# Patent

## Title
Gene expression markers for breast cancer prognosis

## Abstract

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

## Background
This application claims priority under 35 U.S.C. § 119(e) to provisional application Ser. No. 60/440,861 filed on Jan. 15, 2003, the entire disclosure of which is hereby expressly incorporated by reference.


BACKGROUND OF THE INVENTION
1. 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.
2. Description of the Related Art
Oncologists have a number of treatment options available to them, including different combinations of chemotherapeutic drugs that are characterized as “standard of care,” and a number of drugs that do not carry a label claim for particular cancer, but for which there is evidence of efficacy in that cancer. Best likelihood of good treatment outcome requires that patients be assigned to optimal available cancer treatment, and that this assignment be made as quickly as possible following diagnosis.
Currently, diagnostic tests used in clinical practice are single analyte, and therefore do not capture the potential value of knowing relationships between dozens of different markers. Moreover, diagnostic tests are frequently not quantitative, relying on immunohistochemistry. This method often yields different results in different laboratories, in part because the reagents are not standardized, and in part because the interpretations are subjective and cannot be easily quantified. RNA-based tests have not often been used because of the problem of RNA degradation over time and the fact that it is difficult to obtain fresh tissue samples from patients for analysis. Fixed paraffin-embedded tissue is more readily available and methods have been established to detect RNA in fixed tissue. However, these methods typically do not allow for the study of large numbers of genes (DNA or RNA) from small amounts of material. Thus, traditionally fixed tissue has been rarely used other than for immunohistochemistry detection of proteins.
Recently, several groups have published studies concerning the classification of various cancer types by microarray gene expression analysis (see, e.g. Golub et al., Science 286:531-537 (1999); Bhattacharjae et al., Proc. Natl. Acad. Sci. USA 98:13790-13795 (2001); Chen-Hsiang et al., Bioinformatics 17 (Suppl. 1):S316-S322 (2001); Ramaswamy et al., Proc. Natl. Acad. Sci. USA 98:15149-15154 (2001)). Certain classifications of human breast cancers based on gene expression patterns have also been reported (Martin et al., Cancer Res. 60:2232-2238 (2000); West et al., Proc. Natl. Acad. Sci. USA 98:11462-11467 (2001); Sorlie et al., Proc. Natl. Acad. Sci. USA 98:10869-10874 (2001); Yan et al., Cancer Res. 61:8375-8380 (2001)). However, these studies mostly focus on improving and refining the already established classification of various types of cancer, including breast cancer, and generally do not provide new insights into the relationships of the differentially expressed genes, and do not link the findings to treatment strategies in order to improve the clinical outcome of cancer therapy.
Although modern molecular biology and biochemistry have revealed hundreds of genes whose activities influence the behavior of tumor cells, state of their differentiation, and their sensitivity or resistance to certain therapeutic drugs, with a few exceptions, the status of these genes has not been exploited for the purpose of routinely making clinical decisions about drug treatments. One notable exception is the use of estrogen receptor (ER) protein expression in breast carcinomas to select patients to treatment with anti-estrogen drugs, such as tamoxifen. Another exceptional example is the use of ErbB2 (Her2) protein expression in breast carcinomas to select patients with the Her2 antagonist drug Herceptin® (Genentech, Inc., South San Francisco, Calif.).
Despite recent advances, the challenge of cancer treatment remains to target specific treatment regimens to pathogenically distinct tumor types, and ultimately personalize tumor treatment in order to maximize outcome. Hence, a need exists for tests that simultaneously provide predictive information about patient responses to the variety of treatment options. This is particularly true for breast cancer, the biology of which is poorly understood. It is clear that the classification of breast cancer into a few subgroups, such as ErbB2+ subgroup, and subgroups characterized by low to absent gene expression of the estrogen receptor (ER) and a few additional transcriptional factors (Perou et al., Nature 406:747-752 (2000)) does not reflect the cellular and molecular heterogeneity of breast cancer, and does not allow the design of treatment strategies maximizing patient response.
SUMMARY OF THE INVENTION
The present invention provides a set of genes, the expression of which has prognostic value, specifically with respect to disease-free survival.
The present invention accommodates the use of archived paraffin-embedded biopsy material for assay of all markers in the set, and therefore is compatible with the most widely 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, Chk1, IGFBP2, BAG1, CEGP1, STK15, GSTM1, FHIT, RIZ1, AIB1, SURV, BBC3, IGF1R, p27, GATA3, ZNF217, EGFR, CD9, MYBL2, HIF1α, pS2, ErbB3, TOP2B, MDM2, RAD51C, KRT19, TS, Her2, KLK10, β-Catenin, γ-Catenin, MCM2, PI3KC2A, IGF1, TBP, CCNB1, FBXO5, and DR5,

    
    
        wherein expression of one or more of GRB7, CD68, CTSL, Chk1, AIB1, CCNB1, MCM2, FBXO5, Her2, STK15, SURV, EGFR, MYBL2, HIF1α, 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, β-Catenin, γ-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: α-Catenin, AIB1, AKT1, AKT2, β-actin, BAG1, BBC3, Bc12, CCNB1, CCND1, CD68, CD9, CDH1, CEGP1, Chk1, CIAP1, cMet.2, Contig 27882, CTSL, DR5, EGFR, EIF4E, EPHX1, ErbB3, EstR1, FBXO5, FHIT1 FRP1, GAPDH, GATA3, G-Catenin, GRB7, GRO1, GSTM1, GUS, HER2, HIF1A, HNF3A, IGF1R, IGFBP2, KLK10, KRT14, KRT17, KRT18, KRT19, KRT5, Maspin, MCM2, MCM3, MDM2, MMP9, MTA1, MYBL2, P14ARF, p27, P53, PI3KC2A, PR, PRAME, pS2, RAD51C, 3RB1, RIZ1, STK15, STMY3, SURV, TGFA, TOP2B, TP53BP2, TRAIL, TS, upa, VDR, VEGF, and ZNF217.
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, Chk1, IGFBP2, BAG1, CEGP1, STK15, GSTM1, FHIT, RIZ1, AIB1, SURV, BBC3, IGF1R, p27, GATA3, ZNF217, EGFR, CD9, MYBL2, HIF1α, pS2, RIZ1, ErbB3, TOP2B, MDM2, RAD51C, KRT19, TS, Her2, KLK10, β-Catenin, γ-Catenin, MCM2, PI3KC2A, IGF1, TBP, CCNB1, FBXO5 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, PDGFRβ, 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, ID1, PPM1D, MCM6, and WISP1;
    (e) Bc12, TP53BP2, BAD, EPHX1, PDGFRβ, DIABLO, XIAP, YB1, CA9, and KRT8;
    (f) KRT14, KRT5, PRAME, TP53BP2, GUS1, AIB1, 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) Chk1, PRAME, TP53BP2, GRB7, CA9, CTSL, CCNB1, TOP2A, tumor size, and IGFBP2;
    (l) IGFBP2, GRB7, PRAME, DIABLO, CTSL, β-Catenin, PPM1D, Chk1, WISP1, and LOT1;
    (m) HER2, TP53BP2, Bc12, DIABLO, TIMP1, EPHX1, TOP2A, TRAIL, CA9, and AREG;
    (n) BAG1, TP53BP2, PRAME, IL6, CCNB1, PAI1, AREG, tumor size, CA9, and Ki67;
    (o) CEGP1, TP53BP2, PRAME, DIABLO, 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) AIB1, 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) HIF1α, PR, DIABLO, PRAME, Chk1, AKT2, GRB7, CCNE1, TOP2A, and CCNB1;
    (y) MDM2, TP53BP2, DIABLO, Bc12, AIB1, 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 ID1;
    (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, TIMP1, AIB1, CA9, p53, KRT8, and BAD;
    (ae) ZNF217, GRB7, TP53BP2, PRAME, DIABLO, Bc12, COX2, CCNE1, APC4, and β-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; FBXO5; SURV; CCNB1; MCM2; Chk1; MYBL2; HIF1A; cMET; EGFR; TS; STK15, IGFR1; BC12; HNF3A; TP53BP2; GATA3; BBC3; RAD51C; BAG1; IGFBP2; PR; CD9; RB1; EPHX1; CEGP1; TRAIL; DR5; p27; p53; MTA; RIZ1; ErbB3; TOP2B; EIF4E, wherein expression of the following genes in ER-positive cancer is indicative of a reduced likelihood of survival without cancer recurrence following surgery: CD68; CTSL; FBXO5; SURV; CCNB1; MCM2; Chk1; MYBL2; HIF1A; cMET; EGFR; TS; STK15, and wherein expression of the following genes is indicative of a better prognosis for survival without cancer recurrence following surgery: IGFR1; BC12; HNF3A; TP53BP2; GATA3; BBC3; RAD51C; BAG1; IGFBP2; PR; CD9; RB1; EPHX1; CEGP1; TRAIL; DR5; p27; p53; MTA; RIZ1; ErbB3; TOP2B; EIF4E.
        (2) subjecting the data obtained in step (1) to statistical analysis; and
        (3) determining whether the likelihood of said long-term survival has increased or decreased.
    
    


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; α-Catenin; VDR; GRO1; KT14; KLK10; Maspin, TGFα, and FRP1, wherein expression of the following genes is indicative of a reduced likelihood of survival without cancer recurrence: CCND1; UPA; HNF3A; CDH1; Her2; GRB7; AKT1; STMY3; α-Catenin; VDR; GRO1, and wherein expression of the following genes is indicative of a better prognosis for survival without cancer recurrence: KT14; KLK10; Maspin, TGFα, 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, Chk1, AIB1, CCNB1, MCM2, FBXO5, Her2, STK15, SURV, EGFR, MYBL2, HIF1α, 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:

    
    
        (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, β-Catenin, γ-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.



BRIEF DESCRIPTION OF THE DRAWINGS
Table 1 is a list of genes, expression of which correlate with breast cancer survival. Results from a retrospective clinical trial. Binary statistical analysis.
Table 2 is a list of genes, expression of which correlates with breast cancer survival in estrogen receptor (ER) positive patients. Results from a retrospective clinical trial. Binary statistical analysis.
Table 3 is a list of genes, expression of which correlates with breast cancer survival in estrogen receptor (ER) negative patients. Results from a retrospective clinical trial. Binary statistical analysis.
Table 4 is a list of genes, expression of which correlates with breast cancer survival. Results from a retrospective clinical trial. Cox proportional hazards statistical analysis.
Tables 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 same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992), provide one skilled in the art with a general guide to many of the terms used in the present application.
One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.
The term “microarray” refers to an ordered arrangement of hybridizable array elements, preferably polynucleotide probes, on a substrate.
The term “polynucleotide,” when used in singular or plural, generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Thus, for instance, polynucleotides as defined herein include, without limitation, single- and double-stranded DNA, DNA including single- and double-stranded regions, single- and double-stranded RNA, and RNA including single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or include single- and double-stranded regions. In addition, the term “polynucleotide” as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or 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% Ficoll/0.1% polyvinylpyrrolidone/50 mM 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×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodium citrate) and 50% formamide at 55° C., followed by a high-stringency wash consisting of 0.1×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×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1×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. Calos, eds., 1987); “Current Protocols in Molecular Biology” (F. M. Ausubel et al., eds., 1987); and “PCR: The Polymerase Chain Reaction”, (Mullis et al., eds., 1994).
1. Gene Expression Profiling
In general, methods of gene expression profiling can be divided into two large groups: methods based on hybridization analysis of polynucleotides, and methods based on sequencing of polynucleotides. The most commonly used methods known in the art for the quantification of mRNA expression in a sample include northern blotting and in situ hybridization (Parker & Barnes, Methods in Molecular Biology 106:247-283 (1999)); RNAse protection assays (Hod, Biotechniques 13:852-854 (1992)); and reverse transcription polymerase chain reaction (RT-PCR) (Weis et al., Trends in Genetics 8:263-264 (1992)). Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Representative methods for sequencing-based gene expression analysis include Serial Analysis of Gene Expression (SAGE), and gene expression analysis by massively parallel signature sequencing (MPSS).
2. Reverse Transcriptase PCR (RT-PCR)
Of the techniques listed above, the most sensitive and most flexible quantitative method is RT-PCR, which can be used to compare mRNA levels in different sample populations, in normal and tumor tissues, with or without drug treatment, to characterize patterns of gene expression, to discriminate between closely related mRNAs, and to analyze RNA structure.
The first step is the isolation of mRNA from a target sample. The starting material is typically total RNA isolated from human tumors or tumor cell lines, and corresponding normal tissues or cell lines, respectively. Thus RNA can be isolated from a variety of primary tumors, including breast, lung, colon, prostate, brain, liver, kidney, pancreas, spleen, thymus, testis, ovary, uterus, etc., tumor, or tumor cell lines, with pooled DNA from healthy donors. If the source of mRNA is a primary tumor, mRNA can be extracted, for example, from frozen or archived paraffin-embedded and fixed (e.g. formalin-fixed) tissue samples.
General methods for mRNA extraction are well known in the art and are disclosed in standard textbooks of molecular biology, including Ausubel et al., Current Protocols of Molecular Biology, John Wiley and Sons (1997). Methods for RNA extraction from paraffin embedded tissues are disclosed, for example, in Rupp and Locker, Lab Invest. 56:A67 (1987), and De Andrés et al., BioTechniques 18:42044 (1995). In particular, RNA isolation can be performed using purification kit, buffer set and protease from commercial manufacturers, such as Qiagen, according to the manufacturer's instructions. For example, total RNA from cells in culture can be isolated using Qiagen RNeasy mini-columns. Other commercially available RNA isolation kits include MasterPureTM Complete DNA and RNA Purification Kit (EPICENTRE®, Madison, Wis.), and Paraffin Block RNA Isolation Kit (Ambion, Inc.). Total RNA from tissue samples can be isolated using RNA Stat-60 (Tel-Test). RNA prepared from tumor can be isolated, for example, by cesium chloride density gradient centrifugation.
As RNA cannot serve as a template for PCR, the first step in gene expression profiling by RT-PCR is the reverse transcription of the RNA template into cDNA, followed by its exponential amplification in a PCR reaction. The two most commonly used reverse transcriptases are avilo myeloblastosis virus reverse transcriptase (AMV-RT) and Moloney murine leukemia virus reverse transcriptase (MMLV-RT). The reverse transcription step is typically primed using specific primers, random hexamers, or oligo-dT primers, depending on the circumstances and the goal of expression profiling. For example, extracted RNA can be reverse-transcribed using a GeneAmp RNA PCR kit (Perkin Elmer, Calif., USA), following the manufacturer's instructions. The derived cDNA can then be used as a template in the subsequent PCR reaction.
Although the PCR step can use a variety of thermostable DNA-dependent DNA polymerases, it typically employs the Taq DNA polymerase, which has a 5′-3′ nuclease activity but lacks a 3′-5′ proofreading endonuclease activity. Thus, TaqMan® PCR typically utilizes the 5′-nuclease activity of Taq or Tth polymerase to hydrolyze a hybridization probe bound to its target amplicon, but any enzyme with equivalent 5′ nuclease activity can be used. Two oligonucleotide primers are used to generate an amplicon typical of a PCR reaction. A third oligonucleotide, or probe, is designed to detect nucleotide sequence located between the two PCR primers. The probe is non-extendible by Taq DNA polymerase enzyme, and is labeled with a reporter fluorescent dye and a quencher fluorescent dye. Any laser-induced emission from the reporter dye is quenched by the quenching dye when the two dyes are located close together as they are on the probe. During the amplification reaction, the Taq DNA polymerase enzyme cleaves the probe in a template-dependent manner. The resultant probe fragments disassociate in solution, and signal from the released reporter dye is free from the quenching effect of the second fluorophore. One molecule of reporter dye is liberated for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data.
TaqMan® RT-PCR can be performed using commercially available equipment, such as, for example, ABI PRISM 7700TM Sequence Detection SystemTM (Perkin-Elmer-Applied Biosystems, Foster City, Calif., USA), or Lightcycler (Roche Molecular Biochemicals, Mannheim, Germany). In a preferred embodiment, the 5′ nuclease procedure is run on a real-time quantitative PCR device such as the ABI PRISM 7700TM Sequence Detection SystemTM. The system consists of a thermocycler, laser, charge-coupled device (CCD), camera and computer. The system amplifies samples in a 96-well format on a thermocycler. During amplification, laser-induced fluorescent signal is collected in real-time through fiber optics cables for all 96 wells, and detected at the CCD. The system includes software for running the instrument and for analyzing the data.
5′-Nuclease assay data are initially expressed as Ct, or the threshold cycle. As discussed above, fluorescence values are recorded during every cycle and represent the amount of product amplified to that point in the amplification reaction. The point when the fluorescent signal is first recorded as statistically significant is the threshold cycle (Ct).
To minimize errors and the effect of sample-to-sample variation, RT-PCR is usually performed using an internal standard. The ideal internal standard is expressed at a constant level among different tissues, and is unaffected by the experimental treatment. RNAs most frequently used to normalize patterns of gene expression are mRNAs for the housekeeping genes glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and β-actin.
A more recent variation of the RT-PCR technique is the real time quantitative PCR, which measures PCR product accumulation through a dual-labeled fluorigenic probe (i.e., TaqMan® probe). Real time PCR is compatible both with quantitative competitive PCR, where internal competitor for each target sequence is used for normalization, and with quantitative comparative PCR using a normalization gene contained within the sample, or a housekeeping gene for RT-PCR. For further details see, e.g. Held et al., Genome Research 6:986-994 (1996).
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 μm 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, N.J., pp 365-386)
The most important factors considered in PCR primer design include primer length, melting temperature (Tm), and G/C content, specificity, complementary primer sequences, and 3′-end sequence. In general, optimal PCR primers are generally 17-30 bases in length, and contain about 20-80%, such as, for example, about 50-60% G+C bases. Tm's between 50 and 80° C., e.g. about 50 to 70° C. are typically preferred.
For further guidelines for PCR primer and probe design see, e.g. Dieffenbach, C. W. et al., “General Concepts for PCR Primer Design” in: PCR Primer, A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1995, pp. 133-155; Innis and Gelfand, “Optimization of PCRs” in: PCR Protocols, A Guide to Methods and Applications, CRC Press, London, 1994, pp. 5-11; and Plasterer, T. N. Primerselect: Primer and probe design. Methods Mol. Biol. 70:520-527 (1997), the entire disclosures of which are hereby expressly incorporated by reference.
3. Microarrays
Differential gene expression can also be identified, or confirmed using the microarray technique. Thus, the expression profile of breast cancer-associated genes can be measured in either fresh or paraffin-embedded tumor tissue, using microarray technology. In this method, polynucleotide sequences of interest (including cDNAs and oligonucleotides) are plated, or arrayed, on a microchip substrate. The arrayed sequences are then hybridized with specific DNA probes from cells or tissues of interest. Just as in the RT-PCR method, the source of mRNA typically is total RNA isolated from human tumors or tumor cell lines, and corresponding normal tissues or cell lines. Thus RNA can be isolated from a variety of primary tumors or tumor cell lines. If the source of mRNA is a primary tumor, mRNA can be extracted, for example, from frozen or archived paraffin-embedded and fixed (e.g. formalin-fixed) tissue samples, which are routinely prepared and preserved in everyday clinical practice.
In a specific embodiment of the microarray technique, PCR amplified inserts of cDNA clones are applied to a substrate in a dense array. Preferably at least 10,000 nucleotide sequences are applied to the substrate. The microarrayed genes, immobilized on the microchip at 10,000 elements each, are suitable for hybridization under stringent conditions. Fluorescently labeled cDNA probes may be generated through incorporation of fluorescent nucleotides by reverse transcription of RNA extracted from tissues of interest. Labeled cDNA probes applied to the chip hybridize with specificity to each spot of DNA on the array. After stringent washing to remove non-specifically bound probes, the chip is scanned by confocal laser microscopy or by another detection method, such as a CCD camera. Quantitation of hybridization of each arrayed element allows for assessment of corresponding mRNA abundance. With dual color fluorescence, separately labeled cDNA probes generated from two sources of RNA are hybridized pairwise to the array. The relative abundance of the transcripts from the two sources corresponding to each specified gene is thus determined simultaneously. The miniaturized scale of the hybridization affords a convenient and rapid evaluation of the expression pattern for large numbers of genes. Such methods have been shown to have the sensitivity required to detect rare transcripts, which are expressed at a few copies per cell, and to reproducibly detect at least approximately two-fold differences in the expression levels (Schena et al., Proc. Natl. Acad. Sci. USA 93(2):106-149 (1996)). Microarray analysis can be performed by commercially available equipment, following manufacturer's protocols, such as by using the Affymetrix GenChip technology, or Incyte's microarray technology.
The development of microarray methods for large-scale analysis of gene expression makes it possible to search systematically for molecular markers of cancer classification and outcome prediction in a variety of tumor types.
4. Serial Analysis of Gene Expression (SAGE)
Serial analysis of gene expression (SAGE) is a method that allows the simultaneous and quantitative analysis of a large number of gene transcripts, without the need of providing an individual hybridization probe for each transcript. First, a short sequence tag (about 10-14 bp) is generated that contains sufficient information to uniquely identify a transcript, provided that the tag is obtained from a unique position within each transcript. Then, many transcripts are linked together to form long serial molecules, that can be sequenced, revealing the identity of the multiple tags simultaneously. The expression pattern of any population of transcripts can be quantitatively evaluated by determining the abundance of individual tags, and identifying the gene corresponding to each tag. For more details see, e.g. Velculescu et al., Science 270:484-487 (1995); and Velculescu et al., Cell 88:243-51 (1997).
5. MassARRAY Technology
The MassARRAY (Sequenom, San Diego, Calif.) technology is an automated, high-throughput method of gene expression analysis using mass spectrometry (MS) for detection. According to this method, following the isolation of RNA, reverse transcription and PCR amplification, the cDNAs are subjected to primer extension. The cDNA-derived primer extension products are purified, and dipensed on a chip array that is pre-loaded with the components needed for MALTI-TOF MS sample preparation. The various cDNAs present in the reaction are quantitated by analyzing the peak areas in the mass spectrum obtained.
6. Gene Expression Analysis by Massively Parallel Signature Sequencing (MPSS)
This method, described by Brenner et al., Nature Biotechnology 18:630-634 (2000), is a sequencing approach that combines non-gel-based signature sequencing with in vitro cloning of millions of templates on separate 5 μm diameter microbeads. First, a microbead library of DNA templates is constructed by in vitro cloning. This is followed by the assembly of a planar array of the template-containing microbeads in a flow cell at a high density (typically greater than 3×106 microbeads/cm2). The free ends of the cloned templates on each microbead are analyzed simultaneously, using a fluorescence-based signature sequencing method that does not require DNA fragment separation. This method has been shown to simultaneously and accurately provide, in a single operation, hundreds of thousands of gene signature sequences from a yeast cDNA library.
7. Immunohistochemistry
Immunohistochemistry methods are also suitable for detecting the expression levels of the prognostic markers of the present invention. Thus, antibodies or antisera, preferably polyclonal antisera, and most preferably monoclonal antibodies specific for each marker are used to detect expression. The antibodies can be detected by direct labeling of the antibodies themselves, for example, with radioactive labels, fluorescent labels, hapten labels such as, biotin, or an enzyme such as horse radish peroxidase or alkaline phosphatase. Alternatively, unlabeled primary antibody is used in conjunction with a labeled secondary antibody, comprising antisera, polyclonal antisera or a monoclonal antibody specific for the primary antibody. Immunohistochemistry protocols and kits are well known in the art and are commercially available.
8. Proteomics
The term “proteome” is defined as the totality of the proteins present in a sample (e.g. tissue, organism, or cell culture) at a certain point of time. Proteomics includes, among other things, study of the global changes of protein expression in a sample (also referred to as “expression proteomics”). Proteomics typically includes the following steps: (1) separation of individual proteins in a sample by 2-D gel electrophoresis (2-D PAGE); (2) identification of the individual proteins recovered from the gel, e.g. my mass spectrometry or N-terminal sequencing, and (3) analysis of the data using bioinformatics. Proteomics methods are valuable supplements to other methods of gene expression profiling, and can be used, alone or in combination with other methods, to detect the products of the prognostic markers of the present invention.
9. 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 μm 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 Cyp1. 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 II 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, paraffin-embedded tissue samples, and prepared for gene expression analysis as described in section 9 above.
Molecular assays of quantitative gene expression were performed by RT-PCR, using the ABI PRISM 7900TM Sequence Detection SystemTM (Perkin-Elmer-Applied Biosystems, Foster City, Calif., USA). ABI PRISM 7900TM consists of a thermocycler, laser, charge-coupled device (CCD), camera and computer. The system amplifies samples in a 384-well format on a thermocycler. During amplification, laser-induced fluorescent signal is collected in real-time through fiber optics cables for all 384 wells, and detected at the CCD. The system includes software for running the instrument and for analyzing the data.
Analysis and Results
Tumor tissue was analyzed for 185 cancer-related genes and 7 reference genes. The threshold cycle (CT) values for each patient were normalized based on the 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






















Bcl2
−0.15748
−1.22816
4.00034
75
0.000147
35
42


PR
−2.67225
−5.49747
3.61540
75
0.000541
35
42


IGF1R
−0.59390
−1.71506
3.49158
75
0.000808
35
42


BAG1
0.18844
−0.68509
3.42973
75
0.000985
35
42


CD68
−0.52275
0.10983
−3.41186
75
0.001043
35
42


EstR1
−0.35581
−3.00699
3.32190
75
0.001384
35
42


CTSL
−0.64894
−0.09204
−3.26781
75
0.001637
35
42


IGFBP2
−0.81181
−1.78398
3.24158
75
0.001774
35
42


GATA3
1.80525
0.57428
3.15608
75
0.002303
35
42


TP53BP2
−4.71118
−6.09289
3.02888
75
0.003365
35
42


EstR1
3.67801
1.64693
3.01073
75
0.003550
35
42


CEGP1
−2.02566
−4.25537
2.85620
75
0.005544
35
42


SURV
−3.67493
−2.96982
−2.70544
75
0.008439
35
42


p27
0.80789
0.28807
2.55401
75
0.012678
35
42


Chk1
−3.37981
−2.80389
−2.46979
75
0.015793
35
42


BBC3
−4.71789
−5.62957
2.46019
75
0.016189
35
42


ZNF217
1.10038
0.62730
2.42282
75
0.017814
35
42


EGFR
−2.88172
−2.20556
−2.34774
75
0.021527
35
42


CD9
1.29955
0.91025
2.31439
75
0.023386
35
42


MYBL2
−3.77489
−3.02193
−2.29042
75
0.024809
35
42


HIF1A
−0.44248
0.03740
−2.25950
75
0.026757
35
42


GRB7
−1.96063
−1.05007
−2.25801
75
0.026854
35
42


pS2
−1.00691
−3.13749
2.24070
75
0.028006
35
42


RIZ1
−7.62149
−8.38750
2.20226
75
0.030720
35
42


ErbB3
−6.89508
−7.44326
2.16127
75
0.033866
35
42


TOP2B
0.45122
0.12665
2.14616
75
0.035095
35
42


MDM2
1.09049
0.69001
2.10967
75
0.038223
35
42


PRAME
−6.40074
−7.70424
2.08126
75
0.040823
35
42


GUS
−1.51683
−1.89280
2.05200
75
0.043661
35
42


RAD51C
−5.85618
−6.71334
2.04575
75
0.044288
35
42


AIB1
−3.08217
−2.28784
−2.00600
75
0.048462
35
42


STK15
−3.11307
−2.59454
−2.00321
75
0.048768
35
42


GAPDH
−0.35829
−0.02292
−1.94326
75
0.055737
35
42


FHIT
−3.00431
−3.67175
1.86927
75
0.065489
35
42


KRT19
2.52397
2.01694
1.85741
75
0.067179
35
42


TS
−2.83607
−2.29048
−1.83712
75
0.070153
35
42


GSTM1
−3.69140
−4.38623
1.83397
75
0.070625
35
42


G-
0.31875
−0.15524
1.80823
75
0.074580
35
42


Catenin


AKT2
0.78858
0.46703
1.79276
75
0.077043
35
42


CCNB1
−4.26197
−3.51628
−1.78803
75
0.077810
35
42


PI3KC2A
−2.27401
−2.70265
1.76748
75
0.081215
35
42


FBXO5
−4.72107
−4.24411
−1.75935
75
0.082596
35
42


DR5
−5.80850
−6.55501
1.74345
75
0.085353
35
42


CIAP1
−2.81825
−3.09921
1.72480
75
0.088683
35
42


MCM2
−2.87541
−2.50683
−1.72061
75
0.089445
35
42


CCND1
1.30995
0.80905
1.68794
75
0.095578
35
42


EIF4E
−5.37657
−6.47156
1.68169
75
0.096788
35
42









In the foregoing Table 1, negative t-values indicate higher expression, associated with worse outcomes, and, inversely, higher (positive) t-values indicate higher expression associated with better outcomes. Thus, for example, elevated expression of the CD68 gene (t-value=−3.41, CT mean alive<CT mean deceased) indicates a reduced likelihood of disease free survival. Similarly, elevated expression of the 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, Chk1, Her2, STK15, AIB1, SURV, EGFR, MYBL2, HIF1α.
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, β Catenin, GSTM1, FHIT, Riz1, IGF1, BBC3, IGFR1, TBP, p27, IRS1, IGF1R, GATA3, CEGP1, ZNF217, CD9, pS2, ErbB3, TOP2B, MDM2, RAD51, and KRT19.
Analysis of ER Positive Patients by Binary 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
30
27


Bcl2
0.15345
−0.70480
3.55488
55
0.000786
30
27


CD68
−0.54779
0.19427
−3.41818
55
0.001193
30
27


HNF3A
0.39617
−0.63802
3.20750
55
0.002233
30
27


CTSL
−0.66726
0.00354
−3.20692
55
0.002237
30
27


TP53BP2
−4.81858
−6.44425
3.13698
55
0.002741
30
27


GATA3
2.33386
1.40803
3.02958
55
0.003727
30
27


BBC3
−4.54979
−5.72333
2.91943
55
0.005074
30
27


RAD51C
−5.63363
−6.94841
2.85475
55
0.006063
30
27


BAG1
0.31087
−0.50669
2.61524
55
0.011485
30
27


IGFBP2
−0.49300
−1.30983
2.59121
55
0.012222
30
27


FBXO5
−4.86333
−4.05564
−2.56325
55
0.013135
30
27


EstR1
0.68368
−0.66555
2.56090
55
0.013214
30
27


PR
−1.89094
−3.86602
2.52803
55
0.014372
30
27


SURV
−3.87857
−3.10970
−2.49622
55
0.015579
30
27


CD9
1.41691
0.91725
2.43043
55
0.018370
30
27


RB1
−2.51662
−2.97419
2.41221
55
0.019219
30
27


EPHX1
−3.91703
−5.85097
2.29491
55
0.025578
30
27


CEGP1
−1.18600
−2.95139
2.26608
55
0.027403
30
27


CCNB1
−4.44522
−3.35763
−2.25148
55
0.028370
30
27


TRAIL
0.34893
−0.56574
2.20372
55
0.031749
30
27


EstR1
4.60346
3.60340
2.20223
55
0.031860
30
27


DR5
−5.71827
−6.79088
2.14548
55
0.036345
30
27


MCM2
−2.96800
−2.48458
−2.10518
55
0.039857
30
27


Chk1
−3.46968
−2.85708
−2.08597
55
0.041633
30
27


p27
0.94714
0.49656
2.04313
55
0.045843
30
27


MYBL2
−3.97810
−3.14837
−2.02921
55
0.047288
30
27


GUS
−1.42486
−1.82900
1.99758
55
0.050718
30
27


P53
−1.08810
−1.47193
1.92087
55
0.059938
30
27


HIF1A
−0.40925
0.11688
−1.91278
55
0.060989
30
27


cMet
−6.36835
−5.58479
−1.88318
55
0.064969
30
27


EGFR
−2.95785
−2.28105
−1.86840
55
0.067036
30
27


MTA1
−7.55365
−8.13656
1.81479
55
0.075011
30
27


RIZ1
−7.52785
−8.25903
1.79518
55
0.078119
30
27


ErbB3
−6.62488
−7.10826
1.79255
55
0.078545
30
27


TOP2B
0.54974
0.27531
1.74888
55
0.085891
30
27


EIF4E
−5.06603
−6.31426
1.68030
55
0.098571
30
27


TS
−2.95042
−2.36167
−1.67324
55
0.099959
30
27


STK15
−3.25010
−2.72118
−1.64822
55
0.105010
30
27









For each gene, a classification 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; FBXO5; SURV; CCNB1; MCM2; Chk1; MYBL2; HIF1A; cMET; EGFR; TS; STK15. Many of these genes (CD68, CTSL, SURV, CCNB1, MCM2, Chk1, 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; EIF4E. 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) <0 (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 to 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 <.118. The first column of mean expression values pertains to patients who neither had a metastatic recurrence nor died from breast cancer. The second column of mean expression values pertains to patients who either had a metastatic recurrence of or died from breast cancer.














TABLE 3






Mean
Mean
t-value
df
p
Valid N
Valid N






















KRT14
−1.95323
−6.69231
4.03303
18
0.000780
5
15


KLK10
−2.68043
−7.11288
3.10321
18
0.006136
5
15


CCND1
−1.02285
0.03732
−2.77992
18
0.012357
5
15


Upa
−0.91272
−0.04773
−2.49460
18
0.022560
5
15


HNF3A
−6.04780
−2.36469
−2.43148
18
0.025707
5
15


Maspin
−3.56145
−6.18678
2.40169
18
0.027332
5
15


CDH1
−3.54450
−2.34984
−2.38755
18
0.028136
5
15


HER2
−1.48973
1.53108
−2.35826
18
0.029873
5
15


GRB7
−2.55289
0.00036
−2.32890
18
0.031714
5
15


AKT1
−0.36849
0.46222
−2.29737
18
0.033807
5
15


TGFA
−4.03137
−5.67225
2.28546
18
0.034632
5
15


FRP1
1.45776
−1.39459
2.27884
18
0.035097
5
15


STMY3
−1.59610
−0.26305
−2.23191
18
0.038570
5
15


Contig
−4.27585
−7.34338
2.18700
18
0.042187
5
15


27882


A-Catenin
−1.19790
−0.39085
−2.15624
18
0.044840
5
15


VDR
−4.37823
−2.37167
−2.15620
18
0.044844
5
15


GRO1
−3.65034
−5.97002
2.12286
18
0.047893
5
15


MCM3
−3.86041
−5.55078
2.10030
18
0.050061
5
15


B-actin
4.69672
5.19190
−2.04951
18
0.055273
5
15


HIF1A
−0.64183
−0.10566
−2.02301
18
0.058183
5
15


MMP9
−8.90613
−7.35163
−1.88747
18
0.075329
5
15


VEGF
0.37904
1.10778
−1.87451
18
0.077183
5
15


PRAME
−4.95855
−7.41973
1.86668
18
0.078322
5
15


AIB1
−3.12245
−1.92934
−1.86324
18
0.078829
5
15


KRT5
−1.32418
−3.62027
1.85919
18
0.079428
5
15


KRT18
1.08383
2.25369
−1.83831
18
0.082577
5
15


KRT17
−0.69073
−3.56536
1.78449
18
0.091209
5
15


P14ARF
−1.87104
−3.36534
1.63923
18
0.118525
5
15









Based on the data set forth in Table 3, expression of the following genes in ER-negative cancer above a defined expression level is indicative of a reduced likelihood of survival without cancer recurrence (p<0.05): CCND1; UPA; HNF3A; CDH1; Her2; GRB7; AKT1; STMY3; α-Catenin; VDR; GRO1. Only 2 of these genes (Her2 and Grb7) were also identified as indicators of poor prognosis in the previous analysis, not limited to ER-negative breast cancer. Based on the data set forth in Table 3, expression of the following genes in ER-negative cancer above a defined expression level is indicative of a better prognosis for survival without cancer recurrence (KT14; KLK10; Maspin, TGFα, 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)
z
p




















TP53BP2
−0.21892
0.803386
0.068279
−3.20625
0.00134


GRB7
0.235697
1.265791
0.073541
3.204992
0.00135


PR
−0.10258
0.90251
0.035864
−2.86018
0.00423


CD68
0.465623
1.593006
0.167785
2.775115
0.00552


Bcl2
−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
0.87358
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)×Ct(geneA)+coef(geneB)×Ct(geneB)+coef(geneC)×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 ΔCts, 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 ΔCts 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, PDGFRβ, 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, ID1, PPM1D, MCM6, and WISP1.
    (e) Bc12, TP53BP2, BAD, EPHX1, PDGFRβ, DIABLO, XIAP, YB1, CA9, and KRT8.
    (f) KRT14, KRT5, PRAME, TP53BP2, GUS1, AIB1, 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) Chk1, PRAME, p53BP2, GRB7, CA9, CTSL, CCNB1, TOP2A, tumor size, and IGFBP2.
    (l) IGFBP2, GRB7, PRAME, DIABLO, CTSL, β-Catenin, PPM1D, Chk1, WISP1, and LOT1.
    (m) 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) AIB1, 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) HIF1α, PR, DIABLO, PRAME, Chk1, AKT2, GRB7, CCNE1, TOP2A, and CCNB1.
    (y) MDM2, TP53BP2, DIABLO, Bc12, AIB1, 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 ID1.
    (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, TIMP1, AIB1, CA9, p53, KRT8, and BAD.
    (ae) ZNF217, GRB7, p53BP2, PRAME, DIABLO, Bc12, COX2, CCNE1, APC4, and β-Catenin.


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










TABLE 5A








SEQ


Gene
Accession
Seq
ID NO:


















AIB1
NM_006534
GCGGCGAGTTTCCGATTTAAAGCTGAGCTGCGAGGAAAATGGCGGCGGGAGGATCAAAATACTTGCTG-
1




GATGGTGGACTCA


AKT1
NM_005163
CGCTTCTATGGCGCTGAGATTGTGTCAGCCCTGGACTACCTGCACTCGGAGAAGAACGTGGTGTACCG-
2




GGA


AKT2
NM_001626
TCCTGCCACCCTTCAAACCTCAGGTCACGTCCGAGGTCGACACAAGGTACTTCGATGATGAATTTACCG-
3




CC


APC
NM_000038
GGACAGCAGGAATGTGTTTCTCCATACAGGTCACGGGGAGCCAATGGTTCAGAAACAAATCGAGTGGG-
4




T


AREG
NM_001657
TGTGAGTGAAATGCCTTCTAGTAGTGAACCGTCCTCGGGAGCCGACTATGACTACTCAGAAGAGTATG-
5




ATPACGAACCACAA


B-actin
NM_001101
CAGCAGATGTGGATCAGCAAGCAGGAGTATGACGAGTCCGGCCCCTCCATCGTCCACCGCAAATGC
6


B-Catenin
NM_001904
GGCTCTTGTGCGTACTGTCCTTCGGGCTGGTGACAGGGAAGACATCACTGAGCCTGCCATCTGTGCTCT-
7




TCGTCATCTGA


BAD
NM_032989
GGGTCAGGTGCCTCGAGATCGGGCTTGGGCCCAGAGCATGTTCCAGATCCCAGAGTTTGAGCCGAGTG-
8




AGCAG


BAG1
NM_004323
CGTTGTCAGCACTTGGAATACAAGATGGTTGCCGGGTCATGTTAATTGGGAAAAAGAACAGTCCACAG-
9




GAAGAGGTTGAAC


BBC3
NM_014417
CCTGGAGGGTCCTGTACAATCTCATCATGGGAGTCCTGCCCTTACCCAGGGGCCACAGAGCCCCCGAG-
10




ATGGAGCCCAATTAG


Bcl2
NM_000633
CAGATGGACCTAGTACCCACTGAGATTTCCACGGCGAAGGACAGCGATGGGAAAAATGCCCTTAAATC-
11




ATAGG


CA9
NM_001216
ATCCTAGCCCTGGTTTTTGGCCTCCTTTTTGCTGTCACCAGCGTCGCGTTCCTTGTGCAGAAAGGCAG
12


CCNB1
NM_031966
TTCAGGTTGTTGCAGGAGACCATGTACATGACTGTCTCCATTATTGATCGGTTCATGCAGAATAATTGT-
13




GTGCCCAAGAAGATG


CCND1
NM_001758
GCATGTTCGTGGCCTCTAAGATGAAGGAGACCATCCCCCTGACGGCCGAGAAGCTGTGCATCTACACCG
14


CCNE1
NM_001238
AAAGAAGATGATGACCGGGTTTACCCAAACTCAACGTGCAAGCCTCGGATTATTGCACCATCCAGAGG-
15




CTC


CCNE2
NM_057749
ATGCTGTGGCTCCTTCCTAACTGGGGCTTTCTTGACATGTAGGTTGCTTGGTAATAACCTTTTTGTATAT-
16




CACAATTTGGGT


CD3z
NM_000734
AGATGAAGTGGAAGGCGCTTTTCACCGCGGCCATCCTGCAGGCACAGTTGCCGATTACAGAGGCA
17


CD68
NM_001251
TGGTTCCCAGCCCTGTGTCCACCTCCAAGCCCAGATTCAGATTCGAGTCATGTACACAACCCAGGGTGG-
18




AGGAG


CD9
NM_001769
GGGCGTGGAACAGTTTATCTCAGACATCTGCCCCAAGAAGGACGTACTCGAAACCTTCACGTG
19


CDH1
NM_004360
TGAGTGTCCCCCGGTATCTTCCCCGCCCTGCCAATCCCGATGAAATTGGAAATTTTATTGATGAAAATC-
20




TGAAAGCGGCTG


CEGP1
NM_020974
TGACAATCAGCACACCTGCATTCACCGCTCGGAAGAGGGCCTGAGCTGCATGAATAAGGATCACGGCT-
21




GTAGTCACA


Chk1
NM_001274
GATAAATTGGTACAAGGGATCAGCTTTTCCCAGCCCACATGTCCTGATCATATGCTTTTGAATAGTCAG-
22




TTACTTGGCACCC


CIAP1
NM_001166
TGCCTGTGGTGGGAAGCTCAGTAACTGGGAACCAAAGGATGATGCTATGTCAGAACACCGGAGGCATT-
23




TTCC


cIAP2
NM_001165
GGATATTTCCGTGGCTCTTATTCAAACTCTCCATCAAATCCTGTAAACTCCAGAGCAAATCAAGATTTT-
24




TCTGCCTTGATGAGAAG


cMet
NM_000245
GACATTTCCAGTCCTGCAGTCAATGCCTCTCTGCCCCACCCTTTGTTCAGTGTGGCTGGTGCCACGACA-
25




AATGTGTGCGATCGGAG


Contig
AK000618
GGCATCCTGGCCCAAAGTTTCCCAAATCCAGGCGGCTAGAGGCCCACTGCTTCCCAACTACCAGCGAG-
26


27882

GGGGTC


COX2
NM_000963
TCTGCAGAGTTGGAAGCACTCTATGGTGACATCGATGCTGTGGAGCTGTATCCTGCCCTTCTGGTAGAA-
27




AAGCCTCGGC


CTSL
NM_001912
GGGAGGCTTATCTCACTGAGTGAGCAGAATCTGGTAGACTGCTCTGGGCCTCAAGGCAATGAAGGCTG-
28




CAATGG


CTSL2
NM_001333
TGTCTCACTGAGCGAGCAGAATCTGGTGGACTGTTCGCGTCCTCAAGGCAATCAGGGCTGCAATGGT
29


DAPK1
NM_004938
CGCTGACATCATGAATGTTCCTCGACCGGCTGGAGGCGAGTTTGGATATGACAAAGACACATCGTTGCT-
30




GAAAGAGA


DIABLO
NM_019887
CACAATGGCGGCTCTGAAGAGTTGGCTGTCGCGCAGCGTAACTTCATTCTTCAGGTACAGACAGTGTTT-
31




GTGT



















TABLE 5B








SEQ ID


Gene
Accession
Seq
NO:


















DR5
NM_003842
CTCTGAGACAGTGCTTCGATGACTTTGCAGACTTGGTGCCCTTTGACTCCTGGGAGCCGCTCATGAGGA-
32




AGTTGGGCCTCATGG


EGFR
NM_005228
TGTCGATGGACTTCCAGAACCACCTGGGCAGCTGCCAAAAGTGTGATCCAAGCTGTCCCAAT
33


EIF4E
NM_001968
GATCTAAGATGGCGACTGTCGAACCGGAAACCACCCCTACTCCTAATCCCCCGACTACAGAAGAGGAG-
34




AAAACGGAATCTAA


EMS1
NM_005231
GGCAGTGTCACTGAGTCCTTGAAATCCTCCCCTGCCCCGCGGGTCTCTGGATTGGGACGCACAGTGCA
35


EpCAM
NM_002354
GGGCCCTCCAGAACAATGATGGGCTTTATGATCCTGACTGCGATGAGAGCGGGCTCTTTAAGGCCAAG-
36




CAGTGCA


EPHX1
NM_000120
ACCGTAGGCTCTGCTCTGAATGACTCTCCTGTGGGTCTGGCTGCCTATATTCTAGAGAAGTTTTCCACC-
37




TGGACCA


ErbB3
NM_001982
CGGTTATGTCATGCCAGATACACACCTCAAAGGTACTCCCTCCTCCCGGGAGGCACCCTTTCTTCAGTG-
38




GGTCTCAGTTC


EstR1
NM_000125
CGTGGTGCCCCTCTATGACCTGCTGCTGGAGATGCTGGACGCCCACCGCCTACATGCGCCCACTAGCC
39


FBXO5
NM_012177
GGCTATTCCTCATTTTCTCTACAAAGTGGCCTCAGTGAACATGAAGAAGGTAGCCTCCTGGAGGAGAAT-
40




TTCGGTGACAGTCTAGAATCC


FGF18
NM_003862
CGGTAGTCAAGTCCGGATCAAGGGCAAGGAGACGGAATTCTACCTGTGCATGAACCGCAAAGGCAAGC
41


FGFR1
NM_023109
CACGGGACATTCACCACATCGACTACTATAAAAAGACAACCAACGGCCGACTGCCTGTGAAGTGGATG-
42




GCACCC


FHIT
NM_002012
CCAGTGGAGCGCTTCCATGACCTGCGTCCTGATGAAGTGGCCGATTTGTTTCAGACGACCCAGAGAG
43


FRP1
NM_003012
TTGGTACCTGTGGGTTAGCATCAAGTTCTCCCCAGGGTAGAATTCAATCAGAGCTCCAGTTTGCATTTG-
44




GATGTG


G-Catenin
NM_002230
TCAGCAGCAAGGGCATCATGGAGGAGGATGAGGCCTGCGGGCGCCAGTACACGCTCAAGAAAACCACC
45


GAPDH
NM_002046
ATTCCACCCATGGCAAATTCCATGGCACCGTCAAGGCTGAGAACGGGAAGCTTGTCATCAATGGAAAT-
46




CCCATC


GATA3
NM_002051
CAAAGGAGCTCACTGTGGTGTCTGTGTTCCAACCACTGAATCTGGACCCCATCTGTGAATAAGCCATTC-
47




TGACTC


GRB7
NM_005310
CCATCTGCATCCATCTTGTTTGGGCTCCCCACCCTTGAGAAGTGCCTCAGATAATACCCTGGTGGCC
48


GRO1
NM_001511
CGAAAAGATGCTGAACAGTGACAAATCCAACTGACCAGAAGGGAGGAGGAAGCTCACTGGTGGCTGT-
49




TCCTGA


GSTM1
NM_000561
AAGCTATGAGGAAAAGAAGTACACGATGGGGGACGCTCCTGATTATGACAGAAGCCAGTGGCTGAAT-
50




GAAAAATTCAAGCTGGGCC


GUS
NM_000181
CCCACTCAGTAGCCAAGTCACAATGTTTGGAAAACAGCCCGTTTACTTGAGCAAGACTGATACCACCT-
51




GCGTG


HER2
NM_004448
CGGTGTGAGAAGTGCAGCAAGCCCTGTGCCCGAGTGTGCTATGGTCTGGGCATGGAGCACTTGCGAGA-
52




GG


HIF1A
NM_001530
TGAACATAAAGTCTGCAACATGGAAGGTATTGCACTGCACAGGCCACATTCACGTATATGATACCAAC-
53




AGTAACCACCTCA


HNF3A
NM_004496
TCCAGGATGTTAGGAACTGTGAAGATGGAAGGGCATGAAACCAGCGACTGGAACAGCTACTACGCAG-
54




ACACGC


ID1
NM_002165
AGAACCGCAAGGTGAGCAAGGTGGAGATTCTCCAGCACGTCATCGACTACATCAGGGACCTTCAGTTG-
55




GA


IGF1
NM_000618
TCCGGAGCTGTGATCTAAGGAGGCTGGAGATGTATTGCGCAGCCCTCAAGCCTGCCAAGTCAGCTCGC-
56




TCTGTCCG


IGF1R
NM_000875
GCATGGTAGCCGAAGATTTCACAGTCAAAATCGGAGATTTTGGTATGACGCGAGATATCTATGAGACA-
57




GACTATTACCGGAAA


IGFBP2
NM_000597
GTGGACAGCACCATGAACATGTTGGGCGGGGGAGGCAGTGCTGGCCGGAAGCCCCTCAAGTCGGGTAT-
58




GAAGG


IL6
NM_000600
CCTGAACCTTCCAAAGATGGCTGAAAAAGATGGATGCTTCCAATCTGGATTCAATGAGGAGACTTGCC-
59




TGGT


IRS1
NM_005544
CCACAGCTCACCTTCTGTCAGGTGTCCATCCCAGCTCCAGCCAGCTCCCAGAGAGGAAGAGACTGGCA-
60




CTGAGG


Ki-67
NM_002417
CGGACTTTGGGTGCGACTTGACGAGCGGTGGTTCGACAAGTGGCCTTGCGGGCCGGATCGTCCCAGTG-
61




GAAGAGTTGTAA


KLK10
NM_002776
GCCCAGAGGCTCCATCGTCCATCCTCTTCCTCCCCAGTCGGCTGAACTCTCCCCTTGTCTGCACTGTTCA-
62




AACCTCTG



















TABLE 5C








SEQ ID


Gene
Accession
Seq
NO:







KRT14
NM_000526
GGCCTGCTGAGATCAAAGACTACAGTCCCTACTTCAAGACCATTGAGGACCTGAGGAACAAGATTCTC-
63




ACAGCCACAGTGGAC


KRT17
NM_000422
CGAGGATTGGTTCTTGAGCAAGACAGAGGAACTGAACCGCGAGGTGGCCACCAACAGTGAGCTGGTGC-
64




AGAGT


KRT18
NM_000224
AGAGATCGAGGCTCTCAAGGAGGAGCTGCTCTTCATGAAGAAGAACCACGAAGAGGAAGTAAAAGGCC
65


KRT19
NM_002276
TGAGCGGCAGAATCAGGAGTACCAGCGGCTCATGGACATCAAGTCGCGGCTGGAGCAGGAGATTGCC-
66




ACCTACCGCA


KRT5
NM_000424
TCAGTGGAGAAGGAGTTGGACCAGTCAACATCTCTGTTGTCACAAGCAGTGTTTCCTCTGGATATGGCA
67


KRT8
NM_002273
GGATGAAGCTTACATGAACAAGGTAGAGCTGGAGTCTCGCCTGGAAGGGCTGACCGACGAGATCAACT-
68




TCCTCAGGCAGCTATATG


LOT1
NM_002656
GGAAAGACCACCTGAAAAACCACCTCCAGACCCACGACCCCAACAAAATGGCCTTTGGGTGTGAGGAG-
69


variant 1

TGTGGGAAGAAGTAC


Maspin
NM_002639
CAGATGGCCACTTTGAGAACATTTGAGAACATTTTAGCTGACAACAGTGTGAACGACCAGACCAAAAT-
70




CCTTGTGGTTAATGCTGCC


MCM2
NM_004526
GACTTTTGCCCGCTACCTTTCATTCCGGCGTGACAACAATGAGCTGTTGCTCTTCATACTGAAGCAGTT-
71




ACTGGC


MCM3
NM_002388
GGAGAACAATCCCCTTGAGACAGAATATGGCCTTTCTGTCTACAAGGATCACCAGACCATCACCATCC-
72




AGGAGAT


MCM6
NM_005915
TGATGGTCCTATGTGTCACATTCATCACAGGTTTCATACCAACACAGGCTTCAGCACTTCCTTTGGTGTG-
73




TTTCCTGTCCCA


MDM2
NM_002392
CTACAGGGACAGCCATCAGAATCCGGATCTTGATGCTGGTGTAAGTGAACATTCAGGTGATTGGTTGG-
74




AT


MMP9
NM_004994
GAGAACCAATCTCACCGACAGGCAGCTGGCAGAGGAATACCTGTACCGCTATGGTTACACTCGGGTG
75


MTA1
NM_004689
CCGCCCTCACCTGAAGAGAAACGCGCTCCTTGGCGGACACTGGGGGAGGAGAGGAAGAAGCGCGGCT-
76




AACTTATTCC


MYBL2
NM_002466
GCCGAGATCGCCAAGATGTTGCCAAGATGTTGCCAGGGAGGACAGACAATGCTGTGAAGAATCACTGG-
77




AACTCTACCATCAAAAG


P14ARF
S78535
CCCTCGTGCTGATGCTACTGAGGAGCCAGCGTCTAGGGCAGCAGCCGCTTCCTAGAAGACCAGGTCAT-
78




GATG


p27
NM_004064
CGGTGGACCACGAAGAGTTAACCCGGGACTTGGAGAAGCACTGCAGAGACATGGAAGAGGCGAGCC
79


P53
NM_000546
CTTTGAACCCTTGCTTGCAATAGGTGTGCGTCAGAAGCACCCAGGACTTCCATTTGCTTTGTCCCGGG
80


PAI1
NM_000602
CCGCAACGTGGTTTTCTCACCCTATGGGGTGGCCTCGGTGTTGGCCATGCTCCAGCTGACAACAGGAGG-
81




AGAAACCCAGCA


PDGFRb
NM_002609
CCAGCTCTCCTTCCAGCTACAGATCAATGTCCCTGTCCGAGTGCTGGAGCTAAGTGAGAGCCACCC
82


PI3KC2A
NM_002645
ATACCAATCACCGCACAAACCCAGGCTATTTGTTAAGTCCAGTCACAGCGCAAAGAAACATATGCGGA-
83




GAAAATGCTAGTGTG


PPM1D
NM_003620
GCCATCCGCAAAGGCTTTCTCGCTTGTCACCTTGCCATGTGGAAGAAACTGGCGGAATGGCC
84


PR
NM_000926
GCATCAGGCTGTCATTATGGTGTCCTTACCTGTGGGAGCTGTAAGGTCTTCTTTAAGAGGGCAATGGAA-
85




GGGCAGCACAACTACT


PRAME
NM_006115
TCTCCATATCTGCCTTGCAGAGTCTCCTGCAGCACCTCATCGGGCTGAGCAATCTGACCCACGTGC
86


pS2
NM_003225
GCCCTCCCAGTGTGCAAATAAGGGCTGCTGTTTCGACGACACCGTTCGTGGGGTCCCCTGGTGCTTCTA-
87




TCCTAATACCATCGACG


RAD51C
NM_058216
GAACTTCTTGAGCAGGAGCATACCCAGGCTTCATAATCACCTTCTGTTCAGCACTAGAGTGATATTCTT-
88




GGGGGTGGA


RB1
NM_000321
CGAAGCCCTTACAAGTTTCCTAGTTCACCCTTACGGATTCCTGGAGGGAACATCTATATTTCACCCCTG-
89




AAGAGTCC


RIZ1
NM_012231
CCAGACGAGCGATTAGAAGCGGCAGCTTGTGAGGTGAATGATTTGGGGGAAGAGGAGGAGGAGGAAG-
90




AGGAGGA


STK15
NM_003600
CATCTTCCAGGAGGACCACTCTCTGTGGCACCCTGGACTACCTGCCCCCTGAAATGATTGAAGGTCGGA
91


STMY3
NM_005940
CCTGGAGGCTGCAACATACCTCAATCCTGTCCCAGGCCGGATCCTCCTGAAGCCCTTTTCGCAGCACTG-
92




CTATCCTCCAAAGCCATTGTA


SURV
NM_001168
TGTTTTGATTCCCGGGCTTACCAGGTGAGAAGTGAGGGAGGAAGAAGGCAGTGTCCCTTTTGCTAGAG-
93




CTGACAGCTTTG



















TABLE 5D








SEQ ID


Gene
Accession
Seq
NO:


















TBP
NM_003194
GCCCGAAACGCCGAATATAATCCCAAGCGGTTTGCTGCGGTAATCATGAGGATAAGAGACCACG
94


TGFA
NM_003236
GGTGTGCCACAGACCTTCCTACTTGGCCTGTAATCACCTGTGCAGCCTTTTGTGGGCCTTCAAAACTCT-
95




GTCAAGAACTCCGT


TIMP1
NM_003254
TCCCTGCGGTCCCAGATAGCCTGAATCCTGCCCGGAGTGGAACTGAAGCCTGCACAGTGTCCACCCTGT-
96




TCCCAC


TOP2A
NM_001067
AATCCAAGGGGGAGAGTGATGACTTCCATATGGACTTTGACTCAGCTGTGGCTCCTCGGGCAAAATCT-
97




GTAC


TOP2B
NM_001068
TGTGGACATCTTCCCCTCAGACTTCCCTACTGAGCCACCTTCTCTGCCACGAACCGGTCGGGCTAG
98


TP
NM_001953
CTATATGCAGCCAGAGATGTGACAGCCACCGTGGACAGCCTGCCACTCATCACAGCCTCCATTCTCAGT-
99




AAGAAACTCGTGG


TP53BP2
NM_005426
GGGCCAAATATTCAGAAGCTTTTATATCAGAGGACCACCATAGCGGCCATGGAGACCATCTCTGTCCC-
100




ATCATACCCATCC


TRAIL
NM_003810
CTTCACAGTGCTCCTGCAGTCTCTCTGTGTGGCTGTAACTTACGTGTACTTTACCAACGAGCTGAAGCA-
101




GAGATG


TS
NM_001071
GCCTCGGTGTGCCTTTCAACATCGCCAGCTACGCCCTGCTCACGTACATGATTGCGCACATCACG
102


upa
NM_002658
GTGGATGTGCCCTGAAGGACAAGCCAGGCGTCTACACGAGAGTCTCACACTTCTTACCCTGGATCCGC-
103




AG


VDR
NM_000376
GCCCTGGATTTCAGAAAGAGCCAAGTCTGGATCTGGGACCCTTTCCTTCCTTCCCTGGCTTGTAACT
104


VEGF
NM_003376
CTGCTGTCTTGGGTGCATTGGAGCCTTGCCTTGCTGCTCTACCTCCACCATGCCAAGTGGTCCCAGGCT-
105




GC


VEGFB
NM_003377
TGACGATGGCCTGGAGTGTGTGCCCACTGGGCAGCACCAAGTCCGGATGCAGATCCTCATGATCCGGT-
106




ACC


WISP1
NM_003882
AGAGGCATCCATGAACTTCACACTTGCGGGCTGCATCAGCACACGCTCCTATCAACCCAAGTACTGTGG-
107




AGTTTG


XIAP
NM_001167
GCAGTTGGAAGACACAGGAAAGTATCCCCAAATTGCAGATTTATCAACGGCTTTTATCTTGAAAATAGT-
108




GCCACGCA


YB-1
NM_004559
AGACTGTGGAGTTTGATGTTGTTGAAGGAGAAAAGGGTGCGGAGGCAGCAAATGTTACAGGTCCTGGT-
109




GGTGTTCC


ZNF217
NM_006526
ACCCAGTAGCAAGGAGAAGCCCACTCACTGCTCCGAGTGCGGCAAAGCTTTCAGAACCTACCACCAGC-
110




TG





















TABLE 6A










SEQ ID


Gene
Accession
Probe Name
Seq
Length
NO:







AIB1
NM_005534
S1994/A1B1.f3
GCGGCGAGTTTCCGATTTA
19
111


AIB1
NM_006534
S1995/AIB1.r3
TGAGTCCACCATCCAGCAAGT
21
112


AIB1
NM_006534
S5055/AIB1.p3
ATGGCGGCGGGAGGATCAAAA
21
113


AKT1
NM_005163
S0010/AKT1.f3
CGCTTCTATGGCGCTGAGAT
20
114


AKT1
NM_005163
S0012/AKT1.r3
TCCCGGTACACCACGTTCTT
20
115


AKT1
NM_005163
S4776/AKT1.p3
CAGCCCTGGACTACCTGCACTCGG
24
116


AKT2
NM_001626
S0828/AKT2.f3
TCCTGCCACCCTTCAAACC
19
117


AKT2
NM_001626
S0829/AKT2.r3
GGCGGTAAATTCATCATCGAA
21
118


AKT2
NM_001626
S4727/AKT2.p3
CAGGTCACGTCCGAGGTCGACACA
24
119


APC
NM_000038
S0022/APC.f4
GGACAGCAGGAATGTGTTTC
20
120


APC
NM_000038
S0024/APC.r4
ACCCACTCGATTTGTTTCTG
20
121


APC
NM_000038
S4888/APC.p4
CATTGGCTCCCCGTGACCTGTA
22
122


AREG
NM_001657
S0025/AREG.f2
TGTGAGTGAAATGCCTTCTAGTAGTGA
27
123


AREG
NM_001657
S0027/AREG.r2
TTGTGGTTCGTTATCATACTCTTCTGA
27
125


AREG
NM_001657
S4889/AREG.p2
CCGTCCTCGGGAGCCGACTATGA
23
124


B-actin
NM_001101
S0034/B-acti.f2
CAGCAGATGTGGATCAGCAAG
21
126


B-actin
NM_001101
S0036/B-acti.r2
GCATTTGCGGTGGACGAT
18
127


B-actin
NM_001101
S4730/B-acti.p2
AGGAGTATGACGAGTCCGGCCCC
23
128


B-Catenin
NM_001904
S2150/B-Cate.f3
GGCTCTTGTGCGTACTGTCCTT
22
129


B-Catenin
NM_001904
S2151/B-Cate.r3
TCAGATGACGAAGAGCACAGATG
23
130


B-Catenin
NM_001904
S5046/B-Cate.p3
AGGCTCAGTGATGTCTTCCCTGTCACCAG
29
131


BAD
NM_032989
S2011/BAD.f1
GGGTCAGGTGCCTCGAGAT
19
132


BAD
NM_032989
S2012/BAD.r1
CTGCTCACTCGGCTCAAACTC
21
133


BAD
NM_032989
S5058/BAD.p1
TGGGCCCAGAGCATGTTCCCAGATC
24
134


BAG1
NM_004323
S1386/BAG1.f2
CGTTGTCAGCACTTGGAATACAA
23
135


BAG1
NM_004323
S1387/BAG1.r2
GTTCAACCTCTTCCTGTGGACTGT
24
135


BAG1
NM_004323
S4731/BAG1.p2
CCCAATTAACATGACCCGGCAACCAT
26
137


BBC3
NM_014417
S1584/BBC3.f2
CCTGGAGGGTCCTGTACAAT
20
138


BBC3
NM_014417
S1585/BBC3.r2
CTAATTGGGCTCCATCTCG
19
139


BBC3
NM_014417
S4890/BBC3.p2
CATCATGGGACTCCTGCCCTTACC
24
140


Bcl2
NM_000633
S0043/Bcl2.f2
CAGATGGACCTAGTACCCACTGAGA
25
141


Bcl2
NM_000633
S0045/Bcl2.r2
CCTATGATTTAAGGGCATTTTTCC
24
143


Bcl2
NM_000633
S4732/Bcl2.p2
TTCCACGCCGAAGGACAGCGAT
22
142


CA9
NM_001216
S1398/CA9.f3
ATCCTAGCCCTGGTTTTTGG
20
144


CA9
NM_001216
S1399/CA9.r3
CTGCCTTCTCATCTGCACAA
20
145


CA9
NM_001216
S4938/CA9.p3
TTTGCTGTCACCAGCGTCGC
20
146


CCNB1
NM_031966
S1720/CCNB1.f2
TTCAGGTTGTTGCAGGAGAC
20
147


CCNB1
NM_031966
S1721/CCNB1.r2
CATCTTCTTGGGCACACAAT
20
148


CCNB1
NM_031966
S4733/CCNB1.p2
TGTCTCCATTATTGATCGGTTCATGCA
27
149


CCND1
NM_001758
S0058/CCND1.f3
GCATGTTCGTGGCCTCTAAGA
21
150


CCND1
NM_001758
S0060/CCND1.r3
CGGTGTAGATGCACAGCTTCTC
22
151


CCND1
NM_001758
S4986/CCND1.p3
AAGGAGACCATCCCCCTGACGGC
23
152


CCNE1
NM_001238
S1446/CCNE1.f1
AAAGAAGATGATGACCGGGTTTAC
24
153


CCNE1
NM_001238
S1447/CCNE1.r1
GAGCCTCTGGATGGTGCAAT
20
154


CCNE1
NM_001238
S4944/CCNE1.p1
CAACTCAACGTGCAAGCCTCGGA
24
155





















TABLE 6B










SEQ ID


Gene
Accession
Probe Name
Seq
Length
NO:




















CCNE2
NM_057749
S1458/CCNE2.f2
ATGCTGTGGCTCCTTCCTAACT
22
156


CCNE2
NM_057749
S1459/CCNE2.r2
ACCCAAATTGTGATATACAAAAAGGTT
27
157


CCNE2
NM_057749
S4945/CCNE2.p2
TACCAAGCAACCTACATGTCAAGAAAGCCC
30
158


CD3z
NM_000734
S0064/CD3z.f1
AGATGAAGTGGAAGGCGCTT
20
159


CD3z
NM_000734
S0066/CD3z.r1
TGCCTCTGTAATCGGCAACTG
21
161


CD3z
NM_000734
S4988/CD3z.p1
CACCGCGGCCATCCTGCA
18
160


CD68
NM_001251
S0067/CD68.f2
TGGTTCCCAGCCCTGTGT
18
162


CD68
NM_001251
S0069/CD68.r2
CTCCTCCACCCTGGGTTGT
19
164


CD68
NM_001251
S4734/CD68.p2
CTCCAAGCCCAGATTCAGATTCGAGTCA
28
163


CD9
NM_001769
S0686/CD9.f1
GGGCGTGGAACAGTTTATCT
20
165


CD9
NM_001769
S0687/CD9.r1
CACGGTGAAGGTTTCGAGT
19
166


CD9
NM_001769
S4792/CD9.p1
AGACATCTGCCCCAAGAAGGACGT
24
167


CDH1
NM_004360
S0073/CDH1.f3
TGAGTGTCCCCCGGTATCTTC
21
168


CDH1
NM_004360
S0075/CDH1.r3
CAGCCGCTTTCAGATTTTCAT
21
169


CDH1
NM_004360
S4990/CDH1.p3
TGCCAATCCCGATGAAATTGGAAATTT
27
170


CEGP1
NM_020974
S1494/CEGP1.f2
TGACAATCAGCACACCTGCAT
21
171


CEGP1
NM_020974
S1495/CEGP1.r2
TGTGACTACAGCCGTGATCCTTA
23
172


CEGP1
NM_020974
S4735/CEGP1.p2
CAGGCCCTCTTCCGAGCGGT
20
173


Chk1
NM_001274
S1422/Chk1.f2
GATAAATTGGTACAAGGGATCAGCTT
26
174


Chk1
NM_001274
S1423/Chk1.r2
GGGTGCCAAGTAACTGACTATTCA
24
175


Chk1
NM_001274
S4941/Chk1.p2
CCAGCCCACATGTCCTGATCATATGC
26
176


CIAP1
NM_001166
S0764/CIAP1.f2
TGCCTGTGGTGGGAAGCT
18
177


CIAP1
NM_001166
S0765/CIAP1.r2
GGAAAATGCCTCCGGTGTT
19
178


CIAP1
NM_001166
S4802/CIAP1.p2
TGACATAGCATCATCCTTTGGTTCCCAGTT
30
179


cIAP2
NM_001165
S0076/cIAP2.f2
GGATATTTCCGTGGCTCTTATTCA
24
180


cIAP2
NM_001165
S0078/cIAP2.r2
CTTCTCATCAAGGCAGAAAAATCTT
25
182


cIAP2
NM_001165
S4991/cIAP2.p2
TCTCCATCAAATCCTGTAAACTCCAGAGCA
30
181


cMet
NM_000245
S0082/cMet.f2
GACATTTCCAGTCCTGCAGTCA
22
183


cMet
NM_000245
S0084/cMet.r2
CTCCGATCGCACACATTTGT
20
185


cMet
NM_000245
S4993/cMet.p2
TGCCTCTCTGCCCCACCCTTTGT
23
184


Contig
AK000618
S2633/Contig.f3
GGCATCCTGGCCCAAAGT
18
186


27882


Contig
AK000618
S2634/Contig.r3
GACCCCCTCAGCTGGTAGTTG
21
187


27882


Contig
AK000618
S4977/Contig.p3
CCCAAATCCAGGCGGCTAGAGGC
23
188


27882


COX2
NM_000963
S0088/COX2.f1
TCTGCAGAGTTGGAAGCACTCTA
23
189


COX2
NM_000963
S0090/COX2.r1
GCCGAGGCTTTTCTACCAGAA
21
191


COX2
NM_000963
S4995/COX2.p1
CAGGATACAGCTCCACAGCATCGATGTC
28
190


CTSL
NM_001912
S1303/CTSL.f2
GGGAGGCTTATCTCACTGAGTGA
23
192


CTSL
NM_001912
S1304/CTSL.r2
CCATTGCAGCCTTCATTGC
19
193


CTSL
NM_001912
S4899/CTSL.p2
TTGAGGCCCAGAGCAGTCTACCAGATTCT
29
194


CTSL2
NM_001333
S4354/CTSL2.f1
TGTCTCACTGAGCGAGCAGAA
21
195


CTSL2
NM_001333
S4355/CTSL2.r1
ACCATTGCAGCCCTGATTG
19
196


CTSL2
NM_001333
S4356/CTSL2.p1
CTTGAGGACGCGAACAGTCCACCA
24
197





















TABLE 60










SEQ ID


Gene
Accession
Probe Name
Seq
Length
NO:







DAPK1
NM_004938
S1768/DAPK1.f3
CGCTGACATCATGAATGTTCCT
22
198


DAPK1
NM_004938
S1769/DAPK1.r3
TCTCTTTCAGCAACGATCTCTCTT
24
199


DAPK1
NM_004938
S4927/DAPK1.p3
TCATATCCAAACTCGCCTCCAGCCG
25
200


DIABLO
NM_019887
S0808/DIABLO.f1
CACAATGGCGGCTCTGAAG
19
201


DIABLO
NM_019887
S0809/DIABLO.r1
ACACAAACACTGTCTGTACCTGAAGA
26
202


DIABLO
NM_019887
S4813/DIABLO.p1
AAGTTACGCTGCGCGACAGCCAA
23
203


DR5
NM_003842
S2551/DR5.f2
CTCTGAGACAGTGCTTCGATGACT
24
204


DR5
NM_003842
S2552/DR5.r2
CCATGAGGCCCAACTTCCT
19
205


DR5
NM_003842
S4979/DR5.p2
CAGACTTGGTGCCCTTTGACTCC
23
208


EGFR
NM_005228
S0103/EGFR.f2
TGTCGATGGACTTCCAGAAC
20
207


EGFR
NM_005228
S0105/EGFR.r2
ATTGGGACAGCTTGGATCA
19
209


EGFR
NM_005228
S4999/EGFR.p2
CACCTGGGCAGCTGCCAA
18
208


EIF4E
NM_001968
S0106/EIF4E.f1
GATCTAAGATGGCGACTGTCGAA
23
210


EIF4E
NM_001968
S0108/EIF4E.r1
TTAGATTCCGTTTTCTCCTCTTCTG
25
211


EIF4E
NM_001968
S5000/EIF4E.p1
ACCACCCCTACTCCTAATCCCCCGACT
27
212


EMS1
NM_005231
S2663/EMS1.f1
GGCAGTGTCACTGAGTCCTTGA
22
213


EMS1
NM_005231
S2664/EMS1.r1
TGCACTGTGCGTCCCAAT
18
214


EMS1
NM_005231
S4956/EMS1.p1
ATCCTCCCCTGCCCCGCG
18
215


EpCAM
NM_002354
S1807/EpCAM.f1
GGGCCCTCCAGAACAATGAT
20
216


EpCAM
NM_002354
S1808/EpCAM.r1
TGCACTGCTTGGCCTTAAAGA
21
217


EpCAM
NM_002354
S4984/EpCAM.p1
CCGCTCTCATCGCAGTCAGGATCAT
25
218


EPHX1
NM_000120
S1865/EPHX1.f2
ACCGTAGGCTCTGCTCTGAA
20
219


EPHX1
NM_000120
S1866/EPHX1.r2
TCCTGGAGGTGGAAAACTTC
20
220


EPHX1
NM_000120
S4754/EPHX1.p2
AGGCAGCCAGACCCACAGGA
20
221


ErbB3
NM_001982
S0112/ErbB3.f1
CGGTTATGTCATGCCAGATACAC
23
222


ErbB3
NM_001982
S0114/ErbB3.r1
GAACTGAGACCCACTGAAGAAAGG
24
224


ErbB3
NM_001982
S5002/ErbB3.p1
CCTCAAAGGTACTCCCTCCTCCCGG
25
223


EstR1
NM_000125
S0115/EstR1.f1
CGTGGTGCCCCTCTATGAC
19
225


EstR1
NM_000125
S0117/EstR1.r1
GGCTAGTGGGCGCATGTAG
19
227


EstR1
NM_000125
S4737/EstR1.p1
CTGGAGATGCTGGACGCCC
19
226


FBXO5
NM_012177
S2017/FBXO5.r1
GGATTGTAGACTGTCACCGAAATTC
25
228


FBXO5
NM_012177
S2018/FBXO5.f1
GGCTATTCCTCATTTTCTCTACAAAGTG
28
229


FBXO5
NM_012177
S5061/FBXO5.p1
CCTCCAGGAGGCTACCTTCTTCATGTTCAC
30
230


FGF18
NM_003862
S1665/FGF18.f2
CGGTAGTCAAGTCCGGATCAA
21
231


FGF18
NM_003862
S1666/FGF18.r2
GCTTGCCTTTGCGGTTCA
18
232


FGF18
NM_003862
S4914/FGF18.p2
CAAGGAGACGGAATTCTACCTGTGC
25
233


FGFR1
NM_023109
S0818/FGFR1.f3
CACGGGACATTCACCACATC
20
234


FGFR1
NM_023109
S0819/FGFR1.r3
GGGTGCCATCCACTTCACA
19
235


FGFR1
NM_023109
S4816/FGFR1.p3
ATAAAAAGACAACCAACGGCCGACTGC
27
236


FHIT
NM_002012
S2443/FHIT.f1
CCAGTGGAGCGCTTCCAT
18
237


FHIT
NM_002012
S2444/FHIT.r1
CTCTCTGGGTCGTCTGAAACAA
22
238


FHIT
NM_002012
S2445/FHIT.p1
TCGGCCACTTCATCAGGACGCAG
23
239


FHIT
NM_002012
S4921/FHIT.p1
TCGGCCACTTCATCAGGACGCAG
23
239


FRP1
NM_003012
S1804/FRP1.f3
TTGGTACCTGTGGGTTAGCA
20
240


FRP1
NM_003012
S1805/FRP1.r3
CACATCCAAATGCAAACTGG
20
241





















TABLE 6D










SEQ ID


Gene
Accession
Probe Name
Seq
Length
NO:







FRP1
NM_003012
S4983/FRP1.p3
TCCCCAGGGTAGAATTCAATCAGAGC
26
242


G-Catenin
NM_002230
S2153/G-Cate.f1
TCAGCAGCAAGGGCATCAT
19
243


G-Catenin
NM_002230
S2154/G-Cate.r1
GGTGGTTTTCTTGAGCGTGTACT
23
244


G-Catenin
NM_002230
S5044/G-Cate.p1
CGCCCGCAGGCCTCATCCT
19
245


GAPDH
NM_002046
S0374/GAPDH.f1
ATTCCACCCATGGCAAATTC
20
246


GAPDH
NM_002046
S0375/GAPDH.r1
GATGGGATTTCCATTGATGACA
22
247


GAPDH
NM_002046
s4738/GAPDH.p1
CCGTTCTCAGCCTTGACGGTGC
22
248


GATA3
NM_002051
S0127/GATA3.f3
CAAAGGAGCTCACTGTGGTGTCT
23
249


GATA3
NM_002051
S0129/GATA3.r3
GAGTCAGAATGGCTTATTCACAGATG
26
251


GATA3
NM_002051
S5005/GATA3.p3
TGTTCCAACCACTGAATCTGGACC
24
250


GRB7
NM_005310
S0130/GRB7.f2
CCATCTGCATCCATCTTGTT
20
252


GRB7
NM_005310
S0132/GR87.r2
GGCCACCAGGGTATTATCTG
20
254


GRB7
NM_005310
S4726/GRB7.p2
CTCCCCACCCTTGAGAAGTGCCT
23
253


GR01
NM_001511
S0133/GRO1.f2
CGAAAAGATGCTGAACAGTGACA
23
255


GRO1
NM_001511
S0135/GRO1.r2
TCAGGAACAGCCACCAGTGA
20
256


GRO1
NM_001511
S5006/GRO1.p2
CTTCCTCCTCCCTTCTGGTCAGTTGGAT
28
257


GSTM1
NM_000561
S2026/GSTM1.r1
GGCCCAGCTTGAATTTTTCA
20
258


GSTM1
NM_000561
S2027/GSTM1.f1
AAGCTATGAGGAAAAGAAGTACACGAT
27
259


GSTM1
NM_000561
S4739/GSTM1.p1
TCAGCCACTGGCTTCTGTCATAATCAGGAG
30
260


Gus
NM_000181
S0139/GUS.f1
CCCACTCAGTAGCCAACTCA
20
261


GUS
NM_000181
S0141/GUS.r1
CACGCAGGTGGTATCAGTCT
20
263


GUS
NM_000181
S4740/GUS.p1
TCAAGTAAACGGGCTGTTTTCCAAACA
27
262


HER2
NM_004448
S0142/HER2.f3
CGGTGTGAGAAGTGCAGCAA
20
264


HER2
NM_004448
S0144/HER2.r3
CCTCTCGCAAGTGCTCCAT
19
266


HER2
NM_004448
S4729/HER2.p3
CCAGACCATAGCACACTCGGGCAC
24
265


HIF1A
NM_001530
S1207/HIF1A.f3
TGAACATAAAGTCTGCAACATGGA
24
267


HIF1A
NM_001530
S1208/HIF1A.r3
TGAGGTTGGTTACTGTTGGTATCATATA
28
268


HIF1A
NM_001530
S4753/HIF1A.p3
TTGCACTGCACAGGCCACATTCAC
24
269


HNF3A
NM_004496
S0148/HNF3A.f1
TCCAGGATGTTAGGAACTGTGAAG
24
270


HNF3A
NM_004496
S0150/HNF3A.r1
GCGTGTCTGCGTAGTAGCTGTT
22
271


HNF3A
NM_004496
S5008/HNF3A.p1
AGTCGCTGGTTTCATGCCCTTCCA
24
272


ID1
NM_002165
S0820/ID1.f1
AGAACCGCAAGGTGAGCAA
19
273


ID1
NM_002165
S0821/ID1.r1
TCCAACTGAAGGTCCCTGATG
21
274


ID1
NM_002165
S4832/ID1.p1
TGGAGATTCTCCAGCACGTCATCGAC
26
275


IGF1
NM_000618
S0154/IGF1.f2
TCCGGAGCTGTGATCTAAGGA
21
276


IGF1
NM_000618
S0156/IGF1.r2
CGGACAGAGCGAGCTGACTT
20
278


IGF1
NM_000618
S5010/IGF1.p2
TGTATTGCGCACCCCTCAAGCCTG
24
277


IGF1R
NM_000875
S1249/IGF1R.f3
GCATGGTAGCCGAAGATTTCA
21
279


IGF1R
NM_000875
S1250/IGF1R.r3
TTTCCGGTAATAGTCTGTCTCATAGATATC
30
280


IGF1R
NM_000875
S4895/IGF1R.p3
CGCGTCATACCAAAATCTCCGATTTTGA
28
281


IGFBP2
NM_000597
S1128/IGFBP2.f1
GTGGACAGCACCATGAACA
19
282


IGFBP2
NM_000597
S1129/IGFBP2.r1
CCTTCATACCCGACTTGAGG
20
283


IGFBP2
NM_000597
S4837/IGFBP2.p1
CTTCCGGCCAGCACTGCCTC
20
284


IL6
NM_000600
S0760/IL6.f3
CCTGAACCTTCCAAAGATGG
20
285





















TABLE 6E










SEQ ID


Gene
Accession
Probe Name
Seq
Length
NO:







IL6
NM_000600
S0761/IL6.r3
ACCAGGCAAGTCTCCTCATT
20
286


IL6
NM_000600
S4800/IL6.p3
CCAGATTGGAAGCATCCATCTTTTTCA
27
287


IRS1
NM_005544
S1943/IRS1.f3
CCACAGCTCACCTTCTGTCA
20
288


IRS1
NM_005544
S1944/IRS1.r3
CCTCAGTGCCAGTCTCTTCC
20
289


IRS1
NM_005544
S5050/IRS1.p3
TCCATCCCAGCTCCAGCCAG
20
290


Ki-67
NM_002417
S0436/Ki-67.f2
CGGACTTTGGGTGCGACTT
19
292


Ki-67
NM_002417
S0437/Ki-67.r2
TTACAACTCTTCCACTGGGACGAT
24
293


Ki-67
NM_002417
S4741/Ki-67.p2
CCACTTGTCGAACCACCGCTCGT
23
291


KLK10
NM_002776
S2624/KLK10.f3
GCCCAGAGGCTCCATCGT
18
294


KLK10
NM_002776
S2625/KLK10.r3
CAGAGGTTTGAACAGTGCAGACA
23
295


KLK10
NM_002776
S4978/KLK10.p3
CCTCTTCCTCCCCAGTCGGCTGA
23
296


KRT14
NM_000526
S1853/KRT14.f1
GGCCTGCTGAGATCAAAGAC
20
297


KRT14
NM_000526
S1854/KRT14.r1
GTCCACTGTGGCTGTGAGAA
20
298


KRT14
NM_000526
S5037/KRT14.p1
TGTTCCTCAGGTCCTCAATGGTCTTG
26
299


KRT17
NM_000422
S0172/KRT17.f2
CGAGGATTGGTTCTTCAGCAA
21
300


KRT17
NM_000422
S0174/KRT17.r2
ACTCTGCACCAGCTCACTGTTG
22
301


KRT17
NM_000422
S5013/KRT17.p2
CACCTCGCGGTTCAGTTCCTCTGT
24
302


KRT18
NM_000224
S1710/KRT18.f2
AGAGATCGAGGCTCTCAAGG
20
303


KRT18
NM_000224
S1711/KRT18.r2
GAGCCTTTTACTTCCTCTTCG
20
304


KRT18
NM_000224
S4762/KRT18.p2
TGGTTCTTCTTCATGAAGAGCAGCTCC
27
305


KRT19
NM_002276
S1515/KRT19.f3
TGAGCGGCAGAATCAGGAGTA
21
306


KRT19
NM_002276
S1516/KRT19.r3
TGCGGTAGGTGGCAATCTC
19
307


KRT19
NM_002276
S4866/KRT19.p3
CTCATGGACATCAAGTCGCGGCTG
24
308


KRT5
NM_000424
S0175/KRT5.f3
TCAGTGGAGAAGGAGTTGGA
20
309


KRT5
NM_000424
S0177/KRT5.r3
TGCCATATCCAGAGGAAACA
20
311


KRT5
NM_000424
S5015/KRT5.p3
CCAGTCAACATCTCTGTTGTCACAAGCA
28
310


KRT8
NM_002273
S2588/KRT8.f3
GGATGAAGCTTACATGAACAAGGTAGA
27
312


KRT8
NM_002273
S2589/KRT8.r3
CATATAGCTGCCTGAGGAAGTTGAT
25
313


KRT8
NM_002273
S4952/KRT8.p3
CGTCGGTCAGCCCTTCCAGGC
21
314


LOT1 variant
NM_002656
S0692/LOT1 v.f2
GGAAAGACCACCTGAAAAACCA
22
315


1


LOT1 variant
NM_002656
S0693/LOT1 v.r2
GTACTTCTTCCCACACTCCTCACA
24
316


1


LOT1 variant
NM_002656
S4793/LOT1 v.p2
ACCCACGACCCCAACAAAATGGC
23
317


1


Maspin
NM_002639
S0836/Maspin.f2
CAGATGGCCACTTTGAGAACATT
23
318


Maspin
NM_002639
S0837/Maspin.r2
GGCAGCATTAACCACAAGGATT
22
319


Maspin
NM_002639
S4835/Maspin.p2
AGCTGACAACAGTGTGAACGACCAGACC
28
320


MCM2
NM_004526
S1602/MCM2.f2
GACTTTTGCCCGCTACCTTTC
21
321


MCM2
NM_004526
S1603/MCM2.r2
GCCACTAACTGCTTCAGTATGAAGAG
26
322


MCM2
NM_004526
S4900/MCM2.p2
ACAGCTCATTGTTGTCACGCCGGA
24
323


MCM3
NM_002388
S1524/MCM3.f3
GGAGAACAATCCCCTTGAGA
20
324


MCM3
NM_002388
S1525/MCM3.r3
ATCTCCTGGATGGTGATGGT
20
325


MCM3
NM_002388
S4870/MCM3.p3
TGGCCTTTCTGTCTACAAGGATCACCA
27
326


MCM6
NM_005915
S1704/MCM6.f3
TGATGGTCCTATGTGTCACATTCA
24
327


MCM6
NM_005915
S1705/MCM6.r3
TGGGACAGGAAACACACCAA
20
328





















TABLE 6F










SEQ ID


Gene
Accession
Probe Name
Seq
Length
NO:







MCM6
NM_005915
S4919/MCM6.p3
CAGGTTTCATACCAACACAGGCTTCAGCAC
30
329


MDM2
NM_002392
S0830/MDM2.f1
CTACAGGGACGCCATCGAA
19
330


MDM2
NM_002392
S0831/MDM2.r1
ATCCAACCAATCACCTGAATGTT
23
331


MDM2
NM_002392
S4834/MDM2.p1
CTTACACCAGCATCAAGATCCGG
23
332


MMP9
NM_004994
S0656/MMP9.f1
GAGAACCATCTCACCGACA
20
333


MMP9
NM_004994
S0657/MMP9.r1
CACCCGAGTGTAACCATAGC
20
334


MMP9
NM_004994
S4760/MMP9.p1
ACAGGTATTCCTCTGCCAGCTGCC
24
335


MTA1
NM_004689
S2369/MTA1.f1
CCGCCCTCACCTGAAGAGA
19
336


MTA1
NM_004689
S2370/MTA1.r1
GGAATAAGTTAGCCGCGCTTCT
22
337


MTA1
NM_004689
S4855/MTA1.p1
CCCAGTGTCCGCCAAGGAGCG
21
338


MYBL2
NM_002466
S3270/MYBL2.f1
GCCGAGATCGCCAAGATG
18
339


MYBL2
NM_002466
S3271/MYBL2.r1
CTTTTGATGGTAGAGTTCCAGTGATTC
27
340


MYBL2
NM_002466
S4742/MYBL2.p1
CAGCATTGTCTGTCCTCCCTGGCA
24
341


P14ARF
S78535
S2842/P14ARF.f1
CCCTCGTGCTGATGCTACT
19
342


P14ARF
S78535
S2843/P14ARF.r1
CATCATGACCTGGTCTTCTAGG
22
343


P14ARF
S78535
S4971/P14ARF.p1
CTGCCCTAGACGCTGGCTCCTC
22
344


p27
NM_004064
S0205/p27.f3
CGGTGGACCACGAAGAGTTAA
21
345


p27
NM_004064
S0207/p27.r3
GGCTCGCCTCTTCCATGTC
19
347


p27
NM_004064
S4750/p27.p3
CCGGGACTTGGAGAAGCACTGCA
23
346


P53
NM_000546
S0208/P53.f2
CTTTGAACCCTTGCTTGCAA
20
348


P53
NM_000546
S0210/P53.r2
CCCGGGACAAAGCAAATG
18
350


P53
NM_000546
S5065/P53.p2
AAGTCCTGGGTGCTTCTGACGCACA
25
349


PAI1
NM_000602
S0211/PAI1.f3
CCGCAACGTGGTTTTCTCA
19
351


PAI1
NM_000602
S0213/PAI1.r3
TGCTGGGTTTCTCCTCCTGTT
21
353


PAI1
NM_000602
S5066/PAI1.p3
CTCGGTGTTGGCCATGCTCCAG
22
352


PDGFRb
NM_002609
S1346/PDGFRb.f3
CCAGCTCTCCTTCCAGCTAC
20
354


PDGFRb
NM_002609
S1347/PDGFRb.r3
GGGTGGCTCTCACTTAGCTC
20
355


PDGFRb
NM_002609
S4931/PDGFRb.p3
ATCAATGTCCCTGTCCGAGTGCTG
24
356


PI3KC2A
NM_002645
S2020/PI3KC2.r1
CACACTAGCATTTTCTCCGCATA
23
357


PI3KC2A
NM_002645
S2021/PI3KC2.f1
ATACCAATCACCGCACAAACC
21
358


PI3KC2A
NM_002645
S5062/PI3KC2.p1
TGCGCTGTGACTGGACTTAACAAATAGCCT
30
359


PPM1D
NM_003620
S3159/PPM1D.f1
GCCATCCGCAAAGGCTTT
18
360


PPM1D
NM_003620
S3160/PPM1D.r1
GGCCATTCCGCCAGTTTC
18
361


PPM1D
NM_003620
S4856/PPM1D.p1
TCGCTTGTCACCTTGCCATGTGG
23
362


PR
NM_000926
S1336/PR.f6
GCATCAGGCTGTCATTATGG
20
363


PR
NM_000926
S1337/PR.r6
AGTAGTTGTGCTGCCCTTCC
20
364


PR
NM_000926
S4743/PR.p6
TGTCCTTACCTGTGGGAGCTGTAAGGTC
28
365


PRAME
NM_006115
S1985/PRAME.f3
TCTCCATATCTGCCTTGCAGAGT
23
366


PRAME
NM_006115
S1986/PRAME.r3
GCACGTGGGTCAGATTGCT
19
367


PRAME
NM_006115
S4756/PRAME.p3
TCCTGCAGCACCTCATCGGGCT
22
368


pS2
NM_003225
S0241/pS2.f2
GCCCTCCCAGTGTGCAAAT
19
369


pS2
NM_003225
S0243/pS2.r2
CGTCGATGGTATTAGGATAGAAGCA
25
371


pS2
NM_003225
S5026/pS2.p2
TGCTGTTTCGACGACACCGTTCG
23
370


RAD51C
NM_058216
S2606/RAD51C.f3
GAACTTCTTGAGCAGGAGCATACC
24
372





















TABLE 6G










SEQ ID


Gene
Accession
Probe Name
Seq
Length
NO:







RAD51C
NM_058216
S2607/RAD51C.r3
TCCACCCCCAAGAATATCATCTAGT
25
373


RAD51C
NM_058216
S4764/RAD51C.p3
AGGGCTTCATAATCACCTTCTGTTC
25
374


RB1
NM_000321
S2700/RB1.f1
CGAAGCCCTTACAAGTTTCC
20
375


RB1
NM_000321
S2701/RB1.r1
GGACTCTTCAGGGGTGAAAT
20
376


RB1
NM_000321
S4765/RB1.p1
CCCTTACGGATTCCTGGAGGGAAC
24
377


RIZ1
NM_012231
S1320/RIZ1.f2
CCAGACGAGCGATTAGAAGC
20
378


RIZ1
NM_012231
S1321/RIZ1.r2
TCCTCCTCTTCCTCCTCCTC
20
379


RIZ1
NM_012231
S4761/RIZ1.p2
TGTGAGGTGAATGATTTGGGGGA
23
380


STK15
NM_003600
S0794/STK15.f2
CATCTTCCAGGAGGACCACT
20
381


STK15
NM_003600
S0795/STK15.r2
TCCGACCTTCAATCATTTCA
20
382


STK15
NM_003600
S4745/STK15.p2
CTCTGTGGCACCCTGGACTACCTG
24
383


STMY3
NM_005940
S2067/STMY3.f3
CCTGGAGGCTGCAACATACC
20
384


STMY3
NM_005940
S2068/STMY3.r3
TACAATGGCTTTGGAGGATAGCA
23
385


STMY3
NM_005940
S4746/STMY3.p3
ATCCTCCTGAAGCCCTTTTCGCAGC
25
386


SURV
NM_001168
S0259/SURV.f2
TGTTTTGATTCCCGGGCTTA
20
387


SURV
NM_001168
S0261/SURV.r2
CAAAGCTGTCAGCTCTAGCAAAAG
24
389


SURV
NM_001168
S4747/SURV.p2
TGCCTTCTTCCTCCCTCACTTCTCACCT
28
388


TBP
NM_003194
S0262/TBP.f1
GCCCGAAACGCCGAATATA
19
390


TBP
NM_003194
S0264/TBP.r1
CGTGGCTCTCTTATCCTCATGAT
23
392


TBP
NM_003194
S4751/T8P.p1
TACCGCAGCAAACCGCTTGGG
21
391


TGFA
NM_003236
S0489/TGFA.f2
GGTGTGCCACAGACCTTCCT
20
393


TGFA
NM_003236
S0490/TGFA.r2
ACGGAGTTCTTGACAGAGTTTTGA
24
394


TGFA
NM_003236
S4768/TGFA.p2
TTGGCCTGTAATCACCTGTGCAGCCTT
27
395


TIMP1
NM_003254
S1695/TIMP1.f3
TCCCTGCGGTCCCAGATAG
19
396


TIMP1
NM_003254
S1696/TIMP1.r3
GTGGGAACAGGGTGGACACT
20
397


TIMP1
NM_003254
S4918/TIMP1.p3
ATCCTGCCCGGAGTGGAACTGAAGC
25
398


TOP2A
NM_001067
S0271/TOP2A.f4
AATCCAAGGGGGAGAGTGAT
20
399


TOP2A
NM_001067
S0273/TOP2A.r4
GTACAGATTTTGCCCGAGGA
20
401


TOP2A
NM_001067
S4777/TOP2A.p4
CATATGGACTTTGACTCAGCTGTGGC
26
400


TOP2B
NM_001068
S0274/TOP2B.f2
TGTGGACATCTTCCCCTCAGA
21
402


TOP2B
NM_001068
S0276/TOP2B.r2
CTAGCCCGACCGGTTCGT
18
404


TOP2B
NM_001068
S4778/TOP2B.p2
TTCCCTACTGAGCCACCTTCTCTG
24
403


TP
NM_001953
S0277/TP.f3
CTATATGCAGCCAGAGATGTGACA
24
405


TP
NM_001953
S0279/TP.r3
CCACGAGTTTCTTACTGAGAATGG
24
407


TP
NM_001953
S4779/TP.p3
ACAGCCTGCCACTCATCACAGCC
23
406


TP53BP2
NM_005426
S1931/TP53BP.f2
GGGCCAAATATTCAGAAGC
19
408


TP53BP2
NM_005426
S1932/TP53BP.r2
GGATGGGTATGATGGGACAG
20
409


TP53BP2
NM_005426
S5049/TP53BP.p2
CCACCATAGCGGCCATGGAG
20
410


TRAIL
NM_003810
S2539/TRAIL.f1
CTTCACAGTGCTCCTGCAGTCT
22
411


TRAIL
NM_003810
S2540/TRAIL.r1
CATCTGCTTCAGCTCGTTGGT
21
412


TRAIL
NM_003810
S4980/TRAIL.p1
AAGTACACGTAAGTTACAGCCACACA
26
413


TS
NM_001071
S0280/TS.f1
GCCTCGGTGTGCCTTTCA
18
414


TS
NM_001071
S0282/TS.r1
CGTGATGTGCGCAATCATG
19
416


TS
NM_001071
S4780/TS.p1
CATCGCCAGCTACGCCCTGCTC
22
415


upa
NM_002658
S0283/upa.f3
GTGGATGTGCCCTGAAGGA
19
417





















TABLE 6H










SEQ ID


Gene
Accession
Probe Name
Seq
Length
NO:




















upa
NM_002658
S0285/upa.r3
CTGCGGATCCAGGGTAAGAA
20
418


upa
NM_002658
S4769/upa.p3
AAGCCAGGCGTCTACACGAGAGTCTCAC
28
419


VDR
NM_000376
S2745/VDR.f2
GCCCTGGATTTCAGAAAGAG
20
420


VDR
NM_000376
S2746/VDR.r2
AGTTACAAGCCAGGGAAGGA
20
421


VDR
NM_000376
S4S62/VDR.p2
CAAGTCTGGATCTGGGACCCTTTCC
25
422


VEGF
NM_003376
S0286/VEGF.f1
CTGCTGTCTTGGGTGCATTG
20
423


VEGF
NM_003376
S0288/VEGF.r1
GCAGCCTGGGACCACTTG
18
424


VEGF
NM_003376
S4782/VEGF.p1
TTGCCTTGCTGCTCTACCTCCACCA
25
425


VEGFB
NM_003377
S2724/VEGFB.f1
TGACGATGGCCTGGAGTGT
19
426


VEGFB
NM_003377
S2725/VEGFB.r1
GGTACCGGATCATGAGGATCTG
22
427


VEGFB
NM_003377
S4960/VEGFB.p1
CTGGGCAGCACCAAGTCCGGA
21
428


WISP1
NM_003882
S1671/WISP1.f1
AGAGGCATCCATGAACTTCACA
22
429


WISP1
NM_003882
S1672/WISP1.r1
CAAACTCCACAGTACTTGGGTTGA
24
430


WISP1
NM_003882
S4915/WISP1.p1
CGGGCTGCATCAGCACACGC
20
431


XIAP
NM_001167
S0289/XIAP.f1
GCAGTTGGAAGACACAGGAAAGT
23
432


XIAP
NM_001167
S0291/XIAP.r1
TGCGTGGCACTATTTTCAAGA
21
434


XIAP
NM_001167
S4752/XIAP.p1
TCCCCAAATTGCAGATTTATCAACGGC
27
433


YB-1
NM_004559
S1194/YB-1.f2
AGACTGTGGAGTTTGATGTTGTTGA
25
435


YB-1
NM_004559
S1195/YB-1.r2
GGAACACCACCAGGACCTGTAA
22
436


YB-1
NM_004559
S4843/YB-1.p2
TTGCTGCCTCCGCACCCTTTTCT
23
437


ZNF217
NM_006526
S2739/ZNF217.f3
ACCCAGTAGCAAGGAGAAGC
20
438


ZNF217
NM_006526
S2740/ZNF217.r3
CAGCTGGTGGTAGGTTCTGA
20
439


ZNF217
NM_006526
S4961/ZNF217.p3
CACTCACTGCTCCGAGTGCGG
21
440

## Claims

1. A method of predicting the likelihood of long-term survival without recurrence of breast cancer for a patient having estrogen receptor (ER)-positive breast cancer, the method comprising:
(a) assaying an expression level of an RNA transcript or its expression product in a biological sample comprising a breast cancer cell obtained from the patient, wherein the RNA transcript is a MYBL2 transcript:
(b) determining a normalized expression level of the MYBL2 transcript or its expression product, wherein the normalized expression level of the MYBL2 transcript or its expression product positively correlates with an increased likelihood of breast cancer recurrence in the patient; and
(c) providing information regarding the likelihood of breast cancer recurrence for the patient, wherein the information comprises the normalized expression level of the MYBL2 transcript or its expression product.

2. The method of claim 1 wherein breast cancer is invasive breast carcinoma.

3. The method of claim 1 wherein said the biological sample is a fixed, wax-embedded breast cancer tissue specimen.

4. The method of claim 1 wherein the biological sample is isolated from core biopsy tissue or fine needle aspirated cells.

5. A method of preparing a personalized genomics profile for a patient with estrogen receptor (ER)-positive breast cancer, comprising the steps of:
(a) assaying an expression level of an RNA transcript or its expression product in a biological sample comprising a breast cancer cell obtained from the patient, wherein the RNA transcript is a MYBL2 transcript;
(b) determining a normalized expression level of the MYBL2 transcript or its expression product, wherein the normalized expression level of the MYBL2 transcript or its expression product positively correlates with an increased likelihood of breast cancer recurrence in the patient; and
(c) creating a report summarizing data obtained from the normalized expression level and containing an estimate of likelihood of long-term survival without breast cancer recurrence in said patient.

6. The method of claim 5, wherein the biological sample is a fixed, paraffin-embedded biopsy sample.

7. The method of claim 5, wherein the RNA transcript is fragmented.

8. The method of claim 5 further comprising identifying a treatment option for the patient based on the normalized expression level.

9. The method of claim 1 wherein the expression level of the MYLB2 transcript, or its expression product, is normalized against a reference set comprising RNA transcripts of two or more housekeeping genes, or their expression products.

10. The method of claim 9 wherein the two or more housekeeping genes are selected from the group consisting of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Cyp1, albumin, actins, tubulins, cyclophilin, hypoxantine phosphoribosyltransferase (HRPT), L32, 28S, and 18S.

11. The method of claim 1, further comprising
(a) assaying an expression level of at least one RNA transcript or its expression product in a biological sample comprising at least one breast cancer cell obtained from the patient, wherein the at least one RNA transcript is the transcript of a gene selected from the group consisting of: GRB7, CTSL, CD68, Chk1, AIB1, CCNB1, MCM2, FBXO5, STK15, SURV, EGFR, HIF1α, and TS:
(b) determining a normalized expression level of the RNA transcript or its expression product wherein the normalized expression level of the RNA transcript or its expression product positively correlates with an increased likelihood of breast cancer recurrence; and
(c) providing information comprising the likelihood of long-term survival without breast cancer recurrence for the patient, wherein the information comprises the normalized expression level of the RNA transcript or its expression product.

12. The method of claim 1, further comprising
(a) assaying an expression level of at least one RNA transcript or its expression product in a biological sample comprising at least one breast cancer cell obtained from the patient, wherein the at least one RNA transcript is the transcript of a gene selected from the group consisting of: TP53BP2, Bc12, KRT14, EstR1, IGFBP2, BAG1, CEGP1, KLK10, β-Catenin, γ-Catenin, DR5, P13KCA2, RAD51C, GSTM1, FHIT, RIZ1, BBC3, TBP, p27, IRS1, IGF1R, GATA3, ZNF217, CD9, pS2, ErbB3, TOP2B, MDM2, IGF1, and KRT19;
(b) determining a normalized expression level of the at least one RNA transcript or its expression product, wherein the normalized expression level of the at least one RNA transcript or its expression product negatively correlates with a an increased likelihood of breast cancer recurrence.

13. The method of claim 1 further comprising determining the normalized expression level of a PR RNA transcript or its expression product, wherein the the normalized expression level of PR negatively correlates with an increased likelihood of breast cancer recurrence.

14. The method of claim 1 further comprising determining a normalized expression level of Her2 RNA transcript or its expression product, wherein the normalized expression level of Her2negatively correlates with an increased likelihood of long-term survival without breast cancer recurrence.

15. The method of claim 1 further comprising in step (a) determining the expression level of the PR RNA transcript or its expression product in a breast cancer tissue sample obtained from said patient, normalized against the expression levels of all RNA transcripts or their expression products in said breast cancer tissue sample, or of a reference set of RNA transcripts or their products.

16. The method of claim 1 or claim 15 further comprising in step (a) determining the expression level of the Her2 RNA transcript or its expression product in a breast cancer tissue sample obtained from said patient, normalized against the expression levels of all RNA transcripts or their expression products in said breast cancer tissue sample, or of a reference set of RNA transcripts or their products.

17. The method of claim 5 further comprising in step (b) determining the expression level of PR, 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.

18. The method of claim 5 or claim 17 further comprising in step (b) determining the expression level of Her2 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.

19. The method of claim 1, wherein the information is provided in the form of a report.

20. The method of claim 11, wherein the information is provided in the form of a report.