CA2506066A1 - Gene expression profiling of egfr positive cancer - Google Patents

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 EGFRexpressing cancer, which allow a physician to predict whether a patient is likely to respond well to treatmen t with an EGFR inhibitor.

Description

GENE EXPRESSION PROFILING OF EGFR POSITIVE CANCER
Background of the Invention 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.
Description of the Related Art Oncologists have a nuyber 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., Scies2ce 286:531-537 (1999); Bhattacharjae et al., Pr~oc. Natl. Acad. Sci. USA 98:13790-13795 (2001); Chen-Hsiang et al., Bioiv~fo~°matics 17 (Suppl. 1):5316-5322 (2001); Ramaswa~.ny et al., P~°oc. Natl. Acad. eSci.

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., Pf~oc. Natl. Acad. Sci. USA 98:11462-11467 (2001); Sorlie et al., Proc. Natl. Acad.
Sci. USA 98:10869-10874 (2001); Yan et ~al., Cahcer~ 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, CA).
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..
_2_ Summar~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; Cadl7;
CCND3; CD105;
CD44s; CD82; CD9; CGA;; CTSL; EGFRd27; ErbB3; EREG; GPC3; GUS; HGF; IDl;
IGFBP3; ITGB3; ITGB3; p27; P53; PTPD1; RB1; RPLPO; STK15; SURV; TERC; TGFBR2;
TIMP2; TITF1; XIAP; YB-1; A-Catenin; AKTl; AKT2; APC; Bax; B-Catenin; BTC;
CA9;
CCNA2; CCNEl; CCNE2; CD134; CD44E; CD44v3; CD44v6; CD68; CDC25B; CEACAM6;
Chk2; cMet; COX2; cripto; DCR3; DIABLO; DPYD; DRS; EDN1 endothelin; EGFR;
EIF4E;
ERBB4; ERKl ; fas; FRP 1; GRO 1; HB-EGF; HER2; IGF 1 R; IRS 1; ITGA3; ~KRT 17;
LAMC2;
MTAl; NMYC; P14ARF; PAI1; PDGFA; PDGFB; PGKl; PLAUR; PPARG; RANBP2;
RASSF1; RIZ1; SPRY2; Src; TFRC; TP53BPl;UPA; and VEGFC, wherein (a) the patient is unlikely to benefit from treatment with an EGFR inlubitor if the normalized levels of any of the following genes A-Catenin; AKTl; AKT2; APC; Bax; B-Catenin; BTC; CA9; CCNA2;
CCNEl;
CCNE2; CD134; CD44E; CD44v3; CD44v6; CD68; CDC25B; CEACAM6; Chk2; cMet;
COX2; cripto; DCR3; DIABLO; DPYD; DRS; EDN1 endothelin; EGFR; EIF4E; ERBB4;
ERKl ; fas; FRP 1; GRO 1; HB-EGF; HER2; IGF 1 R; IRS 1; ITGA3; KRT 17; LAMC2;
MTA 1;
NMYC; P14ARF; PAI1; PDGFA; PDGFB; PGKl; PLAUR; PPARG; RANBP2; RASSFl;
RIZl; 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; Cadl7;
CCND3; CD105; -CD44s; CD82; CD9; CGA;; CTSL; EGFRd27; ErbB3; EREG; GPC3; GUS;
HGF; ID1; IGFBP3; ITGB3; ITGB3; p27; P53; PTPDl; RBl; RPLPO; STK15; SURV;
TERC;
TGFBR2; TIMP2; TITFl; 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; DRS; GROl; 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 Ievel 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; RBl;
TIMP2; YB-l; A-Catenin; AKTI; AKT2; APC; Bax; B-Catenin; BTC; CCNA2; CCNE1;
CCNE2; CD105; CD44v3; CD44v6; CD68; CEACAM6; Chk2; cMet; COX2; cripto; DCR3;
DIABLO; DPYD; DRS; EDN1 endothelin; EGFR; EIF4E; ERBB4; ERKl; fas; FRPl; GRO1;
HB-EGF; HER2; IGF 1 R; IRS 1; ITGA3; KRT 17; ~ LAMC2; MTA 1; NMYC; PAI l ;
PDGFA;
PGKl; PTPD1; RANBP2; SPRY2; TP53BP1; and VEGFC, wherein (a) normalized expression of one or more of A-Catenin; AKTl; AKT2; APC; Bax; B-Catenin; BTC; CCNA2;
CCNE1;
CCNE2; CD105; CD44v3; CD44v6; CD68; CEACAM6; Chk2; cMet; COX2; cripto; DCR3;
DIABLO; DPYD; DRS; EDNl endothelin; EGFR; EIF4E; ERBB4; ERKl; fas; FRPl; GROl HB-EGF; HER2; IGF1R; IRS1; ITGA3; KRT17; LAMC2; MTAl; NMYC; PAIL; PDGFA;
PGKl; 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 ofCD44s;
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 inlubitor.
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 i11 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;.Cadl7; CCND3; CCNEl; CCNE2;
CD105;
CD9; COX2; DIABLO; ErbB3; EREG; FRP1; GPC3; GUS; HER2; HGF; ID1; ITGB3; PTPD1;
RPLPO; STK15; SURV; TERC; TGFBR2; TITF1XIAP; CA9; CD134; CD44E; CD44v3;
CD44v6; CDC25B; CGA; DRS; GRO1; KRT17; LAMC2; P14ARF; PDGFB; PLAUR; PPARG;
RAS SF 1; RIZ 1; Src; TFRC; and UPA, wherein (a) elevated expression of one or more of CA9;
CD134; CD44E; CD44v3; CD44v6; CDC25B; CGA; DRS; GROl; 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; BRAE; BRK; Cadl7; CCND3; CCNE1; CCNE2; CD105; CD9; COX2; DIABLO;
ErbB3; EREG; _FRPl; GPC3; GUS; HER2; HGF; ID1; ITGB3; PTPD1; RPLPO; STK15;
SURV; TERC; TGFBR2; TITFl; 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 aalother 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; BRAE; BRK; Cadl7; CCND3;
CD105;
CD44s; CD82; GD9; CGA;; CTSL; EGFRd27; ErbB3; EREG; GPC3; GUS; HGF; IDl;
IGFBP3; ITGB3; ITGB3; p27; P53; PTPD1; RBl; RPLPO; STK15; SURV; TERC; TGFBR2;
TIMP2; TITFl; XIAP; YB-1; A-Catenin; AKTl; AKT2; APC; Bax; B-Catenin; BTC;
GA9;
CCNA2; CCNEl; CCNE2; CD134; CD44E; CD44v3; CD44v6; CD68; CDC25B; CEACAM6;
Chk2; cMet; COX2; cripto; DCR3; DIABLO;.DPYD; DRS;.EDNl endothelia; EGFR;
EIF4E;
ERBB4; ERKl ; fas; FRP 1; GRO 1; HB-EGF; HER2; IGF 1 R; IRS 1; ITGA3; KRT 17;
LAMC2;
MTAl; NMYC; P14ARF; PAIL; PDGFA; PDGFB; PGKl; PLAUR; PPARG; RANBP2;
RASSFl; RIZ1; SPRY2; Src; TFRC; TP53BP1; UPA; and VEGFC genes, or the corresponding gene products in the cancer, with an effective amount of an EGFR-inlubitor, 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 detei~nined 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-l; A-Catenin; AKT1; AKT2; APC; Bax;
B-Catenin; BTC; CCNA2; CCNEl; CCNE2; CD105; CD44v3; CD44v6; CD68; CEACAM6;
Chk2; cMet; COX2; cripto; DCR3; DIABLO; DPYD; DRS; EDNl endothelia; EGFR;
EIF4E;

ERBB4; ERKl; fas; FRP1; GRO1; HB-EGF; HER2; IGF1R; IRS1; ITGA3; KRT17; LAMC2;
MTAl; 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-inlubitor, 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; BRKCadl7;
CCND3;
CCNEl; CCNE2; CD105; CD9; COX2; DIABLO; ErbB3; EREG; FRP1; GPC3; GUS; HER2;
HGF; ID1; ITGB3; PTPDl; RPLPO; STK15; SURV; TERC; TGFBR2; TITF1; XIAP; CA9;
CD134; CD44E; CD44v3; CD44v6; CDC25B; CGA; DRS; GRO1; KRT17; LAMC2; P14ARF;
PDGFB; PLAUR; PPARG; RASSF1; RIZl; 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; Cadl7; CCND3; CD105; CD44s; CD82; CD9;
CGA;;
CTSL; EGFRd27; ErbB3; EREG; GPC3; GUS; HGF; ID1; IGFBP3; ITGB3; ITGB3; p27;
P53;
PTPD1; RBl; RPLPO; STK15; SURV; TERC; TGFBR2; TIMP2; TITFl; 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; DRS; EDNl endothelin; EGFR; EIF4E; ERBB4; ERKl; fas; FRPl; GROl;
HB-EGF; HER2; IGF 1 R; IRS 1; ITGA3; KRT 17; . LAMC2; MTA 1; NMYC; P 14ARF;
PAI 1;
PDGFA; PDGFB; PGKl; PLAUR; PPARG; RANBP2; RASSF1; RIZ1; SPRY2; Src; TFRC;
TP53BP1;UPA; VEGFC; or (b)' an array comprising polynucleotides hybridizing to the following genes: CD44v3; CD44v6; DRS; GROI; 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; AKTl;
AKT2; APC; Bax; B-Catenin; BTC; CCNA2; CCNEl; CCNE2; CD105; CD44v3; CD44v6;
CD68; CEACAM6; Chlc2; cMet; COX2; cripto; DCR3; DIABLO; DPYD; DRS; EDNl 3 0 endothelin; EGFR; EIF4E; ERBB4; ERKl ; fas; FRP 1; GRO 1; HB-EGF; HER2;
IGF 1 R; IRS 1;
ITGA3; KRT17; LAMC2; MTA1; NMYC; PAI1; PDGFA; PGKl; PTPD1; RANBP2; SPRY2;
TP53BP1; and VEGFC, immobilized on a solid surface, or (d) an array comprising polynucleotides hybridizing to the following genes: Bak; Bclx; BRAE; BRK;
Cadl7; CCND3;

CCNE1; CCNE2; CD105; CD9; COX2; DIABLO; ErbB3; EREG; FRPl; GPC3; GUS; HER2;
HGF; ID1; ITGB3; PTPD1; RPLPO; STK15; SURV; TERC; TGFBR2; TITF1; XIAP; CA9;
CD134; CD44E; CD44v3; CD44v6; CDC25B; CGA; DRS; GRO1; KRT17; LAMC2; P14ARF;
PDGFB; PLAUR; PPARG; RASSFl; RIZ1; Src; TFRC; and UPA, immobilized on a solid surface.
In a further aspect, the invention concerns a method in wluch 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 SA and SB by quantitative RT-PCR (qRT-PCR) reaction, by using an amplicon listed in Tables SA and SB and a corresponding primer-probe set listed in Tables 6A-6F.
Brief Description of the Drawings , Figure 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 RiboGreenT"" RNA Quantitation Reagent and Protocol.
Figure 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 _7_ & Sons (New York, NY 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, NY 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 from 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 aal 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 modif ed 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 _g_ oligonucleotide synthesizers that are commercially available. However, oligonucleotides can be made by a variety of other methods, including iri vit~~o 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 lugher or lower level in a subject suffering from a disease, specifically cancer, such as breast cancer, relative to its expression in a nornlal 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 subj ect 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 l 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 axe 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, z. 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 tez~n "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 terns "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 turrior 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 marmnals 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, hepatocellulax 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), Cypl, albumin, actins, e.g. ~i-actin, tubulins, cyclophilin, hypoxantine phsophoribosyltransferase (HRPT), L32. 28S, and 185. .
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", 2°d 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 Innnunology", 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 Exm°essiofz Pnofilin~
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 lcnown in the art for the quantification of mRNA .expression in a sample include northern blotting and in situ hybridization (Parker &
Barnes, Methods in Moleculaf° Biology 106:247-283 (1999)); RNAse protection assays (Hod, Biotechraiques 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. Reoense Ti~ahscy-~tase PCR ART 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. fonnalin-fixed) tissue samples General methods for mRNA extraction are well known in the art and axe disclosed in standard textbooks of molecular biology, including Ausubel et al., Current Protocols of Molecular Biolo~y, 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 Andres et al., BioTeclZniques 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 MasterPureT"" Complete DNA and RNA Purification Kit (EPICENTREO, Madison, WI), and Paraffin Block RNA Isolation Kit (Atnbion, 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 marine 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 Ehner, 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 endoriuclease 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, CA, 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.instruinent 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 patterls of gene expression are mRNAs for the housekeeping genes glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and (3-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., Gefzo~2e Reseanel2 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 Dent, 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 ca~.i 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) Bioinfonmatics Methods and P~°otocols: Methods in Moleculaf°
Biology. Humana Press, Totowa, NJ, pp 365-386) The most important factors considered in PCR primer design include primer length, melting temperature (Tm), and GlC 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 P~°i~2er, A
Labor°ato~ y Manual, Cold Spring Harbor Laboratory Press, New York, 1995, pp. 133-155; Tnnis and Gelfand, "Optimization of PCRs" in: PCR P~°otocols, A Guide to Metlzods aszd Applications, CRC Press, London, 1994, pp. 5-11; and Plasterer, T.N. Primerselect: Primer and probe design. Methods Mol. Biol. 70:520-527 (1997), the~entire disclosures of which are hereby expressly incorporated by reference.
3. Mic~oa~°~°ays 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 tlus 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., Ps°oc. 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. See°ial Anal sy iS O~'Ge~ze Exy°ession 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. MassARRAYTechnolo~-y The MassARR.AY (Sequenom, San Diego, California) 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 Exp~°essioh Afzalysis by Massively PaT°allel Si~natm°e Sequencin~(MPSS
This method, described by Brenner et al., Natm°e BiotechtZOlogy 18:630-634 (2000), is a sequencing approach that combines non-gel-based signature sequencing with ih vitro cloning of millions of templates on separate 5 ~,m diameter microbeads. First, a microbead library of DNA
templates is constructed by ifz 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 x 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. I~zmunohistochemist~°y 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 axe well known in the art and are commercially available.

8. Proteoniics 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 ,~of Isolation of Nucleic Acid fi°om 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". Paraffm-embedded, fonnalin-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. Cancinog. 25:86-91 (1999); Starita and Bonin, Biotechhiques 24:271-276 (1998); Godfrey et al., J. Mol. Diag~rostics 2:84 (2000); Specht et al., J. Mol. Med. 78:B27 (2000); Specht et al., Am. J. Patl2ol. 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 lcits and protocols for RNA
extraction from FPET [MasterPureT"" Complete DNA and RNA Purification Kit (EPICENTRE~, Madison, WI); Paraffin Block RNA Isolation Kit (Ambion, Inc.) and RNeasyT""
Mini kit (Qiagen, Chatsworth, CA)] 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.
Figure 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 iri 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 tlus 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. Wlule 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 he 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, wluch 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 Ge~ze Specific P~°irnin~ofRever~se Ti°ansc~°iption .
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. Nonr~aalization St~~ateQ-y 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 Cypl. ~ Alternatively or in adddition, 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 Inlzibito~s 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 OSI774 (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 b owth. 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 22:3-17 (2003).
While the listed examples of EGFR irilubitors 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 neclc cancer patients who responded or did not respond to treatment with an EGFR inhibitor.
The results are based ow the use of five different EGFR inhibitor drugs.
Study design Molecular assays were performed on para~n-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, CA, 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 thennocycler. 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 + e~~iercept+SlopexReferenceNormalized 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 inlubitor treatment: A-Catenin; AKTl; AKT2; APC; Bax;
B-Catenin;
BTC; CCNA2; CCNE1; CCNE2; CD105; CD44v3; CD44v6; CD68; CEACAM6; Chk2; cMet;
COX2; cripto; DCR3; DIABLO; DPYD; DRS; EDN1 endothelin; EGFR; EIF4E; ERBB4;
ERK 1; fas; FRP 1; GRO 1; HB-EGF; HER2; ' IGF 1 R; IRS 1; ITGA3 ; KRT 17;
LAMC2; MTA 1;
NMYC; PAI1; PDGFA; PGKl; PTPDl; 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 Stud~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 shov~m 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;
DRS; GRO1; KRT17; LAMC2; P14ARF; PDGFB; PLAUR; PPARG; RASSFl; 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; DRS; GRO 1; I~RT 17; LAMC2.
In similar experiments, the elevated expression of LAMC2, B-Catenn, 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; ERKl; FRPl. Elevated expression of the following genes in EGFR-expressing colon cancer correlated with resistance to treatment: APN;
CA9; CCND1; CDC25B; CD134; LAMC2; PDGFB; CD44v6; CYPl; DRS; GAPDH; IGFBP2;
PLAUR; RASSFl; 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 appaxent 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 Likelihood Logistic Ratio Test Discriminat Function Gene Name Response Intercept Slope R2 P Value __ Negative 26.5168713 4.57143179 0.6662 0.0011 cMet 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 GR01 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 .

VEGFG 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 0.0853 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 endothelinNegative 6.06858911 0.87185553 0.1688 0.0998 IGF1 R 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 7_ Likelihood Logistic Ratio Test Discriminat Function Gene Name Response Intercept Slope R2 P Value '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 CD10 5 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 _~8_ Table 2: Clinical Benefit Genes for Head and Neck Study Logistic Discriminat Likelihood Function Ratio Test Gene Name Res onse Interce Slo ~e RZ P Value t cMet.2 Negative 23.583252 4.4082875 0.6444 0.0007 GR01.2 Negative 10.10717 2.46904056 0.5388 0.0019 A-Catenin.2 Negative 5.132986512:60834812 0.3628 0.0107 AKT1.3 Negative 7.7652606 2.83068092 0.3044 0.0194 DCR3.3 Negative 10.29571411.85012996 0.293 0.0219 B-Catenin.3 Negative 4.212672791.5417788 0.2791 0.0252 EDN1 endothelin.1Negative 6.830228141.14550062 0.2758 0.0261 CCNE1.1 Negative 7.437313991.21270723 0.2661 0.0289 LAMC2.2 Negative 1.796598620.56623898 0.2498 0.0342 .

CD44v6.1 Negative 2.550505771.87838162 0,2071 0.0539 DIABL0.1 Negative 16.50518412.99910512 0.2066 0.0542 CD44v3.2 Negative 3.024926192.05469571 0.2002 0.058 NMYC.2 Negative 23.20103273.20767305 0.1955 0.061 CD82.3 Positive -2.7521937-1.1692268 0.188 0.0662 RANBP2.3 Negative 2.020767880.42173233 0.1807 ~ 0.0718 RB1.1 Positive -5.7352964-1.7540651 0.1761 0.0754 HER2.3 Negative 3.875641581.11486016 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 1.665969670.59307792 0.1602 0.0899 PTPD1.2 Negative 5.512427631.18616068 0.1601 0.0901 ERK1.3 Negative 2.4144706 0.72072834 0.154 0.0964 Bax.1 Negative 2.913382560.76334619 0.152 0.0987 STMY3.3 Positive -0.9946728-0.6053981 0.1483 0.1028 COX2.1 Negative 5.792796161.0312018 0.1478 0.1034 EIF4E.1 Negative 2.080053970.55985052 0.1468 0.1045 YB-1.2 Positive 0.45158771-2.2935538 0.1'426 0.1096 fas.1 Negative 4.05538424'0.8686042 0.1397 0.1134 PDGFA.3 Negative 2.433882750.53168307 0.1371 0.1168 FRP1.3 Negative 2.173202450.41529609 0.137 0.1169 PGK1.1 Negative 1.864167031.92395917 0.1338 0.1212 AKT2.3 Negative 1.451312061.43341036 0.1281 0.1294 BTC.3 Negative 12.11537341.67411928 0.1281 0.1294 APC.4 Negative 2.507919380.92506412 0.128 0.1296 CCNE2.2 . Negative 3.98727145'0.89372321 0.1267 0.1315 OPN, osteopontin.3Positive -0.522697 -0.5069258 0.1225 0.1382 ITGA3.2 Negative 2.233817630.3800099 0.1203 0.1417 KRT17.2 Negative -0.48611690.43917211 0.1184 0.1449 CD44s.1 Positive -0.9768133-0.8896223 0.118 0.1456 EGFR.2 Ne ative 0.432583540.46719029 0.1162 0.1487 Table 3: Partial Response Genes for Colon Study Logistic Likelihood Discriminat Function Ratio Test Gene Name Response Intercept Slope RZ P Value 2 Positive 2.04896151 -2.1025144 0.172 0.0801 Bclx _ Positive -2.5305788 -3.0987684 0.2532 0.0337 BRAF_2 BRK_2 Positive -2.6096501 -1.577388 0.2998 0.0209 CA9_3 Negative 2.65287578 0.83720397 0.2'758 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 . -1.81848981.12771829 0.2064 0.0552 CDC2~B_1 Negative 10.4351019 1.59196005 0.2455 0.0365 .

DR5_2 Negative -1.7399226 1.60177588 0.1759 0.0767 1 Positive 3.65681435 -0.760436 0.1222 0.1401 ErbB3 _ Positive -2.3409861 -1.1217612. 0.2542 0.0333 EREG_1 GPC3_1 Positive 4.03889935 -1.9097648 Ø3752 0.0097 GR01.2 Negative 2.77545378 0.74734483 0.124 0.1359 1 Positive 8.29578416 -1.9015759 0.2105 0.0529 GUS

_ Positive 5.10609383 -1.1947949 0.2361. 0.0403 HGF_4 _ 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 1 Negative 4.36456658 4.10859002 0.2946 0.022 _ Negative -4.7055966 1.96517114 0.3299 0.0154 PDGFB 3 ~

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 Likelihood Discriminat Fun ction Ratio Test Gene 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 O
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d a a. d a n. d ~ ~ ~ en cn cn fn !- - I- i- I- I- I- ~ > X >-Tables 6A - 6F
Gene Accession Name Sequence Length, Seq ID.

A-CateninNM 001903S21381A-Cate.f2CGTTCCGATCCTCTATACTGCAT 23 94 A-CateninNM 001903S2139/A-Cate.r2AGGTCCCTGTTGGCCTTATAGG 22 95 .

A-CateninNM 001903S4725/A-Cate.p2ATGCCTACAGCACCCTGATGTCGCA 25 96 AKT1 NM 005163S00101AKT1.f3GCTTCTATGGCGCTGAGAT 20 97 C

AKT1 NM 005163S0012/AKT1.r3TCCCGGTACACCACGTTCTT 20 98 AKT1 NM 005163S4776/AKT1.p3AGCCCTGGACTACCTGCACTCGG 24 99 C

AKT2 NM 001626S0828/AKT2.f3TCCTGCCACCCTTCAAACC 19 100 AKT2 NM_ 001626S0829/AKT2.r3GGCGGTAAATTCATCATCGAA 21 101 AKT2 NM 001626S47271AKT2.p3CAGGTCACGTCCGAGGTCGACACA 24 102 APC NM 000038S0022IAPC.f4GGACAGCAGGAATGTGTTTC 20 103 APC NM_ 000038S0024/APC.r4ACCCACTCGATTTGTTTCTG 20 104 APC NM 000038S4888/APC.p4CATTGGCTCCCCGTGACCTGTA 22 105 B-CateninNM 001904S2150/B-Cate.f3GCTCTTGTGCGTACTGTCCTT 22 106 G

B-CateninNM 001904S2151/B-Cate.r3TCAGATGACGAAGAGCACAGATG 23 107 B-CateninNM 001904S5046/B-Cate.p3AGGCTCAGTGATGTCTTCCCTGTCACCAG29 .

Bak NM_ 001188S0037/Bak.f2CCATTCCCACCATTCTACCT 20 109 Bak NM_ 001188S00391Bak.r2GGGAACATAGACCCACCAAT 20 110 Bak NM 001188S4724/Bak.p2w P,CACCCCAGACGTCCTGGCCT 21 111 ~

Bax NM_ 004324S0040/Bax.f1CCGCCGTGGACACAGACT 18 112 Bax NM_ 004324S0042/Bax.r1TTGCCGTCAGAAAACATGTCA 21 113 Bax NM 004324S4897/Bax.p1TGCCACTCGGAAAAAGACCTCTCGG 25 114 Bclx NM_ 001191~ S0046/Bclx.f2CTTTTGTGGAACTCTATGGGAACA 24 115 Bclx NM_ 001191S0048/Bclx.r2CAGCGGTTGAAGCGTTCCT 19 116 Bclx NM_ 001191S4898/Bclx.p2TTCGGCTCTCGGCTGCTGCA 20 117 BRAF NM _004333S3027/BRAF.f2CCTTCCGACCAGCAGATGAA 20 118 BRAF NM _004333S3028/BRAF.r2TTTATATGCACATTGGGAGCTGAT 24 119 BRAE NM 004333S4818/BRAF.p2CAATTTGGGCAACGAGACCGATCCT 25 ~ 120 BRK NM _005975S0678/BRK.f2GTGCAGGAAAGGTTCACAAA 20 121 BRK NM 005975S06791BRK.r2GCACACACGATGGAGTAAGG 20 122 BRK NM 005975S4789/BRK.p2AGTGTCTGCGTCCAATACACGCGT 24 123 BTC NM _001729S1216/BTC.f3GGGAGATGCCGCTTCGT 18 124 A

BTC NM 001729S1217/BTC.r3CTCTCACACCTTGCTCCAATGTA 23 125 BTC NM S4844/BTC.p3CCTTCATCACAGACACAGGAGGGCG 25 126 CA9 NM_001216 S1398/CA9.f3ATCCTAGCCCTGGTTTTTGG 20 127 CA9 NM_001216 S1399/CA9.r3CTGCCTTCTCATCTGCACAA 20 128 CA9 NM S4938/CA9.p3TTTGCTGTCACCAGCGTCGC 20 129 Cad17 NM_004063 S2186/Cad17.f1GAAGGCCAAGAACCGAGTCA 20 130 Cad17 NM S2187/Cad17.r1TCCCCAGTTAGTTCAAAAGTCACA 24 131 Cad17 NM 004063S5038/Cad17~.p1TTATATTCCAGTTTAAGGCCAATCCTC 27 132 .

CCNA2 NM_001237 S3039/CCNA2.f1CATACCTCAAGTATTTGCCATCAG 25 133 C

CCNA2 NM _001237S3040/CCNA2.r1AGCTTTGTCCCGTGACTGTGTA 22 134 CCNA2 NM_001237 S4820/CCNA2.p1ATTGCTGGAGCTGCCTTTCATTTAGCACT29 135 CCND3 NM _001760.S2799/CCND3.f1CCTCTGTGCTACAGATTATACCTTTGC 27 136 CCND3 NM_001760 S2800/CCND3.r1CACTGCAGCCCCAATGCT 18 137 CCND3 NM _001760S4966/CCND3.p1TACCCGCCATCCATGATCGCCA 22 138 CCNE1 NM_001238 S1446lCCNE1.f1AAAGAAGATGATGACCGGGTTTAC 24 139 CCNE1 NM S1447ICCNE1.r1GAGCCTCTGGATGGTGCAAT 20 140 CCNE1 NM 00123854944/CCNE1.p1CAAACTCAACGTGCAAGCCTCGGA 24 141 CCNE2 NM_057749S1458/CCNE2.f2ATGCTGTGGCTCCTTCCTAACT 22 142 CCNE2 NM 057749S1459/CCNE2.r2ACCCAAATTGTGATATACAAAAAGGTT 27 143 CCNE2 NM 057749S4945/CCNE2.p2TACCAAGCAACCTACATGTCAAGAAAGCCC30 144 CD105 NM_000118S1410/CD105.f1GCAGGTGTCAGCAAGTATGATCAG 24 145 CD105 NM 000118S1411/CD105.r1TTTTTCCGCTGTGGTGATGA 20 146 CD105 NM 000118S4940/CD105.p1CGACAGGATATTGACCACCGCCTCATT 27 147 CD134 NM_003327S3138/CD134.f2GCCCAGTGCGGAGAACAG 18 148 CD134- NM_003327S3139/CD134.r2AATCACACGCACCTGGAGAAC 21 149 CD134 NM 00332753241/CD134.p2CCAGCTTGATTCTCGTCTCTGCACTTAAGC30 150 CD44E X55150 S3267/CD44E.f1ATCACCGACAGCACAGACA 19 151 CD44E X55150 S3268/CD44E.r1ACCTGTGTTTGGATTTGCAG 20 152 CD44E X55150 S4767/CD44E.p1CCCTGCTACCAATATGGACTCCAGTCA 27 153 CD44s M5904D S3102/CD44s.f1GACGAAGACAGTCCCTGGAT 20 154 ~

CD44s M59040 S3103/CD44s.r1ACTGGGGTGGAATGTGTCTT 20 155 CD44s M59040 S4826/CD44s.p1CACCGACAGCACAGACAGAATCCC 24 156 CD44v3 AJ251595v3S2997/CD44v3.f2CACACAAAACAGAACCAGGACT 22 157 CD44v3 AJ251595v3S2998/CD44v3.r2CTGAAGTAGCACTTCCGGATT 21 157 CD44v3 AJ251595v3S4814/CD44v3.p2ACCCAGTGGAACCCAAGCCATTC 23 159.

CD44v6 AJ251595v6S3003/CD44v6.f1CTCATACCAGCCATCCAATG 20 160 CD44v6 AJ251595v6S3004/CD44v6.r1TTGGGTTGAAGAAATCAGTCC 21 161 CD44v6 AJ251595v6S4815/CD44v6.p1CACCAAGCCCAGAGGACAGTTCCT 24 162 CD68 NM_001251S0067/CD68.f2TGGTTCCCAGCCCTGTGT 18 163 CD68 NM_001251S0069/CD68.r2CTCCTCCACCCTGGGTTGT 19 164 CD68 NM 001251S4734/CD68.p2CTCCAAGCCCAGATTCAGATTCGAGTCA 28 165 CD82 NM_002231S0684/CD82.f3GTGCAGGCTCAGGTGAAGTG 20 166 ~

CD82 NM_002231S0685/CD82.r3GACCTCAGGGCGATTCATGA 20 167 CD82 NM 002231S4790/CD82.p3TCAGCTTCTACAACTGGACAGACAACGCTG30 168 .

CD9 NM_001769S06861CD9.f1 GGGCGTGGAACAGTTTATCT 20 168 CD9 NM 001769S0687/CD9.r1 CACGGTGAAGGTTTCGAGT 19 170 CD9 NM 001769S4792/CD9.p1 AGACATCTGCCCCAAGAAGGACGT 24 171 CDC25B NM_021874S1160/CDC25B.f1AAACGAGCAGTTTGCCATCAG 21 172 CDC25B NM_021874S1161/CDC25B.r1GTTGGTGATGTTCCGAAGCA 20 176 .

CDC25B NM 021874S4842/CDC25B.p1CCTCACCGGCATAGACTGGAAGCG 24. 174 CEACAM6NM_002483S3197/CEACAM.f1ACAGCCTCACTTCTAACCTTCTG 24 175 C

CEACAM6NM_002483S3198/CEACAM.r1TTGAATGGCGTGGATTCAATAG 22 176 CEACAM6NM_002483S3261/CEACAM.p1ACCCACCCACCACTGCCAAGCTC 23 177 CGA NM_001275S32211CGA.f3 CTGAAGGAGCTCCAAGACCT 20 178 CGA NM 001275S3222/CGA.r3 CAAAACCGCTGTGTTTCTTC 20 179 CGA NM 001275S3254/CGA.p3 TGCTGATGTGCCCTCTCCTTGG 22 180 , Chk2 NM_007194S14341Chk2.f3ATGTGGAACCCCCACCTACTT 21 181 Chk2 NM_007194S14351Chk2.r3CAGTCCACAGCACGGTTATACC 22 182 Chk2 NM 007194S49421Chk2.p3AGTCCCAACAGAAACAAGAACTTCAGGCG 29 183 cMet NM_000245S0082/cMet.f2ACATTTCCAGTCCTGCAGTCA 22 184 G

cMet NM_000245S0084/cMet.r2CTCCGATCGCACACATTTGT 20 185 cMet NM_000245S49931cMet.p2TGCCTCTCTGCCCCACCCTTTGT 23 186 COX2 NM_000963S0088/COX2:f1TCTGCAGAGTTGGAAGCACTCTA 23 187 COX2 NM_000963S00901COX2.r1GCCGAGGCTTTTCTACCAGAA 21 188 COX2 NM 000963S49951COX2.p1CAGGATACAGCTCCACAGCATCGATGTC 28 189 cripto NM 003212S3117/cripto.f1GGGTCTGTGCCCCATGAC 18 190 cripto NM 003212S3118/cripto.r1TGACCGTGCCAGCATTTACA 20 191 cripto NM 003212S3237lcripto.p1CCTGGCTGCCCAAGAAGTGTTCCCT 25 192 CTSL NM 001912S1303lCTSL.f2GGGAGGCTTATCTCACTGAGTGA 23 193 CTSL NM_001912S1304/CTSL.r2CCATTGCAGCCTTCATTGC 19 194 CTSL NM 001912S4899/CTSL.p2TTGAGGCCCAGAGCAGTCTACCAGATTCT 29 195 DCR3 NM_016434S17861DCR3.f3GACCAAGGTCCTGGAATGTC 20 196 DCR3 NM 016434S17871DCR3.r3GTCTTCCCTGTACCCGTAGG 20 197 ~DCR3 NM 016434S4982/DCR3.p3CAGGATGCCATTCACCTTCTGCTG 24 198 DIABLO NM_019887S0808/DIABLO.f1CACAATGGCGGCTCTGAAG 19 199 DIABLO NM_019887S0809/DIABLO.r1ACACAAACACTGTCTGTACCTGAAGA 26 200 DIABLO NM 019887S4813/DIABLO.p1AAGTTACGCTGCGCGACAGCCAA 23 201 DPYD NM_000110S0100/DPYD.f2AGGACGCAAGGAGGGTTTG 19 202 DPYD NM 000110S0102/DPYD.r2GATGTCCGCCGAGTCCTTACT 21 203 DPYD ~NM 000110S4998/DPYD.p2CAGTGCCTACAGTCTCGAGTCTGCCAGTG 29 204 DR5 NM_003842S2551/DR5.f2CTCTGAGACAGTGCTTCGATGACT 24 205 DR5 NM_003842S2552/DR5.r2CCATGAGGCCCAACTTCCT 19 206 DR5 NM 003842S4979/DR5.p2CAGACTTGGTGCCCTTTGACTCC 23 207 endothelinNM_001955S0774/EDN1 TGCCACCTGGACATCATTTG 20 208 e.f1 endothelinNM_001955S0775/EDN1 TGGACCTAGGGCTTCCAAGTC 21 209 e.r1 endothelinNM 001955S4806/EDN1 CACTCCCGAGCACGTTGTTCCGT 23 210 e.p1 EGFR NM_005228S0103IEGFR.f2TGTCGATGGACTTCCAGAAC 20 211 EGFR NM 005228S0105lEGFR.r2ATTGGGACAGCTTGGATCA ~ 19 212 EGFR NM 005228S4999lEGFR.p2CACCTGGGCAGCTGCCAA 18 213 EGFRd27 EGFRd27 S2484/EGFRd2.f2GAGTCGGGCTCTGGAGGAAAAG 22 214 EGFRd27 EGFRd27 S2485/EGFRd2.r2CCACAGGCTCGGACGCAC ~18 215 EGFRd27 EGFRd27 S4935/EGFRd2.p2AGCCGTGATCTGTCACCACATAATTACC 28 216 EIF4E NM_001968S01061EIF4E.f1GATCTAAGATGGCGACTGTCGAA 23 217 EIF4E NM 001968S0108/EIF4E.r1TTAGATTCCGTTTTCTCCTCTTCTG - 25 218 EIF4E NM 001968S5000/EIF4E.p1ACCACCCCTACTCCTAATCCCCCGACT 27 219 ErbB3 NM_001982S0112/ErbB3.f1CGGTTATGTCATGCCAGATACAC 23 220 ErbB3 NM 001982S0114/ErbB3.r1GAACTGAGACCCACTGAAGAAAGG 24 221 ErbB3 NM D01982S5002/ErbB3.p1CCTCAAAGGTACTCCCTCCTCCCGG 25 222 ERBB4 NM_005235S1231/ERBB4:f3TGGCTCTTAATCAGTTTCGTTACCT 25 223 ERBB4 NM 005235S1232/ERBB4.r3CAAGGCATATCGATCCTCATAAAGT 25 224 ERBB4 NM 005235S4891lERBB4.p3TGTCCCACGAATAATGCGTAAATTCTCCAG 30 225 EREG NM_001432S0670/EREG.f1ATAACAAAGTGTAGCTCTGACATGAATG 28 226 EREG NM 001432S0671/EREG.r1CACACCTGCAGTAGTTTTGACTCA 24 227 EREG NM 001432S4772/EREG.p1TTGTTTGCATGGACAGTGCATCTATCTGGT 30 228 ERK1 211696 S1560/ERK1.f3ACGGATCACAGTGGAGGAAG ' 20 229 ERK1 211696 S1561/ERK1.r3CTCATCCGTCGGGTCATAGT 20 230 ERK1 211696 S4882/ERK1.p3CGCTGGCTCACCCCTACCTG 20 231 ' fas NM_000043S0118/fas.f1GGATTGCTCAACAACCATGCT 21 232 fas NM 000043S0120/fas.r1GGCATTAACACTTTTGGACGATAA 24 233 fas NM 000043S5003/fas.p1TCTGGACCCTCCTACCTCTGGTTCTTACGT 30 234 FRP1 NM_003012S18D4/FRP1.f3TTGGTACCTGTGGGTTAGCA 20 235 FRP1 NM 003012S18051FRP1.r3CACATCCAAATGCAAACTGG 20 236 FRP1 NM 003012S4983/FRP1.p3TCCCCAGGGTAGAATTCAATCAGAGC 26 237 GPC3 NM 004484S1835/GPC3.f1TGATGCGCCTGGAAACAGT 19 238 GPC3 NM 004484 S1836/GPC3.r1CGAGGTTGTGAAAGGTGCTTATC 23 239 GPC3 NM 004484 S5036/GPC3.p1AGCAGGCAACTCCGAAGGACAACG 24 240 GR01 NM_001511 0133/GR01.f2CGAAAAGATGCTGAACAGTGACA 23 241 GR01 NM 001511 S0135/GR01.r2TCAGGAACAGCCACCAGTGA 20 242 GRO1 NM 001511 S5006/GR01.p2CTTCCTCCTCCCTTCTGGTCAGTTGGAT 28 243 GUS NM_000181 S0139/GUS.f1CCCACTCAGTAGCCAAGTCA 20 244 GUS NM_000181 S0141/GUS.r1CACGCAGGTGGTATCAGTCT 20 245 GUS NM 000181 S4740/GUS.p1TCAAGTAAACGGGCTGTTTTCCAAACA 27 246 HB-EGF NM_001945 S0662/HB-EGF.f1GACTCCTTCGTCCCCAGTTG 20 247 HB-EGF NM_001945 S0663/HB-EGF.r1TGGCACTTGAAGGCTCTGGTA 21 248 HB-EGF NM 001945 S4787/HB-EGF.p1TTGGGCCTCCCATAATTGCTTTGCC 25 249 HER2 NM_004448 S0142/HER2.f3CGGTGTGAGAAGTGCAGCAA 20 250 HER2 NM_004448 S0144/HER2.r3CCTCTCGCAAGTGCTCCAT 19 251 HER2 NM 004448 S4729/HER2.p3CCAGACCATAGCACACTCGGGCAC . 24 242 HGF M29145 S1327/HGF.f4CCGAAATCCAGATGATGATG 20 253 HGF M29145 S1328/HGF.r4~ CCCAAGGAATGAGTGGATTT 20 254 HGF M29145 S4901/HGF.p4CTCATGGACCCTGGTGCTACACG 23 255 ID1 NM_002165 S0820/ID1.f1AGAACCGCAAGGTGAGCAA 19 256 ID1 NM_002165 50821/ID1.r1TCCAACTGAAGGTCCCTGATG 21 257 ID1 NM 002165 S4832/ID1.p1TGGAGATTCTCCAGCACGTCATCGAC 26 258 IGF1 NM_000875 S12491IGF1 GCATGGTAGCCGAAGATTTCA 21 259 R R.f3 R R.r3 IGF1R NM 000875 S4895/IGF1R.p3CGCGTCATACCAAAATCTCCGATTTTGA 28 261 IGFBP3 NM_000598 S0157/IGFBP3.f3ACGCACCGGGTGTCTGA 17 262 IGFBP3 NM 000598 S0159/IGFBP3.r3TGCCCTTTCTTGATGATGATTATC 24 263 IGFBP3 NM 000598 S5011/IGFBP3.p3CCCAAGTTCCACCCCCTCCATTCA 24 264 IRS1 NM_005544 S1943/IRS1.f3CCACAGCTCACCTTCTGTCA 20 265 IRS1 NM 005544 S1944/IRS1.r3CCTCAGTGCCAGTCTCTTCC 20 266 IRS1 NM 005544 S50501IRS1.p3TCCATCCCAGCTCCAGCCAG 20 267 , ITGA3 NM_002204 S2347/ITGA3.f2CCATGATCCTCACTCTGCTG 20 268 ITGA3 NM 002204 S2348/ITGA3.r2GAAGCTTTGTAGCCGGTGAT 20 269 ITGA3 NM 002204 S4852/ITGA3.p2CACTCCAGACCTCGCTTAGCATGG 24 270 ~

ITGB3 NM_000212 S3126/ITGB3.f1ACCGGGAGCCCTACATGAC 19 271 ITGB3 NM 000212 S3127/ITGB3.r1CCTTAAGCTCTTTCACTGACTCAATCT 27 272 ITGB3 NM 000212 S3243/ITGB3.p1AAATACCTGCAACCGTTACTGCCGTGAC 28 273 KRT17 NM_000422 S0172/KRT17.f2CGAGGATTGGTTCTTCAGCAA 21 274 KRT17 NM 000422 S0174/KRT17.r2ACTCTGCACCAGCTCACTGTTG 22 275 KRT17 NM 000422 S5013/KRT17.p2CACCTCGCGGTTCAGTTCCTCTGT 24 276 LAMC2 NM_005562 S2826/LAMC2.f2ACTCAAGCGGAAATTGAAGCA 21 277 LAMC2 NM 005562 S2827/LAMC2.r2ACTCCCTGAAGCCGAGACACT 21 278 LAMC2 NM 005562 S4969/LAMC2.p2AGGTCTTATCAGCACAGTCTCCGCCTCC 28 278 MTA1 NM_004689 S2369/MTA1.f1CCGCCCTCACCTGAAGAGA 19 280 ~

MTA1 NM 004689 S2370/MTA1.r1GGAATAAGTTAGCCGCGCTTCT 22 281 MTA1 NM 004689 S4855/MTA1.p1CCCAGTGTCCGCCAAGGAGCG 21 282 NMYC NM_005378 S2884/NMYC.f2TGAGCGTCGCAGAAACCA 18 283 NMYC NM_005378 S2885/NMYC.r2TCCCTGAGCGTGAGAAAGCT 20 284 NMYC NM 005378 S4976/NMYC.p2CCAGCGCCGCAACGACCTTC 20 285 p14ARF NM 000077 S0199/p14ARF.f3GCGGAAGGTCCCTCAGACA 19 286 p14ARF NM 000077 S0201/pl4ARF.r3TCTAAGTTTCCCGAGGTTTCTCA 23 297 p14ARF NM 000077 S5068/p14ARF.p3CCCCGATTGAAAGAACCAGAGAGGCT 26 288 -3 ~-p27 NM 004064S0205/p27.f3 CGGTGGACCACGAAGAGTTAA 21 289 p27 NM 004064S02071p27.r3 GGCTCGCCTCTTCCATGTC 19 290 p27 , 004064S4750Ip27.p3 CCGGGACTTGGAGAAGCACTGCA 23 291 NM

P53 NM_ 000546S0208/P53.f2 CTTTGAACCCTTGCTTGCAA 20 292 P53 NM 000546S0210/P53.r2 CCCGGGACAAAGCAAATG 18 293 P53 NM 000546S50651P53.p2 AAGTCCTGGGTGCTTCTGACGCACA 25 294 PAI1 NM_ 000602S0211IPAI1.f3CGCAACGTGGTTTTCTCA 19 295 C

PAI1 NM_ 000602S0213IPAI1.r3TGCTGGGTTTCTCCTCCTGTT , 21 296 PAI1 NM 000602S5066/PAI1.p3CTCGGTGTTGGCCATGCTCCAG 22 297 -PDGFA NM_ 002607S0214/PDGFA.f3'TTGTTGGTGTGCCCTGGTG 19 298 PDGFA NM_ 002607S02161PDGFA.r3TGGGTTCTGTCCAAACACTGG 21 299 ~PDGFA NM 002607S5067/PDGFA.p3TGGTGGCGGTCACTCCCTCTGC 22 300 PDGFB NM 002608S0217/PDGFB.f3CTGAAGGAGACCCTTGGAG 20 301 A

PDGFB NM_ 002608S0219/PDGFB.r3TAAATAACCCTGCCCACACA 20 302 PDGFB NM 002608S5014/PDGFB.p3TCTCCTGCCGATGCCCCTAGG 21 303 PGK1 NM_ 000291S0232IPGK1.f1AGAGCCAGTTGCTGTAGAACTCAA 24 304 PGK1 NM_ 000291S0234/PGK1.r1CTGGGCCTACACAGTCCTTCA 21 305 PGK1 NM_ 000291S5022/PGK1.p1TCTCTGCTGGGCAAGGATGTTCTGTTC 27 306 PLAUR NM_ 002659S1976/PLAUR.f3CCCATGGATGCTCCTCTGAA 20 307 PLAUR NM_ 002659S1977/PLAUR.r3CCGGTGGCTACCAGACATTG 20 308 PLAUR NM 002659S5054IPLAUR.p3CATTGACTGCCGAGGCCCCATG 22 309 PPARG NM_ 005037S3090/PPARG.f3TGACTTTATGGAGCCCAAGTT 21 310 PPARG NM_ 005037S3091/PPARG.r3GCCAAGTCGCTGTCATCTAA 20 31'I

PPARG NM 005037S4824/PPARG.p3TTCCAGTGCATTGAACTTCACAGCA 25 312 PTPD1 NM_ 007039S3069IPTPD1.f2CGCTTGCCTAACTCATACTTTCC 23 313 PTPD1 NM _007039S3070/PTPD1.r2CCATTCAGACTGCGCCACTT 20 314 PTPD1 NM 007039S4822/PTPD1.p2TCCACGCAGCGTGGCACTG 19 315 RANBP2 NM _006267S3081/RANBP2.f3CCTTCAGCTTTCACACTGG 20 316 T

RANBP2 NM 006267S3082/RANBP2.r3AAATCCTGTTCCCACCTGAC 20 317 RANBP2 NM 006267S4823/RANBP2.p3CCAGAAGAGTCATGCAACTTCATTTCTG 29 318 T

RASSF1 NM _007182S2393/RASSF1.f3AGTGGGAGACACCTGACCTT 20 319 RASSF1 NM _007182S2394/RASSF1.r3TGATCTGGGCATTGTACTCC 20 320 RASSF1 NM 007182S4909lRASSF1.p3TTGATCTTCTGCTCAATCTCAGCTTGAGA29 321 -RB1 NM _00032152700/RB1.fl~CGAAGCCCTTACAAGTTTCC 20 322 RB1 NM S2701/RB1.r1 GGACTCTTCAGGGGTGAAAT 20 323 RB1 NM S4765/RB1.p1 CCCTTACGGATTCCTGGAGGGAAC 24 324 RIZ1 NM_012231 S1320/RIZ1.f2CAGACGAGCGATTAGAAGC 20 325 C

RIZ1 NM_012231 S1321/RIZ1.r2TCCTCCTCTTCCTCCTCCTC 20 326 RIZ1 NNI_012231 S47611RIZ1.p2TGTGAGGTGAATGATTTGGGGGA 23 327 RPLPO NM_001002 S0256/RPLPO.f2CCATTCTATCATCAACGGGTACAA 24 328 ' RPLPO NM_001002 . S0258/RPLPO.r2TCAGCAAGTGGGAAGGTGTAATC 23 329 RPLPO NM S4744lRPLPO.p2TCTCCACAGACAAGGCCAGGACTCG 25 330 SPRY2 NM_005842 S2985/SPRY2.f2TGTGGCAAGTGCAAATGTAA 20 331 SPRY2 NM_005842 S2986lSPRY2.r2GTCGCAGATCCAGTCTGATG 20 332 SPRY2 NM_005842 S4811/SPRY2.p2' CAGAGGCCTTGGGTAGGTGCACTC 24 333 Src NM_004383 S1820/Src.f2 CCTGAACATGAAGGAGCTGA 20 334 Src NM_004383 S1821/Src.r2 CATCACGTCTCCGAACTCC 19 335 Src NM _004383S5034/Src.p2 TCCCGATGGTCTGCAGCAGCT 21 336 STK15 NM_003600 S0794/STK15.f2CATCTTCCAGGAGGACCACT 20 337 STK15 NM S07951STK15.r2TCCGACCTTCAATCATTTCA 20 338 STK15 NM 003600S4745/STK15.p2CTCTGTGGCACCCTGGACTACCTG 24 339 SURV NM 001168S0259/SURV.f2TGTTTTGATTCCCGGGCTTA ' 20 340 SURV NM 001168S0261/SURV.r2CAAAGCTGTCAGCTCTAGCAAAAG 24 341 SURV NM 001168S4747/SURV.p2TGCCTTCTTCCTCCCTCACTTCTCACCT 28 342 TERC 086046 S2709/TERC.f2AAGAGGAACGGAGCGAGTC 19 343 ..

TERC 086046 S2710/TERC.r2ATGTGTGAGCCGAGTCCTG 19 344 TERC 086 046 S4958/TERC.p2CACGTCCCACAGCTCAGGGAATC 23 345 TFRC NM_ 003234S1352/TFRC.f3GCCAACTGCTTTCATTTGTG 20 346 TFRC NM 003234S1353/TFRC.r3ACTCAGGCCCATTTCCTTTA 20 347 TFRC NM 003234S4748/TFRC.p3AGGGATCTGAACCAATACAGAGCAGACA 28 348 TGFBR2 NM_ 003242S2422/TGFBR2.f3AACACCAATGGGTTCCATCT 20 349 TGFBR2 NM 003242S2423ITGFBR2.r3CCTCTTCATCAGGCCAAACT 20 350 TGFBR2 NM 003242S4913/TGFBR2.p3TTCTGGGCTCCTGATTGCTCAAGC 24 351 TIMP2 NM_ 003255S1680/TIMP2.f1TCACCCTCTGTGACTTCATCGT 22 352 TIMP2 NM 003255S1681/TIMP2.r1TGTGGTTCAGGCTCTTCTTCTG 22 353 TIMP2 NM S4916/TIMP2.p1CCCTGGGACACCCTGAGCACCA 22 354 TITF1 NM_ 003317S2224ITITF1.f1CGACTCCGTTCTGAGTGTCTGA 22 355 ~

TITF1 NM_ 003317S2225/TITF1.r1CCCTCCATGCCCACTTTCT 19 356 TITF1 NM 003317S4829/TITF1.p1ATCTTGAGTCCCCTGGAGGAAAGC 24 357 TP53BP1NM_ 005657S1747/TP53BP.f2TGCTGTTGCTGAGTCTGTTG 20 358 TP53BP1NM 005657S1748/TP53BP.r2CTTGCCTGGCTTCACAGATA 20 359 TP53BP1NM 005657S4924/TP53BP.p2CCAGTCCCCAGAAGACCATGTCTG 24 360 .

upa NM 002658S0283/upa.f3GTGGATGTGCCCTGAAGGA 19 361 upa NM 002658S0285/upa.r3CTGCGGATCCAGGGTAAGAA 20 362 upa NM 002658S47691upa.p3AAGCCAGGCGTCTACACGAGAGTCTCAC 28 363 VEGFC NM _005429S2251/VEGFC.f1CCTCAGCAAGACGTTATTTGAAATT 25 364 VEGFC NM _005429S2252/VEGFC.r1AAGTGTGATTGGCAAAACTGATTG 24 365 VEGFC NM 005429S4758/VEGFC.p1CCTCTCTCTCAAGGCCCCAAACCAGT 26 366 XIAP NM _001167S0289/XIAP.f1GCAGTTGGAAGACACAGGAAAGT 23 367 XIAP NM 001167S0291/XIAP.r1TGCGTGGCACTATTTTCAAGA 21 368 XIAP NM 001167S4752/XIAP.p1TCCCCAAATTGCAGATTTATCAACGGC 27 369 YB-1 NM _004559S1194/YB-1.f2AGACTGTGGAGTTTGATGTTGTTGA - 25 370 YB-1 NM 004559S1195/YB-1.r2GGAACACCACCAGGACCTGTAA 22 371 YB-1 NM 004559S4843/YB-1.p2TTGCTGCCTCCGCACCCTTTTCT 23 372 39740-0005 PCT.TXT
SEQUENCE LISTING
<110> Genomic Health vall d' Hebron university Hostipal Baker, ~offre Cronin, Maureen shak, Steve Baselga, lose <120> GENE EXPRESSION PROFILING OF EGFR
POSITIVE CANCER
<130> 39740-0005 <140> Unassigned <141> 2003-11-15 <150> 60/427090 <151> 2003-11-15 <160> 372 <170> FastsEQ for Windows Version 4.0 <210> 1 <211> 78 <212> DNA
<213> Homo Sapiens <400> 1 cgttccgatc ctctatactg catcccaggc atgcctacag caccctgatg tcgcagccta 60 taaggccaac agggacct 78 <210> 2 <211> 71 <212> DNA
<213> Homo Sapiens <400> 2 cgcttctatg gcgctgagat tgtgtcagcc ctggactacc tgcactcgga gaagaacgtg 60 gtgtaccggg a 71 <210> 3 <211> 71 <212> DNA
<213> Homo Sapiens <400> 3 tcctgccacc cttcaaacct caggtcacgt ccgaggtcga cacaaggtac ttcgatgatg 60 aatttaccgc c 71 <210> 4 <211> 69 <212> DNA
<213> Homo Sapiens <400> 4 ggacagcagg aatgtgtttc tccatacagg tcacggggag ccaatggttc agaaacaaat 60 cgagtgggt <210> 5 <211> 80 <212> DNA
<213> Homo Sapiens <400> 5 ggctcttgtg cgtactgtcc ttcgggctgg tgacagggaa gacatcactg agcctgccat 60 39740-0005 PCT.TXT
ctgtgctctt cgtcatctga 80 <210> 6 <211> 66 <212> DNA
<213> Homo Sapiens <400> 6 ccattcccac cattctacct gaggccagga cgtctggggt gtggggattg gtgggtctat 60 gttccc 66 <210> 7 <211> 70 <212> DNA
<213> Homo Sapiens <400> 7 ccgccgtgga cacagactcc ccccgagagg tctttttccg agtggcagct gacatgtttt 60 ctgacggcaa 70 <210> 8 <211> 70 <212> DNA
<213> Homo Sapiens <400> 8 cttttgtgga actctatggg aacaatgcag cagccgagag ccgaaagggc caggaacgct 60 tcaaccgctg 70 <210> 9 <211> 82 <212> DNA
<213> Homo Sapiens <400> 9 ccttccgacc agcagatgaa gatcatcgaa atcaatttgg gcaacgagac cgatcctcat 60 cagctcccaa tgtgcatata as 82 <210> 10 <211> 79 <212> DNA
<213> Homo Sapiens <400> 10 gtgcaggaaa ggttcacaaa tgtggagtgt ctgcgtccaa tacacgcgtg tgctcctctc 60 cttactccat cgtgtgtgc 79 <210> 11 <211> 81 <212> DNA
<213> Homo Sapiens <400> 11 agggagatgc cgcttcgtgg tggccgagca gacgccctcc tgtgtctgtg atgaaggcta 60 cattggagca aggtgtgaga g 81 <210> 12 <211> 72 .
<212> DNA
<213> Homo Sapiens <400> 12 atcctagccc tggtttttgg cctccttttt gctgtcacca gcgtcgcgtt ccttgtgcag 60 atgagaaggc ag <210> 13 <211> 77 <212> DNA

39740-0005 PCT.TXT
<213> Homo Sapiens <400> 13 gaaggccaag aaccgagtca aattatattc cagtttaagg ccaatcctcc tgctgtgact 60 tttgaactaa ctgggga 77 <210> 14 <211> 79 <212> DNA
<213> Homo Sapiens <400> 14 ccatacctca agtatttgcc atcagttatt gctggagctg cctttcattt agcactctac 60 acagtcacgg gacaaagct 79 <210> 15 <211> 76 <212> DNA
<213> Homo Sapiens <400> 15 cctctgtgct acagattata cctttgccat gtacccgcca tccatgatcg ccacgggcag 60 cattggggct gcagtg 76 <210> 16 <211> 71 <212> DNA
<213> Homo Sapiens <400> 16 aaagaagatg atgaccgggt ttacccaaac tcaacgtgca agcctcggat tattgcacca 60 tccagaggct c 71 <210> 17 <211> 82 <212> DNA
<213> Homo Sapiens <400> 17 atgctgtggc tccttcctaa ctggggcttt cttgacatgt aggttgcttg gtaataacct 60 ttttgtatat cacaatttgg gt 82 <210> 18 <211> 75 <212> DNA
<213> Homo Sapiens <400> 18 gcaggtgtca gcaagtatga tcagcaatga ggcggtggtc aatatcctgt cgagctcatc 60 accacagcgg aaaaa 75 <210> 19 <211> 72 <212> DNA
<213> Homo Sapiens <400> 19 gcccagtgcg gagaacaggt ccagcttgat tctcgtctct gcacttaagc tgttctccag 60 gtgcgtgtga tt 72 <210> 20 <211> 90 <212> DNA
<213> Homo Sapiens <400> 20 atcaccgaca gcacagacag aatccctgct accaatatgg actccagtca tagtacaacg 60 cttcagccta ctgcaaatcc aaacacaggt 90 39740-0005 PCT.TxT
<210> 21 <211> 78 <212> DNA
<213> Homo Sapiens <400> 21 gacgaagaca gtccctggat caccgacagc acagacagaa tccctgctac cagagaccaa 60 gacacattcc accccagt 78 <210> 22 <211> 69 <212> DNA
<213> Homo Sapiens <400> 22 cacacaaaac agaaccagga ctggacccag tggaacccaa gccattcaaa tccggaagtg 60 ctacttcag 6g <210> 23 <211> 78 <212> DNA
<213> Homo Sapiens <400> 23 ctcataccag ccatccaatg caaggaagga caacaccaag cccagaggac agttcctgga 60 ctgatttctt caacccaa 78 <210> 24 <211> 74 <212> DNA
<213> Homo Sapiens <400> 24 tggttcccag ccctgtgtcc acctccaagc ccagattcag attcgagtca tgtacacaac 60 ccagggtgga ggag 74 <210> 25 <211> 84 <212> DNA
<213> Homo Sapiens <400> 25 gtgcaggctc aggtgaagtg ctgcggctgg gtcagcttct acaactggac agacaacgct 60 gagctcatga atcgccctga ggtc 84 <210> 26 <211> 64 <212> DNA
<213> Homo Sapiens <400> 26 gggcgtggaa cagtttatct cagacatctg ccccaagaag gacgtactcg aaaccttcac 60 cgtg <210> 27 <211> 85 <212> DNA ' <213> Homo Sapiens <400> 27 aaacgagcag tttgccatca gacgcttcca gtctatgccg gtgaggctgc tgggccacag 60 ccccgtgctt cggaacatca ccaac 85 <210> 28 <211> 72 <212> DNA
<213> Homo Sapiens 39740-0005 PCT.TXT
<400> 28 cacagcctca cttctaacct tctggaaccc acccaccact gccaagctca ctattgaatc 60 cacgccattc as 72 <210> Z9 <211> 76 <212> DNA
<213> Homo Sapiens <400> 29 ctgaaggagc tccaagacct cgctctccaa ggcgccaagg agagggcaca tcagcagaag 60 aaacacagcg gttttg 76 <210> 30 <211> 78 <212> DNA
<213> Homo Sapiens <400> 30 atgtggaacc cccacctact tggcgcctga agttcttgtt tctgttggga ctgctgggta 60 taaccgtgct gtggactg 78 <210> 31 <211> 86 <212> DNA
<213> Homo Sapiens <400> 31 gacatttcca gtcctgcagt caatgcctct ctgccccacc ctttgttcag tgtggctggt 60 gccacgacaa atgtgtgcga tcggag 86 <210> 32 <211> 79 <212> DNA
<213> Homo Sapiens <400> 32 tctgcagagt tggaagcact ctatggtgac atcgatgctg tggagctgta tcctgccctt 60 ctggtagaaa agcctcggc 79 <210> 33 <211> 65 <212> DNA
<213> Homo Sapiens <400> 33 gggtctgtgc cccatgacac ctggctgccc aagaagtgtt ccctgtgtaa atgctggcac 60 ggtca <210> 34 <211> 74 <212> DNA
<213> Homo Sapiens <400> 34 gggaggctta tctcactgag tgagcagaat ctggtagact gctctgggcc tcaaggcaat 60 gaaggctgca atgg 74 <210> 35 <211> 72 <Z12> DNA .
<213> Homo Sapiens <400> 35 gaccaaggtc ctggaatgtc tgcagcagaa ggtgaatggc atcctggaga gccctacggg 60 tacagggaag ac 72 39740-0005 PCT.TXT
<210> 36 <211> 73 <212> DNA
<213> Homo Sapiens <400> 36 cacaatggcg gctctgaaga gttggctgtc gcgcagcgta acttcattct tcaggtacag 60 acagtgtttg tgt 73 <210> 37 <211> 87 <212> DNA
<213> Homo Sapiens <400> 37 aggacgcaag gagggtttgt cactggcaga ctcgagactg taggcactgc catggcccct 60 gtgctcagta aggactcggc ggacatc 87 <210> 38 <211> 84 <212> DNA
<213> Homo Sapiens <400> 38 ctctgagaca gtgcttcgat gactttgcag acttggtgcc ctttgactcc tgggagccgc 60 tcatgaggaa gttgggcctc atgg 84 <210> 39 <211> 73 <212> DNA
<213> Homo Sapiens <400> 39 tgccacctgg acatcatttg ggtcaacact cccgagcacg ttgttccgta tggacttgga 60 agccctaggt cca 73 <210> 40 <211> 62 <212> DNA
<213> Homo Sapiens <400> 40 tgtcgatgga cttccagaac cacctgggca gctgccaaaa gtgtgatcca agctgtccca 60 at 62 <210> 41 <211> 72 <212> DNA
<213> Homo Sapiens <400> 41 gagtcgggct ctggaggaaa agaaaggtaa ttatgtggtg acagatcacg gctcgtgcgt 60 ccgagcctgt gg <Z10> 42 <211> 82 <212> DNA
<213> Homo Sapiens <400> 42 gatctaagat ggcgactgtc gaaccggaaa ccacccctac tcctaatccc ccgactacag 60 aagaggagaa aacggaatct as 82 <210> 43 <211> 81 <212> DNA
<213> Homo Sapiens 39740-0005 Pcr.Txr <400> 43 cggttatgtc atgccagata cacacctcaa aggtactccc tcctcccggg aaggcaccct 60 ttcttcagtg ggtctcagtt c 81 <210> 44 <211> 86 <212> DNA
<213> Homo Sapiens <400> 44 tggctcttaa tcagtttcgt tacctgcctc tggagaattt acgcattatt cgtgggacaa 60 aactttatga ggatcgatat gccttg 86 <210> 45 <211> 91 <212> DNA
<213> Homo Sapiens <400> 45 ataacaaagt gtagctctga catgaatggc tattgtttgc atggacagtg catctatctg 60 gtggacatga gtcaaaacta ctgcaggtgt g 91 <210> 46 <211> 67 <212> DNA
<213> Homo Sapiens <400> 46 acggatcaca gtggaggaag cgctggctca cccctacctg gagcagtact atgacccgac 60 ggatgag <210> 47 <211> 91 <212> DNA
<213> Homo Sapiens <400> 47 ggattgctca acaaccatgc tgggcatctg gaccctccta cctctggttc ttacgtctgt 60 tgctagatta tcgtccaaaa gtgttaatgc c , 91 <210> 48 <211> 75 <212> DNA
<213> Homo Sapiens <400> 48 ttggtacctg tgggttagca tcaagttctc cccagggtag aattcaatca gagctccagt 60 ttgcatttgg atgtg 75 <210> 49 <211> 68 <212> DNA
<213> Homo Sapiens <400> 49 tgatgcgcct ggaaacagtc agcaggcaac tccgaaggac aacgagataa gcacctttca 60 caacctcg 68 <210> 50 <211> 73 <212> DNA
<213> Homo Sapiens <400> 50 cgaaaagatg ctgaacagtg acaaatccaa ctgaccagaa gggaggagga agctcactgg 60 tggctgttcc tga 73 <210> 51 39740-0005 PCT.TxT
<211> 73 <212> DNA
<213> Homo Sapiens <400> 51 cccactcagt agccaagtca caatgtttgg aaaacagccc gtttacttga gcaagactga 60 taccacctgc gtg 73 <210> 52 <211> 80 <212> DNA
<213> Homo Sapiens <400> 52 gactccttcg tccccagttg ccgtctagga ttgggcctcc cataattgct ttgccaaaat 60 accagagcct tcaagtgcca 80 <210> 53 <211> 70 <212> DNA
<213> Homo Sapiens <400> 53 cggtgtgaga agtgcagcaa gccctgtgcc cgagtgtgct atggtctggg catggagcac 60 ttgcgagagg <210> 54 <211> 65 <212> DNA
<213> Homo Sapiens <400> 54 ccgaaatcca gatgatgatg ctcatggacc ctggtgctac acgggaaatc cactcattcc 60 ttggg <210> 55 <211> 70 <212> DNA
<213> Homo Sapiens <400> 55 agaaccgcaa ggtgagcaag gtggagattc tccagcacgt catcgactac atcagggacc 60 ttcagttgga 70 <210> 56 <211> 83 <212> DNA
<213> Homo Sapiens <400> 56 gcatggtagc cgaagatttc acagtcaaaa tcggagattt tggtatgacg cgagatatct 60 atgagacaga ctattaccgg aaa 83 <210> 57 <211> 68 <Z12> DNA
<213> Homo Sapiens <400> 57 acgcaccggg tgtctgatcc caagttccac cccctccatt caaagataat catcatcaag 60 aaagggca 68 <210> 58 <211> 74 <212> DNA
<213> Homo Sapiens <400> 58 39740-0005 PCT.TxT
ccacagctca ccttctgtca ggtgtccatc ccagctccag ccagctccca gagaggaaga 60 gactggcact gagg 74 <210> 59 <211> 77 <212> DNA
<213> Homo Sapiens <400> 59 ccatgatcct cactctgctg gtggactata cactccagac ctcgcttagc atggtaaatc 60 accggctaca aagcttc 77 <210> 60 <211> 78 <212> DNA
<213> Homo Sapiens <400> 60 accgggagcc ctacatgacc gaaaatacct gcaaccgtta ctgccgtgac gagattgagt 60 cagtgaaaga gcttaagg 78 <210> 61 <211> 73 <212> DNA
<213> Homo Sapiens <400> 61 cgaggattgg ttcttcagca agacagagga actgaaccgc gaggtggcca ccaacagtga 60 gctggtgcag agt 73 <210> 62 <211> 80 <212> DNA
<213> Homo Sapiens <400> 62 actcaagcgg aaattgaagc agataggtct tatcagcaca gtctccgcct cctggattca 60 gtgtctcggc ttcagggagt 80 <210> 63 <211> 77 <212> DNA
<213> Homo Sapiens <400> 63 ccgccctcac ctgaagagaa acgcgctcct tggcggacac tgggggagga gaggaagaag 60 cgcggctaac ttattcc 77 <210> 64 <211> 78 <212> DNA
<213> Homo Sapiens <400> 64 tgagcgtcgc agaaaccaca acatcctgga gcgccagcgc cgcaacgacc ttcggtccag 6$
ctttctcacg ctcaggga <210> 65 <211> 70 <212> DNA
<213> Homo Sapiens <400> 65 gcggaaggtc cctcagacat ccccgattga aagaaccaga gaggctctga gaaacctcgg 600 gaaacttaga <210> 66 <211> 66 39740-0005 PCT.TxT
<212> DNA
<213> Homo sapiens~
<400> 66 cggtggacca cgaagagtta acccgggact tggagaagca ctgcagagac atggaagagg 60 cgagcc 66 <210> 67 <211> 68 <212> DNA
<213> Homo Sapiens <400> 67 ctttgaaccc ttgcttgcaa taggtgtgcg tcagaagcac ccaggacttc catttgcttt 60 gtcccggg 68 <210> 68 <211> 81 <212> DNA
<213> Homo Sapiens <400> 68 ccgcaacgtg gttttctcac cctatggggt ggcctcggtg ttggccatgc tccagctgac 60 aacaggagga gaaacccagc a 81 <210> 69 <211> 67 <212> DNA
<213> Homo Sapiens <400> 69 ttgttggtgt gccctggtgc cgtggtggcg gtcactccct ctgctgccag tgtttggaca 60 gaaccca 67 a <210> 70 <211> 62 <212> DNA
<213> Homo Sapiens <400> 70 actgaaggag acccttggag cctaggggca tcggcaggag agtgtgtggg cagggttatt 60 to 62 <Z10> 71 <211> 74 <212> DNA
<213> Homo Sapiens <400> 71 agagccagtt gctgtagaac tcaaatctct gctgggcaag gatgttctgt tcttgaagga 60 ctgtgtaggc ccag 74 <210> 72 <211> 76 <212> DNA
<213> Homo Sapiens <400> 72 cccatggatg ctcctctgaa gagactttcc tcattgactg ccgaggcccc atgaatcaat 60 °gtctggtagc caccgg 76 <210> 73 <211> 72 <212> DNA
<213> Homo Sapiens <400> 73 tgactttatg gagcccaagt ttgagtttgc tgtgaagttc aatgcactgg aattagatga 60 39740-0005 PCT.TXT
cagcgacttg gc 72 <210> 74 <211> 81 <212> DNA
<213> Homo Sapiens <400> 74 cgcttgccta actcatactt tcccgttgac acttgatcca cgcagcgtgg cactgggacg 60 taagtggcgc agtctgaatg g 81 <210> 75 <211> 73 <212> DNA
<213> Homo Sapiens <400> 75 tccttcagct ttcacactgg gctcagaaat gaagttgcat gactcttctg gaagtcaggt 60 gggaacagga ttt 73 <210> 76 <211> 69 <212> DNA
<213> Homo Sapiens <400> 76 agtgggagac acctgacctt tctcaagctg agattgagca gaagatcaag gagtacaatg 69 cccagatca <210> 77 <211> 77 <212> DNA
<213> Homo Sapiens <400> 77 cgaagccctt acaagtttcc tagttcaccc ttacggattc ctggagggaa catctatatt 60 tcacccctga agagtcc 77 <210> 78 <211> 74 <212> DNA
<213> Homo Sapiens <400> 78 ccagacgagc gattagaagc ggcagcttgt gaggtgaatg atttggggga agaggaggag 60 gaggaagagg agga 74 <210> 79 <211> 75 <212> DNA
<213> Homo Sapiens <400> 79 ccattctatc atcaacgggt acaaacgagt cctggccttg tctgtggaga cggattacac 60 cttcccactt gctga .75 <210> 80 <211> 66 <212> DNA
<213> Homo Sapiens <400> 80 tgtggcaagt gcaaatgtaa ggagtgcacc tacccaaggc ctctgccatc agactggatc 60 tgcgac 66 <210> 81 <211> 64 <212> DNA

39740-0005 PCT.TxT
<213> Homo Sapiens <400> 81 cctgaacatg aaggagctga agctgctgca gaccatcggg aagggggagt tcggagacgt 60 gatg 64 <210> 82 <211> 69 <212> DNA
<213> Homo Sapiens <400> 82 catcttccag gaggaccact ctctgtggca ccctggacta cctgccccct gaaatgattg 60 aaggtcgga 69 <210> 83 <211> 80 <212> DNA
<213> Homo Sapiens <400> 83 tgttttgatt cccgggctta ccaggtgaga agtgagggag gaagaaggca gtgtcccttt 60 tgctagagct gacagctttg 80 <210> 84 <211> 79 <212> DNA
<213> Homo Sapiens <400> 84 aagaggaacg gagcgagtcc ccgcgcgcgg cgcgattccc tgagctgtgg gacgtgcacc 60 caggactcgg ctcacacat 79 <210> 85 <211> 68 , <212> DNA
<213> Homo Sapiens <400> 85 gccaactgct ttcatttgtg agggatctga accaatacag agcagacata aaggaaatgg 60 gcctgagt <210> 86 <211> 66 <212> DNA
<213> Homo Sapiens <400> 86 aacaccaatg ggttccatct ttctgggctc ctgattgctc aagcacagtt tggcctgatg 60 aagagg <210> 87 <211> 69 <212> DNA
<213> Homo Sapiens <400> 87 tcaccctctg tgacttcatc gtgccctggg acaccctgag caccacccag aagaagagcc 60 tgaaccaca 69 <210> 88 <211> 70 <212> DNA
<213> Homo Sapiens <400> 88 cgactccgtt ctcagtgtct gacatcttga gtcccctgga ggaaagctac aagaaagtgg 600 gcatggaggg 39740-0005 PCT.TxT
<210> 89 <211> 74 <212> DNA
<213> Homo Sapiens <400> 89 tgctgttgct gagtctgttg ccagtcccca gaagaccatg tctgtgttga gctgtatctg 6~
tgaagccagg caag <210> 90 <211> 70 <212> DNA
<213> Homo Sapiens <400> 90 gtggatgtgc cctgaaggac aagccaggcg tctacacgag agtctcacac ttcttaccct 60 ggatccgcag <210> 91 <211> 83 <212> DNA
<213> Homo Sapiens <400> 91 cctcagcaag acgttatttg aaattacagt gcctctctct caaggcccca aaccagtaac 60 aatcagtttt gccaatcaca ctt 83 <210> 92 <211> 77 <212> DNA
<213> Homo Sapiens <400> 92 gcagttggaa gacacaggaa agtatcccca~aattgcagat ttatcaacgg cttttatctt 60 gaaaatagtg ccacgca 77 <210> 93 <211> 76 <212> DNA
<213> Homo Sapiens <400> 93 agactgtgga gtttgatgtt gttgaaggag aaaagggtgc ggaggcagca aatgttacag 60 gtcctggtgg tgttcc 76 <210> 94 <211> 23 <212> DNA
<213> Artificial Sepuence <220>
<223> primer <400> 94 cgttccgatc ctctatactg cat 23 <210> 95 <211> 22 <212> DNA
<213> Artificial sepuence <220>
<223> primer <400> 95 aggtccctgt tggccttata gg 22 39740-0005 PCT.TXT
<210> 96 <211> 25 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 96 atgcctacag caccctgatg tcgca 25 <210> 97 <211> 20 <212> DNA

<213> Artificial sequence <220>

<Z23> primer <400> 97 cgcttctatg gcgctgagat 20 <210> 98 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 98 tcccggtaca ccacgttctt 20 <210> 99 <211> 24 <21Z> DNA

<213> Artificial sequence <220>

<223> primer <400> 99 cagccctgga ctacctgcac tcgg 24 <210> 100 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 100 tcctgccacc cttcaaacc 19 <210> 101 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 101 ggcggtaaat tcatcatcga a 21 <210> 102 <211> 24 39740-0005 PCT.TxT
<212> DNA

<213> Artificial sequence <220>

<223> primer <400> 102 caggtcacgt ccgaggtcga caca 24 <210> 103 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 103 ggacagcagg aatgtgtttc 20 <210> 104 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 104 acccactcga tttgtttctg 20 <210> 105 <Z11> 22 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 105 cattggctcc ccgtgacctg to 22 <210> 106 <211> 22 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 106 ggctcttgtg cgtactgtcc tt 22 <210> 107 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 107 tcagatgacg aagagcacag atg 23 <210> 108 <211> 29 <212> DNA

<213> Artificial sequence 39740-0005 PCT.TXT
<220>

<223> primer <400> 108 aggctcagtg atgtcttccc tgtcaccag 2g <210> 109 <211> 20 <212> DNA

<213> Artificial sequence <220> ' <223> primer <400> 109 ccattcccac cattctacct 20 <210> 110 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 110 gggaacatag acccaccaat 20 <210> 111 <211> 21 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 111 acaccccaga cgtcctggcc t 21 <210> 112 <211> 18 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 112 ccgccgtgga cacagact 18 <210> 113 <211> 21 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 113 ttgccgtcag aaaacatgtc a 21 <210> 114 <211> 25 <212> DNA

<213> Artificial Sequence <220>

39740-0005 PCT.TXT
<223> primer <400> 114 tgccactcgg aaaaagacct ctcgg 25 <210> 115 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 115 cttttgtgga actctatggg aaca 24 <210> 116 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 116 cagcggttga agcgttcct 19 <210> 117 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 117 ttcggctctc ggctgctgca 20 <210> 118 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 118 ccttccgacc agcagatgaa 20 <210> 119 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 119 tttatatgca cattgggagc tgat 24 <210> 120 <211> 25 <212> DNA

<213> Artificial Sequence <220>

<223> primer 39740-0005 PCT.TxT

<400> 120 caatttgggc aacgagaccg atcct 25 <210> 121 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 121 2~

gtgcaggaaa ggttcacaaa <210> 122 <211> ZO

<212> DNA

<213> Artificial sequence <220>

<223> primer <400> 122 gcacacacga tggagtaagg 20 <210> 123 <211> 24 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 123 agtgtctgcg tccaatacac gcgt <210> 124 <211> 18 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 124 agggagatgc cgcttcgt <210> 125 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 125 ctctcacacc ttgctccaat gta <210> 126 <211> 25 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 126 ccttcatcac agacacagga gggcg 39740-0005 PCT.TxT
<210> 127 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 127 atcctagccc tggtttttgg 20 <210> 128 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 128 ctgccttctc atctgcacaa 20 <210> 129 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 129 tttgctgtca ccagcgtcgc 20 <210> 130 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 130 gaaggccaag aaccgagtca 20 <210> 131 <211> 24 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 131 tccccagtta gttcaaaagt caca 24 <210> 132 <211> 27 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 132 ttatattcca gtttaaggcc aatcctc 27 <210> 133 39740-0005 PCT.TxT
<211> 25 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 133 ccatacctca agtatttgcc atcag 25 <210> 134 <211> 22 -<212> DNA

<213> Artificial sequence <220>

<223> primer <400> 134 agctttgtcc cgtgactgtg to 22 <210> 135 <211> 29 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 135 attgctggag ctgcctttca tttagcact 2g <210> 136 <211> 27 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 136 cctctgtgct acagattata cctttgc 27 <210> 137 <211> 18 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 137 cactgcagcc ccaatgct 18 <210> 138 <211> 22 <212> DNA ' <213> Artificial sequence <220>

<223> primer <400> 138 tacccgccat ccatgatcgc ca 22 <210> 139 <211> 24 <212> DNA

39740-0005 PCT.TXT

<213> Artificial sequence <220>

<223> primer <400> 139 aaagaagatg atgaccgggt ttac 24 <210> 140 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 140 gagcctctgg atggtgcaat 20 <210> 141 <211> 24 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 141 caaactcaac gtgcaagcct cgga 24 <210> 142 <211> 22 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 142 atgctgtggc tccttcctaa ct 22 <210> 143 <211> 27 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 143 acccaaattg tgatatacaa aaaggtt 27 <210> 144 <211> 30 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 144 taccaagcaa cctacatgtc aagaaagccc 30 <210> 145 <211> 24 <212> DNA

<213> Artificial Sequence 39740-0005 PCT.TXT
<220>

<223> primer <400> 145 gcaggtgtca gcaagtatga tcag <210> 146 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 146 tttttccgct gtggtgatga <210> 147 <211> 27 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 147 cgacaggata ttgaccaccg cctcatt <210> 148 <211> 18 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 148 gcccagtgcg gagaacag <210> 149 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 149 aatcacacgc acctggagaa c <210> 150 <211> 30 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 150 ccagcttgat tctcgtctct gcacttaagc <210> 151 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> primer 39740-0005 PCT.TxT
<400> 151 1g atcaccgaca gcacagaca <210> 152 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 152 ~ 20 acctgtgttt ggatttgcag <210> 153 <211> 27 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 153 ccctgctacc aatatggact ccagtca <210> 154 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 154 gacgaagaca gtccctggat <210> 155 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 155 actggggtgg aatgtgtctt <210> 156 <211> 24 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 156 caccgacagc acagacagaa tccc <210> 157 <211> 22 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 157 39740-0005 PCT.TXT

cacacaaaac agaaccagga ct 22 <210> 158 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 158 ctgaagtagc acttccggat t 21 <210> l59 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 159 acccagtgga acccaagcca ttc 23 <210> 160 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 160 ctcataccag ccatccaatg <210> 161 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 161 ttgggttgaa gaaatcagtc c <210> 162 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 162 caccaagccc agaggacagt tcct <210> 163 <211> 18 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 163 tggttcccag ccctgtgt 18 39740-0005 PCT.TxT
<210> 164 <211> 19 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 164 ctcctccacc ctgggttgt <210> 165 <211> 28 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 165 ctccaagccc agattcagat tcgagtca <210> 166 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 166 gtgcaggctc aggtgaagtg <210> 167 <211> 20 <212> DNA

<213> Artificial sequence a.

<220>

<223> primer <400> 167 gacctcaggg cgattcatga <210> 168 <211> 30 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 168 tcagcttcta caactggaca gacaacgctg <210> 169 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 169 gggcgtggaa cagtttatct <210> 170 <211> 19 39740-0005 PCT.TXT
<212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 170 cacggtgaag gtttcgagt <210> 171 <211> 24 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 171 agacatctgc cccaagaagg acgt <210> 172 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 172 aaacgagcag tttgccatca g <210> 173 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 173 gttggtgatg ttccgaagca <210> 174 <211> 24 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 174 cctcaccggc atagactgga agcg <210> 175 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 175 cacagcctca cttctaacct tctg <210> 176 <211> 22 <212> DNA

<213> Artificial sequence 39740-0005 PCT.TXT
<220>

<223> primer <400> 176 attcaat ag ttgaatggcg tgg <210> 177 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 177 acccacccac cactgccaag ctc <210> 178 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 178 ctgaaggagc tccaagacct <210> 179 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 179 caaaaccgct gtgtttcttc <210> 180 <211> 22 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 180 tgctgatgtg ccctctcctt gg <210> 181 <211> 21 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 181 atgtggaacc cccacctact t <210> 182 <211> 22 <212.> DNA

<213> Artificial Sequence <220>

39740-0005 PCT.TXT

<223> primer <400> 182 cagtccacag cacggttata cc 22 <210> 183 <211> 29 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 183 agtcccaaca gaaacaagaa cttcaggcg 29 <210> 184 <211> 22 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 184 gacatttcca gtcctgcagt ca 22 <210> 185 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 185 ctccgatcgc acacatttgt 20 <210> 186 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 186 tgcctctctg ccccaccctt tgt 23 <210> 187 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 187 tctgcagagt tggaagcact cta 23 <210> 188 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> primer 39740-0005 PCT.TxT

<400> 188 gccgaggctt ttctaccaga a 21 <210> 189 <211> 28 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 189 caggatacag ctccacagca tcgatgtc 28 <210> 190 <211> 18 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 190 gggtctgtgc cccatgac <210> 191 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 191 tgaccgtgcc agcatttaca <Z10> 192 <211> 25 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 192 cctggctgcc caagaagtgt tccct <210> 193 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 193 gggaggctta tctcactgag tga <210> 194 <211> 19 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 194 ccattgcagc cttcattgc 39740-0005 PCT.TXT
<210> 195 <211> 29 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 195 ttgaggccca gagcagtcta ccagattct <210> 196 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 196 gaccaaggtc ctggaatgtc <Z10> 197 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 197 gtcttccctg tacccgtagg <210> 198 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 198 caggatgcca ttcaccttct gctg <210> 199 <211> 19 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 199 cacaatggcg gctctgaag <210> 200 <211> 26 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 200 acacaaacac tgtctgtacc tgaaga <210> 201 39740-0005 PCT.TXT
<211> 23 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 201 aagttacgct gcgcgacagc caa 23 <210> 202 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 202 aggacgcaag gagggtttg 19 <210> 203 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 203 gatgtccgcc gagtccttac t 21 <210> 204 <211> 29 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 204 cagtgcctac agtctcgagt ctgccagtg 2g <210> 205 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 205 ctctgagaca gtgcttcgat gact 24 <210> 206 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 206 ccatgaggcc caacttcct 19 <210> 207 <211> 23 <212> DNA

39740-0005 PCT.TxT
<213> Artificial sequence <220>

<223> primer <400> 207 cagacttggt gccctttgac tcc 23 <210> 208 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 208 tgccacctgg acatcatttg 20 <210> 209 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 209 tggacctagg gcttccaagt c 21 <210> 210 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 210 cactcccgag cacgttgttc cgt <210> 211 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 211 tgtcgatgga cttccagaac <Z10> 212 <211> 19 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 212 attgggacag cttggatca <210> 213 <211> 18 <212> DNA

<213> Artificial sequence 39740-0005 PCT.TXT
<220>

<223> primer <400> 213 cacctgggca gctgccaa <210> 214 <211> 22 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 214 gagtcgggct ctggaggaaa ag <210> 215 <211> 18 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 215 ccacaggctc ggacgcac <210> 216 <211> 28 <212> DNA

<213> Artificialsequence <220>

<223> primer <400> 216 agccgtgatc tgtcaccaca taattacc <210> 217 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 217 gatctaagat ggcgactgtc gaa <210> 218 <211> 25 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 218 ttagattccg ttttctcctc ttctg <210> 219 <211> 27 <212> DNA

<213> Artificial Sequence <220>

<223> primer 39740-0005 PCT.TxT
<400> 219 accaccccta ctcctaatcc cccgact 27 <210> 220 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 220 cggttatgtc atgccagata cac 23 <210> 221 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 221 gaactgagac ccactgaaga aagg 24 <210> 222 <211> 25 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 222 cctcaaaggt actccctcct cccgg <210> 223 <211> 25 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 223 tggctcttaa tcagtttcgt tacct <210> 224 <211> 25 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 224 caaggcatat cgatcctcat aaagt <210> 225 <211> 30 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 225 39740-0005 PCT.TXT

tgtcccacga ataatgcgta aattctccag 30 <210> 226 <211> 28 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 226 ataacaaagt gtagctctga catgaatg ~

<210> 227 <211> 24 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 227 cacacctgca gtagttttga ctca <210> 228 <211> 30 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 228 ttgtttgcat ggacagtgca tctatctggt <210> 229 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 229 acggatcaca gtggaggaag <210> 230 <211> ZO

<212> DNA

<213> Artificial sequence <220>

<223> primer <400> 230 ctcatccgtc gggtcatagt <210> 231 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 231 cgctggctca cccctacctg 39740-0005 PCT.TXT
<210> 232 <211> 21 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 232 ggattgctca acaaccatgc t 21 <210> 233 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 233 ggcattaaca cttttggacg ataa <210> 234 <211> 30 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 234 tctggaccct cctacctctg gttcttacgt <210> 235 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 235 ttggtacctg tgggttagca <210> 236 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 236 cacatccaaa tgcaaactgg <210> 237 <211> 26 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 237 tccccagggt agaattcaat cagagc <210> 238 <211> 19 39740-0005 PcT.TxT
<212> DNA

<213> Artificial sequence <220>

<223> primer <400> 238 tgatgcgcct ggaaacagt <210> 239 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 239 cgaggttgtg aaaggtgctt atc <210> 240 <211> 24 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 240 agcaggcaac tccgaaggac aacg <210> 241 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 241 cgaaaagatg ctgaacagtg aca <210> 242 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 242 tcaggaacag ccaccagtga <210> 243 <211> 28 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 243 cttcctcctc ccttctggtc agttggat <210> 244 <211> 20 <212> DNA

<213> Artificial Sequence 39740-0005 PCT.TxT
<220>

<223> primer <400> 244 cccactcagt agccaagtca <210> 245 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 245 cacgcaggtg gtatcagtct <210> 246 <211> 27 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 246 tcaagtaaac gggctgtttt ccaaaca <210> 247 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 247 gactccttcg tccccagttg <210> 248 <211> 21 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 248 tggcacttga aggctctggt a <210> 249 <211> 25 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 249 ttgggcctcc cataattgct ttgcc <210> 250 <211> 20 <212> DNA

<213> Artificial sequence <220>

39740-0005 PCT.TxT

<223> primer <400> 250 cggtgtgaga agtgcagcaa <210> 251 <211> 19 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 251 cctctcgcaa gtgctccat <210> 252 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 252 ccagaccata gcacactcgg gcac <210> 253 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 253 ccgaaatcca gatgatgatg <210> 254 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 254 cccaaggaat gagtggattt <210> 255 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 255 ctcatggacc ctggtgctac acg <210> 256 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> primer 39740-0005 PCT.TXT

<400> 256 agaaccgcaa ggtgagcaa <210> 257 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 257 tccaactgaa ggtccctgat g <210> 258 <211> 26 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 258 tggagattct ccagcacgtc atcgac <210> 259 <211> 21 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 259 gcatggtagc cgaagatttc a <210> 260 <211> 30 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 260 tttccggtaa tagtctgtct catagatatc <210> 261 <211> 28 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 261 cgcgtcatac caaaatctcc gattttga <210> 262 <211> 17 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 262 acgcaccggg tgtctga 39740-0005. PCT.TXT
<210> 263 <Z11> 24 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 263 tgccctttct tgatgatgat tatc 24 <210> 264 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 264 cccaagttcc accccctcca ttca 24 <210> 265 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 265 2~

ccacagctca ccttctgtca <210> 266 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 266 cctcagtgcc agtctcttcc <210> 267 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 267 2~

tccatcccag ctccagccag <210> 268 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 268 ccatgatcct cactctgctg <210> 269 39740-0005 PCT.TXT
<211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 269 gaagctttgt agccggtgat 20 <210> 270 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 270 cactccagac ctcgcttagc atgg <210> 271 <211> 19 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 271 accgggagcc ctacatgac <210> 272 <211> 27 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 272 ccttaagctc tttcactgac tcaatct <210> 273 <211> 28 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 273 aaatacctgc aaccgttact gccgtgac <210> 274 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 274 cgaggattgg ttcttcagca a <210> 275 <211> 22 <212> DNA

39740-0005 PCT.TxT
<213> Artificial Sequence <220>

<223> primer <400> 275 actctgcacc agctcactgt tg <210> 276 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 276 cacctcgcgg ttcagttcct ctgt <210> 277 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 277 actcaagcgg aaattgaagc a <210> 278 <211> 21 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 278 actccctgaa gccgagacac t <210> 279 <211> 28 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 279 aggtcttatc agcacagtct ccgcctcc <210> 280 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 280 ccgccctcac ctgaagaga <210> 281 <211> 22 <212> DNA

<213> Artificial Sequence 39740-0005 PCT.TxT

<220>

<223> primer <400> 281 ggaataagtt agccgcgctt ct 22 <210> 282 <211> 2l <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 282 cccagtgtcc gccaaggagc g 21 <210> 283 <211> 18 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 283 tgagcgtcgc agaaacca 18 <210> 284 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 284 tccctgagcg tgagaaagct 20 <210> 285 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 285 ccagcgccgc aacgaccttc 20 <210> 286 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 286 gcggaaggtc cctcagaca 1g <210> 287 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> primer 39740-0005 PCT.TXT
<400> 287 tctaagtttc ccgaggtttc tca <210> 288 <211> 26 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 288 ccccgattga aagaaccaga gaggct <210> 289 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 289 cggtggacca cgaagagtta a <210> 290 <211> 19 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 290 ggctcgcctc ttccatgtc <210> 291 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 291 ccgggacttg gagaagcact gca <210> 292 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 292 ctttgaaccc ttgcttgcaa <210> 293 <211> 18 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 293 39740-0005 PCT.TxT

cccgggacaa agcaaatg 18 <210> 294 <211> 25 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 294 aagtcctggg tgcttctgac gcaca 25 <210> 295 <211> 19 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> Z95 ccgcaacgtg gttttctca <210> 296 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 296 tgctgggttt ctcctcctgt t <210> 297 <211> 22 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 297 ctcggtgttg gccatgctcc ag <210> 298 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 298 ttgttggtgt gccctggtg <210> 299 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 299 tgggttctgt ccaaacactg g 39740-0005 PCT.TXT
<210> 300 <211> 22 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 300 tggtggcggt cactccctct gc 22 <210> 301 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 301 actgaaggag acccttggag 20 <210> 302 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 302 taaataaccc tgcccacaca <210> 303 <211> 21 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 303 tctcctgccg atgcccctag g <210> 304 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 304 agagccagtt gctgtagaac tcaa <210> 305 <211> 21 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 305 ctgggcctac acagtccttc a <210> 306 <211> 27 39740-0005 PCT.TxT
<212> DNA

<213> Artificial sequence <220>

<223> primer <400> 306 tctctgctgg gcaaggatgt tctgttc 27 <210> 307 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 307 cccatggatg ctcctctgaa <210> 308 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 308 ccggtggcta ccagacattg <210> 309 <211> 22 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 309 cattgactgc cgaggcccca tg <210> 310 <211> 21 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 310 tgactttatg gagcccaagt t <210> 311 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 311 gccaagtcgc tgtcatctaa <210> 312 <211> 25 <212> DNA

<213> Artificial Sequence 39740-0005 PCT.TxT
<220>

<223> primer <400> 312 ttccagtgca ttgaacttca cagca 25 <210> 313 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 313 cgcttgccta actcatactt tcc 23 <210> 314 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 314 ccattcagac tgcgccactt <210> 315 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 315 tccacgcagc gtggcactg <210> 316 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 316 tccttcagct ttcacactgg <210> 317 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 317 aaatcctgtt cccacctgac 20 <210> 318 <211> 29 <212> DNA

<213> Artificial sequence <220>

39740-0005 PCT.TxT
<223> primer <400> 318 tccagaagag tcatgcaact tcatttctg <210> 319 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 319 agtgggagac acctgacctt <210> 320 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 320 tgatctgggc attgtactcc <210> 321 <211> 29 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 321 ttgatcttct gctcaatctc agcttgaga <210> 322 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 322 cgaagccctt acaagtttcc <210> 323 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 323 ggactcttca ggggtgaaat <210> 324 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> primer 39740-0005 PCT.TxT

<400> 324 cccttacgga ttcctggagg gaac 24 <210> 325 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 325 ccagacgagc gattagaagc 20 <210> 326 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 326 tcctcctctt cctcctcctc <210> 327 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 327 tgtgaggtga atgatttggg gga <210> 328 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 328 ccattctatc atcaacgggt acaa <210> 329 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 329 tcagcaagtg ggaaggtgta atc <210> 330 <211> 25 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 330 tctccacaga caaggccagg actcg 39740-0005 PCT.TxT
<210> 331 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 331 tgtggcaagt gcaaatgtaa 20 <210> 332 <211> 20 <212> DNA

<213> Artificial sequence <Z20>

<223> primer <400> 332 gtcgcagatc cagtctgatg <210> 333 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 333 cagaggcctt gggtaggtgc actc <210> 334 <211> 40 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 334 cctgaacatg aaggagctga cctgaacatg aaggagctga <210> 335 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 335 catcacgtct ccgaactcc <210> 336 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 336 tcccgatggt ctgcagcagc t <210> 337 39740-0005 PCT.TXT
<211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 337"

catcttccag gaggaccact <210> 338 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 338 tccgaccttc aatcatttca <210> 339 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 339 ctctgtggca ccctggacta cctg <210> 340 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 340 tgttttgatt cccgggctta <210> 341 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 341 caaagctgtc agctctagca aaag <210> 342 <211> 28 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 342 tgccttcttc ctccctcact tctcacct <210> 343 <211> 19 <212> DNA

39740-0005 PCT.TxT
<213> Artificial sequence <220>

<223> primer <400> 343 aagaggaacg gagcgagtc <210> 344 <211> 19 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 344 atgtgtgagc cgagtcctg <210> 345 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 345 cacgtcccac agctcaggga atc <210> 346 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 346 gccaactgct ttcatttgtg <210> 347 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 347 actcaggccc atttccttta <210> 348 <211> 28 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 348 agggatctga accaatacag agcagaca <210> 349 <211> 20 <212> DNA

<213> Artificial Sequence 39740-0005 PCT.TxT
<220>

<223> primer <400> 349 aacaccaatg ggttccatct 20 <210> 350 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 350 cctcttcatc aggccaaact 20 <210> 351 <211> 24 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 351 ttctgggctc ctgattgctc aagc <210> 352 <211> 22 <212> DNA

<213> Artificial sequence .

<220>

<223> primer <400> 352 tcaccctctg tgacttcatc gt <210> 353 <211> 22 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 353 tgtggttcag gctcttcttc tg <210> 354 <211> 22 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 354 ccctgggaca ccctgagcac ca <210> 355 <211> 22 <212> DNA

<213> Artificial sequence <220>

<223> primer 39740-0005 PCT.TXT
<400> 355 cgactccgtt ctcagtgtct ga <210> 356 <211> 19 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 356 ccctccatgc ccactttct <210> 357 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 357 atcttgagtc ccctggagga aagc <210> 358 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 358 tgctgttgct gagtctgttg <210> 359 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 359 cttgcctggc ttcacagata <210> 360 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 360 ccagtcccca gaagaccatg tctg <210> 361 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 361 39740-0005 PCT.TxT

gtggatgtgc cctgaagga 19 <210> 362 <211> 20 <212> DNA

<213>_Artificial sequence <220>

<223> primer <400> 362 ctgcggatcc agggtaagaa 20 <210> 363 <211> 28 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 363 2~

aagccaggcg tctacacgag agtctcac <210> 364 <211> 25 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 364 cctcagcaag acgttatttg aaatt <210> 365 <211> 24 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 365 aagtgtgatt ggcaaaactg attg <210> 366 <211> 26 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 366 cctctctctc aaggccccaa accagt <210> 367 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 367 gcagttggaa gacacaggaa agt 39740-0005 PCT.TXT
<210> 368 <211> 21 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 368 tgcgtggcac tattttcaag a 21 <210> 369 <211> 27 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 369 tccccaaatt gcagatttat caacggc 27 <210> 370 <211> 25 <212> DNA

<213> Artificial Sequence <220>

<223> primer <400> 370 agactgtgga gtttgatgtt gttga 25 <210> 371 <211> 22 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 371 ggaacaccac caggacctgt as 22 <210> 372 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> primer <400> 372 ttgctgcctc cgcacccttt tct 23

Claims (55)

WHAT IS CLAIMED IS:

1. 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 the RNA
transcript of at least one gene selected from the group consisting of CD44v3; CD44v6; DR5;
GRO1;
KRT17; and LAMC2, or their product, and (b) identifying the patient as likely to show resistance to treatment with an EGFR-inhibitor if the normalized expression levels of said gene or genes, or their products, are elevated above a defined expression threshold.

2. The method of claim 1 wherein the patient is identified as likely to show resistance to treatment with an EGFR-inhibitor if the expression level of the RNA
transcript of LAMC2 is elevated above a defined expression threshold.

3. The method of claim 1 wherein the levels of the RNA transcripts of said genes are normalized relative to the mean level of the RNA transcript or the product of two or more housekeeping genes.

4. The method of claim 3 wherein the housekeeping genes are selected from the group consisting of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Cyp1, albumin, actins, tubulins, cyclophilin hypoxantine phosphoribosyltransferase (HRPT), L32, 28S, and 18S.

5. The method of claim 3 wherein the sample is subjected to global gene expression analysis of all genes present above the limit of detection.

6. The method of claim 5 wherein the levels of the RNA transcripts of said genes are normalized relative to the mean signal of the RNA transcripts or the products of all assayed genes or a subset thereof.

7. The method of claim 6 wherein the level of RNA transcripts is determined by quantitative RT-PCR (qRT-PCR), and the signal is a Ct value.

8. The method of claim 7 wherein the assayed genes include at least 50 cancer related genes:

9. The method of claim 7 wherein the assayed genes includes at least 100 cancer related genes.

10. The method of claim 1 wherein said patient is human.

11. The method of claim 10 wherein said sample is a fixed, paraffin-embedded tissue (FPET) sample, or fresh or frozen tissue sample.

12. The method of claim 10 wherein said sample is a tissue sample from fine needle, core, or other types of biopsy.

13. The method of claim 10 wherein said quantitative analysis is performed by qRT-PCR.

14. The method of claim 10 wherein said quantitative analysis is performed by quantifying the products of said genes.

15. The method of claim 14 wherein said products are quantified by immunohistochemistry or by proteomics technology.

16. The method of claim 10 wherein the EGFR-expressing cancer is selected from the group consisting of head and neck cancer, colon cancer, breast cancer, ovarian cancer, pancreatic cancer, and non-small cell lung carcinoma.

17. The method of claim 16 wherein said cancer is head and neck cancer or colon cancer.

18. The method of claim 10 further comprising the step of preparing a report comprising a statement whether the patient is likely to respond well to treatment with an EGFR inhibitor.

19. The method of claim 10 further comprising the step of preparing a report comprising a statement whether the patient is likely to show resistance to treatment with an EGFR inhibitor.

20. 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 normalized level of one or more prognostic RNA
transcripts or their products in a sample comprising EGFR-expressing cancer cells obtained from said 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 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; NMYG; PAI1; PDGFA; PGK1; PTPD1;
RANBP2; SPRY2; TP53BP1; and VEGFC, or the corresponding gene product, above a defined threshold expression level indicates that the patient is likely to show resistance to treatment with an EGFR inhibitor, and expression of one or more of CD44s;
CD82; CGA;
CTSL; EGFRd27; IGFBP3; p27; P53; RB1; TIMP2; and YB-1, or the corresponding gene product, above a defined threshold expression level indicates that the patient is likely to respond well to treatment with an EGFR inhibitor.

21. The method of claim 20 comprising determining the normalized levels of at least two of said prognostic transcripts or their expression products.

22. The method of claim 20 comprising determining the normalized levels of at least 5 of said prognostic transcripts or their expression products.

23. The method of claim 20 comprising determining the normalized levels of all of said prognostic transcripts or their expression products.

24. The method of claim 20 wherein said sample is a tissue sample.

25. The method of claim 24 wherein said tissue is fixed, paraffin-embedded, or fresh, or frozen.

26. The method of claim 24 wherein said tissue is from fine needle, core, or other types of biopsy.

27. The method of claim 20 further comprising the step of preparing a report comprising a statement whether said patient is likely to respond well to treatment with an EGFR inhibitor.

28. The method of claim 20 further comprising the step of preparing a report comprising a statement whether said patient is likely to show resistance to treatment with an EGFR inhibitor.

29. 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; and YB-1 genes, or the corresponding gene products in said cancer, or decreased normalized expression of one or more of the RNA
transcripts 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; and VEGFC genes, or the corresponding gene products in said cancer, with an effective amount of an EGFR-inhibitor, wherein, for each gene, elevated or decreased normalized expression is defined by a defined expression threshold value.

30. The method of claim 29 wherein said patient has been determined to have elevated or decreased normalized expression of all of said RNA transcripts or the corresponding gene products.

31. 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 normalized level of one or more prognostic RNA
transcripts or their products in a sample comprising EGFR-expressing cancer cells obtained from said patient, wherein the prognostic transcript is the transcript of one or more genes selected from the group consisting of: Bak; Bclx; BRAE; 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 the normalized level 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, when above a defined expression threshold value, indicates that the patient is likely to show resistance to treatment with an EGFR inhibitor, and the normalized level 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; and XIAP, or the corresponding gene product, when above a defined expression threshold value, indicates that the patient is likely to respond well to treatment with an EGFR inhibitor.

32. The method of claim 31 comprising determining the normalized levels of at least two of said prognostic transcripts or their expression products.

33. The method of claim 31 comprising determining the normalized levels of at least 5 of said prognostic transcripts or their expression products.

34. The method of claim 31 comprising determining the normalized levels of all of said prognostic transcripts or their expression products.

35: The method of claim 31 wherein said sample is a tissue sample.

36. The method of claim 35 wherein the tissue is fixed, paraffin-embedded, or fresh, or frozen.

37. The method of claim 35 wherein the tissue is from fine needle, core, or other types of biopsy.

38. The method of claim 31 further comprising the step of preparing a report comprising a statement whether the patient is likely to respond well to treatment with an EGFR inhibitor.

39. The method of claim 31 further comprising the step of preparing a report comprising a statement whether the patient is likely to show resistance to treatment with an EGFR inhibitor.

40. 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; PTPDl; RPLPO; STK15; SURV; TERC; TGFBR2; TITF1; and XIAP genes, or the corresponding gene products in said cancer, or decreased normalized expression of one or more of the RNA transcripts of CA9; CD134; CD44E; CD44v3;
CD44v6; CDC25B; CGA; DRS; GRO1; KRT17; LAMC2; P14ARF; PDGFB; PLAUR;
PPARG; RASSF1; RIZ1; Src; TFRC; and UPA genes, or the corresponding gene products, with an effective amount of an EGFR-inhibitor, wherein for each gene elevated or decreased normalized expression is determined relative to a defined expression threshold.

41. An array comprising polynucleotides hybridizing to the following genes:
Bak; Bclx; BRAF; BRK; Cadl7; CCND3; CD105; CD44s; CD82; CD9; CGA;; GTSL;
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; DRS; EDNI endothelia; 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, immobilized on a solid surface.

42. The array of claim 41 wherein said polynucleotides are cDNAs.

43. The array of claim 42 wherein said cDNAs are about 500 to about 5000 bases.

44. The array of claim 41 wherein said polynucleotides are oligonucleotides.

45. The array of claim 44 wherein said oligonucleotides are about 20 to 80 bases long.

46. The array of claim 45 which comprises about 330,000 oligonucleotides.

47. The array of claim 41 wherein said solid surface is glass.

48. An array comprising polynucleotides hybridizing to the following genes:
CD44v3; CD44v6; DR5; GRO1; KRT17; LAMC2.

49. An array comprising polynucleotides hybridizing to the following genes:
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; CD44s; CD82;
CGA; CTSL; EGFRd27; IGFBP3; p27; P53; RB1; TIMP2; and YB-1.

50. An array comprising polynucleotides hybridizing to the following genes:
CA9; CD134;.CD44E; CD44v3; CD44v6; CDC25B; CGA; DR5; GRO1; KRT17;
LAMC2; P14ARF; PDGFB; PLAUR; PPARG; RASSF1; RIZ1; Src; TFRC; UPA;
CD44s; CD82; CGA; CTSL; EGFRd27; IGFBP3; p27; P53; RB1; TIMP2; and YB-1.

51. The method of any one of claims 1, 20 and 31, wherein RNA is isolated from said tissue by a procedure comprising:
(a) incubating a section of said 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 said lysis solution.

52. 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 method of any one of claims 1, 20 and 30.

53. A method for amplification of a gene listed in Tables 5A and 5B by polymerase chain reaction (PCR), comprising performing said PCR by using an amplicon listed in Table 5A and 5B and a corresponding primer-probe set listed in Tables 6A-6F.

54. A PCR primer-probe set listed in Tables 6A-6F.

55. A PCR amplicon listed in Tables 5A and 5