CA2513117A1 - Gene expression markers for breast cancer prognosis - Google Patents

Gene expression markers for breast cancer prognosis Download PDF

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CA2513117A1
CA2513117A1 CA002513117A CA2513117A CA2513117A1 CA 2513117 A1 CA2513117 A1 CA 2513117A1 CA 002513117 A CA002513117 A CA 002513117A CA 2513117 A CA2513117 A CA 2513117A CA 2513117 A1 CA2513117 A1 CA 2513117A1
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dna
artificial sequence
expression
genes
tp53bp2
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CA2513117C (en
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Melody A. Cobleigh
Steve Shak
Joffre B. Baker
Maureen T. Cronin
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Genomic Health Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Abstract

The present invention provides gene sets the expression of which is important in the diagnosis and/or prognosis of breast cancer.

Description

Gene Exuression Markers for Breast Cancer Prognosis Background of the Invention Field of the Invention The present invention provides genes and gene sets the expression of which is important in the diagnosis and/or prognosis of breast cancer.
Description of the Related Art Oncologists have a number of treatment options available to them, including different combinations of chemotherapeutic drugs that are characterized as "standard of care," and a number of drugs that do not carry a label claim for particular cancer, but for which there is evidence of efficacy in that cancer. Best likelihood of good treatment outcome requires that patients be assigned to optimal available cancer treatment, and that this assignment be made as quickly as possible following diagnosis.
Currently, diagnostic tests used in clinical practice are single analyte, and therefore do not capture the potential value of knowing relationships between dozens of different markers.
Moreover, diagnostic tests are frequently not quantitative, relying on imnnunohistochemistry.
This method often yields different results in different laboratories, in part because the reagents are not standardized, and in part because the interpretations are subjective and cannot be easily quantified. RNA-based tests have not often been used because of the problem of RNA
degradation over time and the fact that it is difficult to obtain fresh tissue samples from patients for analysis. Fixed paraffin-embedded tissue is more readily available and methods have been established to detect RNA in fixed tissue. However, these methods typically do not allow for the study of large numbers of genes (DNA or RNA) from small amounts of material.
Thus, traditionally fixed tissue has been rarely used other than for immunohistochemistry detection of proteins.
Recently, several groups have published studies concerning the classification of various cancer types by microarray gene expression analysis (see, e.g. Golub et al., Science 286:531-537 (1999); Bhattacharjae et al., Proc. Natl. Acad. Sci. LISA 98:13790-13795 (2001);
Chen-Hsiang et al., Bioihfof~matics 17 (Suppl. 1):5316-5322 (2001); Ramaswamy et al., Pr~oc.
Natl. Acad. Sci. LISA 98:15149-15154 (2001)). Certain classifications of human breast cancers based on gene expression patterns have also been reported (Martin et al., Cancer Res.
60:2232-2238 (2000); West et al., Proc. Natl. Acad. Sci. USA 98:11462-11467 (2001); Sorlie et al., Proc. Natl. Acad. Sci. USA 98:10869-10874 (2001); Yan et al., 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 provide new insights into the relationships of the differentially expressed genes, and 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 hundreds of 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 maximize outcome. Hence, a need exists for tests that simultaneously provide predictive information about patient responses to the variety of treatment options.
This is particularly true for breast cancer, the biology of which is poorly understood. It is clear that the classification of breast cancer into a few subgroups, such as ErbB2+ subgroup, and subgroups characterized by low to absent gene expression of the estrogen receptor (ER) and a few additional transcriptional factors (Perou et al., Nature 406:747-752 (2000)) does not reflect the cellular and molecular heterogeneity of breast cancer, and does not allow the design of treatment strategies maximizing patient response.
Summary of the Invention The present invention provides a set of genes, the expression of which has prognostic value, specifically with respect to disease-free survival.
The present invention accommodates the use of archived paxaffin-embedded biopsy material for assay of all markers in the set, and therefore is compatible with the most widely
2
3 PCT/US2004/000985 available type of biopsy material. It is also compatible with several different methods of tumor tissue harvest, for example, via core biopsy or fine needle aspiration.
Further, for each member of the gene set, the invention specifies oligonucleotide sequences that can be used in the test.
In one aspect, the invention concerns a method of predicting the likelihood of long-term survival of a breast cancer patient without the recurrence of breast cancer, comprising determining the expression level of one or more prognostic RNA transcripts or their expression products in a breast cancer tissue sample obtained from the patient, normalized against the expression level of all RNA transcripts or their products in the breast cancer tissue sample, or of a reference set of RNA transcripts or their expression products, wherein the prognostic RNA transcript is the transcript of one or more genes selected from the group consisting of TP53BP2, GRB7, PR, CD68, Bcl2, KRT14, IRS1, CTSL, EstRl, Chkl, IGFBP2, BAG1, CEGP1, STK15, GSTM1, FHIT, RIZl, AIB1, SURV, BBC3, IGF1R, p27, GATA3, ZNF217, EGFR, CD9, MYBL2, HIFla, pS2, ErbB3, TOP2B, MDM2, RADS1C, KRT19, TS, Her2, KLK10, (3-Catenin, y-Catenin, MCM2, PI3KC2A, IGF1, TBP, CCNBl, FBXOS, and DRS, wherein expression of one or more of GRB7, CD68, CTSL, Chkl, AIB1, CCNB1, MCM2, FBXOS, Her2, STK15, SLTRV, EGFR, MYBL2, HIFla, and TS indicates a decreased likelihood of long-term survival without breast cancer recurrence, and the expression of one or more of TP53BP2, PR, Bcl2, KRT14, EstRl, IGFBP2, BAG1, CEGP1, KLK10, (3-Catenin, y-Catenin, DRS, PI3KCA2, RADS1C, GSTM1, FHIT, RIZ1, BBC3, TBP, p27, IRS1, IGF1R, GATA3, ZNF217, CD9, pS2, ErbB3, TOP2B, MDM2, IGF1, and KRT19 indicates an increased likelihood of long-term survival without breast cancer recurrence.
In a particular embodiment, the expression levels of at least two, or at least 5, or at least 10, or at least 15 of the prognostic RNA transcripts or their expression products are determined. In another embodiment, the method comprises the determination of the expression levels of all prognostic RNA transcripts or their expression products.
In another particular embodiment, the breast cancer is invasive breast carcinoma.
In a fixrther embodiment, RNA is isolated from a fixed, wax-embedded breast cancer tissue specimen of the patient. Isolation may be performed by any technique known in the art, for example from core biopsy tissue or fine needle aspirate cells.

In another aspect, the invention concerns an array comprising polynucleotides hybridizing to two or more of the following genes: oc-Catenin, AIB1, AKT1, AKT2, (3-actin, BAGl, BBC3, Bcl2, CCNB1, CCND1, CD68, CD9, CDH1, CEGP1, Chkl, CIAPl, cMet.2, Contig 27882, CTSL, DRS, EGFR, EIF4E, EPHX1, ErbB3, EstRl, FBXOS, FHIT1 FRP1, GAPDH, GATA3, G-Catenin, GRB7, GROl, GSTM1, GUS, HER2, HIF1A, HNF3A, IGF1R, IGFBP2, KLK10, KRT14, KRT17, KRT18, KRT19, KRTS, Maspin, MCM2, MCM3, MDM2, MMP9, MTAl, MYBL2, P14ARF, p27, P53, PI3KC2A, PR, PRAME, pS2, RADS1C,.3RB1, RIZl, STK15, STMY3, SURV, TGFA, TOP2B, TP53BP2, TRAIL, TS, upa, VDR, VEGF, and ZNF217.
In particular embodiments, the array comprises polynucleotides hybridizing to at least 3, or at least 5, or at least 10, or at least 15, or at least 20, or all of the genes listed above.
In another specific embodiment, the array comprises polynucleotides hybridizing to the following genes: TP53BP2, GRB7, PR, CD68, Bcl2, KRT14, IRS1, CTSL, EstRl, Chkl, IGFBP2, BAG1, CEGP1, STK15, GSTM1, FHIT, RIZ1, A1B1, SURV, BBC3, IGF1R, p27, GATA3, ZNF217, EGFR, CD9, MYBL2, HIFla, pS2, RIZl, ErbB3, TOP2B, MDM2, RADS1C, KRT19, TS, Her2, KLK10, (i-Catenin, y-Catenin, MCM2, PI3KC2A, IGF1, TBP, CCNB1, FBXOS and DRS.
The polynucleotides can be cDNAs, or oligonucleotides, and the solid surface on which they are displayed may, for example, be glass.
In another aspect, the invention concerns a method of predicting the likelihood of long-term survival of a patient diagnosed with invasive breast cancer, without the recurrence of breast cancer, comprising the steps of (1) determining the expression levels of the RNA transcripts or the expression products of genes or a gene set selected from the group consisting of (a) TP53BP2, Bcl2, BAD, EPHXl, PDGFR~i, DIABLO, XIAP, YBl, CA9, and KRT8;
(b) GRB7, CD68, TOP2A, Bcl2, DIABLO, CD3, ID1, PPM1D, MCM6, and WISP1;
(c) PR, TP53BP2, PRAME, DIABLO, CTSL, IGFBP2, TI1VIP1, CA9, MMP9, and COX2;
(d) CD68, GRB7, TOP2A, Bcl2, DIABLO, CD3, ll~l, PPM1D, MCM6, and WISP1;
(e) Bcl2, TP53BP2, BAD, EPHX1, PDGFR(3, DIABLO, XIAP, YB1, CA9, and KRT8;
(f) KRT14, KRTS, PRAME, TP53BP2, GUS1, AIB1, MCM3, CCNEl, MCM6, and ID1;
4 (g) PRAME, TP53BP2, EstRl, DIABLO, CTSL, PPM1D, GRB7, DAPKl, BBC3, and VEGFB;
(h) CTSL2, GRB7, TOP2A, CCNB1, Bcl2, DIABLO, PRAME, EMS1, CA9, and EpCAM;
(i) EstRl, TP53BP2, PRAME, DIABLO, CTSL, PPM1D, GRB7, DAPKl, BBC3, and VEGFB;
(k) Chkl, PRAME, TP53BP2, GRB7, CA9, CTSL, CCNB 1, TOP2A, tumor size, and IGFBP2;
(1) IGFBP2, GRB7, PRAME, DIABLO, CTSL, (3-Catenin, PPM1D, Chkl, WISP1, and LOT1;
(m) HER2, TP53BP2, Bcl2, DIABLO, TIMP1, EPHX1, TOP2A, TRAIL, CA9, and AREG;
(n) BAG1, TP53BP2, PRAME, IL6, CCNB1, PAI1, AREG, tumor size, CA9, and Ki67;
(o) CEGP1, TP53BP2, PRAME, DIABLO, Bcl2, COX2, CCNE1, STK15, and AKT2, and FGF18;
(p) STK15, TP53BP2, PRAME, IL6, CCNE1, AKT2, DIABLO, cMet, CCNE2, and COX2;
(q) KLK10, EstRl, TP53BP2, PRAMS, DIABLO, CTSL, PPM1D, GRB7, DAPKl, and BBC3;
(r) AIB1, TP53BP2, Bcl2, DIABLO, TIMP1, CD3, p53, CA9, GRB7, and EPHX1 (s) BBC3, GRB7, CD68, PRAMS, TOP2A, CCNB1, EPHX1, CTSL
GSTM1, and APC;
(t) CD9, GRB7, CD68, TOP2A, Bcl2, CCNB1, CD3, DIABLO, ID1, and PPM1D;
(w) EGFR, KRT14, GRB7, TOP2A, CCNB1, CTSL, Bcl2, TP, KLK10, and CA9;
(x) HIFla, PR, DIABLO, PRAMS, Chkl, AKT2, GRB7, CCNEl, TOP2A, and CCNB1;
(y) MDM2, TP53BP2, DIABLO, Bcl2, AIB1, TllVn'1, CD3, p53, CA9, and HER2;
(z) MYBL2, TP53BP2, PRAMS, Ih6, Bcl2, DIABLO, CCNE1, EPHX1, TI1VVIP1, and CA9;
(aa) p27, TP53BP2, PRAMS, DIABLO, Bcl2, COX2, CCNEl, STK15, AKT2, and ID1;
(ab) RAD51, GRB7, CD68, TOP2A, CIAP2, CCNB1, BAG1, IL6, FGFRl, and TP53BP2;
(ac) SLTRV, GRB7, TOP2A, PRAMS, CTSL, GSTM1, CCNB1, VDR, CA9; and CCNE2;
(ad) TOP2B, TP53BP2, DIABLO, Bcl2, TM'1, AIBl, CA9, p53, KRTB, and BAD;
5 (ae) GNF217, GRB7, TP53BP2, PRAME, DIABLO, Bcl2, COX2, CCNE1, APC4, and (3-Catenin, in a breast cancer tissue sample obtained from the patient, normalized against the expression levels of all RNA transcripts or their expression products in said breast cancer tissue sample, or of a reference set of RNA transcripts or their products;
(2) subjecting the data obtained in step (1) to statistical analysis; and (3) determining whether the likelihood of said long-term survival has increased or decreased.
In a further aspect, the invention concerns a method of predicting the likelihood of long-term survival of a patient diagnosed with estrogen receptor (ER)-positive invasive breast cancer, without the recurrence of breast cancer, comprising the steps of:
(1) determining the expression levels of the RNA transcripts or the expression products of genes of a gene set selected from the group consisting of CD6~;
CTSL; FBXOS;
SURV; CCNB1; MCM2; Chkl; MYBL2; HIFlA; cMET; EGFR; TS; STK15, IGFRl; BC12;
HNF3A; TP53BP2; GATA3; BBC3; RADS1C; BAGl; IGFBP2; PR; CD9; RBl; EPHX1;
CEGP1; TRAIL; DRS; p27; p53; MTA; RIZ1; ErbB3; TOP2B; EIF4E, wherein expression of the following genes in ER-positive cancer is indicative of a reduced likelihood of survival without cancer recurrence following surgery: CD6~; CTSL; FBXOS; SURV; CCNB1;
MCM2; Chkl; MYBL2; HIF1A; cMET; EGFR; TS; STK15, and wherein expression of the following genes is indicative of a better prognosis for survival without cancer recurrence following surgery: IGFR1; BCl2; HNF3A; TP53BP2; GATA3; BBC3; RADS1C; BAG1;
IGFBP2; PR; CD9; RB1; EPHX1; CEGP1; TRAIL; DRS; p27; p53; MTA; RIZ1; ErbB3;
TOP2B; EIF4E.
(2) subjecting the data obtained in step (1) to statistical analysis; and (3) determining whether the likelihood of said long-term survival has increased or decreased.
In yet another aspect, the invention concerns a method of predicting the likelihood of long-term survival of a patient diagnosed with estrogen receptor (ER)-negative invasive breast cancer, without the recurrence of breast cancer, comprising determining the expression levels of the RNA transcripts or the expression products of genes of the gene set CCNDl; UPA;
HNF3A; CDH1; Her2; GRB7; AKT1; STMY3; a-Catenin; VDR; GRO1; KT14; KLK10;
Maspin, TGFa, and FRP1, wherein expression of the following genes is indicative of a
6 reduced likelihood of survival without cancer recurrence: CCNDl; UPA; HNF3A;
CDH1;
Her2; GRB7; AI~T1; STMY3; a-Cateiun; VDR; GRO1, and wherein expression of the following genes is indicative of a better prognosis for survival without cancer recurrence:
I~T 14; KI,Kl 0; Maspin, TGFa, and FRP 1.
In a different aspect, the invention concerns a method of preparing a personalized genomics profile for a patient, comprising the steps of (a) subjecting RNA extracted from a breast tissue obtained from the patient to gene expression analysis;
(b) determining the expression level of one or more genes selected from the breast cancer gene set listed in any one of Tables 1-5, wherein the expression level is normalized against a control gene or genes and optionally is compared to the amount found in a breast cancer reference tissue set; and (c) creating a report summarizing the data obtained by the gene expression analysis.
The report may, for example, include prediction of the likelihood of long term survival of the patient and/or recommendation for a treatment modality of said patient.
In a fixrther aspect, the invention concerns a method for amplification of a gene listed in Tables SA and B by polymerase chain reaction (PCR), comprising performing said PCR by using an amplicon listed in Tables SA and B and a primer-probe set listed in Tables 6A-F.
In a still further aspect, the invention concerns a PCR amplicon listed in Tables SA and B.
In yet another aspect, the invention concerns a PCR primer-probe set listed in Tables 6A-F.
The invention further concerns a prognostic method comprising:
(a) subjecting a sample comprising breast 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 GRB7, CD68, CTSL, Chkl, AIB1, CCNB1, MCM2, FBXOS, Her2, STK15, SURV, EGFR, MYBL2, HIFla, and TS, or their product, and (b) identifying the patient as likely to have a decreased likelihood of long-term survival without breast cancer recurrence if the normalized expression levels of the gene or genes, or their products, are elevated above a defined expression threshold.
In a different aspect, the invention concerns a prognostic method comprising:
7 (a) subjecting a sample comprising breast 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 TP53BP2, PR, Bcl2, KRT14, EstRl, IGFBP2, BAGl, CEGPl, KLK10, (3-Catenin, 'y-Catenin, DRS, PI3KCA2, RADS1C, GSTM1, FHIT, RIZl, BBC3, TBP, p27, IRS1, IGF1R, GATA3, ZNF217, CD9, pS2, ErbB3, TOP2B, MDM2, IGF1, and KRT 19, and (b) identifying the patient as likely to have an increased likelihood of long-term survival without breast cancer recurrence if the normalized expression levels of the gene or genes, or their products, are elevated above a defined expression threshold.
The invention further 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 any of the foregoing methods.
8 Brief Description of the Drawings Table 1 is a list of genes, expression of which correlate with breast cancer survival.
Results from a retrospective clinical trial. Binary statistical analysis.
Table 2 is a list of genes, expression of which correlates with breast cancer survival in estrogen receptor (ER) positive patients. Results from a retrospective clinical trial. Binary statistical analysis.
Table 3 is a list of genes, expression of which correlates with breast cancer survival in estrogen receptor (ER) negative patients. Results from a retrospective clinical trial. Binary statistical analysis.
Table 4 is a list of genes, expression of which correlates with breast cancer survival.
Results from a retrospective clinical trial. Cox proportional hazards statistical analysis.
Tables SA and B show a list of genes, expression of which correlate with breast cancer survival. Results from a retrospective clinical trial. The table includes accession numbers for the genes, and amplicon sequences used for PCR amplification.
Tables 6A-6F The table includes sequences for the forward and reverse primers (designated by "f' and "r", respectively) and probes (designated by "p") used for PCR
amplification of the amplicons listed in Tables SA-B.
Detailed Description of the Preferred Embodiment A. Definitions Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J.
Wiley & Sons (New York, 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.
9 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 an oligonucleotide. The term "polynucleotide"
specifically includes cDNAs. The term includes DNAs (including cDNAs) and RNAs that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritiated bases, are included within the term "polynucleotides" as defined herein. In general, the term "polynucleotide" embraces all chemically, enzymatically and/or metabolically modified forms of unmodified polynucleotides, as well as the chemical forms of DNA and RNA
characteristic of viruses and cells, including simple and complex cells.
The term "oligonucleotide" refers to a relatively short polynucleotide, including, without limitation, single-stranded deoxyribonucleotides, single- or double-stranded ribonucleotides, RNA:DNA hybrids and double-stranded DNAs. Oligonucleotides, such as single-stranded DNA probe oligonucleotides, are often synthesized by chemical methods, for example using automated oligonucleotide synthesizers that are commercially available.
However, oligonucleotides can be made by a variety of other methods, including ih 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 higher or lower level in a subject suffering from a disease, specifically cancer, such as breast cancer, relative to its expression in a normal or control subject. The terms also include genes whose expression is activated to a higher or lower level at different stages of the same disease.
It is also understood that a differentially expressed gene may be either activated or inhibited at the nucleic acid level or protein level, or may be subject to alteniative 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 subj ect.
The phrase "gene amplification" refers to a process by which multiple copies of a gene or gene fragment are formed in a particular cell or cell line. The duplicated region (a stretch of amplified DNA) is often referred to as "amplicon." Usually, the amount of the messenger RNA (mRNA) produced, i.e., the level of gene expression, also increases in the proportion of the number of copies made of the particular gene expressed.
The term "diagnosis" is used herein to refer to the identification of a molecular or pathological state, disease or condition, such as the identification of a molecular subtype of head and neck cancer, colon cancer, or other type of cancer.
The term "prognosis" is used herein to refer to the prediction of the likelihood of cancer-attributable death or progression, including recurrence, metastatic spread, and drug resistance, of a neoplastic disease, such as breast cancer.
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 sugery 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 3 years, more preferably for at least 8 years, most preferably for at least 10 years following surgery or other treatment.
The term "tumor," as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include but are not limited to, breast cancer, colon cancer, lung cancer, prostate cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, 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.
"Stringency" of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so.
For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biolo~y, Wiley Interscience Publishers, (1995).
"Stringent conditions" or "high stringency conditions", as defined herein, typically: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50°C;
(2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1 % bovine serum albumin/0.1 % Ficoll/0.1 % polyvinylpyrrolidone/SOmM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCI, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA
(50 ~,g/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2 x SSC
(sodium chloride/sodium citrate) and SO% fornlamide at 55°C, followed by a high-stringency wash consisting of 0.1 x SSC containing EDTA at 55°C.
"Moderately stringent conditions" may be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and %SDS) less stringent that those described above. An example of moderately stringent conditions is overnight incubation at 37°C in a solution comprising: 20%
formamide, 5 x SSC (150 mM NaCI, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50°C. The skilled artisan will recognize how to adjust the temperature, iouc strength, etc. as necessary to accommodate factors such as probe length and the like.
hi the context of the present invention, reference to "at least one," "at least two," "at least five," etc. of the genes listed in any particular gene set means any one or any and all combinations of the genes listed.
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 survival without cancer recurrence. The threshold is defined experimentally from clinical studies such as those described in the Example below. The expression threshold can be selected either for maximum sensitivity, or for maximum selectivity, or for minimum error. The determination of the expression threshold for any situation is well within the knowledge of those skilled in the art.
B. Detailed Descri tp ion The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Such techniques are explained fully in the literature, such as, "Molecular Cloning:
A Laboratory Manual", 2nd edition (Sambrook et al., 1989); "Oligonucleotide Synthesis"
(M.J. Gait, ed., 1984); "Animal Cell Culture" (R.I. Freshney, ed., 1987); "Methods in Enzymology"
(Academic Press, Inc.); "Handbook of Experimental Immunology", 4th edition (D.M. Weir 8z C.C. Blackwell, eds., Blackwell Science Inc., 1987); "Gene Transfer Vectors for Mammalian Cells" (J.M. Miller & M.P. Calos, eds., 1987); "Current Protocols in Molecular Biology"
(F.M. Ausubel et al., eds., 1987); and "PCR: The Polymerase Chain Reaction", (Mullis et al., eds., 1994).
1. Gene Expression Pro tlin In general, methods of gene expression profiling can be divided into two large groups:
methods based on hybridization analysis of polynucleotides, and methods based on sequencing of polynucleotides. The most commonly used methods known in the art for the quantification of mRNA expression in a sample include northern blotting and in situ hybridization (Parker & Barnes, Methods in Molecular Biology 106:247-283 (1999)); RNAse protection assays (Hod, Biotechniques 13:852-854 (1992)); and reverse transcription polymerase chain reaction (RT-PCR) (Weis et al., Trends in Genetics 8:263-264 (1992)).
Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes.
Representative methods for sequencing-based gene expression analysis include Serial Analysis of Gene Expression (SAGE), and gene expression analysis by massively parallel signature sequencing (MPSS).
2. Reverse Transcriptase PCR nRT 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, etc., tumor, or tumor cell lines, with pooled DNA from healthy donors. If the source of mRNA is a primary tumor, mRNA can be extracted, for example, from frozen or archived paraffin-embedded and fixed (e.g. formalin-fixed) tissue samples.
General methods for mRNA extraction are well known in the art and are disclosed in standard textbooks of molecular biology, including Ausubel et al., Current Protocols of Molecular 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., BioTeclZhic7ues 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 (EPICENTRE~, Madison, WI), and Paraffin Block RNA Isolation Kit (Ambion, Inc.). Total RNA from tissue samples can be isolated using RNA Stat-60 (Tel-Test). RNA
prepared from tumor can be isolated, for example, by cesium chloride density gradient centrifugation.
As RNA cannot serve as a template for PCR, the first step in gene expression profiling by RT-PCR is the reverse transcription of the RNA template into cDNA, followed by its exponential amplification in a PCR reaction. The two most commonly used reverse transcriptases are avilo myeloblastosis virus reverse transcriptase (AMV-RT) and Moloney 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 Elmer, CA, USA), following the manufacturer's instructions. The derived cDNA can then be used as a template in the subsequent PCR reaction.
Although the PCR step can use a variety of thermostable DNA-dependent DNA
polymerases, it typically employs the Taq DNA polymerase, which has a 5'-3' nuclease activity but lacks a 3'-5' proofreading endonuclease activity. Thus, TaqMan~
PCR typically utilizes the 5'-nuclease activity of Taq or Tth polymerase to hydrolyze a hybridization probe bound to its target amplicon, but any enzyme with equivalent 5' nuclease activity can be used.
Two oligonucleotide primers are used to generate an amplicon typical of a PCR
reaction. A
third oligonucleotide, or probe, is designed to detect nucleotide sequence located between the two PCR primers. The probe is non-extendible by Taq DNA polymerase enzyme, and is labeled with a reporter fluorescent dye and a quencher fluorescent dye. Any laser-induced emission from the reporter dye is quenched by the quenching dye when the two dyes are located close together as they are on the probe. During the amplification reaction, the Taq DNA polymerase enzyme cleaves the probe in a template-dependent manner. The resultant probe fragments disassociate in solution, and signal from the released reporter dye is free from the quenching effect of the second fluorophore. One molecule of reporter dye is liberated for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data.
TaqMan~ RT-PCR can be performed using commercially available equipment, such as, for example, ABI PRISM 7700TM Sequence Detection Systems (Perkin-Eliner-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 instrument and for analyzing the data.
5'-Nuclease assay data are initially expressed as Ct, or the threshold cycle.
As discussed above, fluorescence values are recorded during every cycle and represent the amount of product amplified to that point in the amplification reaction. The point when the fluorescent signal is first recorded as statistically significant is the threshold cycle (Ct).

To minimize errors and the effect of sample-to-sample variation, RT-PCR is usually performed using an internal standard. The ideal internal standard is expressed at a constant level among different tissues, and is unaffected by the experimental treatment. RNAs most frequently used to normalize patterns of gene expression are mRNAs for the housekeeping genes glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and (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., Genome Research 6:986-994 (1996).
The steps of a representative protocol for profiling gene expression using fixed, paraffin-embedded tissues as the RNA source, including mRNA isolation, purification, primer extension and amplification are given in various published journal articles for example: T.E.
Godfrey et al,. J. Moles. Diagnostics 2: 84-91 [2000]; K. Specht et al., Am.
J. Pathol. 158:
419-29 [2001]}. Briefly, a representative process starts with cutting about 10 ~.m thick sections of paraffin-embedded tumor tissue samples. The RNA is then extracted, and protein and DNA are removed. After analysis of the RNA concentration, RNA repair and/or amplification steps may be included, if necessary, and RNA is reverse transcribed using gene specific promoters followed by RT-PCR.
According to one aspect of the present invention, PCR primers and probes are designed based upon intron sequences present in the gene to be amplified. In this embodiment, the first step in the primer/probe design is the delineation of intron sequences within the genes. This can be done by publicly available software, such as the DNA BLAT
software developed by Kent, W.J., Genome Res. 12(4):656-64 (2002), or by the BLAST
software including its variations. Subsequent steps follow well established methods of PCR
primer and probe design.
In order to avoid non-specific signals, it is important to mask repetitive sequences within the introns when designing the primers and probes. This can be easily accomplished by using the Repeat Masker program available on-line through the Baylor College of Medicine, which screens DNA sequences against a library of repetitive elements and returns a query sequence in which the repetitive elements are masked. The masked intron sequences can then be used to design primer and probe sequences using any commercially or otherwise publicly available primer/probe design packages, such as Primer Express (Applied Biosystems); MGB assay-by-design (Applied Biosystems); Primer3 (Steve Rozen and Helen J. Skaletsky (2000) Primer3 on the WWW for general users and for biologist programmers.
In: I~rawetz S, Misener S (eds) Bioifaformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ, pp 365-386) The most important factors considered in PCR primer design include primer length, melting temperature (Tm), and G/C content, specificity, complementary primer sequences, and 3'-end sequence. In general, optimal PCR primers are generally 17-30 bases in length, and contain about 20-80%, such as, for example, about 50-60% G+C bases. Tm's between 50 and 80 °C, e.g. about 50 to 70 °C are typically preferred.
For further guidelines for PCR primer and probe design see, e.g. Dieffenbach, C.W. et al., "General Concepts for PCR Primer Design" in: PCR Primer, A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1995, pp. 133-155; Innis and Gelfand, "Optimization of PCRs" in: PCR Protocols, A Guide to Methods afad Applicatioyas, 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. Microarrays Differential gene expression can also be identified, or confirmed using the microarray technique. Thus, the expression profile of breast cancer-associated genes can be measured in either fresh or paraffin-embedded tumor tissue, using microarray technology.
In this method, polynucleotide sequences of interest (including cDNAs and oligonucleotides) are plated, or arrayed, on a microchip substrate. The arrayed sequences are then hybridized with specific DNA probes from cells or tissues of interest. Just as in the RT-PCR method, the source of mRNA typically is total RNA isolated from human tumors or tumor cell lines, and corresponding normal tissues or cell lines. Thus RNA can be isolated from a variety of primary tumors or tumor cell lines. If the source of mRNA is a primary tumor, mRNA can be extracted, for example, from frozen or archived paraffin-embedded and fixed (e.g. formalin-fixed) tissue samples, which are routinely prepared and preserved in everyday clinical practice.

In a specific embodiment of the microarray technique, PCR amplified inserts of cDNA
clones are applied to a substrate in a dense array. Preferably at least 10,000 nucleotide sequences are applied to the substrate. The microarrayed genes, immobilized on the microchip at 10,000 elements each, are suitable for hybridization under stringent conditions.
Fluorescently labeled cDNA probes may be generated through incorporation of fluorescent nucleotides by reverse transcription of RNA extracted from tissues of interest. Labeled cDNA
probes applied to the chip hybridize with specificity to each spot of DNA on the array. After stringent washing to remove non-specifically bound probes, the chip is scanned by confocal laser microscopy or by another detection method, such as a CCD camera.
Quantitation of hybridization of each arrayed element allows for assessment of corresponding mRNA
abundance. With dual color fluorescence, separately labeled cDNA probes generated from two sources of RNA are hybridized pairwise to the array. The relative abundance of the transcripts from the two sources corresponding to each specified gene is thus determined simultaneously. The miniaturized scale of the hybridization affords a convenient and rapid evaluation of the expression pattern for large numbers of genes. Such methods have been shown to have the sensitivity required to detect rare transcripts, which are expressed at a few copies per cell, and to reproducibly detect at least approximately two-fold differences in the expression levels (Schena et al., P~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. Serial Anal sy is of Gene Expression USAGE) 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., Scieiace 270:484-487 (1995); and Velculescu et al., Cell 88:243-51 (1997).
5. MassARRAY Technology The MassARRAY (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 subj ected 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 E~Yession Anal sy is by Massively Parallel Signature Seguencin~ (MPSS) This method, described by Brenner et al., NatuYe Biotechnology 18:630-634 (2000), is a sequencing approach that combines non-gel-based signature sequencing with in vitro cloning of millions of templates on separate 5 ~,m diameter microbeads. First, a microbead library of DNA templates is constructed by in vitro cloning. This is followed by the assembly of a planar array of the template-containing microbeads in a flow cell at a high density (typically greater than 3 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. Irnmunolaistochemisttw hnmunohistochemistry methods are also suitable for detecting the expression levels of the prognostic markers of the present invention. Thus, antibodies or antisera, preferably polyclonal antisera, and most preferably monoclonal antibodies specific for each marker are used to detect expression. The antibodies can be detected by direct labeling of the antibodies themselves, for example, with radioactive labels, fluorescent labels, hapten labels such as, biotin, or an enzyme such as horse radish peroxidase or alkaline phosphatase.
Alternatively, unlabeled primary antibody is used in conjunction with a labeled secondary antibody, comprising antisera, polyclonal antisera or a monoclonal antibody specific for the primary antibody. Immunohistochemistry protocols and kits are well known in the art and are commercially available.
8. Proteomics The term "proteome" is defined as the totality of the proteins present in a sample (e.g.
tissue, organism, or cell culture) at a certain point of time. Proteomics includes, among other things, study of the global changes of protein expression in a sample (also referred to as "expression proteomics"). Proteomics typically includes the following steps:
(1) separation of individual proteins in a sample by 2-D gel electrophoresis (2-D PAGE); (2) identification of the individual proteins recovered from the gel, e.g. my mass spectrometry or N-terminal sequencing, and (3) analysis of the data using bioinformatics. Proteomics methods are valuable supplements to other methods of gene expression profiling, and can be used, alone or in combination with other methods, to detect the products of the prognostic markers of the presentinvention.
9. General Descr~tion of the mRNA Isolation Purification and Amplification The steps of a representative protocol for profiling gene expression using fixed, paraffin-embedded tissues as the RNA source, including mRNA isolation, purification, primer extension and amplification are given in various published journal articles f for example: T.E.
Godfrey et al. J. Molec. Diagnostics 2: 84-91 [2000]; K. specht et al., Am. J.
Pathol. 158:
419-29 [2001]]. Briefly, a representative process starts with cutting about 10 ~m thick sections of paraffin-embedded tumor tissue samples. The RNA is then extracted, and protein and DNA are removed. After analysis of the RNA concentration, RNA repair andlor amplification steps may be included, if necessary, and RNA is reverse transcribed using gene specific promoters followed by RT-PCR. Finally, the data are analyzed to identify the best treatment options) available to the patient on the basis of the characteristic gene expression pattern identified in the tumor sample examined.
10. Breast Cancer Gerae Set Assayed Gene Subsequences and Clinical ~~lication o~'Gene Expression Data An important aspect of the present invention is to use the measured expression of certain genes by breast cancer tissue to provide prognostic information. 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, normalization can be based on the mean or median signal (Ct) 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 breast cancer tissue reference set. The number (I~ of breast 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 breast cancer tissues present in a particular set will have no significant impact on the relative amounts of the genes assayed. Usually, the breast cancer tissue reference set consists of at least about 30, preferably at least about 40 different FPE
breast 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.
Further details of the invention will be described in the following non-limiting Example Example A Phase II Stud~of Gene E~ression in 79 Malignant Breast 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 invasive breast ductal carcinoma, and to explore the correlation between such molecular profiles and disease-free survival.
Study design Molecular assays were performed on paraffin-embedded, formalin-fixed primary breast tumor tissues obtained from 79 individual patients diagnosed with invasive breast cancer. All patients in the study had 10 or more positive nodes. Mean age was 57 years, and mean clinical tumor size was 4.4 cm. 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 and homogeneous pathology.
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, 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 in section 9 above.
Molecular assays of quantitative gene expression were performed by RT-PCR, using the ABI PRISM 7900 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 thermocycler. During amplification, laser-induced fluorescent signal is collected in real-time through fiber optics cables for all 384 wells, and detected at the CCD.
The system includes software for running the instrument and for analyzing the data.
Analysis and Results Tumor tissue was analyzed for 185 cancer-related genes and 7 reference genes.
The threshold cycle (CT) values for each patient were normalized based on the median of the 7 reference genes for that particular patient. Clinical outcome data were available for all patients from a review of registry data and selected patient charts.
Outcomes were classified as:
0 died due to breast cancer or to unknown cause or alive with breast cancer recurrence;

1 alive without breast cancer recurrence or died due to a cause other than breast cancer Analysis was performed by:
1. Analysis of the relationship between normalized gene expression and the binary outcomes of 0 or 1.
2. Analysis of the relationship between normalized gene expression and the time to outcome (0 or 1 as defined above) where patients who were alive without breast cancer recurrence or who died due to a cause other than breast cancer were censored.
This approach was used to evaluate the prognostic impact of individual genes and also sets of multiple genes.
Araalysis o~,patients with iyavasive breast cay~cinoma by binary approach In the first (binary) approach, analysis was performed on all 79 patients with invasive breast carcinoma. A t test was performed on the groups of patients classified as either no recurrence and no breast cancer related death at three years, versus recurrence, or breast cancer-related death at three years, and the p-values for the differences between the groups for each gene were calculated.
Table 1 lists the 47 genes for which the p-value for the differences between the groups was <0.10. The first column of mean expression values pertains to patients who neither had a metastatic recurrence of nor died from breast cancer. The second column of mean expression values pertains to patients who either had a metastatic recurrence of or died from breast cancer.
Table 1 Mean Mean t-value.df p Valid Valid N N

Bcl2 -0.15748-1.228164.00034 75 0.00014735 42 PR -2.67225-5.497473.61540 75 0.00054135 42 IGF1 -0.59390-1.715063.49158 75 0.00080835 42 R

BAG1 0.18844 -0.685093.42973 75 0.00098535 42 CD68 -0.522750.10983-3.4118675 0.00104335 42 EstR1 -0.35581-3.006993.32190 75 0.00138435 42 CTSL -0.64894-0.09204-3.2678175 0.00163735 42 IGFBP2 -0.81181-1.783983.24158 75 0.00177435 42 GATA3 1.80525 0.574283.15608 75 0.00230335 42 TP53BP2-4.71118-6.092893.02888 75 0.00336535 42 EstR1 3.67801 1.646933.01073 75 0.00355035 42 CEGP1 -2.02566-4.255372.85620 75 0.00554435 42 SURV -3.67493-2.96982-2.7054475 0.00843935 42 p27 0.80789 0.288072.55401 75 0.01267835 42 Chk1 -3.37981-2.80389-2.4697975 0.01579335 42 BBC3 -4.71789-5.629572.46019 75 0.01618935 42 ZNF217 1.10038 0.627302.42282 75 0.017814 35 42 EGFR -2.88172-2.20556-2.34774 75 0.021527 35 42 CD9 1.29955 0.910252.31439 75 0.023386 35 42 MYBL2 -3.77489-3.02193-2.29042 75 0.024809 35 42 H I -0.442480.03740-2.25950 75 0.026757 35 42 A

GRB7 -1.96063-1.05007-2.25801 75 0.026854 35 42 pS2 -1.00691-3.137492.24070 75 0.028006 35 42 RIZ1 -7.62149-8.387502.20226 75 0.030720 35 42 ErbB3 -6.89508-7.443262.16127 75 0.033866 35 42 TOP2B 0.45122 0.126652.14616 75 0.035095 35 42 MDM2 1.09049 0.690012.10967 75 0.038223 35 42 PRAME -6.40074-7.704242.08126 75 0.040823 35 42 GUS -1.51683-1.892802.05200 75 0.043661 35 42 RAD51 -5.85618-6.713342.04575 75 0.044288 35 42 C

AIB1 -3.08217-2.28784-2.00600 75 0.048462 35 42 STK15 -3.11307-2.59454-2.00321 75 0.048768 35 42 GAPDH -0.35829-0.02292-1.94326 75 0.055737 35 42 FHIT -3.00431-3.671751.86927 75 0.065489 35 42 KRT19 2.52397 2.016941.85741 75 0.067179 35 42 TS -2.83607-2.29048-1.83712 75 0.070153 35 42 GSTM1 -3.69140-4.386231.83397 75 0.070625 35 42 G- 0.31875 -0.155241.80823 75 0.074580 35 42 Catenin AKT2 0.78858 0.467031.79276 75 0.077043 35 42 CCNB1 -4.26197-3.51628-1.78803 75 0.077810 35 42 P13KC2A-2.27401-2.702651.76748 75 0.081215 35 42 FBXO5 -4.72107-4.24411-1.75935 75 0.082596 35 42 DR5 -5.80850-6.555011.74345 75 0.085353 35 42 CIAP1 -2.81825-3.099211.72480 75 0.088683 35 42 MCM2 -2.87541-2.50683-1.72061 75 0.089445 35 42 CCND1 1.30995 0.809051.68794 75 0.095578 35 42 EIF4E -5.37657-6.471561.68169 75 0.096788 35 42 In the foregoing Table 1, negative t-values indicate higher expression, associated with worse outcomes, and, inversely, higher (positive) t-values indicate higher expression associated with better outcomes. Thus, for example, elevated expression of the CD68 gene (t-value = -3.41, CT mean alive< CT mean deceased) indicates a reduced likelihood of disease free survival. Similarly, elevated expression of the BCl2 gene (t-value =
4.00; CT mean alive> CT mean deceased) indicates an increased likelihood of disease free survival.
Based on the data set forth in Table 1, the expression of any of the following genes in breast cancer above a defined expression threshold indicates a reduced likelihood of survival without cancer recurrence following surgery: Grb7, CD68, CTSL, Chkl, Her2, STK15, AIB1, SURV, EGFR, MYBL2, HIF 1 a.
Based on the data set forth in Table l, the expression of any of the following genes in breast cancer above a defined expression threshold indicates a better prognosis for survival without cancer recurrence following surgery: TP53BP2, PR, Bcl2, KRT14, EstRl, IGFBP2, BAGl, CEGP1, KLK10, (3 Catenin, GSTM1, FHIT, Rizl, IGF1, BBC3, IGFRl, TBP, p27, 1RS1, IGF1R, GATA3, CEGP1, ZNF217, CD9, pS2, ErbB3, TOP2B, MDM2, RAD51, and KRT19.
Ana~sis o~ER-positive patients by binay-y approach 57 patients with normalized CT for estrogen receptor (ER) >0 (i.e., ER
positive patients) were subjected to separate analysis. A t test was performed on the two groups of patients classified as either no recurrence and no breast cancer related death at three years, or recurrence or breast cancer-related death at three years, and the p-values for the differences between the groups for each gene were calculated. Table 2, below, lists the genes where the p-value for the differences between the groups was <0.105. The first column of mean expression values pertains to patients who neither had a metastatic recurrence nor died from breast cancer. The second column of mean expression values pertains to patients who either had a metastatic recurrence of or died from breast cancer.
Table 2 Mean Mean t-value df p Valid Valid N N

IGF1 -0.13975-1.004353.65063 55 0.00058430 27 R

Bcl2 0.15345 -0.704803.55488 55 0.00078630 27 CD68 -0.547790.19427-3.4181855 0.00119330 27 HNF3A 0.39617 -0.638023.20750 55 0.00223330 27 CTSL -0.667260.00354-3.2069255 0.00223730 27 TP53BP2-4.81858-6.444253.13698 55 0.00274130 27 GATA3 2.33386 1.408033.02958 55 0.00372730 27 BBC3 -4.54979-5.723332.91943 55 0.00507430 27 RAD51 -5.63363-6.948412.85475 55 0.00606330 27 C

BAG1 0.31087 -0.506692.61524 55 0.01148530 27 IGFBP2 -0.49300-1.309832.59121 55 0.01222230 27 FBX05 -4.86333-4.05564-2.5632555 0.01313530 27 EstR1 0.68368 -0.665552.56090 55 0.01321430 27 PR -1.89094-3.866022.52803 55 0.01437230 27 SURV -3.87857-3.10970-2.4962255 0.01557930 27 CD9 1.41691 0.917252.43043 55 0.01837030 27 RB 1 -2.51662-2.974192.41221 55 0.01921930 27 EPHX1 -3.91703-5.850972.29491 55 0.02557830 27 CEGP1 -1.18600-2.951392.26608 55 0.02740330 27 CCNB1 -4.44522-3.35763-2.2514855 0.02837030 27 TRAIL 0.34893 -0.565742.20372 55 0.03174930 27 EstR1 4.60346 3.603402.20223 55 0.03186030 27 DR5 -5.71827-6.790882.14548 55 0.03634530 27 MCM2 -2.96800-2.48458-2.1051855 0.03985730 27 Chk1 -3.46968-2.85708-2.0859755 0.04163330 27 p27 0.94714 0.496562.04313 55 0.04584330 27 MYBL2 -3.97810-3.14837-2.0292155 0.04728830 27 GUS -1.42486-1.829001.99758 55 0.05071830 27 P53 -1.08810-1.471931.92087 55 0.059938 30 27 HIF1A -0.409250.11688-1.91278 55 0.060989 30 27 cMet -6.36835-5.58479-1.88318 55 0.064969 30 27 EGFR -2.95785-2.28105-1.86840 55 0.067036 30 27 MTA1 -7.55365-8.136561.81479 55 0.075011 30 27 RIZ1 -7.52785-8.259031.79518 55 0.078119 30 27 ErbB3 -6.62488-7.108261.79255 55 0.078545 30 27 TOP2B 0.54974 0.275311.74888 55 0.085891 30 27 EIF4E -5.06603-6.314261.68030 55 0.098571 30 27 TS -2.95042-2.36167-1.67324 55 0.099959 30 27 STK15 -3.25010-2.72118-1.64822 55 0.105010 30 27 For each gene, a classification algoritlnn was utilized to identify the best threshold value (CT) for using each gene alone in predicting clinical outcome.
Based on the data set forth in Table 2, expression of the following genes in ER-positive cancer above a defined expression level is indicative of a reduced likelihood of survival without cancer recurrence following surgery: CD68; CTSL; FBXOS; SURV;
CCNB1; MCM2; Chkl; MYBL2; HIF1A; cMET; EGFR; TS; STK15. Many of these genes (CD68, CTSL, SURV, CCNB1, MCM2, Chkl, MYBL2, EGFR, and STK15) were also identified as indicators of poor prognosis in the previous analysis, not limited to ER-positive breast cancer. Based on the data set forth in Table 2, expression of the following genes in ER-positive cancer above a defined expression level is indicative of a better prognosis for survival without cancer recurrence following surgery: IGFRl; BCl2; HNF3A;
TP53BP2;
GATA3; BBC3; RADS1C; BAG1; IGFBP2; PR; CD9; RB1; EPHX1; CEGPl; TRAIL; DRS;
p27; p53; MTA; RIZ1; ErbB3; TOP2B; EIF4E. Of the latter genes, IGFRl; BC12;
TP53BP2;
GATA3; BBC3; RADS1C; BAGl; IGFBP2; PR; CD9; CEGP1; DRS; p27; RIZ1; ErbB3;
TOP2B; EIF4E have also been identified as indicators of good prognosis in the previous analysis, not limited to ER-positive breast cancer.
Anal sy is o~ ER ne~ativ~atiefats by bitiary approach Twenty patients with normalized CT for estrogen receptor (ER) <1.6 (i.e., ER
negative patients) were subjected to separate analysis. A t test was performed on the two groups of patients classified as either no recurrence and no breast cancer related death at three years, or recurrence or breast cancer-related death at three years, and the p-values for the differences between the groups for each gene were calculated. Table 3 lists the genes where the p-value for the differences between the groups was <0.118. The first column of mean expression values pertains to patients who neither had a metastatic recurrence nor died from breast cancer. The second column of mean expression values pertains to patients who either had a metastatic recurrence of or died from breast cancer.
Table 3 Mean Mean t-value df p Valid Valid N N

KRT14 -1.95323-6.69231 4.03303 18 0.000780 5 15 KLK10 -2.68043-7.11288 3.10321 18 0.006136 5 15 CCND1 -1.022850.03732 -2.77992 18 0.012357 5 15 Upa -0.91272-0.04773 -2.49460 18 0.022560 5 15 HNF3A -6.04780-2.36469 -2.43148 18 0.025707 5 15 Maspin -3.56145-6.18678 2.40169 18 0.027332 5 15 CDH1 -3.54450-2.34984 -2.38755 18 0.028136 5 15 HER2 -1.489731.53108 -2.35826 18 0.029873 5 15 GRB7 -2.552890.00036 -2.32890 18 0.031714 5 15 AKT1 -0.368490.46222 -2.29737 18 0.033807 5 15 TGFA -4.03137-5.67225 2.28546 18 0.034632 5 15 FRP1 1.45776 -1.39459 2.27884 18 0.035097 5 15 STMY3 -1.59610-0.26305 -2.23191 18 0.038570 5 15 Contig _4,27585-7.34338 2.18700 18 0.042187 5 15 A-Catenin-1.19790-0.39085 -2.15624 18 0.044840 5 15 VDR -4.37823-2.37167 -2.15620 18 0.044844 5 15 G RO -3.65034-5.97002 2.12286 18 0.047893 5 15 MCM3 -3.86041-5.55078 2.10030 18 0.050061 5 15 B-actin4.69672 5.19190 -2.04951 18 0.055273 5 15 H I -0.64183-0.10566 -2.02301 18 0.058183 5 15 A

MMP9 -8.90613-7.35163 -1.88747 18 0.075329 5 15 VEGF 0.37904 1.10778 -1.87451 18 0.077183 5 15 PRAME -4.95855-7.41973 1.86668 18 0.078322 5 15 AIB1 -3.12245-1.92934 -1.86324 18 0.078829 5 15 KRT5 -1.32418-3.62027 1.85919 18 0.079428 5 15 KRT18 1.08383 2.25369 -1.83831 18 0.082577 5 15 KRT17 -0.69073-3.56536 1.78449 18 0.091209 5 15 P14ARF -1.87104-3.36534 1.63923 18 0.118525 5 15 Based on the data set forth in Table 3, expression of the following genes in ER-negative cancer above a defined expression level is indicative of a reduced likelihood of survival without cancer recurrence (p<0.05): CCND1; UPA; HNF3A; CDH1; Her2;
GRB7;
AKT1; STMY3; a-Catenin; VDR; GRO1. Only 2 of these genes (Her2 and Grb7) were also identified as indicators of poor prognosis in the previous analysis, not limited to ER-negative breast cancer. Based on the data set forth in Table 3, expression of the following genes in ER-negative cancer above a defined expression level is indicative of a better prognosis for survival without cancer recurrence (KT14; KL,K10; Maspin, TGFa, and FRPl. Of the latter genes, only KLK10 has been identified as an indicator of good prognosis in the previous analysis, not limited to ER-negative breast cancer.

Anal sy is of mult~l~enes and ihdicato~s o outcome Two approaches were taken in order to determine whether using multiple genes would provide better discrimination between outcomes.
First, a discrimination analysis was performed using a forward stepwise approach.
S Models were generated that classified outcome with greater discrimination than was obtained with any single gene alone.
According to a second approach (time-to-event approach), for each gene a Cox Proportional Hazards model (see, e.g. Cox, D. R., and Oakes, D. (1984), Analysis of Survival Data, Chapman and Hall, London, New York) was defined with time to recurrence or death as the dependent variable, and the expression level of the gene as the independent variable.
The genes that have a p-value < 0.10 in the Cox model were identified. For each gene, the Cox model provides the relative risk (RR) of recurrence or death for a unit change in the expression of the gene. One can choose to partition the patients into subgroups at any threshold value of the measured expression (on the CT scale), where all patients with expression values above the threshold have higher risk, and all patients with expression values below the threshold have lower risk, or vice versa, depending on whether the gene is an indicator of bad (RR>1.01) or good (RR<1.01) prognosis. Thus, any threshold value will define subgroups of patients with respectively increased or decreased risk.
The results are summarized in Table 4. The third column, with the heading: exp(coef), shows RR
values.

Table 4 Gene coef exp(coef) se(coef) z p TP53BP2 -0.21892 0.803386 0.068279 -3.20625 0.00134 GRB7 0.235697 1.265791 0.073541 3.204992 0.00135 PR -0.102580.902510.035864-2.860180.00423 CD68 0.4656231.5930060.1677852.7751150.00552 Bcl2 -0.267690.7651460.100785-2.656030.00791 KRT14 -0.118920.8878770.046938-2.533590.0113 PRAME -0.137070.8719120.054904-2.496490.0125 CTSL 0.4314991.5395640.1852372.3294440.0198 EstR1 -0.076860.9260180.034848-2.205610.0274 Chk1 0.2844661.3290530.1308232.1744410.0297 IGFBP2 -0.2152 0.8063760.099324-2.166690.0303 HER2 0.1553031.1680110.0726332.13818 0.0325 BAG1 -0.226950.7969590.106377-2.133460.0329 CEGP1 -0.078790.9242360.036959-2.131770.033 STK15 0.27947 1.3224280.1327622.1050390.0353 KLK10 -0.110280.8955880.05245-2.102480.0355 B.Catenin-0.165360.8475860.084796-1.950130.0512 EstR1 -0.0803 0.9228420.042212-1.902260.0571 GSTM1 -0.132090.8762660.072211-1.829150.0674 TOP2A -0.111480.8945120.061855-1.802220.0715 AIB1 0.1529681.1652880.0863321.7718610.0764 FHIT -0.155720.8558020.088205-1.7654 0.0775 RIZ1 -0.174670.8397360.099464-1.756090.0791 SURV 0.1857841.2041620.1066251.7423990.0814 IGF1 -0.104990.9003380.060482-1.735810.0826 BBC3 -0.1344 0.8742430.077613-1.731630.0833 IGF1 -0.134840.8738580.077889-1.731150.0834 R

DIABLO 0.2843361.328880.1665561.7071480.0878 TBP -0.344040.7089 0.20564-1.673030.0943 p27 -0.260020.7710330.1564 -1.662560.0964 IRS1 -0.07585Ø9269570.046096-1.645420.0999 The binary and time-to-event analyses, with few exceptions, identified the same genes as prognostic markers. For example, comparison of Tables 1 and 4 shows that 10 genes were represented in the top 15 genes in both lists. Furthermore, when both analyses identified the same gene at [p<0.10], which happened for 21 genes, they were always concordant with respect to the direction (positive or negative sign) of the correlation with survival/recurrence.
Overall, these results strengthen the conclusion that the identified markers have significant prognostic value.
For Cox models comprising more than two genes (multivariate models), stepwise entry of each individual gene into the model is performed, where the first gene entered is pre-selected from among those genes having significant univariate p-values, and the gene selected for entry into the model at each subsequent step is the gene that best improves the fit of the model to the data. This analysis can be performed with any total number of genes. In the analysis the results of which are shown below, stepwise entry was performed for up to 10 genes.
Multivariate analysis is performed using the following equation:
RR=exp[coef(geneA) x Ct(geneA) + coef(geneB) x Ct(geneB) + coef(geneC) x Ct(geneC) + ...............].
In this equation, coefficients for genes that are predictors of beneficial outcome are positive numbers and coefficients for genes that are predictors of unfavorable outcome are negative numbers. The "Ct" values in the equation are ~Cts, i.e. reflect the difference between the average normalized Ct value for a population and the normalized Ct measured for the patient in question. The convention used in the present analysis has been that ~Cts below and above the population average have positive signs and negative signs, respectively (reflecting greater or lesser mRNA abundance). The relative risk (RR) calculated by solving this equation will indicate if the patient has an enhanced or reduced chance of long-term survival without cancer recurrence.
Multivariate gene analysis o~'79 ~atiehts witla invasive breast carcih.oma A multivariate stepwise analysis, using the Cox Proportional Hazards Model, was performed on the gene expression data obtained for all 79 patients with invasive breast carcinoma. The following ten-gene sets have been identified by this analysis as having particularly strong predictive value of patient survival (a) TP53BP2, Bcl2, BAD, EPHX1, PDGFR~i, DIABLO, XIAP, YB1, CA9, and I~RTB.
(b) GRB7, CD68, TOP2A, Bcl2, DIABLO, CD3, ID1, PPM1D, MCM6, and WISP1.
(c) PR, TP53BP2, PRAME, DIABLO, CTSL, IGFBP2, TIMP1, CA9, MMP9, and COX2.
(d) CD68, GRB7, TOP2A, Bcl2, DIABLO, CD3, ID1, PPM1D, MCM6, and WISP1.
(e) Bcl2, TP53BP2, BAD, EPHX1, PDGFR(3, DIABLO, XIAP, YB1, CA9, and I~RTB.
(f) KRT14, KRTS, PRAME, TP53BP2, GUS1, AIB1, MCM3, CCNE1, MCM6, and ID1.
(g) PRAME, TP53BP2, EstRl, DIABLO, CTSL, PPM1D, GRB7, DAPKl, BBC3, and VEGFB.
(h) CTSL2, GRB7, TOP2A, CCNB1, Bcl2, DIABLO, PRAME, EMS1, CA9, and EpCAM.

(i) EstRl, TP53BP2, PRAMS, DIABLO, CTSL, PPM1D, GRB7, DAPKl, BBC3, and VEGFB.
(k) Chkl, PRAMS, p53BP2, GRB7, CA9, CTSL, CCNB1, TOP2A, tumor size, and IGFBP2.
(1) IGFBP2, GRB7, PRAMS, DIABLO, CTSL, [3-Catenin, PPM1D, Chkl, WISP1, and LOT 1.
(m) HER2, TP53BP2, Bcl2, DIABLO, TIIVVIP1, EPHXl, TOP2A, TRAIL, CA9, and AREG.
(n) BAG1, TP53BP2, PRAMS, IL6, CCNB1, PAIL, AREG, tumor size, CA9, and Ki67.
(o) CEGPl, TP53BP2, PRAMS, DIABLO, Bcl2, COX2, CCNE1, STK15, and AKT2, and FGF 18.
(p) STK15, TP53BP2, PRAMS, IL6, CCNE1, AKT2, DIABLO, cMet, CCNE2, and COX2.
(q) KLK10, EstRl, TP53BP2, PRAMS, DIABLO, CTSL, PPM1D, GRB7, DAPKl, and BBC3.
(r) AIB1, TP53BP2, Bcl2, DIABLO, TIMP1, CD3, p53, CA9, GRB7, and EPHX1 (s) BBC3, GRB7, CD68, PRAMS, TOP2A, CCNBl, EPHX1, CTSL
GSTM1, and APC.
(t) CD9, GRB7, CD68, TOP2A, Bcl2, CCNB1, CD3, DIABLO, ID1, and PPM1D.
(w) EGFR, KRT14, GRB7, TOP2A, CCNB1, CTSL, Bcl2, TP, KLK10, and CA9.
(x) HIFla, PR, DIABLO, PRAMS, Chkl, AKT2, GRB7, CCNE1, TOP2A, and CCNB1.
(y) MDM2, TP53BP2, DIABLO, Bcl2, AIB1, TIMP1, CD3, p53, CA9, and HER2.
(z) MYBL2, TP53BP2, PRAMS, IL6, Bcl2, DIABLO, CCNE1, EPHX1, TM'1, and CA9.
(aa) p27, TP53BP2, PRAMS, DIABLO, Bcl2, COX2, CCNE1, STK15, AKT2, and ID1.
(ab) RAD51, GRB7, CD68, TOP2A, CIAP2, CCNB1, BAGl, IL6, FGFRl, and TP53BP2.
(ac) SURV, GRB7, TOP2A, PRAMS, CTSL, GSTM1, CCNB1, VDR, CA9, and CCNE2.
(ad) TOP2B, TP53BP2, DIABLO, Bcl2, TIMP1, AIBl, CA9, p53, KRTB, and BAD.
(ae) ZNF217, GRB7, p53BP2, PRAMS, DIABLO, Bcl2, COX2, CCNE1, APC4, and (3-Catenin.

While the present invention has been described with reference to what are considered to be the specific embodiments, it is to be understood that the invention is not limited to such embodiments. To the contrary, the invention is intended to cover various modifications and equivalents included within the spirit and scope of the appended claims. For example, while the disclosure focuses on the identification of various breast cancer associated genes and gene sets, and on the personalized prognosis of breast cancer, similar genes, gene sets and methods concerning other types of cancer are specifically within the scope herein.
All references cited throughout the disclosure are hereby expressly incorporated by reference.

Gene ' ' Accession Seq AI81 NM_006534 GCGGCGAGTTTCCGATTTAAAGCTGAGCTGCGAGGAAAATGGCGGCGGGAGGATCAAAATACTTGCTGGATGGTGGACT
CA

CGCTTCTATGGCGCTGAGATTGTGTCAGCCCTGGACTACCTGCACTCGGAGAAGAACGTGGTGTACCGGGA

TCCTGCCACCCTTCAAACCTCAGGTCACGTCCGAGGTCGACACAAGGTACTTCGATGATGAATTTACCGCC

GGACAGCAGGAATGTGTTTCTCCATACAGGTCACGGGGAGCCAATGGTTCAGAAACAAATCGAGTGGGT

TGTGAGTGAAATGCCTTCTAGTAGTGAACCGTCCTCGGGAGCCGACTATGACTACTCAGAAGAGTATGATAACGAACCA
CAA
B-actin NM_001101 CAGCAGATGTGGATCAGCAAGCAGGAGTATGACGAGTCCGGCCCCTCCATCGTCCACCGCAAATGC' B-Catenin NM 001904 GGCTCTTGTGCGTACTGTCCTTCGGGCTGGTGACAGGGAAGACATCACTGAGCCTGCCATCTGTGCTCTTCGTCATCTG
A
BAD NM_032989 GGGTCAGGTGCCTCGAGATCGGGCTTGGGCCCAGAGCATGTTCCAGATCCCAGAGTTTGAGCCGAGTGAGCAG
BAG1 NM_004323 CGTTGTCAGCACTTGGAATACAAGATGGTTGCCGGGTCATGTTAATTGGGAAAAAGAACAGTCCACAGGAAGAGGTTGA
AC

CCTGGAGGGTCCTGTACAATCTCATCATGGGACTCCTGCCCTTACCCAGGGGCCACAGAGCCCCCGAGATGGAGCCCAA
TTAG
8c12 NM_000633 CAGATGGACCTAGTACCCACTGAGATTTCCACGCCGAAGGACAGCGATGGGAAAAATGCCCTTAAATCATAGG
CA9 NM_001216 ATCCTAGCCCTGGTTTTTGGCCTCCTTTTTGCTGTCACCAGCGTCGCGTTCCTTGTGCAGATGAGAAGGCAG

TTCAGGTTGTTGCAGGAGACCATGTACATGACTGTCTCCATTATTGATCGGTTCATGCAGAATAAT'fGTGTGCCCAAG
AAGATG
CCND1 NM_001758 GCATGTTCGTGGCCTCTAAGATGAAGGAGACCATCCCCCTGACGGCCGAGAAGCTGTGCATCTACACCG . .

AAAGAAGATGATGACCGGGTTTACCCAAACTCAACGTGCAAGCCTCGGATTATTGCACCATCCAGAGGCTC, ATGCTGTGGCTCCTTCCTAACTGGGGCTTTCTTGACATGTAGGTTGCTTGGTAATAACCTTTTTGTATATCACAATTTG
GGT
CD3z NM_000734 AGATGAAGTGGAAGGCGCTT1'fCACCGCGGCCATCCTGCAGGCACAGTTGCCGATTACAGAGGCA
CD68 NM_001251 TGGTTCCCAGCCCTGTGTCCACCTCCAAGCCCAGATTCAGATTCGAGTCATGTACACAACCCAGGGTGGAGGAG

GGGCGTGGAACAGTTTATCTCAGACATCTGCCCCAAGAAGGACGTACTCGAAACC.TTCACCGTG

TGAGTGTCCCCCGGTATCTTCCCCGCCCTGCCAATCCCGATGAAATTGGAAATTTTATTGATGAAAATCTGAAAGCGGC
TG
CEGP1 NM_020974 TGACAATCAGCACACCTGCATTCACCGCTCGGAAGAGGGCCTGAGCTGCATGAATAAGGATCACGGCTGTAGTCACA
Chki NM_001274 GATAAATTGGTACAAGGGATCAGCTTTTCCCAGCCCACATGTCCTGATCA'fATGCTTTTGAATAGTCAGTTACTTGGC
ACCC
CIAP1 NM_001166 TGCCTGTGGTGGGAAGCTCAGTAACTGGGAACCAAAGGATGATGCTATGTCAGAACACCGGAGGCATTTTCC
cIAP2 NM_001165 TGAGAAG
cMet NM_000245 GACATTTCCAGTCCTGCAGTCAATGCCTCTCTGCCCCACCCTTTGTTCAGTGTGGCTGGTGCCACGACAAATGTGTGCG
ATCGGAG
Contig278AK000618.
GGCATCCTGGCCCAAAGTTTCCCAAATCCAGGCGGCTAGAGGCCCACTGCTTCCCAACTACCAGCTGAGGGGGTC
COX2 NM_000963 TCTGCAGAGTTGGAAGCACTCTATGGTGACATCGATGGTGTGGAGCTGTATCCTGCCCTTCTGGTAGAAAAGCCTCGGC

GGGAGGCTTATCTCACTGAGTGAGCAGAATCTGGTAGACTGCTCTGGGCCTCAAGGCAATGAAGGCTGCAATGG

NM_001333'TGTCTCACTGAGCGAGCAGAATCTGGTGGACTGTTCGCGTCCTCAAGGCAATCAGGGCTGCAATGGT

CGCTGACATCATGAATGTTCCTCGACCGGCTGGAGGCGAGTTTGGATATGACAAAGACACATCGTTGCTGAAAGAGA
DIABLO ~ NM 019887 CACAATGGCGGCTCTGAAGAGTTGGCTGTCGCGCAGCGTAACTTCATTCTTCAGGTACAGACAGTGTTTGTGT
DRS NM_003842 CTCTGAGACAGTGCTTCGATGACTTTGCAGACTTGGTGCCCTTTGACTCCTGGGAGCCGCTCATGAGGAAGTTGGGCCT
CATGG

. .
EIF4E NM_001968 GATCTAAGATGGCGACTGTCGAACCGGAAACCACCCCTACTCCTAATCCCCCGACTACAGAAGAGGAGAAAACGGAATC
TAA
EMS1 NM_005231 GGCAGTGTCACTGAGTCCTTGAAATCCTCCCCTGCCCCGCGGGTCTCTGGATTGGGACGCACAGTGCA
EpCAM NM
002354°GGGCCCTCCAGAACAATGATGGGCTTTATGATCCTGACTGCGATGAGAGCGGGCTCTTTAAGGCC
AAGCAGTGCA
EPHX1 NM_000120 ACCGTAGGCTCTGCTCTGAATGACTCTCCTGTGGGTCTGGCTGCCTATATTCTAGAGAAGTTTTCCACCTGGACCA
ErbB3 NM_001982 CGGTTATGTCATGCCAGATACACACCTCAAAGGTACTCCCTCCTCCCGGGAAGGCACCCTTTCTTCAGTGGGTCTCAGT
TC
EslR1 NM 000125 CGTGGTGCCCCTCTATGACCTGCTGCTGGAGATGCTGGACGCCCACCGCCTACATGCGCCCACTAGCC
FBXOS NM_012177 GGCTATTCCTCATTTTCTCTACAAAGTGGCCTCAGTGAACATGAAGAAGGTAGCCTCCTGGAGGAGAATTTCGGTGACA
GTCTACAATCC
FGF18 NM_003862 CGGTAGTCAAGTCCGGATCAAGGGCAAGGAGACGGAATTCTACCTGTGCATGAACCGCAAAGGCAAGC
FGFR1 NM_023109 CACGGGACATTCACCACATCGACTACTATAAAAAGACAACCAACGGCCGACTGCCTGTGAAGTGGATGGCACCC
FHIT NM_002012 CCAGTGGAGCGCTTCCATGACCTGCGTCCTGATGAAGTGGCCGATTTGTTTCAGACGACCCAGAGAG

TTGGTACCTGTGGGTTAGCATCAAGTTCTCCCCAGGGTAGAATTCAATCAGAGCTCCAGTTTGCATTTGGATGTG
G-Catenin NM_002230 TCAGCAGCAAGGGCATCATGGAGGAGGATGAGGCCTGCGGGCGCCAGTACACGCTCAAGAAAACCACC

ATTCCACCCATGGCAAATTCCATGGCACCGTCAAGGCTGAGAACGGGAAGCTTGTCATCAATGGAAATCCCATC
GATA3 NM_002051 CARP,GGAGCTCACTGTGGTGTCTGTGTTCCAACCACTGAATCTGGACCCCATCTGTGAATAAGCCATTCTGACTC
GRB7 NM_005310 CCATCTGCATCCATCTTGTTTGGGCTCCCCACCCTTGAGAAGTGCCTCAGATAATACCCTGGTGGCC
GR01 NM_001511 CGAAAAGATGCTGAACAGTGACAAATCCAACTGACCAGAAGGGAGGAGGAAGCTCACTGGTGGCTGTTCCTGA
GSTM1 NM_000561 AAGCTATGAGGAAAAGAAGTACACGATGGGGGACGCTCCTGATTATGACAGAAGCCAGTGGCTGAATGAAAAATTCAAG
CTGGGCC

CCCACTCAGTAGCCAAGTCACAATGTTTGGAAAACAGCCCGTTTACTTGAGCAAGACTGATACCACCTGCGTG
HER2 NM_004448 CGGTGTGAGAAGTGCAGCAAGCCCTGTGCCCGAGTGTGCTATGGTCTGGGCATGGAGCACTTGCGAGAGG
HIF1A NM_001530 TGAACATAAAGTCTGCAACATGGAAGGTATTGCACTGCACAGGCCACATTCACGTATATGATACCAACAGTAACCAACC
TCA
HNF3A NM_004496 TCCAGGATGTTAGGAACTGTGAAGATGGAAGGGCATGAAACCAGCGACTGGAACAGCTACTACGCAGACACGC

AGAACCGCAAGGTGAGCAAGGTGGAGATTCTCCAGCACGTCATCGACTACATCAGGGACCTTCAGTTGGA
IGF1 NM_000618 TCCGGAGCTGTGATCTAAGGAGGCTGGAGATGTATTGCGCACCCCTCAAGCCTGCCAAGTCAGCTCGCTCTGTCCG
IGFiR NM 000875 GCATGGTAGCCGAAGATTTCACAGTCAAAATCGGAGATTTTGGTATGACGCGAGATATCTATGAGACAGACTATTACCG
GAAA, IGFBP2 NM_000597 GTGGACAGCACCATGAACATGTTGGGCGGGGGAGGCAGTGCTGGCCGGAAGCCCCTCAAGTCGGGTATGAAGG
IL6 NM_000600 CCTGAACCTTCCAAAGATGGCTGAAAAAGATGGATGCTTCCAATCTGGATTCAATGAGGAGACT1'GCCTGGT
IRS1 NM_005544 CCACAGCTCACCTTCTGTCAGGTGTCCATCCCAGCTCCAGCCAGCTCCCAGAGAGGAAGAGACTGGCACTGAGG

CGGACTTTGGGTGCGACTTGACGAGCGGTGGTTCGACAAGTGGCCTTGCGGGCCGGATCGTCCCAGTGGAAGAGTTGTA
A
KLK10 NM_002776 GCCCAGAGGCTCCATCGTCCATCCTCTTCCTCCCCAGTCGGCTGAACTCTCCCCTTGTCTGCACTGTTCAAACCTCTG

GGCCTGCTGAGATCAAAGACTACAGTCCCTAC'fTCAAGACCATTGAGGACCTGAGGAACAAGATTCTCACAGCCACAG
TGGAC
KRT17 NM_000422 CGAGGATTGGTTCTTCAGCAAGACAGAGGAACTGAACCGCGAGGTGGCCACCAACAGTGAGCTGGTGCAGAGT

AGAGATCGAGGCTCTCAAGGAGGAGCTGCTCTTCATGAAGAAGAACCACGAAGAGGAAGTAAAAGGCC .
kRTl9 NM 002276 TGAGCGGCAGAATCAGGAGTACCAGCGGCTCATGGACATCAAGTCGCGGCTGGAGCAGGAGATTGCCACCTACCGCA
.

KRTS NM_000424 TCAGTGGAGAAGGAGTTGGACCAGTCAACATCTCTGTTGTCACAAGCAGTGTTTCCTCTGGATATGGCA
KRTB NM_002273 GGATGAAGCTTACATGAACAAGGTAGAGCTGGAGTCTCGCCTGGAAGGGCTGACCGACGAGATCAACTTCCTCAGGCAG
CTATATG
LOTlvariaNM 002656 GGAAAGACCACCTGAAAAACCACCTCCAGACCCACGACCCCAACAAAATGGCCTTTGGGTGTGAGGAGTGTGGGAAGAA
GTAC
Maspin NM_002639 CAGATGGCCACTTTGAGAACATTTtAGCTGACAACAGTGTGAACGACCAGACCAAAATCCTTGTGGTTAATGCTGCC
MCMZ NM_004526 GACTTTTGCCCGCTACCTTTCATTCCGGCGTGACAACAATGAGCTGTTGCTCTTCATACTGAAGCAGTTAGTGGC
MCM3 NM_002388 GGAGAACAATCCCCTTGAGACAGAATATGGCCTTTCTGTCTACAAGGATCACCAGACCATCACCATCCAGGAGAT
MCM6 NM_005915 TGATGGTCCTATGTGTCACATTCATCACAGGTTTCATACCAACACAGGCTTCAGCACTTCCTTTGGTGTGTTTCCTGTC
CCA

CTACAGGGACGCCATCGAATCCGGATCT'fGATGCTGGTGTAAGTGAACATTCAGGTGATTGGTTGGAT
MMP9 NM_004994 GAGAACCAATCTCACCGACAGGCAGCTGGCAGAGGAATACCTGTACCGCTATGGTTACACTCGGGTG
MTA1 NM_004689 CCGCCCTCACCTGAAGAGAAACGCGCTCCTTGGCGGACACTGGGGGAGGAGAGGAAGAAGCGCGGCTAACTTATTCC
MYBL2 NM_002466 GCCGAGATCGCCAAGATGTTGCCAGGGAGGACAGACAATGCTGTGAAGAATCACTGGAACTCTACCATCAAAAG

CCCTCGTGCTGATGCTACTGAGGAGCCAGCGTCTAGGGCAGCAGCCGCTTCCTAGAAGACCAGGTCATGATG
p27 NM_004064 CGGTGGACCACGAAGAGTTAACCCGGGACTTGGAGAAGCACTGCAGAGACATGGAAGAGGCGAGCC
P53 NM_000546 CTTTGAACCCTTGCTTGCAATAGGTGTGCGTCAGAAGCACCCAGGACTTCCATTTGCTTTGTCCCGGG
PAIi NM_000602 CCGCAACGTGGTTTTCTCACCCTATGGGGTGGCCTCGGTGTTGGCCATGC.TCCAGCTGACAACAGGAGGAGAAACCCA
GCA
PDGFRb NM_002609 CCAGCTCTCCTTCCAGCTACAGATCAATGTCCCTGTCCGAGTGCTGGAGCTAAGTGAGAGCCACCC
P13KC2A NM_002645 ATACCAATCACCGCACAAACCCAGGCTATTTGTTAAGTCCAGTCACAGCGCAAAGAAACATATGCGGAGAAAATGCTAG
TGTG
PPM1D NM_003620 GCCATCCGCAAAGGCTTTCTCGCTTGTCACCTTGCCATGTGGAAGAAACTGGCGGAATGGCC
PR NM 00092fi GCATCAGGCTGTCATTATGGTGTCCTTACCTGTGGGAGCTGTAAGGTCTTC1'T1'AAGAGGGCAATGGAAGGGCAGCA
CAACTACT
PRAME NM_006115 TCTCCATATCTGCCTTGCAGAGTCTCCTGCAGCACCTCATCGGGCTGAGCAATCTGACCCACGTGC
pS2 NM_003225 GCCCTCCCAGTGTGCAAATAAGGGCTGCTGTTTCGACGACACGGTTCGTGGGGTCCCCTGGTGCTTCTATCCTAATACC
ATCGACG
RADS1C NM_058216 GAACTTCTTGAGCAGGAGCATACCCAGGGCTTCATAATCACCTTCTGTTCAGCACTAGATGATATTCTTGGGGGTGGA
R81 NM_000321 CGAAGCCCTTACAAGTTTCCTAGTTCACCCTTACGGATTCCTGGAGGGAACATCTATATTTCACCCCTGAAGAGTCC

CCAGACGAGCGATTAGAAGCGGCAGCTTGTGAGGTGAATGATTTGGGGGAAGAGGAGGAGGAGGAAGAGGAGGA
STK15 NM_003600 CATCTTCCAGGAGGACCACTCTCTGTGGCACCCTGGACTACCTGCCCCCTGAAATGATTGAAGGTCGGA

CCTGGAGGCTGCAACATACCTCAATCCTGTCCCAGGCCGGATCCTCCTGAAGCCCTTTTCGCAGCACTGCTATCCTCCA
AAGCCATTGTA

Table SB

TGTTTTGATTCCCGGGCTTACCAGGTGAGAAGTGAGGGAGGAAGAAGGCAGTGTCCCTTTTGCTAGAGCTGACAGCTTT
G

GCCCGAAACGCCGAATATAATCCCAAGCGGTTTGCTGCGGTAATCATGAGGATAAGAGAGCCACG' TGFA NM_003236 GGTGTGCCACAGACCTTCCTACTTGGCCTGTAATCACCTGTGCAGCCTTTTGTGGGCCTTCAAAACTCTGTCAAGAACT
CCGT
TIMP1 NM_003254 TCCCTGCGGTCCCAGATAGCCTGAATCCTGCCCGGAGTGGAACTGAAGCCTGCACAGTGTCCACCCTGTTCCCAC

AATCCAAGGGGGAGAGTGATGACTTCCATATGGACTTTGACTCAGCTGTGGCTCCTCGGGCAAAATCTGTAC
TOP2B NM_001068 TGTGGACATCTTCCCCTCAGACTTCCCTACTGAGCCACCTTCTCTGCCACGAACCGGTCGGGCTAG
TP NM_001953 CTATATGCAGCCAGAGATGTGACAGCCACCGTGGACAGCCTGCCACTCATCACAGCCTCCATTCTCAGTAAGAAACTCG
TGG
TP53BP2 NM_005426 GGGCCAAATATTCAGAAGCTTTTATATCAGAGGACCACCATAGCGGCCATGGAGACCATCTCTGTCCCATCATACCCAT
CC
TRAIL NM_003810 CTTCACAGTGCTCCTGCAGTCTCTCTGTGTGGCTGTAACTTACGTGTACTTTACCAACGAGCTGAAGCAGATG
TS NM_001071 GCCTCGGTGTGCCTTTCAACATCGCCAGCTACGCCCTGCTCACGTACATGATTGCGCACATCACG
upa NM 002658 GTGGATGTGCCCTGAAGGACAAGCCAGGCGTCTACACGAGAGTCTCACACTTCTTACCCTGGATCCGCAG
VDR NM_000376 GCCCTGGATTTCAGAAAGAGCCAAGTCTGGATCTGGGACCCTTTCCTTCCTTCCCTGGCTTGTAACT
VEGF NM_003376 CTGCTGTCTTGGGTGCATTGGAGCCTTGCCTTGCTGCTCTACCTCCACCATGCCAAGTGGTCCCAGGCTGC
VEGFB NM_003377 TGACGATGGCCTGGAGTGTGTGCCCACTGGGCAGCACCAAGTCCGGATGCAGATCCTCATGATCCGGTACC
WISP1 NM_003882 AGAGGCATCCATGAACTTCACACTTGCGGGCTGCATCAGCACACGCTCCTATCAACCCAAGTACTGTGGAGTTTG .
XIAP NM_001167 YB-1 NM_004559 AGACTGTGGAGTTTGATGTTGTTGAAGGAGAAAAGGGTGCGGAGGCAGCAAATGTTACAGGTCCTGGTGGTGTTCC
ZNF217 NM_006526 ACCCAGTAGCAAGGAGAAGCCCACTCACTGCTCCGAGTGCGGCAAAGCTTTCAGAACCTACCACCAGCTG

Gene Accession Probe Name Seq Len t AIB1 NM_006534 S1994/AIB1.f3GCGGCGAGTTTCCGATTTA 19 AIB1 NM_006534 S1995/AIB1.r3TGAGTCCACCATCCAGCAAGT 21 AIB1 NM_006534 S5055/AIB1.p3ATGGCGGCGGGAGGATCAAAA 21 AKT1 NM_005163 S0010/AKTl.f3CGCTTCTATGGCGCTGAGAT 20 AKT1 NM_005163 S0012IAKT1.r3TCCCGGTACACCACGTTCTT 20 AKT1 NM_005163 S47761AKT1.p3CAGCCCTGGACTACCTGCACTCGG 24 AKT2 NM_001626 S0828/AKT2.f3TCCTGC'CACCCTTCAAACC 19 AKT2 NM_001626 S0829/AKT2.r3GGCGGTAAATTCATCATCGAA 21 AKT2 NM_001626 S4727/AKT2.p3CAGGTCACGTCCGAGGTCGACACA 24 APC NM_000038 S00221APC.f4GGACAGCAGGAATGTGTTTC. ~ 20 APC NM_000038 S0024/APC.r4ACCCACTCGATTTGTTTCTG 20 APC NM_000038 S48881APC.p4CATTGGCTCCCCGTGACCTGTA ~ 22 AREG NM_001657 S0025/AREG.f2TGTGAGTGAAATGCCTl'CTAGTAGTGA 27 AREG NM_001657 S0027/AREG.r2TTGTGGTTCGTTATCATACTCTTCTGA 27 AREG NM_001657 S4889/AREG.p2CCGTCCTCGGGAGCCGACTATGA 23 B-actinNM_001101 S0034/B-acti.f2CAGCAGATGTGGATCAGCAAG 21 B-actinNM_001101 S00361B-acti.r2GCATTTGCGGTGGACGAT 18 B-actinNM_001101 S4730/B-acti.p2AGGAGTATGACGAGTCCGGCCCC 23 B-CateninNM 001904 S2150/B-Cate.f3GGCTCTTGTGCGTACTGTCCTT 22 B-CateninNM_001904 S2151/B-Cate.r3TCAGATGACGAAGAGCACAGATG 23 B-CateninNM_001904 ' S50461B-Cate.p3AGGCTCAGTGATGTCTTCCCTGTCACCAG 29 BAD NM_032989 S2011/BAD.f1GGGTCAGGTGCCTCGAGAT 19 BAD NM_032989 S2012/BAD.r1CTGCTCACTCGGCTCAAACTC 21 BAD NM 032989 S5058/BAD.p1TGGGCCCAGAGCATGTTCCAGATC 24 BAG1 '. NM_004323S1386/BAG1.f2CGTTGTCAGCACTTGGAATACAA 23 .

BAG1 NM_004323 S1387/BAG1.r2_ 24 GTTCAACCTCTTCCTGTGGACTGT

BAG1 . NM_004323S4731/BAGl.p2CCCAATTAACATGACCCGGCAACCAT 26 BBC3 NM_014417 S1584/BBC3.f2CCTGGAGGGTCCTGTACAAT 20 BBC3 NM_014417 S1585/BBC3.r2CTAATTGGGCTCCATCTCG 19 v ~

BBC3 NM_014417 S4890/BBC3.p2CATCATGGGACTCCTGCCCTTACC 24 Bcl2. NM 000633 S0043/Bcl2.f2CAGATGGACCTAGTACCCACTGAGA 25 Bcl2 NM 000633 S0045/Bcl2.r2CCTATGATTTAAGGGCATTTTTCC 24 Bcl2 NM_000633 S4732/Bcl2.p2TTCCACGCCGAAGGACAGCGAT 22 CA9 NM_001216 S1398/CA9.f3ATCCTAGCCCTGGTTTTTGG 20 CA9. NM_001216 S1399/CA9.r3CTGCCTTCTCATCTGCACAA 20 CA9 NM_001216 S4938/CA9.p3TTTGCTGTCACCAGCGTCGC 20 CCNB1 NM_031966 S1720/CCNB1.f2TTCAGGTTGTTGCAGGAGAC , 20 CCNB1 NM_031966 S1721/CCNB1.r2CATCTTCTTGGGCACACAAT 20 CCNB1 NM 031966 S4733/CCNB1.p2TGTCTCCATTATTGATCGGTTCATGCA 27 r CCND1 NM S0058/CCND1.f3GCATGTTCGTGGCCTCTAAGA 21 _001758 CCND1 NM 001758 S0060ICCND1.r3CGGTGTAGATGCACAGCTTCTC 22 y CCND1 NM S4986lCCND1.p3AAGGAGACCATCCCCCTGACGGC 23 _001758 CCNE NM 001238 1 AAAGAAGATGATGACCGGGTTTAC. 24 r S1446lCCNE1.f1 CCNE1 NM S1447/CCNE1.r1GAGCCTCTGGATGGTGCAAT 20 r CCNE1 NM S4944lCCNE1.p1CAAACTCAACGTGCAAGCCTCGGA 24 _001238 CCNE2 NM 057749 .S1458/CCNE2.f2~ ATGCTGTGGCTCCTTCCTAACT 22 CCNE 2 NM 057749S1459/CCNE2.r2ACCCAAATTGTGATATACAAAAAGGTT 27 CCNE2 NM 057749 S4945/CCNE2.p2TACCAAGCAACCTACATGTCAAGAAAGCCC 30 CD3z NM 000734 S0064/CD3z.f1AGATGAAGTGGAAGGCGCTT 20 CD3z NM_000734 S0066ICD3z.r1TGCCTCTGTAATCGGCAACTG 21 CD3z NM_000734 S49881CD3z.p1CACCGCGGCCATCCTGCA 18 CD68 NM_001251 S0067/CD68.f2TGGTTCCCAGCCCTGTGT 18 CD68 NM_001251 S00691CD68.r2CTCCTCCACCCTGGGTTGT 19 CD68 NM_001251 S4734/CD68.p2CTCCAAGCCCAGATTCAGATTCGAGTCA 28 CD9 NM_001769 S06861CD9.f1GGGCGTGGAACAGTTTATCT 20 CD9 NM_001769 S0687/CD9.r1CACGGTGAAGGTTTCGAGT 19 CD9 NM_001769 S4792/CD9.p1AGACATCTGCCCCAAGAAGGACGT 24 CDH1 NM_004360 S0073/CDH1.f3TGAGTGTCCCCCGGTATCTTC 21 CDH1 NM 004360 S0075/CDH1.r3CQGCCGCTTTCAGATTTTCAT 21 CDH1 NM 004360 S49901CDH1.p3TGCCAATCCCGATGAAATTGGAAAT'1-f' 27 CEGP1 NM 020974 S1494/CEGPI.f2TGACAATCAGCACACCTGCAT 21 ,~.r CEGP1 NM 020974 51495ICEGP1.r2TGTGACTACAGCCGTGATCCTTA 23 CEGP1 ~ 020974 S4735/CEGP1.p2CAGGCCCTCTTCCGAGCGGT 20 NM_ Chk1 NM_001274 S1422/Chk1.f2GATAAATTGGTACAAGGGATCAGCTT 26 Chk1 NM_001274 S14231Chk1.r2GGGTGCCAAGTAACTGACTATTCA 24 Chk1 NM_001274.S4941/Chk1.p2CCAGCCCACATGTCCTGATCATATGC 26 CIAP1 NM_001166 S0764/CIAPI.f2TGCCTGTGGTGGGAAGCT 18 CIAP1 NM_001166 S0765/CIAP1.r2GGAAAATGCCTCCGGTGTT 19 CIAP1 NM_001166 S4802/CIAP1.p2TGACATAGCATCATCCTTTGG'f'T'CCCAGTT30 cIAP2 NM_001165 S0076Ic1AP2.f2GGATATTTCCGTGGCTCTTATTCA 24 ~

cIAP2 NM_001165 S0078/cIAP2.r2CTTCTCATCAAGGCAGAAAAATCTT . 25 cIAP2 NM_001165 S49911cIAP2.p2TCTCCATCAAATCCTGTAAACTCCAGAGCA 30 ' cMet NM_000245 S0082/cMet.f2GACATTTCCAGTCCTGCAGTCA 22 cMet NM_000245 S0084/cNlet.r2CTCCGATCGCACACATTTGT 20 cMet NM_000245 S4993lcMet.p2TGCCTCTCTGCCCCACCCTTTGT 23 Contig AK000618 S2633/Contig.f3GGCATCCTGGCCCAAAGT 18 Contig AK000618 S2634/Contig.r3GACCCCCTCAGCTGGTAGTTG 21 27882 ~

Contig AK000618 S4977IContig.p3CCCAAATCCAGGCGGCTAGAGGC 23 27882 .

COX2 . 000963 S0088/COX2.f1TCTGCAGAGTTGGAAGCACTCTA 23 NM_ COX2 NM_000963 S0090/COX2.r1GCCGAGGCTTTTCTACCAGAA 21 COX2 NM_000963 S4995/COX2.p1CAGGATACAGCTCCACAGCATCGATGTC 28 . .

CTSL NM_001912 S1303lCTSL.f2GGGAGGCTTATCTCACTGAGTGA , 23 CTSL NM 001912 S1304/CTSL.r2CCATTGCAGCCTTCATTGC 19 CTSL NM 001912 S4899/CTSL.p2TTGAGGCCCAGAGCAGTCTACCAGATTCT 29 CTSL2 NM _001333. S4354/CTSL2.f1TGTCTCACTGAGCGAGCAGAA 21 CTSL2 NM 001333 S4355/CTSL2.r1ACCATTGCAGCCCTGATTG 19 CTSL2 ' 001333 S4356/CTSL2.p1CTTGAGGACGCGAACAGTCCACCA 24 NM

DAPK '. _ S1768/DAPK1.f3CGCTGACATCATGAATGTTCCT 22 NM _0049381 ~

DAPK1 NM _004938S1769/DAPK1.r3TCTCTI-fCAGCAACGATGTGTCTT 24 DAPK1 NM 004938 S4927/DAPK1.p3TCATATCCAAACTCGCCTCCAGCCG 25 DIABLO NM 019887 S0808/DIABLO.f1CACAATGGCGGCTCTGAAG 19 DIABLO NM _019887S0809/DIABLO.r1 ACACAAACACTGTCTGTACCTGAAGA 26 ' DIABLO NM _019887S4813/DIABLO.p1AAGTTACGCTGCGCGACAGCCAA 23 ' DR5 NM _003842S25511DR5.f2CTCTGAGACAGTGCTTCGATGACT 24 DR5 NM _003842S2552/DR5.r2CCATGAGGCCCAACTTCCT 19 DR5 NM _003842S4979/DR5.p2CAGACTTGGTGCCCTTTGACTCC 23 EGFR NM _005228S0103/EGFR.f2TGTCGATGGACTTCCAGAAC 20 EGFR NM _005228S0105lEGFR.r2ATTGGGACAGCTTGGATCA 19 EGFR NM _005228S4999/EGFR.p2CACCTGGGCAGCTGCCAA 18 ~

EIF4E NM _001968S0106/EIF4E.f1GATCTAAGATGGCGACTGTCGAA 23 EIF4E NM _001968S01081E1F4E.r1TTAGATTCCGTTTTCTCCTCTTCTG 25 ' EIF4E NM _001968S5000/EIF4E.p1ACCACCCGTACTCCTAATCCCCCGACT 27 EMS1 NM _005231~S2663/EMS1.f1GGCAGTGTCACTGAGTCCTTGA 22 EMS1 NM _005231S2664/EMS1.r1TGCACTGTGCGTCCCAAT 18 EMS1 NM _005231S4956/EMS1.p1ATCCTCCCCTGCCCCGCG 18 EpCAM NM _002354_ GGGCCCTCCAGAACAATGAT 20 S1807lEpCAM.f1 EpCAM NM _002354S1808lEpCAM.r1TGCACTGCTTGGCCTTAAAGA 21 EpCAM NM _~002354S498.41EpCAM.p1CCGCTCTCATCGCAGTCAGGATCAT 25 EPHX1 NM _000120S1865/EPHXI.f2ACCGTAGGCTCTGCTCTGAA 20 EPHX1 NM _000120S1866/EPHX1:r2TGGTCCAGGTGGAAAACTTC 20 EPHX1 NM _000120S4754lEPHX1.p2AGGCAGCCAGACCCACAGGA 20 Erb83 NM _00198250112/ErbB3.f1CGGTTATGTCATGCCAGATACAC 23 ErbB3 NM_001982 S0114lErbB3.r1GAACTGAGACCCACTGAAGAAAGG 24 ErbB3 NM _001982S5002/ErbB3.p1CCTCAAAGGTACTCCCTCCTCCCGG 25 EstR1 NM _000125S0115/EstR1.f1CGTGGTGCCCCTCTATGAC 19 EstR1 NM _000125S0117/EstR1.r1GGCTAGTGGGCGCATGTAG 19 ' EstR1 NM _000125S4737/EstR1.p1CTGGAGATGCTGGACGCCC 19 FBX05 NM _012177S2017/FBX05.r1GGATTGTAGACTGTCACCGAAATTC ~ 25 FBX05 NM S2018/FBX05.f1GGCTATTCCTCATTTTCTCTACAAAGTG 28 FBX05 NM 012177 S5061/FBX05.p1CCTCCAGGAGGCTACCTTCTTCATGTTCAC .30 FGF18 NM _003862S1665/FGF18.f2CGGTAGTCAAGTCCGGATCAA 21 ~

FGF18 NM 003862 S1666/FGF.18.r2GCTTGCCTTTGCGGTTCA 18 ~

FGF18 NM ,003862S4914/FGF18.p2CAAGGAGACGGAATTCTACCTGTGC 25 FGFR1 NM 023109 508181FGFR1.f3CACGGGACATfCACCACATC 20 FGFR1 NM_023109 S0819lFGFR1.r3GGGTGCCATCCACTTCACA 19 FGFR1. NM_023109 S4816/FGFRI.p3ATAAAAAGACAACCAACGGCCGACTGC 27 FHIT NM 002012 S2443lFHIT.f1CCAGTGGAGCGCTi'CCAT ~ 18 FHIT NM 002012 S2444/FHIT.r1CTCTCTGGGTCGTCTGAAACAA ~ 22 FHIT NM_002012'S2445lFHIT.p1TCGGCCACTTCATCAGGACGCAG 23 FHIT NM 002012 S4921/FHIT.p1TCGGCCACI-fCATCAGGACGCAG 23 FRP1 NM 003012 S1804IFRP1.f3TTGGTACCTGTGGGT'1-AGCA 20 FRP1 NM_003012 S1805/FRP1.r3CACATCCAAATGCAAACTGG 20 FRP1 NM_003012 S4983/FRPl.p3TCCCCAGGGTAGAATTCAATCAGAGC 26 G-CateninNM_002230 S2153/G-Cate.f1TCAGCAGCAAGGGCATCAT 19 G-CateninNM_002230 S2154/G-Cate.r1GGTGGTfTTCTTGAGCGTGTACT 23 G-CateninNM_002230 S5044/G-Cate.plCGCCCGCAGGCCTCATCCT 19 GAPDH NM S0374lGAPDH.f1ATTCCACCCATGGCAAATTC 20 GAPDH _ S0375/GAPDH.r1GATGGGATI-fCCATTGAT'GACA 22 NM_002046 GAPDH NM_002046 S4738/GAPDH.p1CCGTTCTCAGCCTTGACGGTGC 22 GATA3 NM_002051 S0127/GATA3.f3CAAAGGAGCTCACTGTGGTGTCT 23 GATA3 : NM_002051S0129/GATA3.r3GAGTCAGAATGGCTTATTCACAGATG 26 GATA3 NM_002051 S5005/GATA3.p3TGTTCCAACCACTGAATCTGGACC 24 GRB7 NM_005310 S0130IGRB7.f2CCATCTGCATCCATCTT'GTT 20 GRB7 NM_00531'0S0132/GRB7.r2GGCCACCAGGGTATTATCTG 20 GRB7 NM_005310 S4726/GRB7.p2CTCCCCACCCTTGAGAAGTGCCT 23.

GR01 NM_001511 S0133/GR01.f2CGAAAAGATGCTGAACAGTGACA 23 GR01 NM_001511 S0135IGR01.r2TCAGGAACAGCCACCAGTGA 20 GR01 NM_001511 S5006/GR01.p2CTTCCTCCTCCCTTCTGGTCAGTTGGAT 28 GSTM1 ~ NM 000561S2026/GSTM1.r1GGCCCAGCTTGAATTTTTCA 20 x GSTM1 ' NM 000561S2027/GSTM1.f1AAGCTATGAGGAAAAGAAGTACACGAT 27 GSTM1 NM 000561 S4739/GSTM1.p1TCAGCCACTGGCTTCTGTCATAATCAGGAG30 GUS 000181 ' S01391GUS.f1CCCACTCAGTAGCCAAGTCA 20 NM

GUS _ S0141/GUS.r1 CACGCAGGTGGTATCAGTCT 20 NM

GUS _ S4740/GUS.p1 TCAAGTAAACGGGCTGTTTTCCAAACA 27 .
NM

HER2 _ S0142/HER2.f3CGGTGTGAGAAGTGCAGCAA 20 NM

HER2 _ S0144/HER2.r3CCTCTCGCAAGTGCTCCAT 19 NM_004448 HER2 NM S47291HER2.p3CCAGACCATAGCACACTCGGGCAC 24 HIF1A _ S1207/HIF1A.f3TGAACATAAAGTCTGCAACATGGA 24 NM_001530 HIF1A NM_001530 S12081HIF1A.r3TGAGGTI'GGTTACTGTt'GGTATCATATA28 HIF1A NM 001530 S4753/HIF1A.p3TTGCACTGCACAGGCCACATTCAC 24 HNF3A NM 004496 S01481HNF3A.f1TCCAGGATGTTAGGAACTGTGAAG 24 HNF3A NM_004496 S0150/HNF3A.r1GCGTGTCTGCGTAGTAGCTGTT 22 ~

HNF3A NM_004496 S5008/HNF3A.p1AGTCGCTGGTTfCATGCCCTTCCA 24 ID1 002165 S0820/ID1.f1 AGAACCGCAAGGTGAGCAA 19 NM

lD1 _ S0821/ID1.r1 TCCAACTGAAGGTCCCTGATG 21 . NM_002165 ID1 002165 S4832/IDl.p1 TGGAGATTCTCCAGCACGTCATCGAC 26 NM

IGF1 _ S0154/IGF1.f2TCCGGAGCTGTGATCTAAGGA 21 NM

IGF1 _ S0156/IGF1.r2CGGACAGAGCGAGCTGACTT 20 NM

IGF1 _ S5010/IGF1.p2TGTATTGCGCACCCCTCAAGCCTG 24 NM

IGF1 _ S1249/IGF1 GCATGGTAGCCGAAGAlTfCA 21 R NM R.f3 IGF1 _ S12501IGF1 TTTCCGGTAATAGTCTGTCTCATAGATATC30 R 000875 R.r3 NM

IGF1 _ S48951IGF1 CGCGTCATACCAAAATCTCCGATm'GA 28 R NM 000875 R.p3 IGFBP2 000597 S1128/IGFBP2.f1GTGGACAGCACCATGAACA 19 NM

IGFBP2 _ S1129/IGFBP2.r1CCTTCATACCCGACTTGAGG 20 ~000597 NM

IGFBP2 _ S4837/IGFBP2.p1CTTCCGGCCAGCACTGCCTC 20 NM

IL6 _ S0760/IL6.f3 CCTGAACCTTCCAAAGATGG 20 NM

IL6 _ S0761/IL6.r3 ACCAGGCAAGTCTCCTCATT 20 NM

IL6 _ S4800/IL6.p3 CCAGATfGGAAGCATCCATCTTTT'fCA 27 NM

IRS1 _ S1943/IRSl.f3CCACAGCTCACCTTCTGTCA 20 NM

1RS1 _ S1944/IRS1.r3CCTCAGTGCCAGTCTCTTCC 20.
NM

IRS1 _ S5050/IRS1.p3TCCATCCCAGCTCCAGCCAG 20 NM_005544 Ki-67 002417 S0436/Ki-67.f2CGGACTTTGGGTGCGACTT 19 NM

Ki-67 _ S0437/Ki-67.r2TTACAACTCTi'CCACTGGGACGAT 24 Ki-67 NM 002417 S4741/Ki-67.p2CCACTTGTCGAACCACCGCTCGT 23 KLK10 NM 002776 S2624/KLK10.f3GCCC.AGAGGCTCCATCGT 18 KLK10 NM 002776 52625/KLK10.r3 23 ' CAGAGGTTTGAACAGTGCAGACA ' KLK10 NM_002776 S4978/KLK10.p3CCTCTTCCTCCCCAGTCGGCTGA 23 KRT14 NM 000526 S1853/KRT14.f1GGCCTGCTGAGATCAAAGAC 20 KRT14 NM 000526 S1854/KRT14.r1GTCCACTGTGGCTGTGAGA.4 20 KRT14 NM_000526 S5037/KRT14.p1TGTTCCTCAGGTCCTCAATGGTCTTG 26 KRT17 NM_000422 S01721KRT17.f2CGAGGATTGGTTCTTCAGCAA 21 KRT17 NM_000422 S0174IKRT17.r2ACTCTGCACCAGCTCACTGTTG 22 KRT17 NM_000422 S5013/KRT17.p2CACCTCGCGGTTCAGTTCCTCTGT 24 KRT18 NM_000224 S1710/KRT18.f2AGAGATCGAGGCTCTC'AAGG 20 KRT18 NM_000224 S1711/KRT18.r2GGCCTTTTACTTCCTCTTCG , . 20 KRT18 NM_000224 S4762/KRT18.p2TGGTTCTTCTTCATGAAGAGCAGCTCC 27 KRT19 NM_002276 S1515/KRT19.f3TGAGCGGCAGAATCAGGAGTA 21 KRT19 NM_002276 51516/KRT19.r3TGCGGTAGGTGGCAATCTC 19 KRT19 NM_002276 S4866IKRT19.p3'CTCATGGACATCAAGTCGCGGCTG 24 ~

KRT5 NM_000424 S01751KRT5.f3TCAGTGGAGAAGGAGTTGGA 20 KRT5 NM_00042.4S0177/KRT5.r3TGCCATATCCAGAGGAAACA 20 KRT5 NM_000424 S5015/KRT5.p3CCAGTCAACATCTCTGTTGTCACAAGCA 28 KRT8 . NM_002273S2588/KRT8.f3GGATGAAGCTTACATGAACAAGGTAGA 27 KRT8 NM 002273 S2589/KRT8.r3CATATAGCTGCCTGAGGAAGTTGAT 25 ' KRT8 NM_002273 S49521KRT8.p3CGTCGGTCAGCCCTTCCAGGC 21 LOT1 NM_002656 S0692/LOT1 GGAAAGACCACCTGAAAAACCA 22 variant v.f2 , variant v.r2 variant ' v.p2 Maspin NM_002639 50836/Maspin.f2CAGATGGCCACTTTGAGAACATT 23 Maspin NM 002639 S0837IMaspin.r2GGCAGCATTAACCACAAGGATT 22 Maspin NM 002639 S4835/Maspin.p2AGCTGACAACAGTGTGAACGACCAGACC 28 .

MCM2 ~ NM_004526S1602/MCM2.f2GACTTTTGCCCGCTACCTTTC ~21 MCM2 .004526 S1603/MCM2.r2GCCACTAACTGCTTCAGTATGAAGAG 26 NM

MCM2 _ S49001MCM2.p2ACAGCTCATTGTTGTCACGCCGGA 24 NM_004526 MCM3 NM_002388 S1524/MCM3.f3.GGAGAACAATCCCCTTGAGA 20 MCM3 NM 002388 S1525/MCM3.r3ATCTCCTGGATGGTGATGGT 20 MCM3 NM_002388 S4870/MCM3.p3TGGCCTTTCTGTCTACAAGGATCACCA 27 MCM6 NM_005915 S1704/MCM6.f3TGATGGTCCTATGTGTCACATTCA 24 .

MCM6 NM_005915 S1705/MCM6.r3TGGGACAGGAAACACACCAA 20 MCM6 NM_005915 S4919/MCM6.p3CAGGTTTCATACCAACACAGGCTTCAGCAC30 MDM2 NM_002392 S0830/MDM2.f1CTACAGGGACGCCATCGAA 19 MDM2 NM_002392 S0831/MDM2.r1ATCCAACCAATCACCTGAATGTT 23 MDM2 NM_002392 S4834/MDM2.p1CTTACACCAGCATCAAGATCCGG , 23 MMP9 NM_'.004994S0656/MMP9.f1GAGAACCAATCTCACCGACA 20 MMP9 004994 S0657/MMP9.r1CACCCGAGTGTAACCATAGC - 20 NM

MMP9 _ S4760/MMP9.p1ACAGGTATTCCTCTGCCAGCTGCC . 24 NM

MTA1 _ S2369/MTA1.f1CCGCCCTCACCTGAAGAGA 19 NM_004689 MTA1 004689 S2370/MTA1.r1GGAATAAGTTAGCCGCGCTTCT 22 NM

MTA1 _ S4855/MTA1.p1CCCAGTGTCCGCCAAGGAGCG 21 NM

MYBL2 _ S3270/MYBL2.f1GCCGAGATCGCCAAGATG 18 NM

MYBL2 _ S3271/MYBL2.r1CTTTTGATGGTAGAGTTCCAGTGATTC 27 MYBL2 NM 002466 S4742/MYBL2.p1CAGCATTGTCTGTCCTCCCTGGCA 24 P14ARF S78535 S2842IP14ARF.f1CCCTCGTGCTGATGCTACT 19 P14ARF S78535 S2843/P14ARF.r1CATCATGACCTGGTCTTCTAGG 22 P14ARF S78535 S4971/P14ARF.p1CTGCCCTAGACGCTGGCTCCTC 22 p27 NM_004064 S0205/p27.f3 CGGTGGACCACGAAGAGTTAA 21 p27 NM_004064 S0207/p27.r3 GGCTCGCCTCTTCCATGTC 19 p27 NM_004064 S4750/p27.p3 CCGGGACTTGGAGAAGCACTGCA . 23 P53 000546 S0208/P53.f2 CTTTGAACCCTTGCTTGCAA 20 NM

P53 _ S0210/P53.r2 CCCGGGACAAAGCAAATG 18 NM

P53 ~ _ S5065/P53.p2 AAGTCCTGGGTGCTTCTGACGCACA 25 NM , PAI1 _ S0211/PAL1.f3CCGCAACGTGGTT'fTCTCA 19 NM_000602 PAI1 NM_000602 S0213/PAIl.r3TGCTGGGTTTCTCCTCCTGTT 21 PAI1 NM 000602 S5066/PAI1.p3CTCGGTGTTGGCCATGCTCCAG 22 PDGFRb NM 0,02609S1346/PDGFRb.f3CCAGCTCTCCTTCCAGCTAC 20 PDGF.Rb NM 002609 S1347/PDGFRb.r3GGGTGGCTCTCACTTAGCTC 20 PDGFRb NM 002609 S4931/PDGFRb.p3ATCAATGTCCCTGTCCGAGTGCTG 24 P13KC2A 002645 52020IP13KC2.r1CACACTAGCATTTTCTCCGCATA 23 NM .

P13KC2A _ S2021/P13KC2.f1ATACCAATCACCGCACAAACC 21 NM

P13KC2A _ S5062/P13KC2.p1TGCGCTGTGACTGGACTTAACAAATAGCCT30 PPM1D 003620 S31591PPM1D.f1GCCATCCGCAAAGGCTTT ~ 18 ~ NM

PPM1D _ S3160/PPM1D.r1GGCCATTCCGCCAGTTTC 18 NM

PPM1 _ S48561PPM1 TCGCTTGTCACCTTGCCATGTGG 23 D 003620 D.p1 NM

PR _ S13361PR.f6 GCATCAGGCTGTCATTATGG . 20 NM_000926 PR 000926 S1337/PR.r6 AGTAGTTGTGCTGCCCTTCC , 20 NM

PR _ S4743IPR.p6 TGTCCTTACCTGTGGGAGCTGTAAGGTC 28 PRAMS NM 006115 S1985/PRAME.f3TCTCCATATCTGCCTTGCAGAGT . 23 PRAMS 006115 S19861PRAME.r3GCACGTGGGTCAGATTGCT . 19 NM

PRAMS _ S4756/PRAME.p3TCCTGCAGCACCTCATCGGGCT 22 NM

pS2 _ S0241/pS2.f2GCCCTCCCAGTGTGCAAAT
NM_003225 pS2 NM_003225 S0243/pS2.r2CGTCGATGGTATTAGGATAGAAGCA 25 pS2 NM 003225 S5026/pS2.p2TGCTGTTTCGACGACACCGTTCG 23 RAD51 058216 . S2606/RAD51 GAACTTCTTGAGCAGGAGCATACC 24 C NM C.f3 RAD51 _ S2607IRAD51 TCCACCCCCAAGAATATCATCTAGT 25 C 058216 C.r3 NM

RAD51 _ S4764/RAD51 AGGGCTTCATAATCACCTTCTGTTC 25 C 058216 C.p3 . NM

RB1 _ S27001RB1.f1CGAAGCCCTTACAAGTTTCC 20 .NM 000321 RB1 NM 000321 S27011RB1.r1GGACTCTTCAGGGGTGAAAT 20 RB1 NM 000321 S4765/RB1.p1CCCTTACGGATTCCTGGAGGGAAC 24 RIZ1 NM 012231 S1320/RIZ1.f2CCAGACGAGCGATTAGAAGC 20 RIZ1 012231 S1321/RIZ1.r2TCCTCCTCTTCCTCCTCCTC 20 NM

RIZ1 _ S4761/RIZ1.p2TGTGAGGTGAATGATTTGGGGGA 23 NM

STK15 _ S0794/STK15.f2CATCTTCCAGGAGGACCACT 20 NM

STK15 _ S07951STK15.r2TCCGACCTTCAATCATTTCA 20 STK15 ' NM 003600S4745/STK15~p2CTCTGTGGCACCCTGGACTACCTG 24 STMY3 005940 ' S2067/STMY3.f3CCTGGAGGCTGCAACATACC 20 NM

STMY3 _ S2068/STMY3.r3TACAATGGCTTTGGAGGATAGCA 23 STMY3 005940 S4746/STMY3.p3ATCCTCCTGAAGCCCTTTTCGCAGC 25 NM

SURV _ 5.02591SURV.f2TGTTTTGATTCCCGGGCTTA 20 NM 001168 ~
-SURV NM 001168 S0261/SURV.r2CAAAGCTGTCAGCTCTAGCAAAAG 24 ~SURV 001168 S4747/SURV.p2TGCCTTCTTCCTCCCTCACTTCTCACCT , TBP _ S0262/TBP.f1GCCCGAAACGCCGAATATA 19 TBP 003194 S0264ITBP.r1CGTGGCTCTCTTATCCTCATGAT .

TBP _ S4751lTBP.p1TACCGCAGCAAACCGCTTGGG 21 NM 003194 ~

TGFA 003236 S0489/TGFA.f2GGTGTGCCACAGACCTTCCI' 20 NM

TGFA _ S0490rfGFA.r2ACGGAGTTCTTGA.CAGAGTTTTGA 24 NM

TGFA _ ~ S4768/TGFA.p2TTGGCCTGTAATCACCTGTGCAGCCTT 27 TIMP1 003254 S1695ITIMPI.f3TCCCTGCGGTCCCAGATAG ' TIMP1 _ S1696/TIMP1.r3GTGGGAACAGGGTGGACACT 20 NM

TIMP1 _ S4918ITIMP1.p3ATCCTGCCCGGAGTGGAACTGAAGC 25 TOP2A 001067 S0271lTOP2A.f4AATCCAAGGGGGAGAGTGAT 20 NM

TOP2A _ S0273ITOP2A.r4GTACAGATTTTGCCCGAGGA 20 TOP2A .NM 001067 S4777rfOP2A.p4CATATGGACTi~'GACTCAGCTGTGGC 26 TOP2B NM 001068 S0274/TOP2B.f2TGTGGACATCTTCCCCTCAGA 21 TOP2B NM 001068 S0276ITOP2B.r2CTAGCCCGACCGGTTCGT 18 TOP2B 001068 S4778/TOP2B.p2TTCCCTACTGAGCCACCTTCTCTG 24 NM

TP _ S0277/TP.f3 CTATATGCAGCCAGAGATGTGACA 24 , NM

TP _ S0279/TP.r3 CCACGAGTTTCTTACTGAGAATGG 24 NM

TP _ S4779/TP.p3 ACAGCCTGCCACTCATCACAGCC 23 NM

TP538P2 _ S1931IT'P53BP.f2GGGCCAAATATTCAGAAGC 19 NM

TP53BP2 _ S1932ITP53BP.r2GGATGGGTATGATGGGACAG ZO

NM

TP53BP2 _ S5049/TP53BP.p2 20 NM

TRAIL _ S2539lTRAlL.f1CTTCACAGTGCTCCTGCAGTCT 22 NM

TRAIL _ S2540lTRAIL.r1CATCTGCTTCAGCTCGTTGGT ' 21 NM

TRAIL _ S4980rfRAIL.p1AAGTACACGTAAGTTACAGCCACACA' 26 NM

TS _ S0280/TS.f1 GCCTCGGTGTGCCTTTCA 18 NM

TS _ S0282/TS.r1 CGTGATGTGCGCAATCATG 19 NM

TS _ S4780/TS.p1 CATCGCCAGCTACGCCCTGCTC 22 NM

upa _ S0283/upa.f3_ 19 upa NM 002658 S0285/upa.r3a 20 CTGCGGATCCAGGGTAAGAA

'fable 6r upa NM 002658 S4769/upa.p3 AAGCCAGGCGTCTACACGAGAGTCTCAC 28 VDR NM_000376 S2745NDR.f2 GCCCTGGAT')-fCAGAAAGAG 20 ~

VDR NM_000376 S2746NDR.r2 AGTTACAAGCCAGGGAAGGA 20 VDR NM_000376 S4962NDR.p2 CAAGTCTGGATCTGGGACCCTTTCC 25 VEGF NM_003376 S0286NEGF,f1 CTGCTGTCTTGGGTGCATTG 20 VEGF NM_003376 S0288NEGF.r1 GCAGCCTGGGACCACTTG 18 VEGF NM_003376 S4782NEGF.p1 TTGCCTTGCTGCTCTACCTCCACCA 25 VEGFB NM_003377 S2724NEGFB.f1TGACGATGGCCTGGAGTGT 19 VEGFB NM_003377 S2725NEGFB.r1GGTACCGGATCATGAGGATCTG 22 VEGFB NM_00337.7S4960NEGFB.p1CTGGGCAGCACCAAGTCCGGA .' 21 WISP1 NM_003882 S1671/WISPI.f1.AGAGGCATCCATGAACTTCACA 22 WISP1 NM_003882 S1672/WISP1.r1CAAACTCCACAGTACTTGGGTTGA 24 WISP1 NM_003882 S4915/WISP1.p1CGGGCTGCATCAGCACACGC 20 XIAP NM_001167 S0289/XIAP.f1GCAGTTGGAAGACACAGGAAAGT 23 ~

XIAP NM_001167 S0291/XIAP.r1TGCGTGGCACTATTTTCAAGA -XIAP NM_001167 S4752/XIAP.p1TCCCCAAATTGCAGAlTfATCAACGGC 27 YB-1 NM_004559 S1194/YB-1.f2AGACTGTGGAGTTTGATGTTGTTGA 25 YB-1 004559 S1195IYB-1.r2GGAACACCACCAGGACCTGTAA 22 NM

YB-1 _ S4843/YB-1.p2TTGCTGCCTCCGCACCCITfTCT 23 NM

ZNF217 _ S2739/ZNF217.f3ACCCAGTAGCAAGGAGAAGC 20 ' NM

ZNF217 _ S2740/ZNF217.r3CAGCTGGTGGTAGGTTCTGA 20 NM

ZNF217 _ S49611ZNF217.p3CACTCACTGCTCCGAGTGCGG 21 .. , :-, 39740-0008 PCT.TXT
SEQUENCE LISTING
<110> GENOMIC HEALTH, INC.
RUSH UNIVERSITY MEDICAL CENTER
COBLEIGH, Melody SHAK, Steven BAKER, Joff re CRONIN, Maureen <120> GENE EXPRESSION MARKERS FOR BREAST
CANCER PROGNOSIS
<130> 39740-0008 PCT
<140> Unassigned <141> 2004-01-14 <150> US 60/440,861 <151> 2003-01-15 <160> 440 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 81 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 1 gcggcgagtt tccgatttaa agctgagctg cgaggaaaat ggcggcggga ggatcaaaat 60 acttgctgga tggtggactc a 81 <210> 2 <211> 71 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 2 cgcttctatg gcgctgagat tgtgtcagcc ctggactacc tgcactcgga gaagaacgtg 60 gtgtaccggg a 71 <210> 3 <211> 71 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 3 tcctgccacc cttcaaacct caggtcacgt ccgaggtcga cacaaggtac ttcgatgatg 60 aatttaccgc c 71 <210> 4 <211> 69 <212> DNA
<213> Artificial Sequence <220>

<223> Amplicon <400> 4 ggacagcagg aatgtgtttc tccatacagg tcacggggag ccaatggttc agaaacaaat 60 cgagtgggt 69 <210> 5 <211> 82 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 5 tgtgagtgaa atgccttcta gtagtgaacc gtcctcggga gccgactatg actactcaga 60 agagtatgat aacgaaccac as 82 <210> 6 <211> 66 <212> DNA
<213> Artificial sequence <220>
<223> amplicon <400> 6 cagcagatgt ggatcagcaa gcaggagtat gacgagtccg gcccctccat cgtccaccgc 60 aaatgc 66 <210> 7 <211> 80 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 7 ggctcttgtg cgtactgtcc ttcgggctgg tgacagggaa gacatcactg agcctgccat 60 ctgtgctctt cgtcatctga 80 <210> 8 <211> 73 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 8 gggtcaggtg cctcgagatc gggcttgggc ccagagcatg ttccagatcc cagagtttga 60 gccgagtgag cag 73 <210> 9 <211> 81 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 9 cgttgtcagc acttggaata caagatggtt gccgggtcat gttaattggg aaaaagaaca 60 gtccacagga agaggttgaa c 81 <210> 10 <211> 83 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 10 cctggagggt cctgtacaat ctcatcatgg gactcctgcc cttacccagg ggccacagag 60 cccccgagat ggagcccaat tag 83 <210> 11 <211> 73 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 11 cagatggacc tagtacccac tgagatttcc acgccgaagg acagcgatgg gaaaaatgcc 60 cttaaatcat agg 73 <210> 12 <211> 72 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 12 atcctagccc tggtttttgg cctccttttt gctgtcacca gcgtcgcgtt ccttgtgcag 60 atgagaaggc ag 72 <210> 13 <211> 84 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 13 ttcaggttgt tgcaggagac catgtacatg actgtctcca ttattgatcg gttcatgcag 60 aataattgtg tgcccaagaa gatg 84 <210> 14 <211> 69 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 14 gcatgttcgt ggcctctaag atgaaggaga ccatccccct gacggccgag aagctgtgca 60 tctacaccg 6g <Z10> 15 <211> 71 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 15 aaagaagatg atgaccgggt ttacccaaac tcaacgtgca agcctcggat tattgcacca 60 tccagaggct c 71 <210> 16 <211> 82 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 16 atgctgtggc tccttcctaa ctggggcttt cttgacatgt aggttgcttg gtaataacct 60 ttttgtatat cacaatttgg gt 82 <210> 17 <211> 65 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 17 agatgaagtg gaaggcgctt ttcaccgcgg ccatcctgca ggcacagttg ccgattacag 60 aggca 65 <210> 18 <211> 74 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 18 tggttcccag ccctgtgtcc acctccaagc ccagattcag attcgagtca tgtacacaac 60 ccagggtgga ggag 74 <210> 19 <211> 64 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 19 gggcgtggaa cagtttatct cagacatctg ccccaagaag gacgtactcg aaaccttcac 60 cgtg 64 <210> 20 <211> 81 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 20 tgagtgtccc ccggtatctt ccccgccctg ccaatcccga tgaaattgga aattttattg 60 atgaaaatct gaaagcggct g 81 <210> 21 <211> 77 <212> DNA

<213> Artificial Sequence <220>
<223> Amplicon <400> 21 tgacaatcag cacacctgca ttcaccgctc ggaagagggc ctgagctgca tgaataagga 60 tcacggctgt agtcaca 77 <210> 22 <211> 82 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 22 gataaattgg tacaagggat cagcttttcc cagcccacat gtcctgatca tatgcttttg 60 aatagtcagt tacttggcac cc 82 <210> 23 <211> 72 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 23 tgcctgtggt gggaagctca gtaactggga accaaaggat gatgctatgt cagaacaccg 60 gaggcatttt cc <210> 24 <211> 86 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 24 ggatatttcc gtggctctta ttcaaactct ccatcaaatc ctgtaaactc cagagcaaat 60 caagattttt ctgccttgat gagaag 86 <210> 25 <211> 86 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 25 gacatttcca gtcctgcagt caatgcctct ctgccccacc ctttgttcag tgtggctggt~60 gccacgacaa atgtgtgcga tcggag 86 <210> 26 <211> 75 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 26 ggcatcctgg cccaaagttt cccaaatcca ggcggctaga ggcccactgc ttcccaacta 60 ccagctgagg gggtc 75 <210> 27 <211> 79 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 27 tctgcagagt tggaagcact ctatggtgac atcgatgctg tggagctgta tcctgccctt 60 ctggtagaaa agcctcggc 79 <210> 28 <211> 74 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 28 gggaggctta tctcactgag tgagcagaat ctggtagact gctctgggcc tcaaggcaat 60 gaaggctgca atgg 74 <210> 29 <211> 67 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 29 tgtctcactg agcgagcaga atctggtgga ctgttcgcgt cctcaaggca atcagggctg 60 caatggt 67 <210> 30 <211> 77 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 30 cgctgacatc atgaatgttc ctcgaccggc tggaggcgag tttggatatg acaaagacac 60 atcgttgctg aaagaga 77 <210> 31 <211> 73 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 31 cacaatggcg gctctgaaga gttggctgtc gcgcagcgta acttcattct tcaggtacag 60 acagtgtttg tgt 73 <210> 32 <211> 84 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 32 ctctgagaca gtgcttcgat gactttgcag acttggtgcc ctttgactcc tgggagccgc 60 tcatgaggaa gttgggcctc atgg 84 <210> 33 <211> 62 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 33 tgtcgatgga cttccagaac cacctgggca gctgccaaaa gtgtgatcca agctgtccca 60 at 62 <210> 34 <211> 82 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 34 gatctaagat ggcgactgtc gaaccggaaa ccacccctac tcctaatccc ccgactacag 60 aagaggagaa aacggaatct as 82 <210> 35 <211> 68 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 35 ggcagtgtca ctgagtcctt gaaatcctcc cctgccccgc gggtctctgg attgggacgc 60 acagtgca 68 <210> 36 <211> 75 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 36 gggccctcca gaacaatgat gggctttatg atcctgactg cgatgagagc gggctcttta 60 aggccaagca gtgca 75 <210> 37 <211> 76 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 37 accgtaggct ctgctctgaa tgactctcct gtgggtctgg ctgcctatat tctagagaag 60 ttttccacct ggacca 76 <210> 38 <211> 81 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 38 cggttatgtc atgccagata cacacctcaa aggtactccc tcctcccggg aaggcaccct 60 ttcttcagtg ggtctcagtt c 81 <210> 39 <211> 68 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 39 cgtggtgccc ctctatgacc tgctgctgga gatgctggac gcccaccgcc tacatgcgcc 60 cactagcc 68 <210> 40 <211> 90 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 40 ggctattcct cattttctct acaaagtggc ctcagtgaac atgaagaagg tagcctcctg 60 gaggagaatt tcggtgacag tctacaatcc 90 <210> 41 <211> 68 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 41 cggtagtcaa gtccggatca agggcaagga gacggaattc tacctgtgca tgaaccgcaa 60 aggcaagc ~ 68 <210> 42 <211> 74 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 42 cacgggacat tcaccacatc gactactata aaaagacaac caacggccga ctgcctgtga 60 agtggatggc acct 74 <210> 43 <211> 67 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 43 ccagtggagc gcttccatga cctgcgtcct gatgaagtgg ccgatttgtt tcagacgacc 60 cagagag <210> 44 <211> 75 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 44 ttggtacctg tgggttagca tcaagttctc cccagggtag aattcaatca gagctccagt 60 ttgcatttgg atgtg 75 <210> 45 <211> 68 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 45 tcagcagcaa gggcatcatg gaggaggatg aggcctgcgg gcgccagtac acgctcaaga 60 aaaccacc 68 <210> 46 <211> 74 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 46 attccaccca tggcaaattc catggcaccg tcaaggctga gaacgggaag cttgtcatca 60 atggaaatcc catc 74 <210> 47 <211> 75 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 47 caaaggagct cactgtggtg tctgtgttcc aaccactgaa tctggacccc atctgtgaat 60 aagccattct gactc 75 <210> 48 <211> 67 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 48 ccatctgcat ccatcttgtt tgggctcccc acccttgaga agtgcctcag ataataccct 60 ggtggcc <210> 49 <211> 73 <212> DNA
<213> Aritificial sequence <220>
<223> Amplicon <400> 49 cgaaaagatg ctgaacagtg acaaatccaa ctgaccagaa gggaggagga agctcactgg 60 tggctgttcc tga 73 <210> 50 <211> 86 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 50 aagctatgag gaaaagaagt acacgatggg ggacgctcct gattatgaca gaagccagtg 60 gctgaatgaa aaattcaagc tgggcc 86 <210> 51 <211> 73 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 51 cccactcagt agccaagtca caatgtttgg aaaacagccc gtttacttga gcaagactga 60 taccacctgc gtg 73 <210> 52 <211> 70 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 52 cggtgtgaga agtgcagcaa gccctgtgcc cgagtgtgct atggtctggg catggagcac 60 ttgcgagagg <210> 53 <211> 82 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 53 tgaacataaa gtctgcaaca tggaaggtat tgcactgcac aggccacatt cacgtatatg 60 ataccaacag taaccaacct ca 82 <210> 54 <211> 73 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 54 tccaggatgt taggaactgt gaagatggaa gggcatgaaa ccagcgactg gaacagctac 60 tacgcagaca cgc 73 <210> 55 <211> 70 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 55 agaaccgcaa ggtgagcaag gtggagattc tccagcacgt catcgactac atcagggacc 60 ttcagttgga 70 <210> 56 <211> 76 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 56 tccggagctg tgatctaagg aggctggaga tgtattgcgc acccctcaag cctgccaagt 60 cagctcgctc tgtccg 76 <210> 57 <211> 83 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 57 gcatggtagc cgaagatttc acagtcaaaa tcggagattt tggtatgacg cgagatatct 60 atgagacaga ctattaccgg aaa 83 <210> 58 <211> 73 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 58 gtggacagca ccatgaacat gttgggcggg ggaggcagtg ctggccggaa gcccctcaag 60 tcgggtatga agg <210> 59 <211> 72 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 59 cctgaacctt ccaaagatgg ctgaaaaaga tggatgcttc caatctggat tcaatgagga 60 gacttgcctg gt 72 <210> 60 <211> 74 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 60 ccacagctca ccttctgtca ggtgtccatc ccagctccag ccagctccca gagaggaaga 60 gactggcact gagg 74 <210> 61 <211> 80 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 61 cggactttgg gtgcgacttg acgagcggtg gttcgacaag tggccttgcg ggccggatcg 60 tcccagtgga agagttgtaa 80 <210> 62 <211> 78 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 62 gcccagaggc tccatcgtcc atcctcttcc tccccagtcg gctgaactct ccccttgtct 60 gcactgttca aacctctg 78 <210> 63 <211> 83 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 63 ggcctgctga gatcaaagac tacagtccct acttcaagac cattgaggac ctgaggaaca 60 agattctcac agccacagtg gac 83 <210> 64 <211> 73 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 64 cgaggattgg ttcttcagca agacagagga actgaaccgc gaggtggcca ccaacagtga 60 gctggtgcag agt 73 <210> 65 <211> 68 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 65 agagatcgag gctctcaagg aggagctgct cttcatgaag aagaaccacg aagaggaagt 60 aaaaggcc 68 <210> 66 <211> 77 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 66 tgagcggcag aatcaggagt accagcggct catggacatc aagtcgcggc tggagcagga 60 gattgccacc taccgca 77 <210> 67 <211> 69 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 67 tcagtggaga aggagttgga ccagtcaaca tctctgttgt cacaagcagt gtttcctctg 60 gatatggca <210> 68 <211> 86 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 68 ggatgaagct tacatgaaca aggtagagct ggagtctcgc ctggaagggc tgaccgacga 60 gatcaacttc ctcaggcagc tatatg 86 <210> 69 <211> 83 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 69 ggaaagacca cctgaaaaac cacctccaga cccacgaccc caacaaaatg gcctttgggt 60 gtgaggagtg tgggaagaag tac 83 <210> 70 <211> 77 <212> DNA
<213> Artificial Sequence <Z20>
<223> Amplicon <400> 70 cagatggcca ctttgagaac attttagctg acaacagtgt gaacgaccag accaaaatcc 60 ttgtggttaa tgctgcc 77 <210> 71 <211> 75 <212> DNA
<213> Artificial Sequence <220>

<223> Amplicon <400> 71 gacttttgcc cgctaccttt cattccggcg tgacaacaat gagctgttgc tcttcatact 60 gaagcagtta gtggc 75 <210> 72 <211> 75 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 72 ggagaacaat ccccttgaga cagaatatgg cctttctgtc tacaaggatc accagaccat 60 caccatccag gagat 75 <210> 73 <211> 82 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 73 tgatggtcct atgtgtcaca ttcatcacag gtttcatacc aacacaggct tcagcacttc 60 ctttggtgtg tttcctgtcc ca 82 <210> 74 <211> 68 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 74 ctacagggac gccatcgaat ccggatcttg atgctggtgt aagtgaacat tcaggtgatt 60 ggttggat <210> 75 <211> 67 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 75 gagaaccaat ctcaccgaca ggcagctggc agaggaatac ctgtaccgct atggttacac 60 tcgggtg <210> 76 <211> 77 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 76 ccgccctcac ctgaagagaa acgcgctcct tggcggacac tgggggagga gaggaagaag 60 cgcggctaac ttattcc 77 <210> 77 <211> 74 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 77 gccgagatcg ccaagatgtt gccagggagg acagacaatg ctgtgaagaa tcactggaac 60 tctaccatca aaag 74 <210> 78 <Z11> 72 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 78 ccctcgtgct gatgctactg aggagccagc gtctagggca gcagccgctt cctagaagac 60 caggtcatga tg 72 <210> 79 <211> 66 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 79 cggtggacca cgaagagtta acccgggact tggagaagca ctgcagagac atggaagagg 60 cgagcc 66 <210> 80 <211> 68 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 80 ctttgaaccc ttgcttgcaa taggtgtgcg tcagaagcac ccaggacttc catttgcttt 60 gtcccggg 68 <210> 81 <211> 81 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 81 ccgcaacgtg gttttctcac cctatggggt ggcctcggtg ttggccatgc tccagctgac 60 aacaggagga gaaacccagc a <210> 82 <211> 66 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 82 ccagctctcc ttccagctac agatcaatgt ccctgtccga gtgctggagc taagtgagag 60 ccaccc 66 <210> 83 <211> 83 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 83 ataccaatca ccgcacaaac ccaggctatt tgttaagtcc agtcacagcg caaagaaaca 60 tatgcggaga aaatgctagt gtg 83 <210> 84 <211> 62 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 84 gccatccgca aaggctttct cgcttgtcac cttgccatgt ggaagaaact ggcggaatgg 60 cc 62 <210> 85 <211> 85 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 85 gcatcaggct gtcattatgg tgtccttacc tgtgggagct gtaaggtctt ctttaagagg 60 gcaatggaag ggcagcacaa ctact 85 <210> 86 <211> 66 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 86 tctccatatc tgccttgcag agtctcctgc agcacctcat cgggctgagc aatctgaccc 60 acgtgc 66 <210> 87 <211> 86 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 87 gccctcccag tgtgcaaata agggctgctg tttcgacgac accgttcgtg gggtcccctg 60 gtgcttctat cctaatacca tcgacg 86 <210> 88 <211> 78 <212> DNA

<213> Artificial Sequence <220>
<223> Amplicon <400> 88 gaacttcttg agcaggagca tacccagggc ttcataatca ccttctgttc agcactagat 60 gatattcttg ggggtgga 78 <210> 89 <211> 77 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 89 cgaagccctt acaagtttcc tagttcaccc ttacggattc ctggagggaa catctatatt 60 tcacccctga agagtcc 77 <210> 90 <211> 74 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 90 ccagacgagc gattagaagc ggcagcttgt gaggtgaatg atttggggga agaggaggag 60 gaggaagagg agga 74 <210> 91 <211> 69 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 91 catcttccag gaggaccact ctctgtggca ccctggacta cctgccccct gaaatgattg 60 aaggtcgga 69 <210> 92 <211> 90 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 92 cctggaggct gcaacatacc tcaatcctgt cccaggccgg atcctcctga agcccttttc 60 gcagcactgc tatcctccaa agccattgta 90 <210> 93 <211> 80 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 93 tgttttgatt cccgggctta ccaggtgaga agtgagggag gaagaaggca gtgtcccttt 60 tgctagagct gacagctttg 80 <210> 94 <211> 65 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 94 gcccgaaacg ccgaatataa tcccaagcgg tttgctgcgg taatcatgag gataagagag 60 ccacg <210> 95 <211> 83 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 95 ggtgtgccac agaccttcct acttggcctg taatcacctg tgcagccttt tgtgggcctt 60 caaaactctg tcaagaactc cgt 83 <210> 96 <211> 75 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 96 tccctgcggt cccagatagc ctgaatcctg cccggagtgg aactgaagcc tgcacagtgt 60 ccaccctgtt cccac 75 <210> 97 <211> 72 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 97 aatccaaggg ggagagtgat gacttccata tggactttga ctcagctgtg gctcctcggg 60 caaaatctgt ac 72 <210> 98 <211> 66 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 98 tgtggacatc ttcccctcag acttccctac tgagccacct tctctgccac gaaccggtcg 60 ggctag <210> 99 <211> 82 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 99 ctatatgcag ccagagatgt gacagccacc gtggacagcc tgccactcat cacagcctcc 60 attctcagta agaaactcgt gg 82 <210> 100 <211> 81 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 100 gggccaaata ttcagaagct tttatatcag aggaccacca tagcggccat ggagaccatc 60 tctgtcccat catacccatc c 81 <210> 101 <211> 73 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 101 cttcacagtg ctcctgcagt ctctctgtgt ggctgtaact tacgtgtact ttaccaacga 60 gctgaagcag atg 73 <210> 102 <211> 65 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 102 gcctcggtgt gcctttcaac atcgccagct acgccctgct cacgtacatg attgcgcaca 60 tcacg <210> 103 <211> 70 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 103 gtggatgtgc cctgaaggac aagccaggcg tctacacgag agtctcacac ttcttaccct 60 ggatccgcag 70 <210> 104 <211> 67 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 104 gccctggatt tcagaaagag ccaagtctgg atctgggacc ctttccttcc ttccctggct 60 tgtaact 67 <210> 105 <211> 71 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 105 ctgctgtctt gggtgcattg gagccttgcc ttgctgctct acctccacca tgccaagtgg 60 tcccaggctg c 71 <210> 106 <211> 71 <212> DNA
<213> Artificial Sequence <Z20>
<223> Amplicon <400> 106 tgacgatggc ctggagtgtg tgcccactgg gcagcaccaa gtccggatgc agatcctcat 60 gatccggtac c 71 <210> 107 <211> 75 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 107 agaggcatcc atgaacttca cacttgcggg ctgcatcagc acacgctcct atcaacccaa 60 gtactgtgga gtttg 75 <210> 108 <211> 77 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 108 gcagttggaa gacacaggaa agtatcccca aattgcagat ttatcaacgg cttttatctt 60 gaaaatagtg ccacgca 77 <210> 109 <211> 76 <212> DNA
<213> Artificial sequence <220>
<223> Amplicon <400> 109 agactgtgga gtttgatgtt gttgaaggag aaaagggtgc ggaggcagca aatgttacag 60 gtcctggtgg tgttcc 76 <210> 110 .
<211> 70 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 110 acccagtagc aaggagaagc ccactcactgctccgagtgc ggcaaagctt tcagaaccta ccaccagctg <210> 111 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 111 gcggcgagtt tccgattta <210> 112 <211> 21 <212> DNA

<213> Artificial Sequence <220>

<Z23> reverse primer <400> 112 tgagtccacc atccagcaag t 21 <210> 113 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 113 atggcggcgg gaggatcaaa a 21 <210> 114 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 114 cgcttctatg gcgctgagat. 20 <210> 115 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 115 tcccggtaca ccacgttctt 20 <210> 116 <211> 24 <212> DNA

<213> Artificial Sequence <220>

<223> probe Page Z1 <400> 116 cagccctgga ctacctgcac tcgg 24 <210> 117 <211> 19 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 117 tcctgccacc cttcaaacc 19 <210> 118 <211> 21 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 118 ggcggtaaat tcatcatcga a 21 <210> 119 <211> 24 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 119 caggtcacgt ccgaggtcga caca 24 <210> 120 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 120 ggacagcagg aatgtgtttc 20 <210> 121 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 121 acccactcga tttgtttctg 20 <210> 122 <211> 22 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 122 cattggctcc ccgtgacctg to 22 <210> 123 <211> 27 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 123 tgtgagtgaa atgccttcta gtagtga 27 <210> 124 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 124 ccgtcctcgg gagccgacta tga 23 <210> 125 <211> 27 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 125 ttgtggttcg ttatcatact cttctga 27 <210> 126 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 126 cagcagatgt ggatcagcaa g 21 <210> 127 <211> 18 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 127 gcatttgcgg tggacgat 18 <210> 128 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 128 aggagtatga cgagtccggc ccc 23 <210> 129 <211> 22 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 129 ggctcttgtg cgtactgtcc tt 22 <210> 130 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 130 tcagatgacg aagagcacag atg 23 <210> 131 <211> 29 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 131 aggctcagtg atgtcttccc tgtcaccag 29 <210> 132 <211> 19 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 132 gggtcaggtg cctcgagat 19 <210> 133 <211> 21 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 133 ctgctcactc ggctcaaact c 21 <210> 134 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 134 tgggcccaga gcatgttcca gate 24 <210> 135 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 135 cgttgtcagc acttggaata caa 23 <210> 136 <211> 24 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 136 gttcaacctc ttcctgtgga ctgt 24 <210> 137 <211> 26 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 137 cccaattaac atgacccggc aaccat 26 <210> 138 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 138 cctggagggt cctgtacaat 20 <210> 139 <211> 19 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 139 ctaattgggc tccatctcg <210> 140 <211> 24 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 140 catcatggga ctcctgccct tact 24 <210> 141 <211> 25 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 141 cagatggacc tagtacccac tgaga 25 <210> 142 <211> 22 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 142 ttccacgccg aaggacagcg at 22 <210> 143 <211> 24 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 143 cctatgattt aagggcattt ttcc 24 <210> 144 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 144 atcctagccc tggtttttgg 20 <210> 145 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 145 ctgccttctc atctgcacaa 20 <210> 146 <211> 20 <212> DNA

<213> Artificial sequence <220>

<Z23> probe <400> 146 tttgctgtca ccagcgtcgc 20 <210> 147 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 147 ttcaggttgt tgcaggagac 20 <210> 148 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 148 catcttcttg ggcacacaat 20 <210> 149 <211> 27 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 149 tgtctccatt attgatcggt tcatgca 27 <210> 150 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 150 gcatgttcgt ggcctctaag a 21 <210> 151 <211> 22 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 151 cggtgtagat gcacagcttc tc 22 <210> 152 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 152 aaggagacca tccccctgac ggc 23 <210> 153 <211> 24 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 153 aaagaagatg atgaccgggt ttac 24 <210> 154 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 154 gagcctctgg atggtgcaat ZO

<210> 155 <211> 24 <212> DNA

<213> Artificial Sequence <220> .

<223> probe <400> 155 caaactcaac gtgcaagcct cgga 24 <210> 156 <211> 22 <Z12> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 156 atgctgtggc tccttcctaa ct 22 <210> 157 <211> 27 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 157 acccaaattg tgatatacaa aaaggtt 27 <210> 158 <211> 30 .

<212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 158 taccaagcaa cctacatgtc aagaaagccc 30 <210> 159 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 159 agatgaagtg gaaggcgctt 20 <210> 160 <211> 18 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 160 caccgcggcc atcctgca 18 <210> 161 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 161 tgcctctgta atcggcaact g 21 <210> 162 <211> 18 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 162 tggttcccag ccctgtgt 18 <210> 163 <211> 28 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 163 ctccaagccc agattcagat tcgagtca 28 <210> 164 <211> 19 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 164 ctcctccacc ctgggttgt 19 <210> 165 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 165 gggcgtggaa cagtttatct 20 <210> 166 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 166 cacggtgaag gtttcgagt 1g <210> 167 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 167 agacatctgc cccaagaagg acgt 24 <210> 168 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 168 tgagtgtccc ccggtatctt c 21 <210> 169 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 169 cagccgcttt cagattttca t 21 <210> 170 <211> 27 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 170 tgccaatccc gatgaaattg gaaattt 27 <210> 171 <211> 21 <212> DNA

<213> Artificial sequence <220>
<223> forward primer <400> 171 tgacaatcag cacacctgca t 21 <210> 172 <211> 23 <212> DNA
<213> Artificial Sequence <220>
<223> reverse primer <400> 172 tgtgactaca gccgtgatcc tta 23 <210> 173 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> probe <400> 173 caggccctct tccgagcggt 20 <210> 174 <211> 26 <212> DNA
<213> Artificial Sequence <220>
<223> forward primer <400> 174 gataaattgg tacaagggat cagctt 26 <210> 175 <211> 24 <212> DNA
<213> Artificial sequence <220>
<223> reverse primer <400> 175 gggtgccaag taactgacta ttca 24 <210> 176 <211> 26 <212> DNA
<213> Artificial Sequence <220>
<223> probe <400> 176 ccagcccaca tgtcctgatc atatgc 26 <210> 177 <211> 18 <212> DNA
<213> Artificial sequence <220>

<223> forward primer <400> 177 tgcctgtggt gggaagct 18 <210> 178 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 178 ggaaaatgcc tccggtgtt 1g <210> 179 <211> 30 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 179 tgacatagca tcatcctttg gttcccagtt 30 <210> 180 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 180 ggatatttcc gtggctctta ttca 24 <210> 181 <211> 30 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 181 tctccatcaa atcctgtaaa ctccagagca 30 <210> 182 <211> 25 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 182 cttctcatca aggcagaaaa atctt 25 <210> 183 <211> 22 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 183 gacatttcca gtcctgcagt ca 22 <210> 184 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 184 tgcctctctg ccccaccctt tgt 23 <210> 185 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 185 ctccgatcgc acacatttgt 20 <210> 186 <211> 18 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 186 ggcatcctgg cccaaagt 18 <210> 187 <211> 21 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 187 gaccccctca gctggtagtt g 21 <210> 188 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 188 cccaaatcca ggcggctaga ggc 23 <210> 189 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 1~9 tctgcagagt tggaagcact cta 23 <210> 190 <211> 2S

<212> DNA

<213> Artificial sequence <220>

<223> probe <400> 190 caggatacag ctccacagca tcgatgtc 28 <210> 191 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 191 gccgaggctt ttctaccaga a 21 <210> 192 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 192 gggaggctta tctcactgag tga 23 <210> 193 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 193 ccattgcagc cttcattgc 1g <210> 194 <211> 29 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 194 ttgaggccca gagcagtcta ccagattct 2g <210> 195 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 195 tgtctcactg agcgagcaga a 21 <210> 196 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 196 accattgcag ccctgattg 19 <210> 197 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 197 cttgaggacg cgaacagtcc acca 24 <210> 198 <211> 22 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 198 cgctgacatc atgaatgttc ct 22 <210> 199 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 199 tctctttcag caacgatgtg tctt 24 <210> 200 <211> 25 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 200 tcatatccaa actcgcctcc agccg 25 <210> 201 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 201 cacaatggcg gctctgaag 19 <210> 202 <211> 26 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 202 acacaaacac tgtctgtacc tgaaga 26 <210> 203 <211> Z3 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 203 aagttacgct gcgcgacagc caa 23 <210> 204 <211> 24 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 204 ctctgagaca gtgcttcgat gact 24 <210> Z05 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 205 ccatgaggcc caacttcct 1g <210> 206 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 206 cagacttggt gccctttgac tcc 23 <210> 207 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 207 tgtcgatgga cttccagaac 20 <210> 208 <211> 18 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 208 cacctgggca gctgccaa 18 <210> 209 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 209 attgggacag cttggatca 19 <210> 210 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 210 gatctaagat ggcgactgtc gaa 23 <210> 211 <211> 25 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 211 ttagattccg ttttctcctc ttctg 25 <210> 212 <211> 27 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 212 accaccccta ctcctaatcc cccgact 27 <210> 213 <211> 22 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 213 ggcagtgtca ctgagtcctt ga 22 <210> 214 <211> 18 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 214 tgcactgtgc gtcccaat 18 <210> 215 <211> 18 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 215 atcctcccct gccccgcg 18 <210> 216 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 216 gggccctcca gaacaatgat 20 <210> 217 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 217 tgcactgctt ggccttaaag a 21 <210> 218 <211> 25 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 218 ccgctctcat cgcagtcagg atcat 25 <210> 219 <211> 20 <212> DNA

<213> Artificial Sequence <Z20>

<223> forward primer <400> 219 accgtaggct ctgctctgaa 20 <210> 220 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 220 tggtccaggt ggaaaacttc 20.

<210> 221 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 221 aggcagccag acccacagga 20 <210> 222 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 222 cggttatgtc atgccagata cac 23 <210> 223 <211> 25 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 223 cctcaaaggt actccctcct cccgg 25 <210> 224 <211> 24 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 224 gaactgagac ccactgaaga aagg 24 <210> 225 <211> 19 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 225 cgtggtgccc ctctatgac 1g <210> 226 <211> 19 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 226 ctggagatgc tggacgccc 1g <210> 227 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 227 ggctagtggg cgcatgtag 1g <210> 228 <211> 25 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 228 ggattgtaga ctgtcaccga aattc 25 <210> 229 <211> 28 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 229 ggctattcct cattttctct acaaagtg 2g <210> 230 <211> 30 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 230 cctccaggag gctaccttct tcatgttcac 30 <210> 231 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 231 cggtagtcaa gtccggatca a <210> 232 <211> 18 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 232 gcttgccttt gcggttca <210> 233 <211> 25 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 233 caaggagacg gaattctacc tgtgc 25 <210> 234 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 234 cacgggacat tcaccacatc 20 <210> 235 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 235 gggtgccatc cacttcaca 1g <210> 236 <211> 27 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 236 ataaaaagac aaccaacggc cgactgc 27 <210> 237 <211> 18 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 237 ccagtggagc gcttccat <210> 238 <211> 22 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 238 ctctctgggt cgtctgaaac as 22 <210> 239 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 239 tcggccactt catcaggacg cag 23 <210> 240 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 240 ttggtacctg tgggttagca 20 <210> 241 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 241 cacatccaaa tgcaaactgg 20 <210> 242 <211> 26 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 242 tccccagggt agaattcaat cagagc 26 <210> 243 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 243 tcagcagcaa gggcatcat <210> 244 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 244 ggtggttttc ttgagcgtgt act 23 <210> 245 <211> 19 <212> DNA

<213> Artificial Sequence <220>

'<223> probe <400> 245 cgcccgcagg cctcatcct 19 <210> 246 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 246 attccaccca tggcaaattc 20 <210> 247 <211> 22 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 247 gatgggattt ccattgatga ca 22 <210> 248 <211> 22 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 248 ccgttctcag ccttgacggt gc 22 <210> 249 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 249 caaaggagct cactgtggtg tct 23 <210> 250 <211> 24 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 250 tgttccaacc actgaatctg gacc 24 <210> 251 <211> 26 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 251 gagtcagaat ggcttattca cagatg 26 <210> 252 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 252 ccatctgcat ccatcttgtt 20 <210> 253 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 253 ctccccaccc ttgagaagtg cct 23 <210> 254 <211> 20 <212> DNA

<213> Artificial Sequence <Z20>

<223> reverse primer <400> 254 ggccaccagg gtattatctg 20 <210> 255 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 255 cgaaaagatg ctgaacagtg aca ~ 23 <210> 256 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 256 tcaggaacag ccaccagtga 20 <210> 257 <211> 28 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 257 cttcctcctc ccttctggtc agttggat 28 <210> 258 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 258 ggcccagctt gaatttttca 20 <210> 259 <211> 27 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 259 aagctatgag gaaaagaagt acacgat 27 <210> 260 <211> 30 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 260 tcagccactg gcttctgtca taatcaggag 30 <210> 261 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 261 cccactcagt agccaagtca 20 <210> 262 <211> 27 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 262 tcaagtaaac gggctgtttt ccaaaca 27 <210> 263 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 263 cacgcaggtg gtatcagtct <210> 264 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 264 cggtgtgaga agtgcagcaa 20 <210> 265 <211> 24 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 265 ccagaccata gcacactcgg gcac <210> 266 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 266 cctctcgcaa gtgctccat 1g <210> 267 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 267 tgaacataaa gtctgcaaca tgga 24 <210> 268 <211> 28 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 268 tgaggttggt tactgttggt atcatata 2g <210> 269 <211> 24 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 269 ttgcactgca caggccacat tcac 24 <210> 270 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 270 tccaggatgt taggaactgt gaag <210> 271 <211> 22 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 271 gcgtgtctgc gtagtagctg tt 22 <210> 272 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 272 agtcgctggt ttcatgccct tcca 24 <210> 273 <211> 19 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 273 agaaccgcaa ggtgagcaa <210> 274 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 274 tccaactgaa ggtccctgat g 21 <220> 275 <211> 26 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 275 tggagattct ccagcacgtc atcgac 26 <210> 276 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 276 tccggagctg tgatctaagg a 21 <210> 277 <211> 24 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 277 tgtattgcgc acccctcaag cctg 24 <210> 278 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 278 cggacagagc gagctgactt 20 <210> 279 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 279 gcatggtagc cgaagatttc a 21 <210> 280 <211> 30 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 280 ~

tttccggtaa tagtctgtct 30 catagatatc <210> 281 <211> 28 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 281 cgcgtcatac caaaatctcc gattttga 2g <210> 282 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 282 gtggacagca ccatgaaca 1g <210> 283 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 283 ccttcatacc cgacttgagg 20 <210> 284 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 284 cttccggcca gcactgcctc <210> 285 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 285 cctgaacctt ccaaagatgg 20 <210> 286 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 286 accaggcaag tctcctcatt 20 <210> 287 <211> 27 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 287 ccagattgga agcatccatc tttttca 27 <210> 288 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 288 ccacagctca ccttctgtca 20 <210> 289 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 289 cctcagtgcc agtctcttcc 20 <210> 290 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 290 tccatcccag ctccagccag 20 <210> 291 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 291 ccacttgtcg aaccaccgct cgt 23 <210> 292 <211> 19 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 292 cggactttgg gtgcgactt 19 <210> 293 <211> 24 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 293 ttacaactct tccactggga cgat 24 <210> 294 <211> 18 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 294 gcccagaggc tccatcgt 18 <210> 295 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 295 cagaggtttg aacagtgcag aca 23 <210> 296 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 296 cctcttcctc cccagtcggc tga 23 <210> 297 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 297 ggcctgctga gatcaaagac 20 <210> 298 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 298 gtccactgtg gctgtgagaa 20 <210> 299 <211> 26 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 299 tgttcctcag gtcctcaatg gtcttg 2g <210> 300 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 300 cgaggattgg ttcttcagca a 21 <210> 301 <211> 22 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 301 actctgcacc agctcactgt tg 22 <210> 302 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 302 cacctcgcgg ttcagttcct ctgt 24 <210> 303 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 303 agagatcgag gctctcaagg <210> 304 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 304 ggccttttac ttcctcttcg 20 <210> 305 <211> 27 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 305 tggttcttct tcatgaagag cagctcc 27 <210> 306 <211> 21 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 306 tgagcggcag aatcaggagt a 21 <210> 307 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 307 tgcggtaggt ggcaatctc 1g <210> 308 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 308 ctcatggaca tcaagtcgcg gctg 2q.

<210> 309 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 309 tcagtggaga aggagttgga 20 <210> 310 <211> 28 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 310 ccagtcaaca tctctgttgt cacaagca 2g <210> 311 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 311 tgccatatcc agaggaaaca <210> 312 <211> 27 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 312 ggatgaagct tacatgaaca aggtaga 27 <210> 313 <211> 25 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 313 catatagctg cctgaggaag ttgat 25 <210> 314 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 314 cgtcggtcag cccttccagg c 21 <210> 315 <211> 22 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 315 ggaaagacca cctgaaaaac ca 22 <210> 316 <211> 24 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 316 gtacttcttc ccacactcct caca 24 <210> 317 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 317 acccacgacc ccaacaaaat ggc 23 <210> 318 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 318 cagatggcca ctttgagaac att 23 <210> 319 <211> 22 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 319 ggcagcatta accacaagga tt 22 <210> 320 <211> 28 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 320 agctgacaac agtgtgaacg accagacc 28 <210> 321 <211> 21 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 321 gacttttgcc cgctaccttt c 21 <210> 322 <211> 26 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 322 gccactaact gcttcagtat gaagag 26 <210> 323 <211> 24 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 323 acagctcatt gttgtcacgc cgga 24 <210> 324 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 324 ggagaacaat ccccttgaga 20 <210> 325 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 325 atctcctgga tggtgatggt 20 <210> 326 <211> 27 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 326 tggcctttct gtctacaagg atcacca 27 <210> 327 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 327 tgatggtcct atgtgtcaca ttca 24 <210> 328 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 328 tgggacagga aacacaccaa <210> 329 <211> 30 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 329 caggtttcat accaacacag gcttcag 30 <210> 330 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 330 ctacagggac gccatcgaa 19 <210> 331 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 331 atccaaccaa tcacctgaat gtt 23 <210> 332 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 332 cttacaccag catcaagatc cgg 23 <210> 333 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 333 gagaaccaat ctcaccgaca 20 <210> 334 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 334 cacccgagtg taaccatagc 20 <210> 335 <211> 24 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 335 acaggtattc ctctgccagc tgcc 24 <210> 336 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 336 ccgccctcac ctgaagaga 19 <210> 337 <211> 22 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 337 ggaataagtt agccgcgctt ct 22 <210> 338 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 338 cccagtgtcc gccaaggagc g <210> 339 <211> 18 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 339 gccgagatcg ccaagatg 1g <210> 340 <211> 27 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 340 cttttgatgg tagagttcca gtgattc 27 <210> 341 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 341 cagcattgtc tgtcctccct ggca 24 <210> 342 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 342 ccctcgtgct gatgctact 19 <210> 343 <211> 22 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 343 catcatgacc tggtcttcta gg 22 <210> 344 <211> 22 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 344 ctgccctaga cgctggctcc tc 22 <210> 345 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 345 cggtggacca cgaagagtta a 21 <210> 346 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 346 ~

ccgggacttg gagaagcact gca 23 <210> 347 <211> 19 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 347 ggctcgcctc ttccatgtc 19 <210> 348 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 348 ctttgaaccc ttgcttgcaa 20 <210> 349 <211> 25 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 349 aagtcctggg tgcttctgac gcaca <210> 350 <211> 18 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 350 cccgggacaa agcaaatg 18 <210> 351 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 351 ccgcaacgtg gttttctca 19 <210> 352 <211> 22 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 352 ctcggtgttg gccatgctcc ag 22 <210> 353 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 353 tgctgggttt ctcctcctgt t 21 <210> 354 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 354 ccagctctcc ttccagctac 20 <210> 355 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 355 gggtggctct cacttagctc 20 <210> 356 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 356 atcaatgtcc ctgtccgagt gctg 24 <210> 357 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 357 cacactagca ttttctccgc ata 23 <210> 358 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 358 ataccaatca ccgcacaaac c 21 <210> 359 <211> 30 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 359 tgcgctgtga ctggacttaa caaatagcct 30 <210> 360 <211> 18 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 360 gccatccgca aaggcttt 18 <210> 361 <211> 18 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 361 ggccattccg ccagtttc 18 <210> 362 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 362 tcgcttgtca ccttgccatg tgg 23 <210> 363 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 363 gcatcaggct gtcattatgg 20 <210> 364 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 364 agtagttgtg ctgcccttcc ZO

<210> 365 <211> 28 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 365 tgtccttacc tgtgggagct gtaaggtc 28 <210> 366 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 366 tctccatatc tgccttgcag agt 23 <210> 367 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 367 gcacgtgggt cagattgct 1g <210> 368 <211> 22 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 368 tcctgcagca cctcatcggg ct 22 <210> 369 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 369 gccctcccag tgtgcaaat 19 <210> 370 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 370 tgctgtttcg acgacaccgt tcg 23 <210> 371 <211> 25 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 371 cgtcgatggt attaggatag aagca 25 <210> 372 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 372 gaacttcttg agcaggagca tacc 24 <210> 373 <211> 25 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 373 tccaccccca agaatatcat ctagt 25 <210> 374 <211> 25 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 374 agggcttcat aatcaccttc tgttc 25 <210> 375 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 375 cgaagccctt acaagtttcc 20 <210> 376 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 376 ggactcttca ggggtgaaat 20 <210> 377 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 377 cccttacgga ttcctggagg gaac Z4 <210> 378 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 378 ccagacgagc gattagaagc 20 <210> 379 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 379 tcctcctctt cctcctcctc 20 <210> 380 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 380 tgtgaggtga atgatttggg gga 23 <210> 381 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 381 catcttccag gaggaccact 20 <210> 382 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 382 tccgaccttc aatcatttca 20 <210> 383 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 383 ctctgtggca ccctggacta cctg 24 <210> 384 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 384 cctggaggct gcaacatacc 20 <210> 385 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 385 tacaatggct ttggaggata gca <210> 386 <211> 25 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 386 atcctcctga agcccttttc gcagc 25 <210> 387 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 387 tgttttgatt cccgggctta 2p <210> 388 <211> 28 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 388 tgccttcttc ctccctcact tctcacct 2g <210> 389 <211> 24 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 389 caaagctgtc agctctagca aaag 24 <210> 390 <211> 19 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 390 gcccgaaacg ccgaatata 1g <210> 391 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 391 taccgcagca aaccgcttgg g 21 <210> 392 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 392 cgtggctctc ttatcctcat gat 23 <210> 393 , <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 393 ggtgtgccac agaccttcct 20 <210> 394 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 394 acggagttct tgacagagtt ttga 24 <210> 395 <211> 27 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 395 ttggcctgta atcacctgtg cagcctt 27 <210> 396 <211> 19 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 396 tccctgcggt cccagatag 19 <210> 397 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 397 gtgggaacag ggtggacact 20 <210> 398 <211> 25 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 398 atcctgcccg gagtggaact gaagc 25 <210> 399 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 399 aatccaaggg ggagagtgat 20 <210> 400 <211> 26 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 400 catatggact ttgactcagc tgtggc 26 <210> 401 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 401 gtacagattt tgcccgagga 20 <210> 402 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 402 tgtggacatc ttcccctcag a 21 <210> 403 <211> 24 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 403 ttccctactg agccaccttc tctg 24 <210> 404 <211> 18 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 404 ctagcccgac cggttcgt 1g <210> 405 <211> 24 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 405 ctatatgcag ccagagatgt gaca 24 <210> 406 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 406 acagcctgcc actcatcaca gcc 23 <210> 407 <211> 24 <212> DNA

<213> Artificial Sequence <Z20>

<223> reverse primer <400> 407 ccacgagttt cttactgaga atgg 24 <210> 408 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 408 gggccaaata ttcagaagc 19 <210> 409 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 409 ggatgggtat gatgggacag 2p <210> 410 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 410 ccaccatagc ggccatggag 20 <210> 411 <211> 22 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 411 cttcacagtg ctcctgcagt ct 22 <210> 412 <211> 21 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 412 catctgcttc agctcgttgg t 21 <210> 413 <211> 26 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 413 aagtacacgt aagttacagc cacaca 26 <210> 414 <211> 18 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 414 gcctcggtgt gcctttca 1g <210> 415 <211> 22 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 415 catcgccagc tacgccctgc tc 22 <210> 416 <211> 19 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 416 cgtgatgtgc gcaatcatg 1g <210> 417 <211> 19 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 417 gtggatgtgc cctgaagga 1g <210> 418 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 418 ctgcggatcc agggtaagaa <210> 419 <211> 28 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 419 aagccaggcg tctacacgag agtctcac 2g <210> 420 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 420 gccctggatt tcagaaagag 20 <210> 421 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 421 agttacaagc cagggaagga 20 <210> 422 <211> 25 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 422 caagtctgga tctgggaccc tttcc 25 <210> 423 <211> 20 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 423 ctgctgtctt gggtgcattg 20 <210> 424 <211> 18 <212> DNA

<213> Artificial sequence <220>

<223> reverse primer <400> 424 gcagcctggg accacttg 18 <210> 425 <211> 25 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 425 ttgccttgct gctctacctc cacca 25 <210> 426 <211> 19 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 426 tgacgatggc ctggagtgt 1g <210> 427 <211> 22 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 427 ggtaccggat catgaggatc tg <210> 428 <211> 21 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 428 ctgggcagca ccaagtccgg a <210> 429 <211> 22 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 429 agaggcatcc atgaacttca ca 22 <210> 430 <211> 24 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 430 caaactccac agtacttggg ttga 24 <210> 431 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 431 cgggctgcat cagcacacgc 20 <210> 432 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 432 gcagttggaa gacacaggaa agt 23 <210> 433 <211> 27 <212> DNA

<213> Artificial sequence <220>

<223> probe <400> 433 tccccaaatt gcagatttat caacggc 27 <210> 434 <211> 21 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 434 tgcgtggcac tattttcaag a 21 <210> 435 <211> 25 <212> DNA

<213> Artificial sequence <220>

<223> forward primer <400> 435 agactgtgga gtttgatgtt gttga 25 <210> 436 <211> 22 <212> DNA

<213> Artificial Sequence <220>

<223> reverse primer <400> 436 ggaacaccac caggacctgt as 22 <210> 437 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> probe <400> 437 ttgctgcctc cgcacccttt tct 23 <210> 438 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> forward primer <400> 438 acccagtagc aaggagaagc 20 <210> 439 <211> 20 <212> DNA

<213> Artificial Sequence <220>
<223> reverse primer <400> 439 cagctggtgg taggttctga 20 <210> 440 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> probe <400> 440 cactcactgc tccgagtgcg g 21

Claims (51)

1. ~A method of predicting the likelihood of long-term survival of a breast cancer patient without the recurrence of breast cancer, comprising determining the expression level of one or more prognostic RNA transcripts or their expression products, in a breast cancer tissue sample obtained from said patient, normalized against the expression level of all RNA
transcripts or their products in said breast cancer tissue sample, or of a reference set of RNA
transcripts or their expression products, wherein the prognostic RNA
transcript is the transcript of one or more genes selected from the group consisting of:
TP53BP2, GRB7, PR, CD68, Bc12, KRT14, IRS1, CTSL, EstR1, Chk1, IGFBP2, BAG1, CEGP1, STK15, GSTM1, FHIT, RIZ1, AIB1, SURV, BBC3, IGF1R, p27, GATA3, ZNF217, EGFR, CD9, MYBL2, HIF1.alpha., pS2, ErbB3, TOP2B, MDM2, RAD51C, KRT19, TS, Her2, KLK10, .beta.-Catenin, .gamma.-Catenin, MCM2, PI3KC2A, IGF1, TBP, CCNB1, FBXO5, and DR5, wherein expression of one or more of GRB7, CD68, CTSL, Chk1, AIB1, CCNB1, MCM2, FBXO5, Her2, STK15, SURV, EGFR, MYBL2, HIF1.alpha., and TS indicates a decreased likelihood of long-term survival without breast cancer recurrence, and the expression of one or more of TP53BP2, PR, Bc12, KRT14, EstR1, IGFBP2, BAG1, CEGP1, KLK10, .beta.-Catenin, .gamma.-Catenin, DR5, PI3KCA2, RAD51C, GSTM1, FHIT, RIZ1, BBC3, TBP, p27, IRS1, IGF1R, GATA3, ZNF217, CD9, pS2, ErbB3, TOP2B, MDM2, IGF1, and KRT19 indicates an increased likelihood of long-term survival without breast cancer recurrence.
2. ~The method of claim 1 comprising determining the expression level of at least two of said prognostic RNA transcripts or their expression products.
3. ~The method of claim 1 comprising determining the expression level of at least of said prognostic RNA transcripts or their expression products.
4. ~The method of claim 1 comprising determining the expression level of at least of said prognostic RNA transcripts or their expression products.
5. ~The method of claim 1 comprising determining the expression level of at least of said prognostic transcripts of their expression products.
6. ~The method of claim 1 wherein the breast cancer is invasive breast carcinoma,
7. ~The method of claim 1 wherein the expression level of one or more prognostic RNA transcripts is determined.~
8. ~The method of claim 1 wherein said RNA is isolated from a fixed, wax-embedded breast cancer tissue specimen of said patient.
9. ~The method of claim 1 wherein said RNA is isolated from core biopsy tissue or fine needle aspirate cells.
10. ~An array comprising polynucleotides hybridizing to two or more of the following genes: .alpha.-Catenin, AIB1, AKT1, AKT2, .beta.-actin, BAG1, BBC3, Bc12, CCNB1, CCND1, CD68, CD9, CDH1, CEGP1, Chk1, CIAP1, cMet.2, Contig 27882, CTSL, DR5, EGFR, EIF4E, EPHX1, ErbB3, EstR1, FBXO5, FHIT1 FRP1, GAPDH, GATA3, G-Catenin, GRB7, GRO1, GSTM1, GUS, HER2, HIF1A, HNF3A, IGF1R, IGFBP2, KLK10, KRT14, KRT17, KRT18, KRT19, KRT5, Maspin, MCM2, MCM3, MDM2, MMP9, MTA1, MYBL2, P14ARF, p27, P53, PI3KC2A, PR, PRAME, pS2, RAD51C, .3RB1, RIZ1, STK15, STMY3, SURV, TGFA, TOP2B, TP53BP2, TRAIL, TS, upa, VDR, VEGF, and ZNF217.
11. ~The array of claim 10 comprising polynucleotides hybridizing to at least 3 of said genes.
12. ~The array of claim 10 comprising polynucleotides hybridizing to at least 5 of said genes.
13. ~The array of claim 10 comprising polynucleotides hybridizing to at least 10 of said genes.
14. ~The array of claim 10 comprising polynucleotides hybridizing to the following genes: TP53BP2, GRB7, PR, CD68, Bcl2, KRT14, IRS1, CTSL, EstR1, Chk1, IGFBP2, BAG1, CEGP1, STK15, GSTM1, FHIT, RIZ1, AIB1, SURV, BBC3, IGF1R, p27, GATA3, ZNF217, EGFR, CD9, MYBL2, HIF1.alpha., pS2, RIZ1, ErbB3, TOP2B, MDM2, RAD51C, KRT19, TS, Her2, KLK10, .beta.-Catenin, .gamma.-Catenin, MCM2, PI3KC2A, IGF1, TBP, CCNB1, FBXO5 and DR5.
15. ~The array of claim 10 or claim 14 wherein said polynucleotides are cDNAs.
16. ~The array of claim 15 wherein said cDNAs are about 500 to 5000 bases long.
17. ~The array of claim 10 or claim 14 wherein said polynucleotides are oligonucleotides.
18. ~The array of claim 17 wherein said oligonucleotides are about 20 to 80 bases long.
19. ~The array of claim 10 or claim 14 wherein the solid surface is glass.
20. ~The array of claim 19 which comprises about 330,000 oligonucleotides.
21. ~A method of predicting the likelihood of long-term survival of a patient diagnosed with invasive breast cancer, without the recurrence of breast cancer, comprising the steps of:
(1) ~determining the expression levels of the RNA transcripts or the expression products of genes or a gene set selected from the group consisting of (a) ~TP53BP2, Bcl2, BAD, EPHX1, PDGFR.beta., DIABLO, XIAP, YB1, CA9, and KRT8;
(b) ~GRB7, CD68, TOP2A, Bcl2, DIABLO, CD3, ID1, PPM1D, MCM6, and WISP1;
(c) ~PR, TP53BP2, PRAME, DIABLO, CTSL, IGFBP2, TIMP1, CA9, MMP9, and COX2;
(d) ~CD68, GRB7, TOP2A, Bcl2, DIABLO, CD3, ID1, PPM1D, MCM6, and WISP1;
(e) ~Bcl2, TP53BP2, BAD, EPHX1, PDGFR.beta., DIABLO, XIAP, YB1, CA9, and KRT8;
(f) ~KRT14, KRT5, PRAMS, TP53BP2, GUST, AIB1, MCM3, CCNE1, MCM6, and ID1;
(g) ~PRAME, TP53BP2, EstR1, DIABLO, CTSL, PPM1D, GRB7, DAPK1, BBC3, and VEGFB;
(h) ~CTSL2, GRB7, TOP2A, CCNB1, Bcl2, DIABLO, PRAMS, EMS1, CA9, and EpCAM;
(i) ~EstR1, TP53BP2, PRAME, DIABLO, CTSL, PPM1D, GRB7, DAPK1, BBC3, and VEGFB;
(k) ~Chk1, PRAME, TP53BP2, GRB7, CA9, CTSL, CCNB1, TOP2A, tumor size, and IGFBP2;
(l) ~IGFBP2, GRB7, PRAME, DIABLO, CTSL, .beta.-Catenin, PPM1D, Chk1, WISP1, and LOT1;
(m) ~HER2, TP53BP2, Bcl2, DIABLO, TIMP1, EPHX1, TOP2A, TRAIL, CA9, and AREG;
(n) ~BAG1, TP53BP2, PRAME, IL6, CCNB1, PAI1, AREG, tumor size, CA9, and Ki67;
(o) ~CEGP1, TP53BP2, PRAME, DIABLO, Bcl2, COX2, CCNE1, STK15, and AKT2, and FGF18;

(p) ~STK15, TP53BP2, PRAMS, IL6, CCNE1, AKT2, DIABLO, cMet, CCNE2, and COX2;
(q)~KLK10, EstR1, TP53BP2, PRAME, DIABLO, CTSL, PPM1D, GRB7, DAPK1, and BBC3;
(r) ~AIB1, TP53BP2, Bcl2, DIABLO, TIMP1, CD3, p53, CA9, GRB7, and EPHX1 (s) ~BBC3, GRB7, CD68, PRAME, TOP2A, CCNB1, EPHX1, CTSL
GSTM1, and APC;
(t) ~CD9, GRB7, CD68, TOP2A, Bcl2, CCNB1, CD3, DIABLO, ID1, and PPM1D;
(w) ~EGFR, KRT14, GRB7, TOP2A, CCNB1, CTSL, Bcl2, TP, KLK10, and CA9;
(x) ~HIF1.alpha., PR, DIABLO, PRAME, Chk1, AKT2, GRB7, CCNE1, TOP2A, and CCNB1;
(y) ~MDM2, TP53BP2, DIABLO, Bcl2, AIB1, TIMP1, CD3, p53, CA9, and HER2;
(z) ~MYBL2, TP53BP2, PRAME, IL6, Bcl2, DIABLO, CCNE1, EPHX1, TIMP1, and CA9;
(aa) ~p27, TP53BP2, PRAME, DIABLO, Bcl2, COX2, CCNE1, STK15, AKT2, and ID1;
(ab) ~RAD51, GRB7, CD68, TOP2A, CIAP2, CCNB1, BAG1, IL6, FGFR1, and TP53BP2;
(ac) ~SURV, GRB7, TOP2A, PRAMS, CTSL, GSTM1, CCNB1, VDR, CA9; and CCNE2;
(ad) ~TOP2B, TP53BP2, DIABLO, Bcl2, TIMP1, AIB1, CA9, p53, KRT8, and BAD;
(ae) ~ZNF217, GRB7, TP53BP2, PRAME, DIABLO, Bcl2, COX2, CCNE1, APC4, and .beta.-Catenin, in a breast cancer tissue sample obtained from said patient, normalized against the expression levels of all RNA transcripts or their expression products in said breast cancer tissue sample, or of a reference set of RNA transcripts or their products;
(2) subjecting the data obtained in step (1) to statistical analysis; and (3) determining whether the likelihood of said long-term survival has increased or decreased.
22. ~A method of predicting the likelihood of long-term survival of a patient diagnosed with estrogen receptor (ER)-positive invasive breast cancer, without the recurrence of breast cancer, comprising the steps of:

(1) determining the expression levels of the RNA transcripts or the expression products of genes of a gene set selected from the group consisting of CD68;
CTSL; FBXO5; SURV; CCNB1; MCM2; Chk1; MYBL2; HIF1A; cMET; EGFR; TS;
STK15, IGFR1; BC12; HNF3A; TP53BP2; GATA3; BBC3; RAD51C; BAG1; IGFBP2; PR;
CD9; RB1; EPHX1; CEGP1; TRAIL; DR5; p27; p53; MTA; RIZ1; ErbB3; TOP2B; EIF4E, wherein expression of the following genes in ER-positive cancer is indicative of a reduced likelihood of survival without cancer recurrence following surgery: CD68;
CTSL; FBXO5;
SURV; CCNB1; MCM2; Chk1; MYBL2; HIF1A; cMET; EGFR; TS; STK15, and wherein expression of the following genes is indicative of a better prognosis for survival without cancer recurrence following surgery: IGFR1; BC12; HNF3A; TP53BP2; GATA3; BBC3;
RAD51C; BAG1; IGFBP2; PR; CD9; RB1; EPHX1; CEGP1; TRAIL; DR5; p27; p53; MTA;
RIZ1; ErbB3; TOP2B; EIF4E.
(2) subjecting the data obtained in step (1) to statistical analysis; and (3) determining whether the likelihood of said long-term survival has increased or decreased.
23. The method of claim 21 or 22 wherein said statistical analysis is performed by using the Cox Proportional Hazards model.
24. A method of predicting the likelihood of long-term survival of a patient diagnosed with estrogen receptor (ER)-negative invasive breast cancer, without the recurrence of breast cancer, comprising determining the expression levels of the RNA
transcripts or the expression products of genes of the gene set CCND1; UPA; HNF3A; CDH1; Her2;
GRB7;
AKT1; STMY3; .alpha.-Catenin; VDR; GRO1; KT14; KLK10; Maspin, TGF.alpha., and FRP1, wherein expression of the following genes is indicative of a reduced likelihood of survival without cancer recurrence: CCND1; UPA; HNF3A; CDH1; Her2; GRB7; AKT1; STMY3; .alpha.-Catenin;
VDR; GRO1, and wherein expression of the following genes is indicative of a better prognosis for survival without cancer recurrence: KT14; KLK10; Maspin, TGF.alpha., and FRP1.
25. A method of preparing a personalized genomics profile for a patient, comprising the steps of:
(a) subjecting RNA extracted from a breast tissue obtained from the patient to gene expression analysis;
(b) determining the expression level of one or more genes selected from the breast cancer gene set listed in any one of Tables 1-5, wherein the expression level is normalized against a control gene or genes and optionally is compared to the amount found in a breast cancer reference tissue set; and (c) ~creating a report summarizing the data obtained by said gene expression analysis.
26. ~The method of claim 25, wherein said breast tissue comprises breast cancer cells.
27. ~The method of claim 26 wherein said breast tissue is obtained from a fixed, paraffin-embedded biopsy sample.
28. ~The method of claim 27 wherein said RNA is fragmented.
29. ~The method of claim 25 wherein said report includes prediction of the likelihood of long term survival of the patient.
30. ~The method of claim 25 wherein said report includes recommendation for a treatment modality of said patient.
31. ~A method for amplification of a gene listed in Tables 5A and B by polymerase chain reaction (PCR), comprising performing said PCR by using an amplicon listed in Tables 5A and B and a primer-probe set listed in Tables 6A-F.
32. ~A PCR amplicon listed in Tables 5A and B.
33. ~A PCR primer-probe set listed in Tables 6A-F.
34. ~A prognostic method comprising:
(a) subjecting a sample comprising breast 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 GRB7, CD68, CTSL, Chk1, AIB1, CCNB1, MCM2, FBXO5, Her2, STK15, SURV, EGFR, MYBL2, HIF1.alpha., and TS, or their product, and (b) identifying the patient as likely to have a decreased likelihood of long-term survival without breast cancer recurrence if the normalized expression levels of said gene or genes, or their products, are elevated above a defined expression threshold.
35. ~A prognostic method comprising:~
(a) subjecting a sample comprising breast 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 TP53BP2, PR, Bcl2, KRT14, EstR1, IGFBP2, BAG1, CEGP1, KLK10, .beta.-Catenin, .gamma.-Catenin, DR5, PI3KCA2, RAD51C, GSTM1, FHIT, RIZ1, BBC3, TBP, p27, IRS1, IGF1R, GATA3, ZNF217, CD9, pS2, ErbB3, TOP2B, MDM2, IGF1, and KRT19, and (b) ~identifying the patient as likely to have an increased likelihood of long-term survival without breast cancer recurrence if the normalized expression levels of said gene or genes, or their products, are elevated above a defined expression threshold.
36. ~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.
37. ~The method of claim 34 or 35 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, 285, and 18S.
38. ~The method of claim 34 or 35 wherein the sample is subjected to global gene expression analysis of all genes present above the limit of detection.
39. ~The method of claim 37 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.
40. ~The method of claim 38 wherein the level of RNA transcripts is determined by quantitative RT-PCR (qRT-PCR), and the signal is a Ct value.
41. ~The method of claim 39 wherein the assayed genes include at least 50 cancer related genes.
42. ~The method of claim 39 wherein the assayed genes includes at least 100 cancer related genes.
43. ~The method of claim 34 or 35 wherein said patient is human.
44. ~The method of claim 42 wherein said sample is a fixed, paraffin-embedded tissue (FPET) sample, or fresh or frozen tissue sample.
45. ~The method of claim 42 wherein said sample is a tissue sample from fine needle, core, or other types of biopsy.
46. ~The method of claim 42 wherein said quantitative analysis is performed by qRT-PCR.
47. ~The method of claim 42 wherein said quantitative analysis is performed by quantifying the products of said genes.
48 48. The method of claim 45 wherein said products are quantified by immunohistochemistry or by proteomics technology.
49. The method of claim 34 further comprising the step of preparing a report indicating that the patient has a decreased likelihood of long-term survival without breast cancer recurrence.
50. The method of claim 35 further comprising the step of preparing a report indicating that the patient has an increased likelihood of long-term survival without breast cancer recurrence.
51. 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, 34 and 35.
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