI
NM_00554
CCACAGCTCACCTTCTGTCAGGTGTCCATCCCAGCTCCAGCCAGCTCCCAGAGAGGAAGAGACTGGCACTG
58




AGG



ITGA3
NM_00220
CCATGATCCTCACTCTGCTGGTGGACTATACACTCCAGACCTCGCTTAGGATGGTAAATCACCGGCTACAA
59




AGCTTC



 


ITGB3
NM_00021
ACCGGGAGCCCTACATGACCGAAAATACCTGCAACCGTTACTGCCGTGACGAGATTGAGTCAGTGAAAGAGC
60




TTAAGG



 


KRT17
NM_00042
CGAGGATTGGTTCTTCAGCAAGACAGAGGAACTGAACCGCGAGGTGGCCACCAACAGTGAGCTGGTGCAGA
61




GT



 


LAMC2
NM_00556
ACTCAAGCGGAAATTGAAGCAGATAGGTCTTATCAGCACAGTCTCCGCCTCCTGGATTCAGTGTCTCGGCT
62




TCAGGGAGT



 


MTA1
NM_00468
CCGCCCTCACCTGAAGAGAAACGCGCTCCTTGGCGGACACTGGGGGAGGAGAGGAAGAAGCGCGGCTAACT
63




TATTCC



 


NMYC
NM_00537
TGAGCGTCGCAGAAACCACAACATCCTGGAGCGCCAGCGCCGCAACGACCTTCGGTCCAGCTTTCTCACGC
64




TCAGGGA



 


p14ARF
NM_00007
GCGGAAGGTCCCTCAGACATCCCCGATTGAAAGAACCAGAGAGGCTCTGAGAAACCTCGGGAAACTTAGA
65


 


p27
NM_00406
CGGTGGACCACGAAGAGTTAACCCGGGACTTGGAGAAGCACTGCAGAGACATGGAAGAGGCGAGCC
66


 


P53
NM_00054
CTTTGAACCCTTGCTTGCAATAGGTGTGCGTCAGAAGCACCCAGGACTTCCATTTGCTTTGTCCCGGG
67


 


PAH
NM_00060
CCGCAACGTGGTTTTCTCACCCTATGGGGTGGCCTCGGTGTTGGCCATGCTCCAGCTGACAACAGGAGGAG
68




AAACCCAGCA



 


PDGFA
NM_00260
TTGTTGGTGTGCCCTGGTGCCGTGGTGGCGGTCACTCCCTCTGCTGCCAGTGTTTGGACAGAACCCA
69


 


PDGFB
NM_00260
ACTGAAGGAGACCCTTGGAGCCTAGGGGCATCGGCAGGAGAGTGTGTGGGCAGGGTTATTTA
70


 


PGK1
NM_00029
AGAGCCAGTTGCTGTAGAACTCAAATCTCTGCTGGGCAAGGATGTTCTGTTCTTGAAGGACTGTGTAGGCC
71




CAG



 


PLAUR
NM_00265
CCCATGGATGCTCCTCTGAAGAGACTTTCCTCATTGACTGCCGAGGCCCCATGAATCAATGTCTGGTAGCC
72




ACCGG



 


PPARG
NM_00503
TGACTTTATGGAGCCCAAGTTTGAGTTTGCTGTGAAGTICAATGCACTGGAATTAGATGACAGCGACTTGG
73




C



 


PTPD1
NM_00703
CGCTTGCCTAACTCATACTTTCCCGTTGACACTTGATCCACGCAGCGTGGCACTGGGACGTAAGTGGCGCA
74




GTCTGAATGG



 


RANBP2
NM_00626
TCCTTCAGCTTTCACACTGGGCTCAGAAATGAAGTFGCATGACTCTTCTGGAAGTCAGGTGGGAACAGGAT
75




TT



 


RASSF1
NM_00718
AGTGGGAGACACCTGACCTTTCTCAAGCTGAGATTGAGCAGAAGATCAAGGAGTACAATGCCCAGATCA
76


 


RB1
NM_00032
CGAAGCCCTTACAAGTTTCCTAGTTCACCCTTACGGATTCCTGGAGGGAACATCTATATTTCACCCCTGAA
77




GAGTCC



 


RIZ1
NM_01223
CCAGACGAGCGATTAGAAGCGGCAGCTTGTGAGGTGAATGATTTGGGGGAAGAGGAGGAGGAGGAAGAGGA
78




GGA



 


RPLPO
NM_00100
CCATTCTATCATCAACGGGTACAAACGAGTCCTGGCCTTGTCTGTGGAGACGGATTACACCTTCCCACTTG
79




CTGA



 


SPRY2
NM_00584
TGTGGCAAGTGCAAATGTAAGGAGTGCACCTACCCAAGGCCTCTGCCATCAGACTGGATCTGCGAC
80


 


Sic
NM_00438
CCTGAACATGAAGGAGCTGAAGCTGCTGCAGACCATCGGGAAGGGGGAGTTCGGAGACGTGATG
81


 


STK15
NM_00360
CATCTTCCAGGAGGACCACTCTCTGTGGCACCCTGGACTACCTGCCCCCTGAAATGATTGAAGGTCGGA
82


 


SURV
NM_00116
TGTTTTGATTCCCGGGCTTACCAGGTGAGAAGTGAGGGAGGAAGAAGGCAGTGTCCCTTTTGCTAGAGCTG
83




ACAGCTTTG



 


TERC
U86046
AAGAGGAACGGAGCGAGTCCCCGCGCGCGGCGCGATTCCCTGAGCTGTGGGACGTGCACCCAGGACTCGGC
84




TCACACAT



 


TFRC
NM_00323
GCCAACTGCTTTCATTTGTGAGGGATCTGAACCAATACAGAGCAGACATAAAGGAAATGGGCCTGAGT
85


 


TGFBR2
NM_00324
AACACCAATGGGTTCCATCTTTCTGGGCTCCTGATTGCTCAAGCACAGTTTGGCCTGATGAAGAGG
86


 


TIMP2
NM_00325
TCACCCTCTGTGACTTCATCGTGCCCTGGGACACCCTGAGCACCACCCAGAAGAAGAGCCTGAACCACA
87


 


TITF1
NM_00331
CGACTCCGTTCTCAGTGTCTGACATCTTGAGTCCCCTGGAGGAAAGCTACAAGAAAGTGGGCATGGAGGG
88


 


TP53BP1
NM_00565
TGCTGTTGCTGAGTCTGTTGCCAGTCCCCAGAAGACCATGTCTGTGTTGAGCTGTATCTGTGAAGCCAGGC
89




AAG



 


upa
NM_00265
GTGGATGTGCCCTGAAGGACAAGCCAGGCGTCTACACGAGAGTCTCACACTTCTTACCCTGGATCCGCAG
90


 


VEGFC
NM_00542
CCTCAGCAAGACGTTATTTGAAATTACAGTGCCTCTCTCTCAAGGCCCCAAACCAGTAACAATCAGTTTTG
91




CCAATCACACTT



 


XIAP
NM_00116
GCAGTTGGAAGACACAGGAAAGTATCCCCAAATTGCAGATTTATCAACGGCTTTTATCTTGAAAATAGTGC
92




CACGCA



 


YB-1
NM_00455
AGACTGTGGAGTTTGATGTTGTTGAAGGAGAAAAGGGTGCGGAGGCAGCAAATGTTACAGGTCCTGGTGGT
93




GTTCC





















TABLES 6A-6F





Gene
Accession
Name
Sequence
Length
Seq ID.




















A-Catenin
NM_001903
S2138/A-Cate.f2
CGTTCCGATCCTCTATACTGCAT
23
94


 


A-Catenin
NM_001903
S2139/A-Cate.r2
AGGTCCCTGTTGGCCTTATAGG
22
95


 


A-Catenin
NM_001903
S4725/A-Cate.p2
ATGCCTACAGCACCCTGATGTCGCA
25
96


 


AKT1
NM_005163
S0010/AKT1.f3
CGCTTCTATGGCGCTGAGAT
20
97


 


AKT1
NM_005163
S00121AKT1.r3
TCCCGGTACACCACGTTCTT
20
98


 


AKT1
NM_005163
S4776/AKT1.p3
CAGCCCTGGACTACCTGCACTCGG
24
99


 


AKT2
NM_001626
S0828/AKT2.f3
TCCTGCCACCCTTCAAACC
19
100


 


AKT2
NM001626
S0829/AKT2.r3
GGCGGTAAATTCATCATCGAA
21
101


 


AKT2
NM_001626
S4727/AKT2.p3
CAGGTCACGTCCGAGGTCGACACA
24
102


 


APC
NM_000038
S0022/APC.f4
GGACAGCAGGAATGTGTTTC
20
103


 


APC
NM_000038
S0024/APC.r4
ACCCACTCGATTTGTTTCTG
20
104


 


APC
NM_000038
S4888/APC.p4
CATTGGCTCCCCGTGACCTGTA
22
105


 


B-Catenin
NM_001904
S2150/B-Cate.f3
GGCTCTTGTGCGTACTGTCCTT
22
106


 


B-Catenin
NM_001904
S2151/B-Cate.r3
TCAGATGACGAAGAGCACAGATG
23
107


 


B-Catenin
NM_001904
S5046/B-Cate.p3
AGGCTCAGTGATGTCTTCCCTGTCACCAG
29
108


 


Bak
NM_001188
S0037/Bak.f2
CCATTCCCACCATTCTACCT
20
109


 


Bak
NM_001188
S0039/Bak.r2
GGGAACATAGACCCACCAAT
20
110


 


Bak
NM_001188
S4724/Bak.p2
ACACCCCAGACGTCCTGGCCT
21
111


 


Bax
NM_004324
S0040/Bax.f1
CCGCCGTGGACACAGACT
18
112


 


Bax
NM_004324
S0042/Bax.r1
TTGCCGTCAGAAAACATGTCA
21
113


 


Bax
NM_004324
S4897/Bax.p1
TGCCACTCGGAAAAAGACCTCTCGG
25
114


 


Bclx
NM_001191
S0046/Bc1x.f2
CTTTTGTGGAACTCTATGGGAACA
24
115


 


Bclx
NM_001191
S0048/Bc1x.r2
CAGCGGTTGAAGCGTTCCT
19
116


 


Bclx
NM_001191
S4898/Bc1x.p2
TTCGGCTCTCGGCTGCTGCA
20
117


 


BRAF
NM_004333
S3027/BRAF.f2
CCTTCCGACCAGCAGATGAA
20
118


 


BRAF
NM_004333
S3028/BRAF.r2
TTTATATGCACATTGGGAGCTGAT
24
119


 


BRAF
NM_004333
S4818/BRAF.p2
CAATTTGGGCAACGAGACCGATCCT
25
120


 


BRK
NM_005975
S0678/BRK.f2
GTGCAGGAAAGGTTCACAAA
20
121


 


BRK
NM_005975
S0679/BRK.r2
GCACACACGATGGAGTAAGG
20
122


 


BRK
NM_005975
S4789/BRK.p2
AGTGTCTGCGTCCAATACACGCGT
24
123


 


BTC
NM_001729
S1216/BTC.f3
AGGGAGATGCCGCTTCGT
18
124


 


BTC
NM_001729
S1217/BTC.r3
CTCTCACACCTTGCTCCAATGTA
23
125


 


BTC
NM_001729
S4844/BTC.p3
CCTTCATCACAGACACAGGAGGGCG
25
126


 


CA9
NM_001216
S1398/CA9.f3
ATCCTAGCCCTGGTTTTTGG
20
127


 


CA9
NM_001216
S1399/CA9.r3
CTGCCTTCTCATCTGCACAA
20
128


 


CA9
NM_001216
S4938/CA9.p3
TTTGCTGTCACCAGCGTCGC
20
129


 


Cad17
NM_004063
S2186/Cad17.f1
GAAGGCCAAGAACCGAGTCA
20
130


 


Cad17
NM_004063
S2187/Cad17.r1
TCCCCAGTTAGTTCAAAAGTCACA
24
131


 


Cad17
NM_004063
S5038/Cad17.p1
TTATATTCCAGTTTAAGGCCAATCCTC
27
132


 


CCNA2
NM_001237
S3039/CCNA2.f1
CCATACCTCAAGTATTTGCCATCAG
25
133


 


CCNA2
NM_001237
S3040/CCNA2.r1
AGCTTTGTCCCGTGACTGTGTA
22
134


 


CCNA2
NM_001237
S4820/CCNA2.p1
ATTGCTGGAGCTGCCTTTCATTTAGCACT
29
135


 


CCND3
NM_001760
S2799/CCND3.f1
CCTCTGTGCTACAGATTATACCTTTGC
27
136


 


CCND3
NM_001760
S2800/CCND3.r1 
CACTGCAGCCCCAATGCT
18
137


 


CCND3
NM_001760
S4966/CCND3.p1
TACCCGCCATCCATGATCGCCA
22
138


 


CCNE1
NM_001238
S1446/CCNE1.f1
AAAGAAGATGATGACCGGGTTTAC
24
139


 


CCNE1
NM_001238
S1447/CCNE1.r1
GAGCCTCTGGATGGTGCAAT
20
140


 


CCNE1
NM_001238
S4944/CCNE1.p1
CAAACTCAACGTGCAAGCCTCGGA
24
141


 


CCNE2
NM_057749
S1458/CCNE2.f2
ATGCTGTGGCTCCTTCCTAACT
22
142


 


CCNE2
NM_057749
S1459/CCNE2.r2
ACCCAAATTGTGATATACAAAAAGGTT
27
143


 


CCNE2
NM_057749
S4945/CCNE2.p2
TACCAAGCAACCTACATGTCAAGAAAGCCC
30
144


 


CD105
NM_000118
S1410/CD105.f1
GCAGGTGTCAGCAAGTATGATCAG
24
145


 


CD105
NM_000118
S1411/CD105.r1
TTTTTCCGCTGTGGTGATGA
20
146


 


CD105
NM_000118
S4940/CD105.p1
CGACAGGATATTGACCACCGCCTCATT
27
147


 


CD134
NM_003327
S3138/CD134.f2
GCCCAGTGCGGAGAACAG
18
148


 


CD134
NM_003327
S3139/CD134.r2
AATCACACGCACCTGGAGAAC
21
149


 


CD134
NM_003327
S3241/CD134.p2
CCAGCTTGATTCTCGTCTCTGCACTTAAGC
30
150


 


CD44E
X55150
S3267/CD44E.f1
ATCACCGACAGCACAGACA
19
151


 


CD44E
X55150
S3268/CD44E.r1
ACCTGTGTTTGGATTTGCAG
20
152


 


CD44E
X55150
S4767/CD44E.p1
CCCTGCTACCAATATGGACTCCAGTCA
27
153


 


CD44s
M59040
S3102/CD44s.f1
GACGAAGACAGTCCCTGGAT
20
154


 


CD44s
M590401
S3103/CD44s.r1
ACTGGGGTGGAATGTGTCTT
20
155


 


CD44s
M59040
S4826/CD44s.p1
CACCGACAGCACAGACAGAATCCC
24
156


 


CD44v3
AJ251595v3
S2997/CD44v3.f2
CACACAAAACAGAACCAGGACT
22
157


 


CD44v3
AJ251595v3
S2998/CD44v3.r2
CTGAAGTAGCACTTCCGGATT
21
157


 


CD44v3
AJ251595v3
S4814/CD44v3.p2
ACCCAGTGGAACCCAAGCCATTC
23
159


 


CD44v6
AJ251595v6
S3003/C044v6.f1
CTCATACCAGCCATCCAATG
20
160


 


CD44v6
AJ251595v6
S3004/C044v6.r1
TTGGGTTGAAGAAATCAGTCC
21
161


 


CD44v6
AJ251595v6
S4815/CD44v6.p1
CACCAAGCCCAGAGGACAGTTCCT
24
162


 


CD68
NM_001251
S0067/CD68.f2
TGGTTCCCAGCCCTGTGT
18
163


 


CD68
NM_001251
S0069/CD68.r2
CTCCTCCACCCTGGGTTGT
19
164


 


CD68
NM_001251
S4734/CD68.p2
CTCCAAGCCCAGATTCAGATTCGAGTCA
28
165


 


CD82
NM_002231
S0684/CD82.f3
GTGCAGGCTCAGGTGAAGTG
20
166


 


CD82
NM_002231
S0685/CD82.r3
GACCTCAGGGCGATTCATGA
20
167


 


CD82
NM_002231
S4790/CD82.p3
TCAGCTTCTACAACTGGACAGACAACGCTG
30
168


 


CD9
NM_001769
S0686/CD9.f1
GGGCGTGGAACAGTTTATCT
20
168


 


CD9
NM_001769
S0687/CD9.r1
CACGGTGAAGGTTTCGAGT
19
170


 


CD9
NM_001769
S4792/CD9.p1
AGACATCTGCCCCAAGAAGGACGT
24
171


 


CDC25B
NM_021874
S1160/CDC25B.f1
AAACGAGCAGTTTGCCATCAG
21
172


 


CDC25B
NM_021874
S1161/CDC25B.r1
GTTGGTGATGTTCCGAAGCA
20
176


 


CDC25B
NM_021874
S4842/CDC25B.p1
CCTCACCGGCATAGACTGGAAGCG
24
174


 


CEACAM6
NM_002483
S3197/CEACAM.f1
CACAGCCTCACTTCTAACCTTCTG
24
175


 


CEACAM6
NM_002483
S3198/CEACAM.r1
TTGAATGGCGTGGATTCAATAG
22
176


 


CEACAM6
NM_002483
S3261/CEACAM.p1
ACCCACCCACCACTGCCAAGCTC
23
177


 


CGA
NM_001275
S3221/CGA.f3
CTGAAGGAGCTCCAAGACCT
20
178


 


CGA
NM_001275
S3222/CGA.r3
CAAAACCGCTGTGTTTCTTC
20
179


 


CGA
NM_001275
S3254/CGA.p3
TGCTGATGTGCCCTCTCCTTGG
22
180


 


Chk2
NM_007194
S1434/Chk2.f3
ATGTGGAACCCCCACCTACTT
21
181


 


Chk2
NM_007194
S1435/Chk2.r3
CAGTCCACAGCACGGTTATACC
22
182


 


Chk2
NM_007194
S4942/Chk2.p3
AGTCCCAACAGAAACAAGAACTTCAGGCG
29
183


 


cMet
NM_000245
S0082/cMet.f2
GACATTTCCAGTCCTGCAGTCA
22
184


 


cMet
NM_000245
S0084/cMet.r2
CTCCGATCGCACACATTTGT
20
185


 


cMet
NM_000245
S4993/cMet.p2
TGCCTCTCTGCCCCACCCITTGT
23
186


 


COX2
NM_000963
S0088/COX2.f1
TCTGCAGAGTTGGAAGCACTCTA
23
187


 


COX2
NM_000963
S0090/COX2.r1
GCCGAGGCTTTTCTACCAGAA
21
188


 


COX2
NM_000963
S4995/COX2.p1
CAGGATACAGCTCCACAGCATCGATGTC
28
189


 


cripto
NM_003212
S3117/cripto.f1
GGGTCTGTGCCCCATGAC
18
190


 


cripto
NM_003212
S3118/cripto.r1
TGACCGTGCCAGCATTTACA
20
191


 


cripto
NM_003212
S3237/cripto.p1
CCTGGCTGCCCAAGAAGTGTTCCCT
25
192


 


CTSL
NM_001912
S1303/CTSL.f2
GGGAGGCTTATCTCACTGAGTGA
23
193


 


CTSL
NM_001912
S1304/CTSL.r2
CCATTGCAGCCTTCATTGC
19
194


 


CTSL
NM_001912
S4899/CTSL.p2
TTGAGGCCCAGAGCAGTCTACCAGATTCT
29
195


 


DCR3
NM_016434
S1786/DCR3.f3
GACCAAGGTCCTGGAATGTC
20
196


 


DCR3
NM_016434
S1787/DCR3.r3
GTCTTCCCTGTACCCGTAGG
20
197


 


DCR3
NM_016434
S4982/DCR3.p3
CAGGATGCCATTCACCTTCTGCTG
24
198


 


DIABLO
NM_019887
S0808/DIABLO.f1
CACAATGGCGGCTCTGAAG
19
199


 


DIABLO
NM_019887
S0809/DIABLO.r1 
ACACAAACACTGTCTGTACCTGAAGA
26
200


 


DIABLO
NM_019887
S4813/DIABLO.p1
AAGTTACGCTGCGCGACAGCCAA
23
201


 


DPYD
NM_000110
S0100/DPYD.f2
AGGACGCAAGGAGGGTTTG
19
202


 


DPYD
NM_000110
S0102/DPYD.r2
GATGTCCGCCGAGTCCTTACT
21
203


 


DPYD
NM_000110
S4998/DPYD.p2
CAGTGCCTACAGTCTCGAGTCTGCCAGTG
29
204


 


DR5
NM_03842
S2551/DR5.f2
CTCTGAGACAGTGCTTCGATGACT
24
205


 


DR5
NM_003842
S2552/DR5.r2
CCATGAGGCCCAACTTCCT
19
206


 


DR5
NM_003842
S4979/DR5.p2
CAGACTTGGTGCCCTTTGACTCC
23
207


 


EDN1
NM_001955
S0774/EDN1e.f1
TGCCACCTGGACATCATTTG
20
208


endothelin







 


EDN1
NM_001955
S0775/EDN1e.r1
TGGACCTAGGGCTTCCAAGTC
21
209


endothelin







 


EDN1
NM_001955
S4806/EDN1e.p1
CACTCCCGAGCACGTTGTTCCGT
23
210


endothelin







 


EGFR
NM_005228
S0103/EGFR.f2
TGTCGATGGACTTCCAGAAC
20
211


 


EGFR
NM_005228
S0105/EGFR.r2
ATTGGGACAGCTTGGATCA
19
212


 


EGFR
NM_005228
S4999/EGFR.p2
CACCTGGGCAGCTGCCAA
18
213


 


EGFRd27
EGFRd27
S2484/EGFRd2.f2
GAGTCGGGCTCTGGAGGAAAAG 
22
214


 


EGFRd27
EGFRd27
S2485/EGFRd2.r2
CCACAGGCTCGGACGCAC 
18
215


 


EGFRd27
EGFRd27
S4935/EGFRd2.p2
AGCCGTGATCTGTCACCACATAATTACC
28
216


 


EIF4E
NM_001968
S0106/EIF4E.f1
GATCTAAGATGGCGACTGTCGAA
23
217


 


EIF4E
NM_001968
S0108/EIF4E.r1
TTAGATTCCGTTTTCTCCTCTTCTG
25
218


 


EIF4E
NM_001968
S5000/EIF4E.p1
ACCACCCCTACTCCTAATCCCCCGACT
27
219


 


ErbB3
NM_001982
S0112/Erbp3.f1
CGGTTATGTCATGCCAGATACAC
23
220


 


ErbB3
NM_001982
S0114/ErbB3.r1
GAACTGAGACCCACTGAAGAAAGG
24
221


 


ErbB3
NM_001982
S5002/ErbB3.p1
CCTCAAAGGTACTCCCTCCTCCCGG
25
222


 


ERBB4
NM005235
S1231/ERBB4.f3
TGGCTCTTAATCAGTTTCGTTACCT
25
223


 


ERBB4
NM_005235
S1232/ERBB4.r3
CAAGGCATATCGATCCTCATAAAGT
25
224


 


ERBB4
NM_005235
S4891/ERBB4.p3
TGTCCCACGAATAATGCGTAAATTCTCCAG
30
225


 


EREG
NM_001432
S0670/EREG.f1
ATAACAAAGTGTAGCTCTGACATGAATG
28
226


 


EREG
NM_001432
S0671/EREG.r1
CACACCTGCAGTAGTTTTGACTCA
24
227


 


EREG
NM_001432
S4772/EREG.p1
TTGTTTGCATGGACAGTGCATCTATCTGGT
30
228


 


ERK1
Z11696
S1560/ERK1.f3
ACGGATCACAGTGGAGGAAG
20
229


 


ERK1
Z11696
S1561/ERK1.r3
CTCATCCGTCGGGTCATAGT
20
230


 


ERK1
Z11696
S4882/ERK1.p3
CGCTGGCTCACCCCTACCTG
20
231


 


fas
NM_000043
S0118/fas.f1
GGATTGCTCAACAACCATGCT
21
232


 


fas
NM_000043
S0120/fas.r1
GGCATTAACACTTTTGGACGATAA
24
233


 


fas
NM_000043
S5003/fas.p1
TCTGGACCCTCCTACCTCTGGTTCTTACGT
30
234


 


FRP1
NM_003012
S1804/FRP1.f3
TTGGTACCTGTGGGTTAGCA
20
235


 


FRP1
NM_003012
S1805/FRP1.r3
CACATCCAAATGCAAACTGG
20
236


 


FRP1
NM_003012
S4983/FRP1.p3
TCCCCAGGGTAGAATTCAATCAGAGC
26
237


 


GPC3
NM_004484
S1835/GPC3.f1
TGATGCGCCTGGAAACAGT
19
238


 


GPC3
NM_004484
S1836/GPC3.r1
CGAGGTTGTGAAAGGTGCTTATC
23
239


 


GPC3
NM_004484
S50361GPC3.p1
AGCAGGCAACTCCGAAGGACAACG
24
240


 


GRO1
NM_001511
S0133/GRO1.12
CGAAAAGATGCTGAACAGTGACA
23
241


 


GRO1
NM_001511
S0135/GRO1.r2
TCAGGAACAGCCACCAGTGA
20
242


 


GRO1
NM_001511
S5006/GRO1.p2
CTTCCTCCTCCCTTCTGGTCAGTTGGAT
28
243


 


GUS
NM_000181
S0139/GUS.f1
CCCACTCAGTAGCCAAGTCA
20
244


 


GUS
NM_000181
S0141/GUS.r1
CACGCAGGTGGTATCAGTCT
20
245


 


GUS
NM_000181
S4740/GUS.p1
TCAAGTAAACGGGCTGTMCCAAACA
27
246


 


HB-EGF
NM_001945
S0662/HB-EGF.f1
GACTCCTTCGTCCCCAGTTG
20
247


 


HB-EGF
NM_001945
S0663/HB-EGF.r1
TGGCACTTGAAGGCTCTGGTA
21
248


 


HB-EGF
NM_001945
S4787/HB-EGF.p1
TTGGGCCTCCCATAATTGCTTTGCC
25
249


 


HER2
NM_004448
S0142/HER2.f3
CGGTGTGAGAAGTGCAGCAA
20
250


 


HER2
NM_004448
S0144/HER2.r3
CCTCTCGCAAGTGCTCCAT
19
251


 


HER2
NM_004448
S4729/HER2.p3
CCAGACCATAGCACACTCGGGCAC
24
242


 


HGF
M29145
S1327/HGF.f4
CCGAAATCCAGATGATGATG
20
253


 


HGF
M29145
S1328/HGF.r4
CCCAAGGAATGAGTGGATTT
20
254


 


HGF
M29145
S4901/HGF.p4
CTCATGGACCCTGGTGCTACACG
23
255


 


ID1
NM_002165
S0820/1D1.f1
AGAACCGCAAGGTGAGCAA
19
256


 


ID1
NM_002165
S0821/1D1.r1
TCCAACTGAAGGTCCCTGATG
21
257


 


ID1
NM_002165
S4832/ID1.p1
TGGAGATTCTCCAGCACGTCATCGAC
26
258


 


IGF1R
NM_000875
S1249/IGF1R.f3
GCATGGTAGCCGAAGATTTCA
21
259


 


IGF1R
NM_000875
S1250/IGF1R.r3
TTTCCGGTAATAGTCTGTCTCATAGATATC
30
260


 


IGF1R
NM_000875
S4895/IGF1R.p3
CGCGTCATACCAAAATCTCCGATTTTGA
28
261


 


IGFBP3
NM_000598
S0157/IGFBP3.f3
ACGCACCGGGTGTCTGA
17
262


 


IGFBP3
NM_000598
S0159/1GFBP3.r3
TGCCCTTTCTTGATGATGATTATC
24
263


 


IGFBP3
NM_000598
S5011/IGFBP3.p3
CCCAAGTTCCACCCCCTCCATTCA
24
264


 


IRS1
NM_005544
S1943/IRS1.f3
CCACAGCTCAGCTTCTGTCA
20
265


 


IRS1
NM_005544
S1944/IRS1.r3
CCTCAGTGCCAGTCTCTTCC
20
266


 


IRS1
NM_005544
S5050/IRS1.p3
TCCATCCCAGCTCCAGCCAG
20
267


 


ITGA3
NM_002204
S2347/ITGA3.f2
CCATGATCCTCACTCTGCTG
20
268


 


ITGA3
NM_002204
S2348/ITGA3.r2
GAAGCTTTGTAGCCGGTGAT
20
269


 


ITGA3
NM_002204
S4852/ITGA3.p2
CACTCCAGACCTCGCTTAGCATGG
24
270


 


ITGB3
NM_000212
S3126/ITGB3.f1
ACCGGGAGCCCTACATGAC
19
271


 


ITGB3
NM_000212
S3127/ITGB3.r1
CCTTAAGCTCTTTCACTGACTCAATCT
27
272


 


ITGB3
NM_060212
S3243/ITGB3.p1
AAATACCTGCAACCGTTACTGCCGTGAC
28
273


 


KRT17
NM_000422
S0172/KRT17.f2
CGAGGATTGGTTCTTCAGCAA
21
274


 


KRT17
NM_000422
S0174/KRT17.r2
ACTCTGCACCAGCTCACTGTTG
22
275


 


KRT17
NM_000422
S5013/KRT17.p2
CACCTCGCGGTTCAGTTCCTCTGT
24
276


 


LAMC2
NM_005562
S2826/LAMC2.f2
ACTCAAGCGGAAATTGAAGCA
21
277


 


LAMC2
NM_005562
S2827/LAMC2.r2
ACTCCCTGAAGCCGAGACACT
21
278


 


LAMC2
NM_005562
S4969/LAMC2.p2
AGGTCTTATCAGCACAGTCTCCGCCTCC
28
278


 


MTA1
NM_004689
S2369/MTA1.f1
CCGCCCTCACCTGAAGAGA
19
280


 


MTA1
NM_004689
S2370/MTA1.r1
GGAATAAGTTAGCCGCGCTTCT
22
281


 


MTA1
NM_004689
S4855/MTA1.p1
CCCAGTGTCCGCCAAGGAGCG
21
282


 


NMYC
NM_005378
S2884/NMYC.f2
TGAGCGTCGCAGAAACCA
18
283


 


NMYC
NM_005378
S2885/NMYC.r2
TCCCTGAGCGTGAGAAAGCT
20
284


 


NMYC
NM_005378
S4976/NMYC.p2
CCAGCGCCGCAACGACCTTC
20
285


 


p14ARF
NM_000077
S0199/p14ARF.f3
GCGGAAGGTCCCTCAGACA
19
286


 


p14ARF
NM_000077
S0201/p14ARF.r3
TCTAAGTTTCCCGAGGTTTCTCA
23
297


 


p14ARF
NM_000077
S5068/p14ARF.p3
CCCCGATTGAAAGAACCAGAGAGGCT
26
288


 


p27
NM_004064
S0205/p27.f3
CGGTGGACCACGAAGAGTTAA
21
289


 


p27
NM_004064
S0207/p27.r3
GGCTCGCCTCTTCCATGTC
19
290


 


p27
NM_004064
S4750/p27.p3
CCGGGACTTGGAGAAGCACTGCA 
23
291


 


P53
NM_000546
S0208/P53.f2
CTTTGAACCCTTGCTTGCAA
20
292


 


P53
NM_000546
S0210/P53.r2 
CCCGGGACAAAGCAAATG
18
293


 


P53
NM_000546
S5065/P53.p2
AAGTCCTGGGTGCTTCTGACGCACA
25
294


 


PAI1
NM_000602
S0211/PA11.f3
CCGCAACGTGGTTTTCTCA
19
295


 


PAI1
NM_000602
S0213/PAI1.r3
TGCTGGGTTTCTCCTCCTGTT
21
296


 


PAI1
NM_000602
S5066/PA11.p3
CTCGGTGTTGGCCATGCTCCAG 
22
297


 


PDGFA
NM_002607
S0214/PDGFA.f3
TTGTTGGTGTGCCCTGGTG
19
298


 


PDGFA
NM_002607
S0216/PDGFA.r3
TGGGTTCTGTCCAAACACTGG
21
299


 


PDGFA
NM_002607
S5067/PDGFA.p3
TGGTGGCGGTCACTCCCTCTGC
22
300


 


PDGFB
NM_002608
S0217/PDGFB.f3
ACTGAAGGAGACCCTTGGAG
20
301


 


PDGFB
NM_002608
S0219/PDGFB.r3
TAAATAACCCTGCCCACACA
20
302


 


PDGFB
NM_602608
S5014/PDGFB.p3
TCTCCTGCCGATGCCCCTAGG
21
303


 


PGK1
NM_000291
S0232/PGK1.f1
AGAGCCAGTTGCTGTAGAACTCAA
24
304


 


PGK1
NM_000291
S0234/PGK1.r1
CTGGGCCTACACAGTCCTTCA
21
305


 


PGK1
NM_000291
S5022/PGK1.p1
TCTCTGCTGGGCAAGGATGTTCTGTTC
27
306


 


PLAUR
NM_002659
S1976/PLAUR.f3
CCCATGGATGCTCCTCTGAA
20
307


 


PLAUR
NM_002659
S1977/PLAUR.r3
CCGGTGGCTACCAGACATTG
20
308


 


PLAUR
NM_002659
S5054/PLAUR.p3
CATTGACTGCCGAGGCCCCATG
22
309


 


PPARG
NM_005037
S3090/PPARG.f3
TGACTTTATGGAGCCCAAGTT 
21
310


 


PPARG
NM_005037
S3091/PPARG.r3
GCCAAGTCGCTGTCATCTAA
20
311


 


PPARG
NM_005037
S4824/PPARG.p3
TTCCAGTG CATTGAACTTCACAG CA
25
312


 


PTPD1
NM_007039
S3069/PTPD1.f2
CGCTTGCCTAACTCATACTTTCC
23
313


 


PTPD1
NM_007039
S3070/PTPD1s2
CCATTCAGACTGCGCCACTT
20
314


 


PTPD1
NM_007039
S4822/PTPD1.p2
TCCACGCAGCGTGGCACTG
19
315


 


RANBP2
NM_006267
S3081/RANBP2.f3
TCCTTCAGCTTTCACACTGG
20
316


 


RANBP2
NM_006267
S3082/RANBP2.r3
AAATCCTGTTCCCACCTGAC
20
317


 


RANBP2
NM_006267
S4823/RANBP2.p3
TPCAGAAGAGTCATGCAACTTCATTTCTG
29
318


 


RASSF1
NM_007182
S2393/RASSF1.f3
AGTGGGAGACACCTGACCTT
20
319


 


RASSF1
NM_007182
S2394/RASSF1.r3
TGATCTGGGCATTGTACTCC
20
320


 


RASSF1
NM_007182
S4909/RASSF1.p3
TTGATCTTCTGCTCAATCTCAGCTTGAGA
29
321


 


RB1
NM_000321
S2700/RB1.f1
CGAAGCCCTTAPAAGTTTCC
20
322


 


RB1
NM_000321
S2701/RB1.r1
GGACTCTTCAGGGGTGAAAT
20
323


 


RB1
NM_000321
S4765/RB1.p1
CCCTTACG GATTCCTGGAGG GAAC
24
324


 


RIZ1
NM_012231
S1320/RIZ1.f2
CCAGACGAGCGATTAGAAGC
20
325


 


RIZ1
NM_012231
S1321/RIZ1.r2
TCCTCCTCTTCCTCCTCCTC
20
326


 


RIZ1
NM_012231
S4761/RIZ1.p2
TGTGAGGTGAATGATTTGGGGGA
23
327


 


RPLPO
NM_001002
S0256/RPLPO.f2
CCATTCTATCATCAACGGGTACAA
24
328


 


RPLPO 
NM_001002
S0258/RPLPO.r2
TCAGCAAGTGGGAAGGTGTAATC
23
329


 


RPLPO
NM_001002
S4744/RPLPO.p2
TCTCCACAGACAAGGCCAGGACTCG
25
330


 


SPRY2
NM_005842
S2985/SPRY2.f2
TGTGGCAAGTGCAAATGTAA
20
331


 


SPRY2
NM_005842
S2986/SPRY2.r2
GTCGCAGATCCAGTCTGATG
20
332


 


SPRY2
NM_005842
S4811/SPRY2.p2
CAGAGGCCTTGGGTAGGTGCACTC
24
333


 


Src
NM_004383
S1820/Src.f2
CCTGAAtATGAAGGAGCTGA
20
334


 


Src
NM_004383
S1821/Src.r2
CATCACGTCTCCGAACTCC
19
335


 


Src
NM_004383
S5034/Src.p2
TCCCGATGGTCTGCAGCAGCT
21
336


 


STK15
NM_003600
S0794/STK15.f2
CATCTTCCAGGAGGACCACT
20
337


 


STK15
NM_003600
S0795/STK15.r2 
TCCGACCTTCAATCATTTCA
20
338


 


STK15
NM_003600
S4745/STK15.p2
CTCTGTGGCACCCTGGACTACCTG
24
339


 


SURV
NM_001168
S0259/SURV.f2
TGTTTTGATTCCCGGGCTTA
20
340


 


SURV
NM_001168
S0261/SURV.r2
CAAAGCTGTCAGCTCTAGCAAAAG
24
341


 


SURV
NM_001168
S4747/SURV.p2
TGCCTTCTTCCTCCCTCACTTCTCACCT
28
342


 


TERC
U86046
S2709/TERC.f2
AAGAGGAACGGAGCGAGTC
19
343


 


TERC
U86046
S2710/TERC.r2
ATGTGTGAGCCGAGTCCTG
19
344


 


TERC
U86046
S4958/TERC.p2
CACGTCCCACAGCTCAGGGAATC
23
345


 


TFRC
NM_003234
S1352/TFRC.f3
GCCAACTGCTTTCATTTGTG
20
346


 


TFRC
NM_003234
S1353/TFRC.r3
ACTCAGGCCCATTTCCTTTA
20
347


 


TFRC
NM_003234
S4748/TFRC.p3
AGGGATCTGAACCAATACAGAGCAGACA
28
348


 


TGFBR2
NM_003242
S2422/TGFBR2.f3
AACACCAATGGGTTCCATCT
20
349


 


TGFBR2
NM_003242
S2423/TGFBR2.r3
CCTCTTCATCAGGCCAAACT
20
350


 


TGFBR2
NM_003242
S4913/TGFBR2.p3
TTCTGGGCTCCTGATTGCTCAAGC
24
351


 


TIMP2
NM_003255
S1680/TIMP2.f1
TCACCCTCTGTGACTTCATCGT
22
352


 


TIMP2
NM_003255
S1681/TIMP2.r1
TGTGGTTCAGGCTCTTCTTCTG
22
353


 


TIMP2
NM_003255
S4916/TIMP2.p1
CCCTGGGACACCCTGAGCACCA
22
354


 


TITF1
NM_003317
S2224/TITF1.f1
CGACTCCGTTCTCAGTGTCTGA
22
355


 


TITF1
NM_003317
S2225/TITF1.r1
CCCTCCATGCCCACTTTCT
19
356


 


TITF1
NM_003317
S4829/TITF1.p1
ATCTTGAGTCCCCTGGAGGAAAGC
24
357


 


TP53BP1
NM_005657
S1747/TP53BP.f2
TGCTGTTGCTGAGTCTGTTG
20
358


 


TP53BP1
NM_005657
S1748/TP53BP.r2
CTTGCCTGGCTTCACAGATA
20
359


 


TP53BP1
NM_005657
S4924/TP53BP.p2
CCAGTCCCCAGAAGACCATGTCTG
24
360


 


upa
NM_002658
S0283/upa.f3
GTGGATGTGCCCTGAAGGA
19
361


 


upa
NM_002658
S0285/upa.r3
CTGCGGATCCAGGGTAAGAA
20
362


 


upa
NM_002658
S4769/upa.p3
AAGCCAGGCGTCTACACGAGAGTCTCAC
28
363


 


VEGFC
NM_005429
S2251NEGFC.f1
CCTCAGCAAGACGTTATTTGAAATT
25
364


 


VEGFC
NM_005429
S2252NEGFC.r1
AAGTGTGATTGGCAAAACTGATTG
24
365


 


VEGFC
NM_005429
S4758NEGFC.p1
CCTCTCTCTCAAGGCCCCAAACCAGT
26
366


 


XIAP
NM_001167
S0289/XIAP.f1
GCAGTTGGAAGACACAGGAAAGT
23
367


 


XIAP
NM_001167
S0291/XIAP.r1
TGCGTGGCACTATTTTCAAGA
21
368


 


XIAP
NM_001167
S4752/XIAP.p1
TCCCCAAATTGCAGATTTATCAACGGC
27
369


 


YB-1
NM_004559
S1194/YB-1.f2
AGACTGTGGAGTTTGATGTTGTTGA
25
370


 


YB-1
NM_004559
S1195/YB-1.r2
GGAACACCACCAGGACCTGTAA
22
371


 


YB-1
NM_004559
S4843/YB-1.p2
TTGCTGCCTCCGCACCCTTTTCT
23
372

## Claims

1. A method for predicting the likelihood that a human colon cancer patient will exhibit a clinically beneficial patient response to treatment with cetuximab, the method comprising:
a) assaying a normalized level of an RNA transcript in a sample comprising EGFR-expressing colon cancer cells obtained from said patient, wherein the RNA transcript is the transcript of EREG;
b) analyzing the normalized level of the EREG RNA transcript; and
c) predicting the likelihood of response of the patient to treatment with cetuximab by comparing the normalized level of the EREG RNA transcript to gene expression data obtained from reference samples derived from patients with colon cancer, wherein an increased normalized level of the EREG RNA transcript correlates with an increased likelihood of response to treatment with cetuximab.

2. The method of claim 1, wherein said sample is a tissue sample.

3. The method of claim 2, wherein the tissue sample is fixed, paraffin-embedded, fresh, or frozen.

4. The method of claim 2, wherein the tissue sample is derived from fine needle, core, or other types of biopsy.

5. The method of claim 1, further comprising the step of preparing a report comprising a prediction of the likelihood that the patient will respond to treatment with cetuximab.

6. The method of claim 1, wherein the normalized level of the EREG RNA transcript is determined using reverse transcription polymerase chain reaction (RT-PCR).

7. The method of claim 1, wherein the normalized level of the EREG RNA transcript is determined using an array comprising polynucleotides hybridizing to a EREG gene immobilized on a solid surface.

8. The method of claim 1, wherein RNA is isolated from colon cancer cells present in a fixed, paraffin-embedded tissue by a procedure comprising:
(a) incubating one or more sections of said fixed, paraffin-embedded tissue at a temperature of about 56° C. to 70° C. in a lysis buffer, in the presence of a protease, without prior dewaxing, to form a lysis solution;
(b) cooling the lysis solution to a temperature where the paraffin solidifies, thereby generating a cooled lysis solution; and
(c) isolating the RNA from said cooled lysis solution.