CA2513117C - Gene expression markers for breast cancer prognosis - Google Patents

Gene expression markers for breast cancer prognosis Download PDF

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CA2513117C
CA2513117C CA2513117A CA2513117A CA2513117C CA 2513117 C CA2513117 C CA 2513117C CA 2513117 A CA2513117 A CA 2513117A CA 2513117 A CA2513117 A CA 2513117A CA 2513117 C CA2513117 C CA 2513117C
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dna
artificial sequence
breast cancer
probe
expression
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CA2513117A1 (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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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 Expression 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 inununohistochemistry.
This method often yields different results in different laboratories, in part because the reagents are not standardized, and in part because the interpretations are subjective and cannot be easily quantified. RNA-based tests have not often been used because of the problem of RNA
degradation over time and the fact that it is difficult to obtain fresh tissue samples from patients for analysis. Fixed paraffin-embedded tissue is more readily available and methods have been established to detect RNA in fixed tissue. However, these methods typically do not allow for the study of large numbers of genes (DNA or RNA) from small amounts of material.
Thus, traditionally fixed tissue has been rarely used other than for immunohistochemistry detection of proteins.
Recently, several groups have published studies concerning the classification of various cancer types by microarray gene expression analysis (see, e.g. Golub et al., Science 286:531-537 (1999); Bhattacharjae et al., Proc. Natl. Acad. Sci. USA 98:13790-13795 (2001);
Chen-Hsiang et al., Bioinformatics 17 (Suppl. 1):S316-S322 (2001); Ramaswamy et al., Proc.
NatL Acad. Sci. USA 98:15149-15154 (2001)). Certain classifications of human breast cancers based on gene expression patterns have also been reported (Martin et al., Cancer Res.
60:2232-2238 (2000); West et al., Proc. Natl. Acad. Sci. USA 98:11462-11467 (2001); Sorlie et al., Proc. Natl. Acad. Sci. USA 98:10869-10874 (2001); Yan et aL, Cancer Res. 61:8375-8380 (2001)). However, these studies mostly focus on improving and refining the already established classification of various types of cancer, including breast cancer, and generally do not 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 modem 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 ante-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.
2 Various embodiments of this invention provide a method of predicting the likelihood of long-term survival of a breast cancer patient without recurrence of breast cancer, comprising:
determining a level of an RNA transcript of MYBL2 or its expression product, in a breast cancer tumor sample from said patient; normalizing said level of the RNA transcript of MYBL2 or its expression product, to obtain a normalized expression level of MYBL2; wherein increased normalized expression level of MYBL2 indicates a decreased likelihood of long-term survival without breast cancer recurrence. The method may further comprise determining a normalized expression level of an RNA transcript of at least one further gene or its expression product. Such a further gene may be: GRB7, CTSL, Chkl, AIB1, CCNB I , MCM2, EBX05, Her2, STK15, SURV, EGFR, HIFI a, or TS; wherein increased normalized expression level of the at least one further gene indicates a decreased likelihood of long-term survival without breast cancer recurrence. The method may further comprise determining a normalized expression level of an RNA transcript of at least one further gene or its expression product. Such a further gene may be:
TP53BP2, PR, Bc12, EstR1, IGFBP2, BAG1, CEGP1, KLK10, p-Catenin, y-Catenin, DR5, PI3KCA2, RAD51C, GSTM1, FHIT, RIZ1, BBC3, TBP, p27, IRS1, IGF1R, GATA3, ZNF217, CD9, pS2, ErbB3, TOP2B, MDM2, IGE1, or KRT19; wherein increased normalized expression level of the at least one further gene indicates an increased likelihood of long-term survival without breast cancer recurrence.
Various embodiments of this invention provide a method of preparing a personalized genomics profile for a patient, comprising the steps of: (a) subjecting RNA
extracted from breast tissue from the patient to gene expression analysis; (b) determining a level of an RNA transcript of MYBL2 or its expression product, wherein the level of the RNA transcript of MYBL2 or its expression product is normalized against at least one control gene to obtain normalized data, and optionally is compared to expression levels found in a breast cancer reference tissue set; and (c) creating a report summarizing the normalized data obtained by said gene expression analysis.
The present invention accommodates the use of archived paraffin-embedded biopsy material for assay of all markers in the set, and therefore is compatible with the most widely 2a
3 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, Bc12, KRT14, IRS1, CTSL, EstR1, Chkl, IGFBP2, BAG1, CEGP1, STK15, GSTM1, FHIT, RIZ1, AlB 1 , SLTRV, BBC3, IGF1R, p27, GATA3, ZNF217, EGFR, CD9, MYBL2, HIF1a, pS2, ErbB3, TOP2B, MDM2, RAD51C, KRT19, TS, Her2, KLK10, 13-Catenin, y-Catenin, MCM2, PI3KC2A, IGF1, TBP, CCNB1, FBX05, and DR5, wherein expression of one or more of GRB7, CD68, CTSL, Chkl, Al}3 1 , CCNB1, MCM2, FBX05, Her2, STK15, SURV, EGFR, MYBL2, HIF1a, 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, 13-Catenin, y-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.
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 further 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: a-Catenin, AlB1, AKT1, AKT2, 13-actin, BAG1, BBC3, Bc12, CCNB1, CCND1, CD68, CD9, CDH1, CEGP1, Chkl, CIAP1, cMet.2, Contig 27882, CTSL, DR5, EGFR, ElF4E, EPHX1, ErbB3, EstR1, FBX05, 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.
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, Bc12, KRT14, IRS1, CTSL, EstR1, Chkl, IGFBP2, BAG1, CEGP1, STK15, GSTM1, FHIT, RIZ1, AlB1, SURV, BBC3, IGF1R, p27, GATA3, ZNF217, EGFR, CD9, MYBL2, HIF1a, pS2, RIZ1, ErbB3, TOP2B, MDM2, RAD51C, KRT19, TS, Her2, KLK10, 13-Catenin, y-Catenin, MCM2, PI3KC2A, IGF1, TBP, CCNB1, FBX05 and DR5.
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, Bc12, BAD, EPHX1, PDGFR13, DIABLO, XIAP, YB1, CA9, and KRT8;
(b) GRB7, CD68, TOP2A, Bc12, DIABLO, CD3, ID1, PPM1D, MCM6, and WISP1;
(c) PR, TP53BP2, PRAME, DIABLO, CTSL, IGFBP2, TIMP1, CA9, MMP9, and COX2;
(d) CD68, GRB7, TOP2A, Bc12, DIABLO, CD3, PPM1D, MCM6, and WISP1;
(e) Bc12, TP53BP2, BAD, EPHX1, PDGFR13, DIABLO, XIAP, YB1, CA9, and KRT8;
(f) KRT14, KRT5, PRA_ME, TP5311P2, GUS1, A1B1, MCM3, CCNE1, MCM6, and 11:301;
4 (g) PRAME, TP53BP2, EstR1, DIABLO, CTSL, PPM1D, GRB7, DAPK1, BBC3, and VEGFB;
(h) CTSL2, GRB7, TOP2A, CCNB1, Bc12, DIABLO, PRAME, EMS1, CA9, and EpCAM;
(i) EstR1, TP53BP2, PRAME, DIABLO, CTSL, PPM1D, GRB7, DAPK1, BBC3, and VEGFB;
(k) Chkl, PRAME, TP53BP2, GRB7, CA9, CTSL, CCNB1, TOP2A, tumor size, and IGFBP2;
(1) IGFBP2, GRB7, PRAME, DIABLO, CTSL, 13-Catenin, PPM1D, Chkl, WISP1, and LOT1;
(m) HER2, TP53BP2, Bc12, DIABLO, TIMP1, EPHX1, TOP2A, TRAIL, CA9, and AREG;
(n) BAG1, TP53BP2, PRAME, 1L6, CCNB1, PAI1, AREG, tumor size, CA9, and Ki67;
(o) CEGP1, TP53BP2, PRAME, DIABLO, Bc12, COX2, CCNE1, STK15, and AKT2, and FGF18;
(p) STK15, TP53BP2, PRAME, IL6, CCNE1, AKT2, DIABLO, cMet, CCNE2, and COX2;
(q) KLK10, EstR1, TP53BP2, PRAME, DIABLO, CTSL, PPM1D, GRB7, DAPK1, and BBC3;
(r) A161, TP53BP2, Bc12, DIABLO, TIMP1, CD3, p53, CA9, GRB7, and EPHX1 (s) BBC3, GRB7, CD68, PRAME, TOP2A, CCNB1, EPHX1, CTSL
GSTM1, and APC;
(0 CD9, GRB7, CD68, TOP2A, Bc12, CCNB1, CD3, DIABLO, 1D1, and PPM1D;
(w) EGFR, KRT14, GRB7, TOP2A, CCNB1, CTSL, Bc12, TP, KLK10, and CA9;
(x) HIF1a, PR, DIABLO, PRAME, Chkl, AKT2, GRB7, CCNE1, TOP2A, and CCNB1;
(y) MDM2, TP53BP2, DIABLO, Bc12, A161, TIMP1, CD3, p53, CA9, and HER2;
(z) MYBL2, TP53BP2, PRAME, I1L6, Bc12, DIABLO, CCNE1, EPHX1, TIMP1, and CA9;
(aa) p27, TP53BP2, PRAME, DIABLO, Bc12, COX2, CCNE1, STK15, AKT2, and ID1;
(ab) RAD51, GRB7, CD68, TOP2A, CIAP2, CCNB1, BAG1, IL6, FGFR1, and TP53BP2;
(ac) SLTRV, GRB7, TOP2A, PRAME, CTSL, GSTM1, CCNB1, VDR, CA9; and CCNE2;
(ad) TOP2B, TP53BP2, DIABLO, Bc12, TIMP1, AIB1, CA9, p53, KRT8, and BAD;
5 (ae) ZNF217, GRB7, TP53BP2, PRAME, DIABLO, Bc12, COX2, CCNE1, APC4, and f3-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 CD68;
CTSL; FBX05;
SURV; CCNB1; MCM2; Chkl; MYBL2; H1F1A; cMET; EGFR; TS; STK15, IGFR1; BC12;
HNF3A; TP53BP2; GATA3 ; BB C3 ; RAD51C ; BAG1 ; IGFBP2; PR; CD9; RBI; 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; FBX05; SURV; CCNB1;
MCM2; Chkl; MYBL2; H1F1A; 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; ElF4E.
(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 CCND1; UPA;
HNF3A; CDH1; Her2 ; GRB7; AKT1 ; STMY3 ; a-Catenin; VDR; GR01; KT14 ; KLK10;
Maspin, TGFa, and FRP1, wherein expression of the following genes is indicative of a
6 reduced likelihood of survival without cancer recurrence: CCND1; UPA; HNF3A;
CDH1;
Her2; GRB7; AKT1; STMY3; a-Catenin; VDR; GRO1, and wherein expression of the following genes is indicative of a better prognosis for survival without cancer recurrence:
KT14; KLK10; Maspin, TGFa, and FRP1.
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 further aspect, the invention concerns 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.
In a still further aspect, the invention concerns a PCR amplicon listed in Tables 5A 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, A1B1, CCNB1, MCM2, FBX05, Her2, STK15, SURV, EGFR, MYBL2, HIFI a, 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, Bc12, KRT14, EstR1, IGFBP2, BAG1, CEGP1, KLK10, f3-Catenin, 7-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 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 Description of the Tables 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 5A 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 5A-B.
Detailed Description of the Preferred Embodiment A. Definitions Unless defined otherwise, technical and scientific terms used herein have the smile 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 defmed 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 in vitro recombinant DNA-mediated techniques and by expression of DNAs in cells and organisms.
The terms "differentially expressed gene," "differential gene expression" and their synonyms, which are used interchangeably, refer to a gene whose expression is activated to a higher or lower level in a subject suffering from a disease, specifically cancer, such as breast cancer, relative to its expression in a normal or control subject. The terms also include genes whose expression is activated to a higher or lower level at different stages of the same disease.
It is also understood that a differentially expressed gene may be either activated or inhibited at the nucleic acid level or protein level, or may be subject to alternative splicing to result in a different polypeptide product. Such differences may be evidenced by a change in mRNA
levels, surface expression, secretion or other partitioning of a polypeptide, for example.
Differential gene expression may include a comparison of expression between two or more genes or their gene products, or a comparison of the ratios of the expression between two or more genes or their gene products, or even a comparison of two differently processed products of the same gene, which differ between normal subjects and subjects suffering from a disease, specifically cancer, or between various stages of the same disease.
Differential expression includes both quantitative, as well as qualitative, differences in the temporal or cellular expression pattern in a gene or its expression products among, for example, normal and diseased cells, or among cells which have undergone different disease events or disease stages. For the purpose of this invention, "differential gene expression" is considered to be present when there is at least an about two-fold, preferably at least about four-fold, more preferably at least about six-fold, most preferably at least about ten-fold difference between the expression of a given gene in normal and diseased subjects, or in various stages of disease development in a diseased subject.
The 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 Biology, 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% Fico11/0.1% polyvinylpyrrolidone/50mM 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 NaC1, 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 tig/m1), 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 50% formamide 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 NaC1, 15 mI\4 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, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
In 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 Description The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Such techniques are explained fully in the literature, such as, "Molecular Cloning:
A Laboratory Manual", 2nd edition (Sambrook et al., 1989); "Oligonucleotide Synthesis"
(M.J. Gait, ed., 1984); "Animal Cell Culture" (R.I. Freshney, ed., 1987); "Methods in Enzymology"
(Academic Press, Inc.); "Handbook of Experimental Immunology", 4th edition (D.M. Weir &
C.C. Blackwell, eds., Blackwell Science Inc., 1987); "Gene Transfer Vectors for Mammalian Cells" (J.M. Miller & M.P. Cabs, eds., 1987); "Current Protocols in Molecular Biology"
(F.M. Ausubel et al., eds., 1987); and "PCR: The Polymerase Chain Reaction", (Mullis et al., eds., 1994).
1. Gene Expression Profiling In general, methods of gene expression profiling can be divided into two large groups:
methods based on hybridization analysis of polynucleotides, and methods based on sequencing of polynucleotides. The most commonly used methods known in the art for the quantification of mRNA expression in a sample include northern blotting and in situ hybridization (Parker & Barnes, Methods in Molecular Biology 106:247-283 (1999)); RNAse protection assays (Hod, Biotechniques 13:852-854 (1992)); and reverse transcription polymerase chain reaction (RT-PCR) (Weis et al., Trends in Genetics 8:263-264 (1992)).
Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes.
Representative methods for sequencing-based gene expression analysis include Serial Analysis of Gene Expression (SAGE), and gene expression analysis by massively parallel signature sequencing (MPS S).
2. Reverse Transcriptase PCR (RT-PCR) Of the techniques listed above, the most sensitive and most flexible quantitative method is RT-PCR, which can be used to compare mRNA levels in different sample populations, in normal and tumor tissues, with or without drug treatment, to characterize patterns of gene expression, to discriminate between closely related mRNAs, and to analyze RNA structure.
The first step is the isolation of mRNA from a target sample. The starting material is typically total RNA isolated from human tumors or tumor cell lines, and corresponding normal tissues or cell lines, respectively. Thus RNA can be isolated from a variety of primary tumors, including breast, lung, colon, prostate, brain, liver, kidney, pancreas, spleen, thymus, testis, ovary, uterus, etc., tumor, or tumor cell lines, with pooled DNA from healthy donors. If the source of mRNA is a primary tumor, mRNA can be extracted, for example, from frozen or archived paraffin-embedded and fixed (e.g. formalin-fixed) tissue samples.
General methods for mRNA extraction are well known in the art and are disclosed in standard textbooks of molecular biology, including Ausubel et al., Current Protocols of Molecular Biology, John Wiley and Sons (1997). Methods for RNA extraction from paraffin embedded tissues are disclosed, for example, in Rupp and Locker, Lab Invest.
56:A67 (1987), and De Andres et al., BioTechniques 18:42044 (1995). In particular, RNA
isolation can be performed using purification kit, buffer set and protease from commercial manufacturers, such as Qiagen, according to the manufacturer's instructions. For example, total RNA from cells in culture can be isolated using Qiagen RNeasy mini-columns. Other commercially available RNA isolation kits include MasterPureTM Complete DNA and RNA
Purification Kit (EPICENTRE , Madison, 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 murine leukemia virus reverse transcriptase (MMLV-RT). The reverse transcription step is typically primed using specific primers, random hexamers, or oligo-dT primers, depending on the circumstances and the goal of expression profiling. For example, extracted RNA can be reverse-transcribed using a GeneAmp RNA PCR kit (Perkin Elmer, CA, USA), following the manufacturer's instructions. The derived cDNA can then be used as a template in the subsequent PCR reaction.
Although the PCR step can use a variety of thermostable DNA-dependent DNA
polymerases, it typically employs the Taq DNA polymerase, which has a 5' -3' nuclease activity but lacks a 3'-5' proofreading endonuclease activity. Thus, TaqMan PCR typically utilizes the 5'-nuclease activity of Taq or Tth polymerase to hydrolyze a hybridization probe bound to its target amplicon, but any enzyme with equivalent 5' nuclease activity can be used.
Two oligonucleotide primers are used to generate an amplicon typical of a PCR
reaction. A
third oligonucleotide, or probe, is designed to detect nucleotide sequence located between the two PCR primers. The probe is non-extendible by Taq DNA polymerase enzyme, and is labeled with a reporter fluorescent dye and a quencher fluorescent dye. Any laser-induced emission from the reporter dye is quenched by the quenching dye when the two dyes are located close together as they are on the probe. During the amplification reaction, the Taq DNA polymerase enzyme cleaves the probe in a template-dependent manner. The resultant probe fragments disassociate in solution, and signal from the released reporter dye is free from the quenching effect of the second fluorophore. One molecule of reporter dye is liberated for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data.
TaqMan RT-PCR can be performed using commercially available equipment, such as, for example, ABI PRISM 7700TM Sequence Detection SystemTm (Perkin-Elmer-Applied Biosystems, Foster City, 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 (CO.

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. 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 [tm 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: Krawetz S, Misener S (eds) Bioinformatics 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 Laboratoly Manual, Cold Spring Harbor Laboratory Press, New York, 1995, pp. 133-155; Innis and Gelfand, "Optimization of PCRs" in: PCR Protocols, A Guide to Methods and Applications, CRC
Press, London, 1994, pp. 5-11; and Plasterer, T.N. Primerselect; Primer and probe design.
Methods MoL Biol. 70:520-527 (1997).
3. MicToan-ays Differential gene expression can also be identified, or confirmed using the microarray technique. Thus, the expression profile of breast cancer-associated genes can be measured in either fresh or paraffin-embedded tumor tissue, using microarray technology.
In this method, polynucleotide sequences of interest (including cDNAs and oligonucleotides) are plated, or arrayed, on a microchip substrate. The arrayed sequences are then hybridized with specific DNA probes from cells or tissues of interest. Just as in the RT-PCR method, the source of mRNA typically is total RNA isolated from human tumors or tumor cell lines, and corresponding normal tissues or cell lines. Thus RNA can be isolated from a variety of primary tumors or tumor cell lines. If the source of mRNA is a primary tumor, mRNA can be extracted, for example, from frozen or archived paraffin-embedded and fixed (e.g. formalin-fixed) tissue samples, which are routinely prepared and preserved in everyday clinical practice.

In a specific embodiment of the microarray technique, PCR amplified inserts of cDNA
clones are applied to a substrate in a dense array. Preferably at least 10,000 nucleotide sequences are applied to the substrate. The microarrayed genes, immobilized on the microchip at 10,000 elements each, are suitable for hybridization under stringent conditions.
Fluorescently labeled cDNA probes may be generated through incorporation of fluorescent nucleotides by reverse transcription of RNA extracted from tissues of interest. Labeled cDNA
probes applied to the chip hybridize with specificity to each spot of DNA on the array. After stringent washing to remove non-specifically bound probes, the chip is scanned by confocal laser microscopy or by another detection method, such as a CCD camera.
Quantitation of hybridization of each arrayed element allows for assessment of corresponding mRNA
abundance. With dual color fluorescence, separately labeled cDNA probes generated from two sources of RNA are hybridized pairwise to the array. The relative abundance of the transcripts from the two sources corresponding to each specified gene is thus determined simultaneously. The miniaturized scale of the hybridization affords a convenient and rapid evaluation of the expression pattern for large numbers of genes. Such methods have been shown to have the sensitivity required to detect rare transcripts, which are expressed at a few copies per cell, and to reproducibly detect at least approximately two-fold differences in the expression levels (Schena et al., Proc. Natl. Acad. Sci. USA 93(2):106-149 (1996)).
Micro array analysis can be performed by commercially available equipment, following manufacturer's protocols, such as by using the Affymetrix GenChip technology, or Incyte's micro array technology.
The development of microarray methods for large-scale analysis of gene expression makes it possible to search systematically for molecular markers of cancer classification and outcome prediction in a variety of tumor types.
4. Serial Analysis of Gene Expression (SAGE) Serial analysis of gene expression (SAGE) is a method that allows the simultaneous and quantitative analysis of a large number of gene transcripts, without the need of providing an individual hybridization probe for each transcript. First, a short sequence tag (about 10-14 bp) is generated that contains sufficient information to uniquely identify a transcript, provided that the tag is obtained from a unique position within each transcript. Then, many transcripts are linked together to form long serial molecules, that can be sequenced, revealing the identity of the multiple tags simultaneously. The expression pattern of any population of transcripts can be quantitatively evaluated by determining the abundance of individual tags, and identifying the gene corresponding to each tag. For more details see, e.g.
Velculescu et aL, Science 270:484-487 (1995); and Velculescu et al., Cell 88:243-51 (1997).
5. MassARRAY Technology The MassARRAY (Sequenom, San Diego, California) technology is an automated, high-throughput method of gene expression analysis using mass spectrometry (MS) for detection. According to this method, following the isolation of RNA, reverse transcription and PCR amplification, the cDNAs are subjected to primer extension. The cDNA-derived primer extension products are purified, and dipensed on a chip array that is pre-loaded with the components needed for MALTI-TOF MS sample preparation. The various cDNAs present in the reaction are quantitated by analyzing the peak areas in the mass spectrum obtained.
6. Gene Expression Analysis by Massively Parallel Signature Sequencing (MPSS) This method, described by Brenner et al., Nature Biotechnology 18:630-634 (2000), is a sequencing approach that combines non-gel-based signature sequencing with in vitro cloning of millions of templates on separate 5 [tm 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. Immunohistochemistry Immunohistochemistry methods are also suitable for detecting the expression levels of the prognostic markers of the present invention. Thus, antibodies or antisera, preferably polyclonal antisera, and most preferably monoclonal antibodies specific for each marker are used to detect expression. The antibodies can be detected by direct labeling of the antibodies themselves, for example, with radioactive labels, fluorescent labels, hapten labels such as, biotin, or an enzyme such as horse radish peroxidase or alkaline phosphatase.
Alternatively, unlabeled primary antibody is used in conjunction with a labeled secondary antibody, comprising antisera, polyclonal antisera or a monoclonal antibody specific for the primary antibody. Immunohistochemistry protocols and kits are well known in the art and are commercially available.
8. Proteomics The term "proteome" is defined as the totality of the proteins present in a sample (e.g.
tissue, organism, or cell culture) at a certain point of time. Proteomics includes, among other things, study of the global changes of protein expression in a sample (also referred to as "expression proteomics"). Proteomics typically includes the following steps:
(1) separation of individual proteins in a sample by 2-D gel electrophoresis (2-D PAGE); (2) identification of the individual proteins recovered from the gel, e.g. my mass spectrometry or N-terminal sequencing, and (3) analysis of the data using bioinformatics. Proteomics methods are valuable supplements to other methods of gene expression profiling, and can be used, alone or in combination with other methods, to detect the products of the prognostic markers of the present invention.
9. General Description 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 {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 pm 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. Finally, the data are analyzed to identify the best treatment option(s) available to the patient on the basis of the characteristic gene expression pattern identified in the tumor sample examined.
10. Breast Cancer Gene Set, Assayed Gene Subsequences, and Clinical Application of 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 (N) 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 11 Study of Gene Expression 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, paraffm-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 79001m 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.
Analysis of patients with invasive breast carcinoma 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 N
Valid N
Bc12 -0.15748 -1.22816 4.00034 75 0.000147 PR -2.67225 -5.49747 3.61540 75 0.000541 IGF1R -0.59390 -1.71506 3.49158 75 0.000808 BAG1 0.18844 -0.68509 3.42973 75 0.000985 35 CD68 -0.52275 0.10983 -3.41186 75 0.001043 EstR1 -0.35581 -3.00699 3.32190 75 0.001384 CTSL -0.64894 -0.09204 -3.26781 75 0.001637 IGFBP2 -0.81181 -1.78398 3.24158 75 0.001774 35 42 GATA3 1.80525 0.57428 3.15608 75 0.002303 35 TP53BP2 -4.71118 -6.09289 3.02888 75 0.003365 35 42 EstR1 3.67801 1.64693 3.01073 75 0.003550 35 CEGP1 -2.02566 -4.25537 2.85620 75 0.005544 SURV -3.67493 -2.96982 -2.70544 75 0.008439 p27 0.80789 0.28807 2.55401 75 0.012678 35 Chk1 -3.37981 -2.80389 -2.46979 75 0.015793 BBC3 -4.71789 -5.62957 2.46019 75 0.016189 ZNF217 1.10038 0.62730 2.42282 75 0.017814 35 EGFR -2.88172 -2.20556 -2.34774 75 0.021527 CD9 1.29955 0.91025 2.31439 75 0.023386 35 MYBL2 -3.77489 -3.02193 -2.29042 75 0.024809 HIF1A -0.44248 0.03740 -2.25950 75 0.026757 GRB7 -1.96063 -1.05007 -2.25801 75 0.026854 pS2 -1.00691 -3.13749 2.24070 75 0.028006 RIZ1 -7.62149 -8.38750 2.20226 75 0.030720 ErbB3 -6.89508 -7.44326 2.16127 75 0.033866 TOP2B 0.45122 0.12665 2.14616 75 0.035095 35 MDM2 1.09049 0.69001 2.10967 75 0.038223 35 PRAM E -6.40074 -7.70424 2.08126 75 0.040823 GUS -1.51683 -1.89280 2.05200 75 0.043661 RAD51C -5.85618 -6.71334 2.04575 75 0.044288 35 42 AIB1 -3.08217 -2.28784 -2.00600 75 0.048462 STK15 -3.11307 -2.59454 -2.00321 75 0.048768 GAPDH -0.35829 -0.02292 -1.94326 75 0.055737 35 42 FHIT -3.00431 -3.67175 1.86927 75 0.065489 KRT19 2.52397 2.01694 1.85741 75 0.067179 35 TS -2.83607 -2.29048 -1.83712 75 0.070153 GSTM1 -3.69140 -4.38623 1.83397 75 0.070625 G- 0.31875 -0.15524 1.80823 75 0.074580 35 Catenin AKT2 0.78858 0.46703 1.79276 75 0.077043 35 CCNB1 -4.26197 -3.51628 -1.78803 75 0.077810 PI3KC2A -2.27401 -2.70265 1.76748 75 0.081215 35 42 FBX05 -4.72107 -4.24411 -1.75935 75 0.082596 DR5 -5.80850 -6.55501 1.74345 75 0.085353 CIAP1 -2.81825 -3.09921 1.72480 75 0.088683 MCM2 -2.87541 -2.50683 -1.72061 75 0.089445 CCND1 1.30995 0.80905 1.68794 75 0.095578 35 El F4E -5.37657 -6.47156 1.68169 75 0.096788 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 BC12 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, HIFla.
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 better prognosis for survival without cancer recurrence following surgery: TP53BP2, PR, Bc12, KRT14, EstR1, IGFBP2, BAG1, CEGP1, KLK10, 13 Catenin, GSTM1, FHIT, Rizl, IGF1, BBC3, IGFR1, TBP, p27, IRS1, IGF1R, GATA3, CEGP1, ZNF217, CD9, pS2, ErbB3, TOP2B, MDM2, RAD51, and KRT19.
Analysis of ER positivepatients bybinaiy 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 N Valid N
IGF1R -0.13975 -1.00435 3.65063 55 0.000584 BcI2 0.15345 -0.70480 3.55488 55 0.000786 CD68 -0.54779 0.19427 -3.41818 55 0.001193 HN F3A 0.39617 -0.63802 3.20750 55 0.002233 CTSL -0.66726 0.00354 -3.20692 55 0.002237 TP53BP2 -4.81858 -6.44425 3.13698 55 0.002741 30 27 GATA3 2.33386 1.40803 3.02958 55 0.003727 30 BBC3 -4.54979 -5.72333 2.91943 55 0.005074 RAD51C -5.63363 -6.94841 2.85475 55 0.006063 30 27 BAG1 0.31087 -0.50669 2.61524 55 0.011485 IGFBP2 -0.49300 -1.30983 2.59121 55 0.012222 30 27 FBX05 -4.86333 -4.05564 -2.56325 55 0.013135 EstR1 0.68368 -0.66555 2.56090 55 0.013214 PR -1.89094 -3.86602 2.52803 55 0.014372 SU RV -3.87857 -3.10970 -2.49622 55 0.015579 CD9 1.41691 0.91725 2.43043 55 0.018370 30 RBI -2.51662 -2.97419 2.41221 55 0.019219 EPHX1 -3.91703 -5.85097 2.29491 55 0.025578 CEGP1 -1.18600 -2.95139 2.26608 55 0.027403 CCN B1 -4.44522 -3.35763 -2.25148 55 0.028370 TRAIL 0.34893 -0.56574 2.20372 55 0.031749 EstR1 4.60346 3.60340 2.20223 55 0.031860 30 D R5 -5.71827 -6.79088 2.14548 55 0.036345 MCM2 -2.96800 -2.48458 -2.10518 55 0.039857 Chk1 -3.46968 -2.85708 -2.08597 55 0.041633 p27 0.94714 0.49656 2.04313 55 0.045843 30 MYBL2 -3.97810 -3.14837 -2.02921 55 0.047288 GUS -1.42486 -1.82900 1.99758 55 0.050718 P53 -1.08810 -1.47193 1.92087 55 0.059938 HIF1A -0.40925 0.11688 -1.91278 55 0.060989 cMet -6.36835 -5.58479 -1.88318 55 0.064969 EGFR -2.95785 -2.28105 -1.86840 55 0.067036 MTA1 -7.55365 -8.13656 1.81479 55 0.075011 RIZ1 -7.52785 -8.25903 1.79518 55 0.078119 ErbB3 -6.62488 -7.10826 1.79255 55 0.078545 TOP2B 0.54974 0.27531 1.74888 55 0.085891 30 ElF4E -5.06603 -6.31426 1.68030 55 0.098571 TS -2.95042 -2.36167 -1.67324 55 0.099959 STK15 -3.25010 -2.72118 -1.64822 55 0.105010 For each gene, a classification algorithm 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; FBX05;
SLTRV;
CCNB1; MCM2; Chkl; MYBL2; HIF1A; cMET; EGFR; TS; STK15. Many of these genes (CD68, CTSL, SLTRV, 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: IGFR1; BC12; HNF3A;
TP53BP2;
GATA3; BBC3; RAD51C; BAG1; IGFBP2; PR; CD9; RB1; EPHX1; CEGP1; TRAIL; DR5;
p27; p53; MTA; RIZ1; ErbB3; TOP2B; ElF4E. Of the latter genes, IGFR1; BC12;
TP53BP2;
GATA3; BBC3; RAD51C; BAG1; IGFBP2; PR; CD9; CEGP1; DR5; 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.
Analysis of ER negative patients by binary 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 13 Valid N
Valid N
KRT14 -1.95323 -6.69231 4.03303 18 0.000780 KLK10 -2.68043 -7.11288 3.10321 18 0.006136 CCND1 -1.02285 0.03732 -2.77992 18 0.012357 Upa -0.91272 -0.04773 -2.49460 18 0.022560 HNF3A -6.04780 -2.36469 -2.43148 18 0.025707 Maspin -3.56145 -6.18678 2.40169 18 0.027332 CDH1 -3.54450 -2.34984 -2.38755 18 0.028136 HER2 -1.48973 1.53108 -2.35826 18 0.029873 5 15 =
GRB7 -2.55289 0.00036 -2.32890 18 0.031714 AKT1 -0.36849 0.46222 -2.29737 18 0.033807 TGFA -4.03137 -5.67225 2.28546 18 0.034632 FRP1 1.45776 -1.39459 2.27884 18 0.035097 STMY3 -1.59610 -0.26305 -2.23191 18 0.038570 Contig 2 _4.27585 -7.34338 2.18700 18 0.042187 A-Catenin -1.19790 -0.39085 -2.15624 18 0.044840 5 15 VDR -4.37823 -2.37167 -2.15620 18 0.044844 GRO1 -3.65034 -5.97002 2.12286 18 0.047893 MCM3 -3.86041 -5.55078 2.10030 18 0.050061 B-actin 4.69672 5.19190 -2.04951 18 0.055273 HIFI A -0.64183 -0.10566 -2.02301 18 0.058183 MMP9 -8.90613 -7.35163 -1.88747 18 0.075329 VEGF 0.37904 1.10778 -1.87451 18 0.077183 PRAM E -4.95855 -7.41973 1.86668 18 0.078322 AlB1 -3.12245 -1.92934 -1.86324 18 0.078829 KRT5 -1.32418 -3.62027 1.85919 18 0.079428 KRT18 1.08383 2.25369 -1.83831 18 0.082577 KRT17 -0.69073 -3.56536 1.78449 18 0.091209 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; NF3A; CDH1; Her2;
GRB7;
AKT1; STMY3; a-Catenin; VDR; GROl. 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; KLK10; Maspin, TGFa, and FRP1. 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.

Analysis of multiple genes and indicators of 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.
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) TP53BP2 -0.21892 0.803386 0.068279 -3.20625 0.00134 GRB7 0.235697 1.265791 0.073541 3.204992 0.00135 PR -0.10258 0.90251 0.035864 -2.86018 0.00423 CD68 0.465623 1.593006 0.167785 2.775115 0.00552 Bc12 -0.26769 0.765146 0.100785 -2.65603 0.00791 KRT14 -0.11892 0.887877 0.046938 -2.53359 0.0113 PRAME -0.13707 0.871912 0.054904 -2.49649 0.0125 CTSL 0.431499 1.539564 0.185237 2.329444 0.0198 EstR1 -0.07686 0.926018 0.034848 -2.20561 0.0274 Chk1 0.284466 1.329053 0.130823 2.174441 0.0297 IGFBP2 -0.2152 0.806376 0.099324 -2.16669 0.0303 HER2 0.155303 1.168011 0.072633 2.13818 0.0325 BAG1 -0.22695 0.796959 0.106377 -2.13346 0.0329 CEGP1 -0.07879 0.924236 0.036959 -2.13177 0.033 STK15 0.27947 1.322428 0.132762 2.105039 0.0353 KLK10 -0.11028 0.895588 0.05245 -2.10248 0.0355 B.Catenin -0.16536 0.847586 0.084796 -1.95013 0.0512 EstR1 -0.0803 0.922842 0.042212 -1.90226 0.0571 GSTM1 -0.13209 0.876266 0.072211 -1.82915 0.0674 TOP2A -0.11148 0.894512 0.061855 -1.80222 0.0715 AlB1 0.152968 1.165288 0.086332 1.771861 0.0764 FHIT -0.15572 0.855802 0.088205 -1.7654 0.0775 RIZ1 -0.17467 0.839736 0.099464 -1.75609 0.0791 SURV 0.185784 1.204162 0.106625 1.742399 0.0814 IGF1 -0.10499 0.900338 0.060482 -1.73581 0.0826 BBC3 -0.1344 0.874243 0.077613 -1.73163 0.0833 IGF1R -0.13484 0.873858 0.077889 -1.73115 0.0834 DIABLO 0.284336 1.32888 0.166556 1.707148 0.0878 TBP -0.34404 0.7089 0.20564 -1.67303 0.0943 p27 -0.26002 0.771033 0.1564 -1.66256 0.0964 IRS1 -0.07585 0.926957 0.046096 -1.64542 0.0999 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 ACts, 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 ACts 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 of 79 patients with invasive breast carcinoma 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, Bc12, BAD, EPHX1, PDGFRJ3, DIABLO, XIAP, YB1, CA9, and KRT8.
(b) GRB7, CD68, TOP2A, Bc12, DIABLO, CD3, EDI, PPM1D, MCM6, and WISP1.
(c) PR, TP53BP2, PRAME, DIABLO, CTSL, IGFBP2, TIMP1, CA9, MMP9, and COX2.
(d) CD68, GRB7, TOP2A, Bc12, DIABLO, CD3, Dl, PPM1D, MCM6, and WISP1.
(e) Bc12, TP53BP2, BAD, EPHX1, PDGFRO, DIABLO, XIAP, YB1, CA9, and KRT8.
(f) KRT14, KRT5, PRAME, TP53BP2, GUS1, ADM, MCM3, CCNE1, MCM6, and ID1.
(g) PRAME, TP53BP2, EstR1, DIABLO, CTSL, PPM1D, GRB7, DAPK1, BBC3, and VEGFB.
(h) CTSL2, GRB7, TOP2A, CCNB1, Bc12, DIABLO, PRAME, EMS1, CA9, and EpCAM.

(i) EstR1, TP53BP2, PRAME, DIABLO, CTSL, PPM1D, GRB7, DAPK1, BBC3, and VEGFB.
(k) Chkl, PRAME, p53BP2, GRB7, CA9, CTSL, CCNB1, TOP2A, tumor size, and IGFBP2.
(1) IGFBP2, GRB7, PRAME, DIABLO, CTSL, 0-Catenin, PPM1D, Chkl, WISP1, and LOT1 .
(in) HER2, TP53BP2, Bc12, DIABLO, TIMP1, EPHX1, TOP2A, TRAIL, CA9, and AREG.
(n) BAG1, TP53BP2, PRAME, IL6, CCNB1, PAIL AREG, tumor size, CA9, and Ki67.
(o) CEGP1, TP53BP2, PRAME, DIABLO, Bc12, COX2, CCNE1, STK15, and AKT2, and FGF18.
(p) STK15, TP53BP2, PRAME, IL6, CCNE1, AKT2, DIABLO, cMet, CCNE2, and COX2.
(q) KLK10, EstR1, TP53BP2, PRAME, DIABLO, CTSL, PPM1D, GRB7, DAPK1, and BBC3.
(r) AD31, TP53BP2, Bc12, 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, Bc12, CCNB1, CD3, DIABLO, ID1, and PPM1D.
(w) EGFR, KRT14, GRB7, TOP2A, CCNB1, CTSL, Bc12, TP, KLK10, and CA9.
(x) HIF1a, PR, DIABLO, PRAME, Chkl, AKT2, GRB7, CCNE1, TOP2A, and CCNB1.
(y) MDM2, TP53BP2, DIABLO, Bc12, AJB1, TIMP1, CD3, p53, CA9, and HER2.
(z) MYBL2, TP53BP2, PRAME, IL6, Bc12, DIABLO, CCNE1, EPHX1, TIMP1, and CA9.
(aa) p27, TP53BP2, PRAME, DIABLO, Bc12, COX2, CCNE1, STK15, AKT2, and D31.
(ab) RAD51, GRB7, CD68, TOP2A, CIAP2, CCNB1, BAG1, IL6, FGFR1, and TP53BP2.
(ac) SURV, GRB7, TOP2A, PRAME, CTSL, GSTM1, CCNB1, VDR, CA9, and CCNE2.
(ad) TOP2B, TP53BP2, DIABLO, Bc12, TEMPI., AD31, CA9, p53, KRT8, and BAD.
(ae) ZNF217, GRB7, p53BP2, PRAME, DIABLO, Bc12, COX2, CCNE1, APC4, and 0-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 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 personali7ed prognosis of breast cancer, similar genes, gene sets and methods concerning other types of cancer are specifically within the scope herein.

=
Table 5A
Gene ¨Accession Seq AlB1 NM_006534GCGGCGAGTTTCCGATTTAAAGCTGAGCTGCGAGGAAAATGGCGGCGGGAGGATCAAAATACTTGCTGGA
TGGTGGACTCA

A =
AKT2 NM_001626TCCTGCCACCCTTCAAACCTCAGGTCACGTCCGAGGTCGACACAAGGTACTTCGATGATGAA1-TTACCGCC =
= APC
NM_000038GGACAGCAGGAATGTGTTTCTCCATACAGGTCACGGGGAGCCAATGGTTCAGAAACAAATCGAGTGGGT
AREG
NM_001657TGTGAGTGAAATGCC1ICTAGTAGTGAACCGTCCTCGGGAGCCGACTATGACTACTCAGAAGAGTATGAT
AACGAACCACAA
6-actin NM_001101 CAGCAGATGTGGATCAGCAAGCAGGAGTATGACGAGTCCGGCCCCTCCATCGTCCACCGCAAATGC' B.Catenin NAL001904 GGCTCTTGTGCGTACTGTCCITCGGGCTGGTGACAGGGAAGACATCACTGAGCCTGCCATCTGTGCTCITCGICATCTG
A
BAD

CAG =

CGTTGTCAGCACTTGGAATACAAGATGGTTGCCGGGTCATGTTAATTGGGAAAAAGAACAGTCCACAGGAAGAGGTTGA
AC

NM_014417CCTGGAGGGTCCTGTACAATCTCATCATGGGACTCCTGCCCITACCCAGGGGCCACAGAGCCCCCGAGAT
GGAGCCCAATTAG
Bc12 NM_000633CAGATGGACCTAGTACCCACTGAGATITCCACGCCGAAGGACAGCGATGGGAAAAATGCCCTIAAATCAT
AGG
'0A9 AG

NM_03196ETTCAGGTTGTTGCAGGAGACCATGTACATGACTGTCTCCATTATTGATCGGTTCATGCAGAATAAITGTG
TGCCCAAGAAGATG
CCND1 NM_001758 GCATGTICGTGGCCTCTAAGATGAAGGAGACCATCCCCCTGACGGCCGAGAAGCTGTGCATCTACACCG .

NM_001238AAAGAAGATGATGACCGGGITTACCCAAACTCAACGTGCAAGCCTCGGATTATTGCACCATCCAGAGGCT
C.
CCNE2 NM_057749ATGCTGIGGCTCCTTCCTAACTGGGGCTITC1-CD3z NM_000734AGATGAAGTGGAAGGCGCTTTTCACCGCGGCCATCCTGCAGGCACAGTTGCCGATTACAGAGGCA
COBB NM_001251 TGGTTCCCAGCCCTGTGTCCACCTCCAAGCCCAGATTCAGATTCGAGTCATGTACACAACCCAGGGTGGAGGAG.
COB
NM_001769GGGCGTGGAACAGITTATCTCAGACATCTGCCCCAAGAAGGACGTACTCGAAACCTTCACCGM
CORI
NM_004360TGAGTGTCCCCCGGTATCTTCCCCG000TGCCAATCCCGATGAAATTGGAAATTTTATTGATGAAAATCT
GAAAGCGGCTG

NM_020974TGACAATCAGCACACCTGCATTCACCGCTCGGAAGAGGGCCTGAGCTGCATGAATAAGGATCACGGCTGT
AGTCACA
Chk1 NM_001274GATAAATTGGTACAAGGGATCAGCT1n-CCCAGCCCACATGTOCTGATCATATGCTTTTGAATAGTCAGTTACTTGGCACCC

NM_001166TGCCTGTGGTGGGAAGCTCAGTAACTGGGAACCAAAGGATGATGCTATGTCAGAACACCGGAGGCATTTT
CC
clAP2 NM_001165GGATATTTCCGTGGCTCT1-A1-cMet NM 000245 GACATTTCCAGTCCTGCAGTCAATGCCTCTCTGCCCCACCCTTTGITCAGIGTGGCTGGTGCCACGACAAATGTGTGCG
ATCGGAG
Contig278AKE00618."GGCATCCIGGCCCAAAGTTTCCCAAATCCAGGCGGCTAGAGGCCCACTGCTTCCCAACTA
CCAGCTGAGGGGGIC
COX2 NM_000963TCTGCAGAGTTGGAAGCACTCTATGGTGACATCGATGCTGTGGAGCTGTATCCTGCCC1-TCTGGTAGAAAAGCCTCGGC ' CTSL
NM..001912GGGAGGCTTATCTCACTGAGTGAGCAGAATCTGGTAGACTGCTCTGGGCCTCAAGGCAATGAAGGCTGC
AATGG

,NM_001333'TGTCTCACTGAGCGAGCAGAATCTGGTGGACTGTTCGCGTCCTCAAGGCAATCAGGGCTGCAATGGT

NM_004938CGCTGACATCATGAATGTTCCTCGACCGGCTGGAGGCGAGTITGGATATGACAAAGACACATCGTTGCTG
AAAGAGA
DIABLO NM_019887 CACAATGGCGGCTCTGAAGAGTTGGCTGTCGCGCAGCGTAACTTCATTCTTCAGGTACAGACAGTGITTGTGT
DRS
NM_003842CTCTGAGACAGTGCTTCGATGACTTTGCAGACTTGGTGCCCTTTGACTCCTGGGAGCCGCTCATGAGGAA

EGFR NN1_005228TGTCGATGGACTTCCAGAACCACCTGGGCAGCTGCCAAAAGTGTGATCCAAGCTGTCCCAAT
ElF4E
NM_001968GATCTAAGATGGCGACTGTCGAACCGGAAACCACCCCTACTCCTAATCCCCCGACTACAGAAGAGGAGAA
AACGGAATCTAA

GGCAGTGTCACTGAGTCCITGAAATCCTCCCCTGCCCCGCGGGTCTCTGGATTGGGACGCACAGTGCA
EpCAM
NM_002354.GGGCCCTCCAGAACAATGATGGGCTTTATGATCCTGACTGCGATGAGAGCGGGCTCTTT4AGGCCAAGC
AGTGCA
EPHX1 NM_000120 'EthB3 NM_001982CGG1TATGTCATGCCAGATACACACCTCAAAGGTACTCCCTCCTCCCGGGAAGGCACCOTTIC1TCAGTG
GGTCTCAGTTC =
EsiR1 NM_000125CGTGGTGCCCCTCTATGACCTGCTGCTGGAGATGCTGGACGCCCACCGCCTACATGCGCCCACTAGCC
=

NM_012177GGCTATTCCTCATTTICTCTACAAAGIGGCCTCAGTGAACATGAAGAAGGTAGCCTCCIGGAGGAGAATT
TCGGTGACAGTCTACAATCC

CACGGGACATTCACCACATCGACTACTATAAAAAGACAACCAACGGCCGACTGCCTGTGAAGTGGATGGCACCC
Fl-HT NM:002012 CCAGTGGAGCGCTTCCATGACCIGCGTCCTGATGAAGTGGCCGATTTGTTTCAGACGACCCAGAGAG
=FRP1 NM_003012TTGGTACCTGTGGGTTAGCATCAAG1TCTCCCCAGGGTAGAAT1-G-Catenin NM_002230 TCAGCAGCAAGGGCATCATGGAGGAGGATGAGGCCTGCGGGCGCCAGTACACGCTCAAGAAAACCACC =
. GAPDH
NM_002046ATTCCACCCATGGCAAATTCCATGGCACCGTCAAGGCTGAGAACGGGAAGCTTGTCATCAATGGAAATCC
CATC
GATA3 NM_002051 CAAAGGAGCTCACTGTGGTGTCTGTGTTCCAACCACTGAATCTGGACCCCATCTGTGAATAAGCCATTCTGACTC

NM_005310CCATCTGCATCCATCTTGTTTGGGCTCCCCACCC1TGAGAAGTGCCTCAGATAATACCCTGGTGGCC
GR01 NM_001511 CGAAAAGATGCTGAACAGTGACAAATCCAACTGACCAGAAGGGAGGAGGAAGCTCACTGGTGGCTGITCCTGA

NM_000561AAGCTATGAGGAAAAGAAGTACACGATGGGGGACGCTCCTGATTATGACAGAAGCCAGTGGCTGAATGA4 AAATTCAAGCTGGGCC
GUS NM_000181 CCCACTCAGTAGCCAAGTCACAATGTTTGGAAAACAGCCCGTTTACTTGAGCAAGACTGATACCACCTGCGTG

NN1_001530TGAACATAAAGTCTGCAACATGGAAGGTATTGCACTGCACAGGCCACATTCACGTATATGATACCAACA
GTAACCAACCTCA

NM_004496TCCAGGATGTTAGGAACTGTGAAGATGGAAGGGCATGAAACCAGCGACTGGAACAGCTACTACGCAGACA
CGC

NM_002165AGAACCGCAAGGTGAGCAAGGTGGAGATTCTCCAGCACGTCATCGACTACATCAGGGACCTTCAGTTGGA

NM_000618TCCGGAGCTGTGATCTAAGGAGGCTGGAGATGTATTGCGCACCCCTCAAGCCTGCCAAGTCAGCTCGCTC
TGTCCG

11M_000875GCATGGTAGCCGAAGATTTCACAGTCAA4ATCGGAGAT1ITGGTATGACGCGAGATATCTATGAGACAG
ACTATTACCGGAAA

NM_000597GTGGACAGCACCATGAACATGTTIGGGCGGGGGAGGCAGTGCTGGCCGGAAGCCCCTCAAGTCGGGTATG
AAGG
ILB
NM_000600CCTGAACCTTCCAAAGATGGCTGAAAAAGATGGATGCTTCCAATCTGGATTCAATGAGGAGACTTGCCTG
GT "

NM_005544CCACAGCTCACCTTCTGTCAGGTGTCCATCCCAGCTCCAGCCAGCTCCCAGAGAGGAAGAGACTGGCACT
GAGG =
1<1-67 NM_002417CGGACITTGGGTGCGACTTGACGAGCGGTGGTTCGACAAGTGGCCTTGCGGGCCGGATCGTCCCAGTGGA
AGAGTTGTA.A.-NM_002776GCCCAGAGGCTCCATCGTCCATCCTC1TCCTCCCCAGTCGGCTGAACTCTCCCCTIGTCTGCACTG1-TCAAACCTCTG

AGCCACAGTGGAC

NM_000422CGAGGATTGGTTCTTCAGCA5GACAGAGGAACTGAACCGCGAGGTGGCCACCAACAGTGAGCTGGTGCAG
AGT =

NM_000224AGAGATCGAGGCTCTCAAGGAGGAGCTGCTCTTCATGAAGAAGAACCACGAAGAGGAAGTAAAAGGCC
=
KRTis NM_002276TGAGCGGCAGAATCAGGAGTACCAGCGGCTCATGGACATCAAGTCGCGGCTGGAGCAGGAGATTGCCACC
TACCGCA
KRT5 A1_000424 TCAGTGGAGAAGGAGTTGGACCAGTCAACATCTCTGTTGICACAAGCAGTGITTCCTCTGGATATGGCA
KRT8 NM_002.273 GGATGAAGCTTACATGAACAAGGTAGAGCTGGAGTCTCGCCTGGAAGGGCTGACCGACGAGATCAACTTCGTCAGGCAG
CTATATG
LOT1earisNM_002656GGAAAGACCACCTGAAAAACCACCTCCAGACCCACGACCCCAACAAAATGGCCTTTGGGTG
TGAGGAGTGIGGGAAGAAGTAC .
Maspin TOCTGCC

NM_004526GAC1TTTGCCCGCTACCITTCATTCCGGCGTGACAACAATGAGCTGTTGCTCTICATACTGAAGCAGTTA
GTGGC

NM_002386GGAGAACAATCCCCTTGAGACAQAATATGGCCITTCTGTCTACAAGGATCACCAGACCATCACCATCCAG
GAGAT

TGATGGICCTATGIGTCACATICATCACAGGTTTCATACCAACACAGGCTICAGCACTTCCTITGGTGTGITTCCTGTC
CCA
MDM2 NM_002392CTACAGGGACGCCATCGAATCCGGATC1-TGATGCTGGIGTAAGTGAACA1-TCAGGTGATTGGTTGGAT

NM_004994GAGAACCAATCTCACCGACAGGCAGCTGGCAGAGGAATACCTGTACCGCTATGGTTACACTCGGGTG

NM_004689CCGCCCTCACCTGAAGAGAAACGCGCTCCTTGGCGGACACTGGGGGAGGAGAGGAAGAAGCGCGGCTAAC

MYBL2 NM_002466 GCCGAGATCGCCAAGATGTTGCCAGGGAGGACAGACAATGCTGTGAAGAATCACTGGAACTCTACCATCAAAAG

CCCTCGTGCTGATGCTACTGAGGAGCCAGCGTCTAGGGCAGGAGCCGCTTCCTAGAAGACCAGGICATGATG
p27 NM_004064 P53 NM_000546 CITTGAACCCTTGCTTGCAAJAGGTGTGCGTCAGAAGCACCCAGGACTTCCATTTGCTTTGTCCCGGG
PAH NM_000602 CCGCAACGTGGlITTCTCACCCTATGGGGTGGCCTCGGIGTTGGCCATGCTCCAGCTGACAACAGGAGGAGAAACCCAG
CA
PDGFRb NM_002609CCAGCTCTCC1TCCAGCTACAGATCAATGTCCCTGTCCGAGTGCTGGAGCTAAGTGAGAGCCACCC

NM_002645ATACCAATCACCGCACAAACCCAGGCTATTTGITAAGTCCAGTCACAGCGCAAAGAAACATATGCGGAGA
AAATGCTAGTGTG
PPM113 NM_003620 GCCATCCGCAAAGGCTTTCTCGCTTGTCACCTTGCCATGTGGAAGAAACTGGCGGAATGGCC
PR
NM_000926GCATCAGGCTGICATTATGGTGTCCTTACCTGTGGGAGCTGTAAGGICTICTTTAAGAGGGCAATGGAAG
GGCAGCACAACTACT
PRAMS

pS2 NM 003225 GCCCTCCCAGTGTGCAAATAAGGGCTGCTGTITCGACGACACCGTTCGTGGGGTCCCCTGGTGCTTCTATCCTAATACC
ATCGACG
RAD51C NM_ 055218 GAACTTCTTGAGCAGGAGCATACCCAGGGCTTCATAATCACCTTCTGTTCAGCACTAGATGATATTCTTGGGGGTGGA
=
RB1 NM_000321 CGAAGCCCTTACAAGITTCCTAGTTCACCCTTACGGATTCCTGGAGGGAACATCTATATTTCACCCCTGAAGAGTCC

STK15 NM_003600 CATCTICCAGGAGGACCACTCTCTGTGGCACCCTGGACTACCTGCCCCCTGAAATGATTGAAGGTCGGA
STMY3 NM_005940 CCTGGAGGCTGCAACATACCTCAATCCTGIOCCAGGCCGGATCCTCCTGAAGCCCITTTCGCAGCACTGCTATCCTCCA
AAGCCATTGTA
=

Table 5B
=
. .
SURV =
NM_0011611TGTTITGATTCCCGGGCTTACCAGGTGAGAAGTGAGGGAGGAAGAAGGCAGTGTCC0TTITGCTAGAGC
TGACAGCTTTG
The NM_003194GCCCGAAACGCCGAATATAATCCCAAGCGG1TTGCTGCGGTAATCATGAGGA1AAGAGAGCCACG
TGFA NM_003236 GGTGTGCCACAGACCTTCCTACTTGGGCTGTAATCACOTGTGOAGCCT-Timpi Nm_003254 TCCOTGCGGTOCCAGATAGCCTGAATOCTGCCCGGAGTGGAACTGAAGCCTGCAGAGTGTGOACCCTGTTOCCAC
TOP2A NM_001087 AATCGAAGGGGGAGAGTGATGACTTCCATATGGACTTTGACTCAGOTGTGGCTOCTOGGGCAAAATOTGTAC

NN1_0010S5TGTGGACATOTTOCCCTCAGACTT000TACTGAGGCAOCTTCTOTGCCACGAA0CGGTCGGGCTAG
TP NM_001B53 CTATATGCAGCCAGAGATGTGACAGCOACCGTGGACAGCCTGCCACTCATCACAGCGTOCATTOTCAGTAAGAAACTCG
TGG

NM_005426GGGCCAAATATTCAGAAGCTTTTATATCAGAGGACCACCATAGGGGOCATGGAGACCATCTCTGTCC0AT
CATACCCATCO
TRAIL NM_003810 CTTCACAGTGOTCCTGCAGTCTCTOTOTGTGGCTGTAACTTACGTGTACTTTACCAAGGAGCTGAAGCAGATG
TS NM_001071 GCCTCGGTGTGOOTTTCAACATCGCCAGOTAGGCCCIGCTCACGTACATGATTGCGCACATCACG
upa W/1_002658 VOR NM_000376 GCGCTGGATTTCAGAAAGAGCCAAGTCTGGATCTGGGACCCITTOCTTCUTTOCCTGGCTTGTAACT =

GTGOTGICTIGGGTGCATTOGAGCCTTGCCTTGCTGCTOTACCTCCACGATGCCAAGTGGTOCCAGGCTGC
VEGFB NM_003377 TGACGATGGCCIGGAGTGTGTGCCCACTGGGCAGCACCAAGTCOGGATGCAGATCOTCATGATCCGGTACC
NASP1 NM_003882 AGAGGCATCCATGAACTTCACACTTGCGGGCTGCATGAGCACACGCTCGTATCAACCCAAGTAGTGTGGAGTTTG .
=

NM_001107GCAGTTGGAAGACACAGGAAAGTATC000AAAITGCAGATTTATCAACGGC1TTTATCTTGAAAATAGTG
CCACGCA

NM_004559AGACTGIGGAGITTGATG7TGTTGAAGGAGAAAAGGGTGCGGAGGCAGCA.AATGTTACA3GICCTGGTG
GIGTTCC

NN1_005526ACCCAGTAGCAAGGAGAAGCCCACTCACTGCTCCGAGTGCGGGAAAGCTTTCAGAACCTACCACCAGCT
G
=
'=
=
=
=
=
=
=
=

, .
Table 6A
. -.
.
Gene Accession = Probe Name S eq Len , .
AIB1 . NM .
_006534 S1994/A1B1f3 GCGGCGAGTTTCCGATTTA 19 ' Al B1 *NM 006534 S1995/AIB1r3 .TGAGTCCACCATCCAGCAAGT
_ . 21 ' A1B1 NM_006534 S5055/AIB1 .p3 AKT1 NM _005163 S0010/AKT1.13 AKT1 NM_005163 S0012/AKT1.r3 TCCCGGTACACCACGTTCTT 20 AKT1 NM 005163 S4776/AKT1.p3 _001626 S0828/AKT2.f3 TCCTGCCACCCTTCAAACC 19 AKT2 NM 001626 S0829/AKT2.r3 GGCGGTAAATTCATCATCGAA '.
_ 21 , AKT2 NM_001626 S4727/AKT2.p3 CAGGTCAC GTC C GA
GGTCGACACA = 24 APC NM_000038 S0022/APC.f4 GGACAGCAGGAATGTGTTTC
APC NM_000038 S0024/APC.r4 ACCCACTCGATTTGTTTCTG 20 APC NM_000038 S4888/APC.p4 CATTGGCTCCCCGTGACCTGTA 22 ' AREG NM_001657 S0025/AREG.f2 TGTGAGTGAAATGCCTTCTAGTAGTGA . 27 .
AREG NM_001657 S0027/AREG.r2 TTGTGGTTCGTTATCATACTCTTCTGA 27 AREG NM_001657 S4889/AREG.p2 CCGTCCTCGGGAGCCGACTATGA 23 B-actin NM_001101 S0034/B-acti.f2_ B-actin . NM_001101 S0036/B-acti.r2 B-actin NM_001101 S4730/B-acti.p2 B-Catenin NM_001904 S2150/B-Cate.f3 GGCTCTTGTGCGTACTGTCCTT 22 B-Catenin NM_001904 S2151/B-Cate.r3 TCAGATGACGAAGAGCACAGATG 23 B-Catenin NM 001904 . ' S5046/B-Cate.p3 AGGCTCAGTGATGTCTTCCCTGTCACCAG 29 BAD NM:032989 S2011/BAD.f1 GGGTCAGGTGCCTCGAGAT 19 BAD NM_032989 S2012/BAD.r1 CTGCTCACTCGGCTCAAACTC . 21 BAD .NM_032989 S5058/BAD.p1 . TGGGCCCAGAGCATGTTCCAGATC . . 24 BAG1 ', NM_004323 .S1386/BAG1.f2 = CGTTGTCAGCACTTGGAATACAA ' . 23 ' BAG1 NM_004323 S1387/BAG1.r2 GTTCAACCTCTTCCTGTGGACTGT 24 BAG1 . NM 004323 34731/BAG1 .p2 = CCCAATTAACATGACCCGGCAACCAT
26 =
BBC3 NM:014417 S1584/BBC3.f2 CCTGGAGGGTCCTGTACAAT = 20 ..
BBC3 NM_014417 = S1585/BBC3.r2 ,.= CTAATTGGGCTCCATCTCG 19 = BBC3 NM_014417 S4890/BBC3.p2 CATCATGGG,ACTCCTGCCCTTACC = - 24 Bc12 NM 000633 S0043/Bc12.f2 _ CAGATGGACCTAGTACCCACTGAGA ' 25 Bc12 NM 000633 S0045/Bc12.r2 CCTATGATTTAAGGGCA II ii I CC = . 24 BcI2 NM 000633 S4732/8c12.p2 CA9 NM:001216 S1398/CA9.f3 ATCCTAGCCCTGG I 1 1 1 1GG .20 CA9 NM 001216 .S1399/CA9.r3 CTGCCTTCTCATCTGCACAA
_ 20 CA9 NM 001216 S4938/CA9.p3 CCNB1 NM:031966 S1720/CCNB1.f2 TTCAGGTTGTTGCAGGAGAC . 20 CCNB1 NM_031966 S1721/CCNB1.r2 . CATCTTCTTGGGCACACAAT 20 CCNB1 NM 031966 S4733/CCNB1.p2 TGTCTCCATTATTGATCGGTTCATGCA 27 CCND1 NM 001758 S0053/CCND1.f3 GCATGTTCGTGGCCTCTAAGA 21 . CCND1 NM_001758 S0060/CCND1.r3 CGGTGTAGATGCACAGCTTCTC 22 CCND1 NM 001758 64986/CaND1.p3 AAGGAGACCATCCCCCTGACGGC 23. .
.
CCNE1 NM_001238 S1446/CCNE1.f1 WGAAGATGATGACCGGGTTTAC. 24 =
CCNE1 NM_001238 = 51447/CCNE1.r1 CCNE1 NM_001238 S4944/CCNE1.p1 CAAACTCAACGTGCAAGCCTCGGA 24 CCNE2 NM057749 .S1458/CCNE2.f2 . ATGCTGTGGCTCCTTCCTAACT 22 CCNE2 NM 057749 S1459/CCNE2.r2 ACCCAAATTGTGATATACAAAAAGGTT 27 CCNE2 NM_057749 S4945/CCNE2.p2 TACCAAGCAACCTACATGTCAAGAAAGCCC 30 CD3z NM 000734 S0064/CD3z.f1 AGATGAAGTGGAAGGCGCTT . 20 .
CD3z NM-000734 S0066/CD3z.r1 TGCCTCTGTAATCGGCAACTG 21 .
' CD3z NM 000734 S4988/CD3z.p1 CD68 NM_001251 S0067/CD68.f2 C D68 NM 001251 S0069/CD68.r2 C D68 NM:001251 S4734/CD68.p2 CD9 NM 001769 .S0686/C09,f1 .

=
CD9 NM 001769 S0687/CD9.r1 CACGGTGAAGGTTTCGAGT 19 .
CD9 NM_001769 34792/CD9.p1 AGACATCTGa cC CAAGAAG GAC GT 24 CDH1 NM_004360 S0073/CDH1.f3 TGAGTGTCCCCCGGTATCTTC 21 . .
=
' CDH1 NM_004360 S0075/CDH 1 .r3 ________ CAGCCGCTTTCAGA I I I I CAT

CDH1 NM_004360 S4990/CDH1.p3 TGCCAATCCCGATGAAATTGGAAATTT 27 CEGP1 NM_020974 S1494/CEGP1.f2 TGACAATCAGCACACCTGCAT 21 ..

_.. .
Table 6B
, .
. , CEGP1 NM_020974 S1495/CEGP1.r2 .TGTGACTACAGCCGTGATCCTTA 23 =
CEGP1 = NM_020974 S4735/CEGP1.p2 CAGGCCCTCTTCCGAGCGGT 20 Chk1 NM 001274 S1422/Chk1.f2 _ GATAAA1TGGTACAAGGGATCAGCTT 26 Chk1 NM 001274 Si23/Chk1.r2 ' GGGTGCCAAGTAACTGACTATTCA
_ 24 =
Chk1 NM 001274 S4941/Chk1.p2 CCAGCCCACATGTCCTGATCATATGC . 26 =
CIAP1 NM 001166 S0764/CIAPl.f2 TGCCTGTGGTGGGAAGCT
_ 18 CIAP1 NM 001166 S0765/CIAP1.r2 GGAAAATGCCTCCGGTGTT
_ 19 =
CIAP1 NM_001166 S4802/CIAP1.p2 TGACATAGCATCATCCITTGGTTCCCAGTT 30 clAP2 NM 001165 S0076/cIAP2.f2 GGATATTTCCGTGGCTCTTATTCA
_ 24 clAP2 . NM_001165 S0078/cIAP2.r2 CTTCTCATCAAGG6AGAAAAATCTT . 25 . clAP2 NM_001165 S4991/cIAP2.p2= TCTCCATCAAATCCTGTAAACTCCAGAGCA 30 cMet NM_000245 S0082./cM et.f2 cMet NM_000245 S0084/cMet.r2 CTCCGATCGCACACATTTGT 20 cMet NM_000245 S4993/cMet.p2 TGCCTCTCTGCCCCAC6CTTTGT 23 Contig 27882 AK00618 S2633/Contig.f3 GGCATCCTGGCCCAAAdT 18 Contig 27882 AK000618 - S2634/Contig.r3 Contig 27882 AK000618 . S4977/Contig.p3 COX2 NM_000963 S0088/C0X2J1 TCTGCAGAGTTGGAAGCACTCTA =23 COX2 NM_000963 S0090/C0X2.r1 GCCGAGGCTTTTCTACCAGAA 21 COX2 NM_000963 S4995/C0X2.p1 = CAGGATACAGCTCCACAGCATCGATGTC . . 28 , CTSL NM_001912 S1303/CTSL.f2 GGGAGGCTTATCTCACTGAGTGA . . 23 CTSL NM_001912 81304/CTSL.r2 CCATTGCAGCCTTCATTGC 19 CTSL NM_001912 = S4899/CTSL.p2 CTSL2 NM 001333 . S4354/CTSL2.f1 CTSL2 NM 001333 S4355/CTSL2.r1 CTSL2 ' NM_001333 S4356/CTS1.2.p1 DAP K1 i NM_004938 S1768/DAPK1.f3 CGCTGACATCATGAATGTTCCT . 22 DAPK1 = NM_004938 S1769/DAPK1.r3 TCTCTTTCAGCAACGATGTGTCTT 24 .DAPK1 NM 004938 84927/DAPK1.p3 TCATATCCAAACTCGCCTCCAGCCG 25 .
' DIABLO . NM:019887 S0808/DIABLO.f1 CACAATGGCGGCTCTGAAG 19 =
DIABLO . NM_019887 S0809/DIABLO.r1 ACACAAACACTGTCTGTACCTGAAGA 26 DIABLO NM_019887 ' S4813/DIABLO.p1 AAGTTACGCTGCGCGACAGCCAA . 23 DR5 NM_003842 S2551/DR5.f2 CTCTGAGACAGTGCTTCGATGACT 24 DR5 NM_003842 S2552/DR5.r2 = CCATGAdGCCCAACTTCCT 19 DR5 NM_003842 S4979/DR5.p2 CAGACTTGGTGCCCTTTGACTCC , 23 EGFR NM_005228 S0103/EGFR.f2 . TGTCGATGGACTTCCAGAAC 20 EGFR NM_005228 S0105/EGFR.r2 ATTGGGACAGCTTGGATCA 19 EGFR NM_005228 S4999/EGFR.p2 ' CACCIGGGCAGCTGCCAA 18 El F4E NM 001968 S0106/E1F4E.f1 ElF4E NM:001968 . S0108/E1F4E.r1 - TTAGATTCCG I 1 i i ElF4E NM_001968 55000/E1F4E.p1 ACCACCCCTACTCCTAATCCCCCGACT 27 , EMS1 NM_005231 =S2663/EMS1.11 GGCAGTGTCACTGAGTCCTTGA 22 ' EMS1 NM_005231 S2664/EMS1.r1 TGCACTGTGCGTCCCAAT 18 .
EMS1 NM_005231 . .S4956/ES1.p1 EpCAM . NM_002354 S1807/EpCAM.f1 GGGCCCTCCAGAACAATGAT 20 EpCAM . NM_002354 S1808/EpCAM.r1 TGCACTGCTTGGCCTTAAAGA 21 EpCAM NM_002354 S4984/EpCAM.p1 CCGCTCTCATCGCAGTCAGGATCAT 25 EPHX1 NM_000120 S 1 865/EPHX1.f2 AC C GTAGGCTCTGCTCTGAA 20 EPHX1 NM_000120 S1866/EPHX1:r2 TGGTCCAGGTGGAAAACTTC 20 EPHX1 = NM_000120 S4754/EPHX1.p2 AGGCAGCCAGACCCACAGGA . 20 =
ErbB3 NM_001982 $0112/ErbB3J1 CGGTTATGTCATGCCAGATACAC 23 ErbB3 NM_001982 S0114/ErbB3.r1 GAACTGAGACCCACTGAAGAAAGG . 24 ErbB3 NM_001982 S5002/ErbB3.pi - CCTCAAAGGTACTCCCTCCTCCCGG 25 EstR1 NM_000125 80115/EstR1.11 EstR1 NM 000125 S0117/EstR1.r1 ' GGCTAGTGGGCGCATGTAG 19 EstR1 NM_000125 S4737/EstR1.p1 CTGGAGATGCTGGACGCCC 19 FBX05 NM 012177 S2017/FBX05.r1 GGATTGTAGACTGTCACCGAAATTC . 25 FBX05 NM 012177 S2018/FBX05.fl FBX05 NM:012177 S5061/FBX05.p1 CCTCCAG GAG GCTACCTTCTTCATGTTCAC .30 .
FGF18 . NM_003862 S1665/FGF18.f2 CGGTAGTCAAGTCCGGATCAA 21 .
FGF18 NM_003862 81666/FGF.18.r2 GCTTOCCTITGCGGTTCA. 18 FGF18 NM_003862 S4914/FGF18.p2 CAAGGAGACGGAATTCTACCTGTGC 25 =
. .

. .
Table 6C
. .
FGFR1 NM _023109 S0818/FGFR1.f3 CACGGGACATTCACCACATC 20 =
FGFR1 NM_023109 S0819/FGFR1.r3 GGGTGCCATCCACTTCACA 19 FGFR1 NM 023109 S4816/FGFR1.p3 ATAAAAAGACAACCAACGGCCGACTGC 27 FHIT NM _002012 S2443/FH1T.f1 CCAGTGGAGCGCTTCCAT = ' 18' FHIT NM_ 002012 S2444/FHIT.r1 CTCTCTGGGTCGTCTGAAACAA .

FHIT NM_002012 S2445/FHIT.p1 TCGGCCACTTCATCAGGACGCAG 23 . FHIT NM_002012 S4921/FHIT.p1 . FRP1 NM_ 003012 S1804/FRP1.f3 FRP1 NM _003012 S1805/FRP 1.r3 FRP1 NM_003012 S4983/FRP1.p3 TCCCCAGGGTAGAATTCAATCAGAGC 26 G-Catenin NM_002230 S2153/G-Cate.f1 TCAGCAGCAAGGGCATCAT . 19 G-Catenin NM_002230 S2154/G-Cate.r1 GGTGG I il G-Catenin NM_002230 S5044/G-Cate.p1 GAP DH NM_002046 S0374/GAPDH.f1 ATTCCACCCATGGCAAATTC 20 GAPDH NM 002046 S0375/GAPDH.r1 GATGGGATTTCCATTGATGACA 22 _ GAPDH NM_002046 S4738/GAPDH.p1 CCGTTCTCAGCCTTGACGGTGC 22 GATA3 NM 002051 S0127/GATA3.f3 CAAAGGAGCTCACTGTGGTGTCT 23 _ GATA3 . NM_002051 S0129/GATA3.r3 GAGTCAGAATGGCTTATTCACAGATG 26 GATA3 NM_002051 S5005/GATA3.p3 TGTTCCAACCACTGAATCTGGACC 24 GRB7 NM_ 005310 50130/GRB7.f2 CCATCTGCATCCATCTTGTT 20 GRB7 NM _00531'0 50132/GRB7.r2 GRB7 NM_005310 S4726/GRB7.p2 CTCCCCACCCTTGAGAAGTGCCT 23.
6R01 NM_001511 S0133/GR01.f2 CGAAAAGATGCTGAACAGTGACA 23 GRO1 NM_001511 S0135/GR01.12 TCAGGAACAGCCACCAGTGA 20 GRO1 NM_001511 =S5006/GR01.p2 CTTCCTCCTCCCTTCTGGTCAGTTGGAT 28 GSTM1 , - NM_000561 S2026/GSTM1.r1 GGCCCAGCTTGAA H H ICA

GSTM1 = NM_000561 S2027/GSTM1.fl AAGCTATGAGGAAAAGAAGTACACGAT 27 GSTM1 NM_000561 54739/GSTM1.p1 TCAGCCACTGGCTTCTGTCATAATCAGGAG 30 GUS NM _000181 ' S0139/GUS.f1 CCCACTCAGTAGCCAAGTCA 20 . .
GUS NM _000181 S0141/GUS.r1 GUS NM_000181 S4740/GUS.p1 _______________________ TCAAGTAAACGGGCTG

HER2 NM _004448 S0142/HER2.f3 HER2 NM_ 004448 S0144/HER2.r3 CCTCTCGCAAGTGCTCCAT 19 HER2 NM 004448 S4729/HER2.p3 CCAGACCATAGCACACTCGGGCAC . 24 .
HIF1A NM:001530 S1207/H1F1A.f3 TGAACATAAAGTCTGCAACATGGA 24 HIF1A NM 001530 S1208/HIF1A.r3 HIF1A NM:001530 S4753/HIF1A.p3 TTGCACTGCACAGGCCACATTCAC 24 HNF3A NM 004496 S0148/HNF3A.f1 TCCAGGATGTTAGGAACTGTGAAG 24 HNF3A NM_ 004496 S0150/HNF3A.r1 HNF3A NM_004496 S5008/HNF3A.p1 AGTCGCTGGTTTCATGCCCTTCCA 24 ID1 NM _002165 SO820/101.11 101 . NM_002165 S0821/1131 .r1 ID-1 NM 002165 S4832/ID1.pl IGF1 NM 000618 S0154/1G.F1.f2 ToCGGAGCTGTGATCTAAGGA 21 ..
IGF1 NM_000618 30156/1GF1 .r2 IGF1 NM_000618 S5010/1GF1.p2 TGTATTGCGCACCCCTCAAGCCTG 24 IGF1R NM_000875 Si 249/1GF1R.f3 IGF1R NM_000875 S1250/1GF1R.r3 TTTCCGGTAATAGTCTGTCTCATAGATATC 30 IGF1R NM 000875 34895/IGF1R.p3 CGCGTCATACCAAAATCTCCGATTTTGA 28 IGFBP2 NM = 000597 S1129/IGFBP2s1 CCTTCATACCCGACTTGAGG 20 _ I GFBP2 NM_000597 S4837/1GF6P2.p1 CTTCCGGCCAGCACTGCCTC 20 IL6 NM 000600 S0760/1L6.f3 IL6 NM 000600 S0761/1L6.r3 11_6 NM 000600 S4800/IL6.p3 _______________________ CCAGATTGGAAGCATCCATC I I I I ICA 27 .
IRS1 NM_ 005544 S1943/IRS1.f3 CCACAGCTCACCTTCTGTCA 20 ' 1RS1 NM 005544 31944/IRS1s3 CCTCAGTGCCAGTCTCTTCC = 20.
1RS1 NM 005544 S5050/IRS1.p 3 Ki-67 NM:002417 S0436/K1-67.f2 CGGACTTTGGGTGCGACTT 19 Ki-67 NM 002417 S0437/K1-67.r2 TTACAACTCTTCCACTGGGACGAT 24 .
KI-67 NM:002417 S4741/11-67.p2 KLK10 NM_002776 32624/KLK10.13 . .
' Table 6D
_ = =
KLK10 NM_002776 S2625/KLK10.r3 ' CAGAGGTTTGAACAGTGCAGACA 23 ' KLK10 NM_002776 S4978/KLK10.p3 = CCTCTTCCTCCCCAGTCGGCTGA 23 KRT14 NM 000526 81853/KRT14f1 GGCCTGCTGAGATCAAAGAC
_ . 20 KRT14 NM 000526 S1854/KRT14.r1 GTCCACTGTGGCTGTGAGAA
_ 20 =
KRT14 NM_000526 S5037/KRT14.p1 TGTTCCTCAGGTCCTCAATGGTCTTG 26 KRT17 NM 000422 S0172/KRT17.f2 CGAGGATTGGTTCTTCAGCAA
_ 21 KRT17 NM 000422 S0174/KRT17.r2 ACTCTGCACCAGCTCACTGTTG
_ 22 KRT17 NM_000422 S5013/KRT17.p2 CACCTCGCGGTTCAGTTCCTCTGT 24 -KRT18 NM 000224 S1710/KRT18.f2 AGAGATCGAGGCTCTCAAGG
_ 20 .
=
KRT18 NM_000224 S1711/KRT18.r2 __ GGCC 1 HI

.
KRT18 NM_000224 S4762/KRT18.p2 TGGTTCTTCTTCATGAAGAGCAGCTCC 27 KRT19 NM_002276 S1515/KRT19.f3 TGAGCGGCAGAATCAGGAGTA
. 21 KRT19 NM_002276 S1516/KRT19.r3 TGCGGTAGGTGGCAATCTC 19 =
KRT19 NM_002276 S4866/KRT19.p3 CTCATGGACATCAAGTCGCGGCTG 24 KRT5 NM 000424 S0175/KRT5.f3 TCAGTGGAGAAGGAGTTGGA
_ 20 KRT5 NM_000424 S0177/KRT5.r3 TGCCATATCCAGAGGAAACA20 =
KRT5 NM_000424 S5015/KRT5.p3 KRT8 . NM_002273 S2588/KRT8.f3 KRT8 .NM_002273 S2589/KRT8.r3 CATATAGCTGCCTGAGGAAGTTGAT 25 =
KRT8 NM_002273 S4952/KRT8.p3 CGTCGGTCAGCCCTTCCAGGC 21 LOT1 variant 1 NM_002656 , S0692/LOT1 v.f2 . GGAAAGACCACCTGAAAAACCA 22 LOT1 variant 1 NM_002656= S0693/LOT1 v.r2 LOT1 variant 1 NM_002656 = S4793/LOT1 v.p2 ACCCACGACCCCAACAAAATGGC 23 Maspin NM_002639 S0836/Maspin.f2 CAGATGGCCACTTTGAGAACATT 23 Maspin NM_002639 S0837/Maspin.r2 GGCAGCATTAACCACAAGGATT 22 Maspin . NM_002639 . S4835/Maspin.p2 . AGCTGACAACAGTGTGAACGACCAG.ACC 28 MCM2 NM_004526 S1602./MCM2.f2 GACTTTTGCCCGCTACCTITC '21 ' MCM2 NM,004526 , . S1603/MCM2.r2 MCM2 NM_004526 54900/MCM2.p2 ACAGCTCATTGTTGTCACGCCG GA =24 , =
. MCM3 NM_002388 S1524/MCM3.f3 = GGAGAACAATCCCCTTGAGA
MCM3 .NM_002388 S1525/MCM3.r3 ' ATCTCCTGGATGGTGATGGT 20 . MCM3 NM_002388 S4870/MCM3.p3 TGGCCTTTCTGTCTACAAGGATCACCA 27 MCM6 ' . NM_005915 S1704/MCM6.f3 TGATGGTCCTATGTGTCACATTCA 24 MCM6 NM 005915 S1705/MCM6.r3 TGGGACAGGAAACACACCAA
_ 20 .
MCM6 NM_005915 S4919/MCM6.p3 CAGGTTTCATACCAACACAGGCTTCAGCAC 30 MDM2 NM 002392 S0830/MDM2.fl MDM2 NM 002392 S0831/MDM2.r1 MDM2 NM_002392 S4834/MDM2.p1 CTTACACCAGCATCAAGATCCGG , ' 23 MMP9 NM004994 S0656/MMP9.fl GAGAACCAATCTCACCGACA 20 .
MMP9 NM_004994 S0657/MMP9.r1 CACCCGAGTGTAACCATAGC 20 MMP9 NM 004994 = S4760/MMP9.p1 ACAGGTATTCCTCTGCCAGCTGCC = = 24 MTA1 NM_ 004689 S2369/MTA1.f1 CCGCCCTCACCTGAAGAGA 19 MTA1 NM_004689 S2370/MTA1.r1 GGAATAAGTTAGCCGOGOTTOT 22 MTA1 NM 004689 54855/MTAl.p1 COCAGTGTCCGCCAAGGAGCG 21 '=
MYBL2 NM_002466 S3270/MYBL2.f1 GCCGAGATCGCCAAGATG 18 MYBL2 NM_002466 S3271/MYBL.2.r1 __ C i 1 i MYBL2 NM 002466 S4742/MYBL2.p1 CAGCATTGTCTGTCCTCCCTGGCA 24 P14ARF S76-535 S2842JP14ARF.f1 CCCTCGTGCTGATGCTACT . 19 ' P14ARF S78535 S2843/P14ARF.r1 CATCATGACCTGGTCTTCTAGG 22 P14ARF S78535 S4971/P14ARF.p1 CTGCCCTAGACGCTGGCTCCTC 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 NM 000546 S0208/P53.f2 CTTTGAACCCTTGCTTGCAA 20 P53 NM_ 000546 S0210/P53.r2 CCCGGGACAAAGCAAATG 18 P53 . NM_000546 S5065/P53.p2 AAGTCCTGGGTGCTTCTGACGCACA , 25 PAH NM_000602 S0211/PAl1 .f3 _______ CCGCAACGTGG I I I I

PAll NM_ 000602 S5066/PAI1.p3 CTCGGTGTTGGCCATGOTCCAG 22 =
PDGFRb NM...0,02609 S1346/PDGFRb.f3 CCAGCTCTCCTTCCAGCTAC 20 =
PDGF.Rb NM 002609 Si47/PDGFRb.r3 GOGTGGCTCTCACTTAGCTC
_ 20 PDGFRb NM_002609 S4931/P DGFR b. p3 ATCAATGTCCCTGTCCGAGTGCTG 24 , * CA 02513117 2013-09-26 . =
Table 6E
= =
PI3KC2A NM_002645 S2020/P13KC2.1-1 ___ CACACTAGCAI i 11CTCCGCATA 23 =
P I3KC2A NM_002645 82021/P13KC2.f1 ATACCAATCACCGCACAAACC - 21 =
PI3KC2A , NM_002645 55062/P13KC2.p1 TGCGCTGTGACTGGACTTAACAAATAGCCT 30 PP MID " NM_003620 S3159/PPM1D.fl GCCATCCGCAAAGGCTTT = 18 P PM1D NM_003620 S3160/PPM1D.r1 GGCCATTCCGCCAGTTTC 18 ' = P PM1D NM_003620 S4856/PPM1D.p1 TCGCTTGTCAbCT7GCCATGTGG 23 PR NM- 000926 S1336/PR.f6 = GCATCAGGCTGTCATTATGG 20 _ PR NM 000926 S1337/PR.r6 AGTAGTTGTGCTGCCCTTCC ' 20 PR NM:000926 S4743/PR.p6 TGTCCITACCTGTGGGAGCTGTAAGGTC 28 FRAME NM 006115 S1985/PRAME.f3 TCTCCATATCTGCCTTGCAGAGT 23 _ .
FRAME NM 006115 S1986/PRAME.r3 GCACGTGGGTCAGATTGCT . 19 _ .
FRAME NM 006115 S4756/PRAME.p3 TCCTGCAGCACCTCATCGGGCT = 22 pS2 - NM:003225 S0241/pS2.f2 GC

pS2 NM_003225 S0243/pS2.r2 CGTCGATGGTATTAGGATAGAAGCA 25 pS2 NM_003225 S5026/pS2.p2 TGCTGTTTCGACGACACCGTTCG 23 RAD51C NM_058216 = S2606/RAD51C.f3 GAACTTCTTGAGCAGGAGCATACC = 24 RAD51C NM_058216 S2607/RAD51C.r3 TCCACCCCCAAGAATATCATCTAGT 25 RAD51C . NM_058216 S4764/RAD51 C. p3 AGGGCTTCATAATCACCTTCTGTTC 25 RB1 .NM_000321 S2700/RB1.fl CGAAGCCCTTACAAGTTTCC 20 RBI NM .000321 S2701/RB1.r1 RBI NM 000321 34765/RB1.p1 R1Z1 NM:012231 S1320/RIZ1J2 RIZ1 NM_012231 S1321/RIZ1.r2 R IZ1 NM_012231 S4761/RIZ1.p2 STK15 NM_ 003600 S0794/STK15.f2 CATCTTCCAGGAGGACCACT 20 STK15 NM 003600 S0795/STK1.5.r2 STK15 - NM 003600 ' S4745/STK15.p2 CTCTGTGGCACCCTGGACTACCTG 24 STMY3 NM:005940 = S2067/STMY3.f3 CCTGGAGGCTGCAACATACC 20 STMY3 NM_005940 S2068/STMY3.r3 TACAATGGCTTTGGAGGATAGCA 23 .
STMY3 = NM_005940 S4746/STMY3.p3 ATCCTCCTGAAGCCCTMCGCAGC = 25 . .
SURV NM_ 001168 S0259/SURV.f2 = TGTTTTGATTCCCGGGCTTA 20 SU RV NM_001168 S0261/SURV.r2 = SURV NM 001168 S4747/S U kv. p 2 TBP NM_ 003194 S 0262/TB P.f1 TB P NM 003194 S0264/TBP.r1 CGTGGCTCTCTTATCCTCATGAT " . 23 .
TBP NM:003194 S4751fTBP.p1 TACCGCAGCAAACCGCTTGGG 21 TGFA NM_003236 = S0489/TGFA.f2 , GGTGTGCCACAGACCTTCCT 20 .
TGFA NM_003236 S0490/TGFA.r2 ACGGAGTTCTTGACAGAG 1 1 i 1 GA 24 TGFA NM_003236 = S4768/TGFA.p2 TIMP1 NM_003254 S1695/11MP i .f3 TCCCTGCGGTCCCAGATAG = 19 TIMP1 NM_003254 S1696/TIMP1.r3 GTGGGAACAGGGTGGACACT 20 TIMP1 NM_003254 S4918/TIMPl.p3 ATCCTGCCCGGAGTGGAACTGAAGC 25 =
TOP2A NM_001067 60271TTOP2A.f4 AATCCAAGGGGGAGAGTGAT 20 =
TOP2A = NM 001067 S0273/TOP2A.r4 GTACAGATTTTGCCCGAGGA 20 .
TOP2A .NM_001067 S4777/TOP2A.p4 CATATGGACTTTGACTCAGCTGTGGC 26 .
TOP2B NM_001068 S0274/TOP2B.f2 TGTGGACATCTTCCCCTCAGA 21 TOP2B = NM 001068 S0276/TOP2B.r2 CTAGCCCGACC GGTTC GT 18 TOP2B NM_001068, S4778/TOP 2B. p2 TTCCCTACTGAGCCACCTTCTCTG 24 TP NM 001953 S0277/TP.13 CTATATGCAGCCAGAGATGTGACA 24 TP NM_001953 S0279/TP.r3 CCACGAGTTraTTACTGAGAATGG ' 24 TP NM_001953 S4779/TP.p3 ACAGCCTGCCACTCATCACAGCC 23 TP538P2 NM_005426 S1931/TP53BP.f2 GGGCCAAATATTCAGAAGC 19 TP533P2 NM 005426 S1932/TP53BP.r2 GGATGGGTATGATGGGACAG 20 TP53BP2 NM:005426 S5049/TP533P.p2 CCACCATAGCGGCCATGGAG 20 TRAIL NM_003810 S2539/TRA1L.f1 CTTCACAGTGCTCCTGCAGTCT 22 TRAIL = NM_003810 S2540/TRAIL.r1 CATCTGCTTCAG CTCGTTG
GT = = . 21 TRAIL NM 003810 S4980/TRAIL.p1 AAGTACACGTAAGTTACAGCCACACA ' 26 TS NM 001071 S0280/TS.f1 GCCTCGGTGTGCCTTTCA 18 TS NM-001071 80282/TS.r1 CGTGATGTGCGCAATCATG 19 TS NM:001071 S4780/TS.pl CATCGCCAGCTACGCCCTGCTC 22 =
up a NM_002658 S0283/upa.f3 GTGGATGTGCCCTGAAGGA. 19 up a NM_002658 S0285/upa.r3 CTGCGGATCCAGGGTAAGAA ' 20 =
40 .

Table 6F
=
upa NM_002658 S4769/upa.p3 AAGCCAGGCGTCTACACGAGAGTCTCAC 28 VDR = NM 000376 S2745NDR.f2 GCCCTGGATTTCAGAAAGAG 20 VDR NM 000376 S2746NDR.r2 AGTTACAAGCCAGGGAAGGA 20 VD R NM_000376 S4962N0 R. p2 VEGF NM 003376 60286NEGF.f1 VEGF NM-003376 60288NEGF.r1 GCAGCCTGGGACCACTTG . 18 VEGF NM_003376 64782NEGF.p1 TTGCCTTGCTGCTCTACCTCCACCA 25 VEGFB NM 003377 S2724NEGFB.f1 VEGFB NM 003377 S2725NEGFB.r1 GGTACCGGATCATGAGGATCTG 22 VEGFB NM_003377 64960NEGFB.p1 CTGGGCAGCACCAAGTCCGGA 21 WISP1 NM 003882 61671NVISP1 .f1 WISP1 NM 003882 S1672/WISP1 .r1 WISP1 NM_003882 S4915/VVISP1.pl CGGGCTGCATCAGCACACGC 20 GCAGTTGGAAGACACAGGAAAGT . 23 XIAP NM 001167 S0291/XIAP.r1 XIAP NM_001167 S4752/XIAP.p1 TCCCCAAATTGCAGATTTATCAACGGC 27 YB-1 NM 004559 S1194/YB-1.f2 YB-1 NM_004559 S1195NB-1.r2 GGAACACCACCAGGACCTGTAA 22 YB-1 NM_004559 S4843NB-1.p2 ______________ TTGCTGCCTCCGCACCC IIIi CT

ZNF217 NM 006526 S2739/ZNF217.f3 ACCCAGTAGCAAGGAGAAGC 20 ZNF217 NM_006526 S2740/ZNF217.r3 CAGCTGGTGGTAGGTTCTGA 20 ZNF2l 7 NM 006526 S4961/ZN F217. p3 CACTCACTGCTCCGAGTGCGG 21 =
=
=

=
SEQUENCE LISTING
<110> GENOMIC HEALTH, INC.
RUSH UNIVERSITY MEDICAL CENTER
COBLEIGH, Melody SHAK, Steven BAKER, Joffre CRONIN, Maureen <120> GENE EXPRESSION MARKERS FOR BREAST
CANCER PROGNOSIS
<130> 39740-0008 PCT
<140> PCT/US2004/000985 <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 aa 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 69 <210> 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 qt 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 72 <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 aa 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 accc 74 <210> 43 <211> 67 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 43 ccagtggagc gcttccatga cctgcgtcct gatgaagtgg ccgatttgtt tcagacgacc 60 cagagag 67 <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 67 <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 70 <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 73 <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 69 <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 a <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 <220>
<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 a <210> 74 <211> 68 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 74 ctacagggac gccatcgaat ccggatcttg atgctggtgt aagtgaacat tcaggtgatt 60 ggttggat 68 <210> 75 <211> 67 <212> DNA
<213> Artificial Sequence <220>
<223> Amplicon <400> 75 gagaaccaat ctcaccgaca ggcagctggc agaggaatac ctgtaccgct atggttacac 60 tcgggtg 67 <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 <211> 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 81 <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 65 <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 66 =
<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 65 <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 <220>
<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 ccactcactg ctccgagtgc ggcaaagctt tcagaaccta 60 ccaccagctg 70 <210> 111 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> forward primer <400> 111 gcggcgagtt tccgattta 19 <210> 112 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> 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 <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 ta 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 gatc 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 19 <210> 140 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> probe <400> 140 catcatggga ctcctgccct tacc 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>
<223> 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 20 <210> 155 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> probe <400> 155 caaactcaac gtgcaagcct cgga 24 <210> 156 <211> 22 <212> 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 19 <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 19 <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> 189 tctgcagagt tggaagcact cta 23 <210> 190 <211> 28 <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 19 <210> 194 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> probe <400> 194 ttgaggccca gagcagtcta ccagattct 29 <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> 23 <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> 205 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> reverse primer <400> 205 ccatgaggcc caacttcct 19 <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 <220>
<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 19 <210> 226 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> probe <400> 226 ctggagatgc tggacgccc 19 <210> 227 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> reverse primer <400> 227 ggctagtggg cgcatgtag 19 <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 28 <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 21 <210> 232 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> reverse primer <400> 232 gcttgccttt gcggttca 18 <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 19 <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 18 <210> 238 <211> 22 <212> DNA
<213> Artificial Sequence <220>
<223> reverse primer <400> 238 ctctctgggt cgtctgaaac aa 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 19 <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 <220>
<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 20 <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 24, <210> 266 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> reverse primer <400> 266 cctctcgcaa gtgctccat 19 <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 28 <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 24 <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 19 <210> 274 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> reverse primer <400> 274 tccaactgaa ggtccctgat g 21 <210> 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 catagatatc 30 <210> 281 <211> 28 <212> DNA
<213> Artificial Sequence <220>
<223> probe <400> 281 cgcgtcatac caaaatctcc gattttga 28 <210> 282 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> forward primer <400> 282 gtggacagca ccatgaaca 19 <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 20 <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 26 <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 20 <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 19 <210> 308 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> probe <400> 308 ctcatggaca tcaagtcgcg gctg 24 <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 28 <210> 311 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> reverse primer <400> 311 tgccatatcc agaggaaaca 20 <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 20 <210> 329 <211> 30 <212> DNA
<213> Artificial Sequence <220>
<223> probe <400> 329 caggtttcat accaacacag gcttcagcac 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 21 <210> 339 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> forward primer <400> 339 gccgagatcg ccaagatg 18 <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 25 <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 20 <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 19 <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 tog 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 taco 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 24 <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 23 <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 20 <210> 388 <211> 28 <212> DNA
<213> Artificial Sequence <220>
<223> probe <400> 388 tgccttcttc ctccctcact tctcacct 28 <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 19 <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 18 <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 <220>
<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 20 <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 18 <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 19 <210> 417 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> forward primer <400> 417 gtggatgtgc cctgaagga 19 <210> 418 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> reverse primer <400> 418 ctgcggatcc agggtaagaa 20 <210> 419 <211> 28 <212> DNA
<213> Artificial Sequence <220>
<223> probe =
<400> 419 aagccaggcg tctacacgag agtctcac 28 <210> 420 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> forward primer <400> 420 gccctggatt tcagaaagag 20 <210> 421 <211> 20 <212> DNA
<212> 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 19 <210> 427 <211> 22 <212> DNA
<213> Artificial Sequence <220>
<223> reverse primer <400> 427 ggtaccggat catgaggatc tg 22 <210> 428 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> probe <400> 428 ctgggcagca ccaagtccgg a 21 <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 aa 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 (12)

WHAT IS CLAIMED IS:
1. A method of predicting the likelihood of long-term survival of a breast cancer patient without recurrence of breast cancer, comprising:
determining a level of an RNA transcript of MYBL2 or its expression product, in a breast cancer tumor sample from said patient;
normalizing said level of the RNA transcript of MYBL2 or its expression product, to obtain a normalized expression level of MYBL2;
wherein increased normalized expression level of MYBL2 indicates a decreased likelihood of long-term survival without breast cancer recurrence.
2. The method of claim 1, further comprising:
determining a normalized expression level of an RNA transcript of at least one further gene or its expression product, wherein the further gene is: GRB7, CTSL, Chk1 , AIB1, CCNB1, MCM2, FBX05, Her2, STK15, SURV, EGFR, HIF1.alpha., or TS;
wherein increased normalized expression level of the at least one further gene indicates a decreased likelihood of long-term survival without breast cancer recurrence.
3. The method of claim 1, further comprising:
determining a normalized expression level of an RNA transcript of at least one further gene or its expression product, wherein the further gene is: TP53BP2, PR, Bcl2, 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, or KRT19;
wherein increased normalized expression level of the at least one further gene indicates an increased likelihood of long-term survival without breast cancer recurrence.
4. The method of claim 1, 2 or 3, wherein the breast cancer is invasive breast carcinoma.
5. The method of any one of claims 1 to 4, wherein the breast cancer is estrogen-receptor positive breast cancer.
6. The method of any one of claims 1 to 5, wherein the breast cancer tumor sample is a fixed, wax-embedded tissue specimen.
7. The method of any one of claims 1 to 6, wherein the level of the RNA
transcript of MYBL2 is determined.
8. The method of claim 7, wherein the level of the RNA transcript of MYBL2 is determined by quantitative reverse-transcription polymerase chain reaction (qRT-PCR).
9. The method of any one of claims 1 to 6, wherein the level of the expression product of the RNA transcript of MYBL2 is determined.
10. The method of claim 9, wherein the level of the expression product is determined by immunohistochemistry or proteomics technology.
11. The method of any one of claims 1 to 10, further comprising providing a report based on the normalized expression level of MYBL2.
12. The method of claim 11, wherein the report comprises a prediction of the likelihood of long-term survival of said patient without breast cancer recurrence.
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