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Predicting outcome with tamoxifen in breast cancer

Abstrict

Methods and compositions are provided for the identification of expression signatures in ER+ breast cancer cases, where the signatures correlate with responsiveness, or lack thereof, to tamoxifen treatment. The signature profiles are identified based upon sampling of reference breast tissue samples from independent cases of breast cancer and provide a reliable set of molecular criteria for predicting the efficacy of treating a subject with ER+ breast cancer with tamoxifen. Additional methods and compositions are provided for predicting tamoxifen responsiveness in cases of ER+ breast cancer by use of three biomarkers. Two biomarkers display increased expression correlated with tamoxifen response while the third biomarker displays decreased expression correlated with tamoxifen response.

Claims

We claim:

1. An array comprising polynucleotide probes, capable of hybridizing to nucleic acid molecules of more than one of the genes in Table 1, 2, 4, and/or 5, hybridized to nucleic acids derived from one or more ER+ breast cancer cell.

2. The array of claim 1 comprising 11 of the genes in Tables 1, 2, and/or 4.

3. The array of claim 2 comprising more than 11 of the genes.

4. The array of claim 1 wherein said one or more ER+ cell is a human cell.

5. The array of claim 1 wherein said nucleic acids derived from one or more ER+ cells are prepared by mRNA amplification or quantitative PCR.

6. The array of claim 1 wherein said one or more ER+ cells are from a section of tissue from a subject or are microdissected from said section.

7. A method to determine the survival outcome of an ER+ breast cancer afflicted subject if treated with tamoxifen, said method comprising assaying a sample of ER+ breast cancer cells from said subject for the expression level(s) of one or more genes in Table 1, 2, 4, and/or 5.

8. The method of claim 7 wherein said one or more genes is 11 or more genes.

9. The method of claim 7 wherein said subject is human.

10. The method of claim 7 wherein said assaying for the expression level of one or more genes comprises detection of nucleic acids derived from said sample of ER+ breast cancer cells.

11. The method of claim 10 wherein said nucleic acids derived from said sample are prepared by mRNA amplification or quantitative PCR.

12. The method of claim 7 wherein said assaying for the expression level of one or more genes comprises detection of proteins encoded by said genes.

13. The method of claim 12 wherein said detection of proteins comprises detection with antibodies which bind said proteins.

14. A method of determining prognosis of a subject having ER+ breast cancer if treated with tamoxifen, or of a subject afflicted with ER+ breast cancer and treated with tamoxifen, said method comprising: assaying for the expression level(s) of one or more genes in Table 1, 2, 4, and/or 5 from a breast cancer cell sample from said subject.

15. The method of claim 14 wherein said one or more genes is 11 or more genes.

16. The method of claim 14 wherein said subject is human.

17. The method of claim 14 wherein said assaying for the expression level of one or more genes comprises detection of nucleic acids derived from said sample of ER+ breast cancer cells or detection of proteins encoded by said genes.

18. The method of claim 17 wherein said nucleic acids derived from said sample are prepared by mRNA amplification or quantitative PCR.

19. The method of claim 17 wherein said detection of proteins comprises detection with antibodies which bind said proteins.

20. The method of claim 14 wherein said assaying comprises using an array.

21. The method of claim 14 wherein said sample is a ductal lavage or fine needle aspiration sample.

22. The method of claim 14 wherein said sample is a section of tissue from a subject or are cells microdissected from said section.

23. A method to determine therapeutic treatment for an ER+ breast cancer patient based upon said patient's expected response to tamoxifen treatment, said method comprising determining an expected response to tamoxifen treatment for said patient by assaying a sample of breast cancer cells from said patient for the expression level(s) of one or more one genes in Table 1, 2, 4, and/or 5; and selecting the appropriate treatment for a patient with such a survival outcome.

24. The method of claim 23 wherein said one or more genes is 11 or more genes.

25. The method of claim 23 wherein said subject is human.

26. The method of claim 23 wherein said assaying comprises detection of nucleic acids derived from said sample of ER+ breast cancer cells or detection of proteins encoded by said genes.

27. The method of claim 26 wherein said nucleic acids derived from said sample are prepared by mRNA amplification or quantitative PCR.

28. The method of claim 26 wherein said detection of proteins comprises detection with antibodies which bind said proteins.

29. The method of claim 23 wherein said assaying comprises using an array.

30. The method of claim 23 wherein said sample is a ductal lavage or fine needle aspiration sample.

31. The method of claim 23 wherein said sample is a section of tissue from a subject or are cells microdissected from said section.

32. A method to determine the survival outcome of a human subject having ER+ breast cancer if treated with tamoxifen, said method comprising assaying a sample of breast cells from said subject for increased expression of one or more human IL17RB or CACNA1D sequences.

33. The method of claim 32 wherein said sample is obtained by solid tissue biopsy or a non-invasive procedure, such as ductal lavage, fine needle aspiration, or a needle biopsy.

34. The method of claim 33 wherein microdissection is used to isolate breast cells from said sample before assaying for nucleic acid expression.

35. The method of claim 32 wherein said assaying is by hybridization to a polynucleotide comprising sequences of at least 24 nucleotides from the 3' untranslated region, the coding region, or the 5' untranslated region, of human IL17RB or CACNA1D.

36. The method of claim 32 wherein said assaying comprises mRNA amplification or PCR amplification, such as quantitative PCR, of said sequences.

37. The method of claim 32 wherein said assaying for increased expression comprises detection of polypeptides encoded by said sequences.

38. The method of claim 37 wherein said detection of polypeptides comprises detection with antibodies which bind said polypeptides.

39. A method to determine the survival outcome of a human subject having ER+ breast cancer if treated with tamoxifen, said method comprising assaying a sample of breast cells from said subject for decreased expression of human HOXB13 sequences.

40. The method of claim 39 wherein said sample is obtained by solid tissue biopsy or a non-invasive procedure, such as ductal lavage, fine needle aspiration, or a needle biopsy.

41. The method of claim 40 wherein microdissection is used to isolate breast cells from said sample before assaying for nucleic acid expression.

42. The method of claim 39 wherein said assaying is by hybridization to a polynucleotide comprising sequences of at least 24 nucleotides from the 3' untranslated region, the coding region, or the 5' untranslated region, of human HOXB13.

43. The method of claim 39 wherein said assaying comprises mRNA amplification or PCR amplification, such as quantitative PCR, of said sequences.

44. The method of claim 39 wherein said assaying is for inactivation or methylation of HOXB13 sequences.

45. The method of claim 39 wherein said assaying comprises detection of increased mRNA degradation.

46. The method of claim 39 wherein said assaying for increased expression comprises detection of polypeptides encoded by said sequences.

47. The method of claim 46 wherein said detection of polypeptides comprises detection with antibodies which bind said polypeptides.

48. A population of singled stranded nucleic acid molecules comprising one or both strands of a human IL17RB or CACNA1D or HOXB13 sequence wherein at least a portion of said population is hybridized to one or both strands of a nucleic acid molecule quantitatively amplified from RNA of an ER+ breast cell.

49. The population of claim 48 wherein the population is immobilized on a solid support, such as a microarray.

50. The population of claim 48 wherein said nucleic acid molecules amplified from an ER+ breast cell are amplified by quantitative PCR (Q-PCR).

51. The population of claim 50 wherein said quantitative PCR is of amplified mRNA from said breast cancer cell.

52. The population of claim 48 wherein said population of single stranded molecules is equal to or in excess of all of one or both strands of the nucleic acid molecules amplified from an ER+ breast cell such that the population is sufficient to hybridize to all of one or both strands.

53. The population of claim 48 wherein said population of single stranded molecules comprising sequences of at least 24 nucleotides from the 3' untranslated region, the coding region, or the 5' untranslated region, of human IL17RB or CACNA1D or HOXB13.

54. A method to determine the survival outcome of a human subject having ER+ breast cancer if treated with tamoxifen, said method comprising assaying a sample of breast cells from said subject for increased expression of a sequence selected from SEQ ID NOS: 8, 9, or 11-17.

55. A method to determine the survival outcome of a human subject having ER+ breast cancer if treated with tamoxifen, said method comprising assaying a sample of breast cells from said subject for decreased expression of a sequence selected from SEQ ID NOS: 10 or 18-31.

Description

RELATED APPLICATIONS

[0001] This application claims benefit of priority from U.S. Provisional Patent Application 60/504,087, filed Sep. 19, 2003, which is hereby incorporated by reference in its entirety as if fully set forth.

FIELD OF THE INVENTION

[0002] The invention relates to the identification and use of gene expression profiles, or patterns, with clinical relevance to the treatment of breast cancer using tamoxifen. In particular, the invention provides the identities of genes that are correlated with patient survival and breast cancer recurrence in women treated with tamoxifen. The gene expression profiles, whether embodied in nucleic acid expression, protein expression, or other expression formats, may be used to select subjects afflicted with breast cancer who will likely respond positively to tamoxifen treatment as well as those who will likely be non-responsive and thus candidates for other treatments. The invention also provides the identities of three sets of sequences from three genes with expression patterns that are strongly predictive of responsiveness to tamoxifen.

BACKGROUND OF THE INVENTION

[0003] Breast cancer is by far the most common cancer among women. Each year, more than 180,000 and 1 million women in the U.S. and worldwide, respectively, are diagnosed with breast cancer. Breast cancer is the leading cause of death for women between ages 50-55, and is the most common non-preventable malignancy in women in the Western Hemisphere. An estimated 2,167,000 women in the United States are currently living with the disease (National Cancer Institute, Surveillance Epidemiology and End Results (NCI SEER) program, Cancer Statistics Review (CSR), www-seer.ims.nci.nih.gov/Publications/CSR1973 (1998)). Based on cancer rates from 1995 through 1997, a report from the National Cancer Institute (NCI) estimates that about 1 in 8 women in the United States (approximately 12.8 percent) will develop breast cancer during her lifetime (NCI's Surveillance, Epidemiology, and End Results Program (SEER) publication SEER Cancer Statistics Review 1973-1997). Breast cancer is the second most common form of cancer, after skin cancer, among women in the United States. An estimated 250,100 new cases of breast cancer are expected to be diagnosed in the United States in 2001. Of these, 192,200 new cases of more advanced (invasive) breast cancer are expected to occur among women (an increase of 5% over last year), 46,400 new cases of early stage (in situ) breast cancer are expected to occur among women (up 9% from last year), and about 1,500 new cases of breast cancer are expected to be diagnosed in men (Cancer Facts & Figures 2001 American Cancer Society). An estimated 40,600 deaths (40,300 women, 400 men) from breast cancer are expected in 2001. Breast cancer ranks second only to lung cancer among causes of cancer deaths in women. Nearly 86% of women who are diagnosed with breast cancer are likely to still be alive five years later, though 24% of them will die of breast cancer after 10 years, and nearly half (47%) will die of breast cancer after 20 years.

[0004] Every woman is at risk for breast cancer. Over 70 percent of breast cancers occur in women who have no identifiable risk factors other than age (U.S. General Accounting Office. Breast Cancer, 1971-1991: Prevention, Treatment and Research. GAO/PEMD-92-12; 1991). Only 5 to 10% of breast cancers are linked to a family history of breast cancer (Henderson I C, Breast Cancer. In: Murphy G P, Lawrence W L, Lenhard R E (eds). Clinical Oncology. Atlanta, Ga.: American Cancer Society; 1995:198-219).

[0005] Each breast has 15 to 20 sections called lobes. Within each lobe are many smaller lobules. Lobules end in dozens of tiny bulbs that can produce milk. The lobes, lobules, and bulbs are all linked by thin tubes called ducts. These ducts lead to the nipple in the center of a dark area of skin called the areola. Fat surrounds the lobules and ducts. There are no muscles in the breast, but muscles lie under each breast and cover the ribs. Each breast also contains blood vessels and lymph vessels. The lymph vessels carry colorless fluid called lymph, and lead to the lymph nodes. Clusters of lymph nodes are found near the breast in the axilla (under the arm), above the collarbone, and in the chest.

[0006] Breast tumors can be either benign or malignant. Benign tumors are not cancerous, they do not spread to other parts of the body, and are not a threat to life. They can usually be removed, and in most cases, do not come back. Malignant tumors are cancerous, and can invade and damage nearby tissues and organs. Malignant tumor cells may metastasize, entering the bloodstream or lymphatic system. When breast cancer cells metastasize outside the breast, they are often found in the lymph nodes under the arm (axillary lymph nodes). If the cancer has reached these nodes, it means that cancer cells may have spread to other lymph nodes or other organs, such as bones, liver, or lungs.

[0007] Major and intensive research has been focused on early detection, treatment and prevention. This has included an emphasis on determining the presence of precancerous or cancerous ductal epithelial cells. These cells are analyzed, for example, for cell morphology, for protein markers, for nucleic acid markers, for chromosomal abnormalities, for biochemical markers, and for other characteristic changes that would signal the presence of cancerous or precancerous cells. This has led to various molecular alterations that have been reported in breast cancer, few of which have been well characterized in human clinical breast specimens. Molecular alterations include presence/absence of estrogen and progesterone steroid receptors, HER-2 expression/amplification (Mark H F, et al. HER-2/neu gene amplification in stages I-IV breast cancer detected by fluorescent in situ hybridization. Genet Med; 1(3):98-103 1999), Ki-67 (an antigen that is present in all stages of the cell cycle except G0 and used as a marker for tumor cell proliferation, and prognostic markers (including oncogenes, tumor suppressor genes, and angiogenesis markers) like p53, p27, Cathepsin D, pS2, multi-drug resistance (MDR) gene, and CD31.

[0008] Adjuvant tamoxifen (TAM) is the most effective systemic treatment for estrogen receptor positive (ER+) breast cancer. ER and progesterone receptor (PR) expression have been the major clinicopathological predictor for response to TAM. However, up to 40% of ER+ tumors fail to respond or develop resistance to TAM. Therefore, better predictive biomarkers for TAM response may be able to identify patients who are unlikely to benefit from TAM so that additional or alternative therapies may be sought.

[0009] van't Veer et al. (Nature 415:530-536, 2002) describe gene expression profiling of clinical outcome in breast cancer. They identified genes expressed in breast cancer tumors, the expression levels of which correlated either with patients afflicted with distant metastases within 5 years or with patients that remained metastasis-free after at least 5 years.

[0010] Ramaswamy et al. (Nature Genetics 33:49-54, 2003) describe the identification of a molecular signature of metastasis in primary solid tumors. The genes of the signature were identified based on gene expression profiles of 12 metastatic adenocarcinoma nodules of diverse origin (lung, breast, prostate, colorectal, uterus) compared to expression profiles of 64 primary adenocarcinomas representing the same spectrum of tumor types from different individuals. A 128 gene set was identified.

[0011] Both of the above described approaches, however, utilize heterogeneous populations of cells found in a tumor sample to obtain information on gene expression patterns. The use of such populations may result in the inclusion or exclusion of multiple genes that are differentially expressed in cancer cells. The gene expression patterns observed by the above described approaches may thus provide little confidence that the differences in gene expression are meaningfully associated with breast cancer recurrence or survival.

[0012] Citation of documents herein is not intended as an admission that any is pertinent prior art. All statements as to the date or representation as to the contents of documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of the documents.

SUMMARY OF THE INVENTION

[0013] The present invention relates to the identification and use of gene expression patterns (or profiles or "signatures") which are clinically relevant to breast cancer. In particular, the identities of genes that are correlated with patient survival and breast cancer recurrence are provided. The gene expression profiles, whether embodied in nucleic acid expression, protein expression, or other expression formats, may be used to predict survival of subjects afflicted with breast cancer and the likelihood of breast cancer recurrence.

[0014] The invention thus provides for the identification and use of gene expression patterns (or profiles or "signatures") which correlate with (and thus able to discriminate between) patients with good or poor survival outcomes. In one embodiment, the invention provides patterns that are able to distinguish patients with estrogen receptor positive (ER+) breast tumors into those with that are responsive, or likely to be responsive, to tamoxifen (TAM) treatment and those that are non-responsive, or likely to be non-responsive, to TAM treatment. Responsiveness may be viewed in terms of better survival outcomes over time. These patterns are thus able to distinguish patients with ER+ breast tumors into at least two subtypes.

[0015] In a first aspect, the present invention provides a non-subjective means for the identification of patients with ER+ breast cancer as likely to have a good or poor survival outcome following TAM treatment by assaying for the expression patterns disclosed herein. Thus where subjective interpretation may have been previously used to determine the prognosis and/or treatment of breast cancer patients, the present invention provides objective gene expression patterns, which may used alone or in combination with subjective criteria to provide a more accurate assessment of ER+ breast cancer patient outcomes or expected outcomes, including survival and the recurrence of cancer, following treatment with TAM. The expression patterns of the invention thus provide a means to determine ER+ breast cancer prognosis. Furthermore, the expression patterns can also be used as a means to assay small, node negative tumors that are not readily assayed by other means.

[0016] The gene expression patterns comprise one or more than one gene capable of discriminating between breast cancer outcomes with significant accuracy. The gene(s) are identified as correlated with ER+ breast cancer outcomes such that the levels of their expression are relevant to a determination of the preferred treatment protocols for a patient. Thus in one embodiment, the invention provides a method to determine the outcome of a subject afflicted with ER+ breast cancer by assaying a cell containing sample from said subject for expression of one or more than one gene disclosed herein as correlated with ER+ breast cancer outcomes following TAM treatment.

[0017] Gene expression patterns of the invention are identified as described below. Generally, a large sampling of the gene expression profile of a sample is obtained through quantifying the expression levels of mRNA corresponding to many genes. This profile is then analyzed to identify genes, the expression of which are positively, or negatively, correlated, with ER+ breast cancer outcome with TAM treatment. An expression profile of a subset of human genes may then be identified by the methods of the present invention as correlated with a particular outcome. The use of multiple samples increases the confidence which a gene may be believed to be correlated with a particular survival outcome. Without sufficient confidence, it remains unpredictable whether expression of a particular gene is actually correlated with an outcome and also unpredictable whether expression of a particular gene may be successfully used to identify the outcome for a ER+ breast cancer patient.

[0018] A profile of genes that are highly correlated with one outcome relative to another may be used to assay an sample from a subject afflicted with ER+ breast cancer to predict the likely responsiveness (or lack thereof) to TAM in the subject from whom the sample was obtained. Such an assay may be used as part of a method to determine the therapeutic treatment for said subject based upon the breast cancer outcome identified.

[0019] The correlated genes may be used singly with significant accuracy or in combination to increase the ability to accurately correlating a molecular expression phenotype with an ER+ breast cancer outcome. This correlation is a way to molecularly provide for the determination of survival outcomes as disclosed herein. Additional uses of the correlated gene(s) are in the classification of cells and tissues; determination of diagnosis and/or prognosis; and determination and/or alteration of therapy.

[0020] The ability to discriminate is conferred by the identification of expression of the individual genes as relevant and not by the form of the assay used to determine the actual level of expression. An assay may utilize any identifying feature of an identified individual gene as disclosed herein as long as the assay reflects, quantitatively or qualitatively, expression of the gene in the "transcriptome" (the transcribed fraction of genes in a genome) or the "proteome" (the translated fraction of expressed genes in a genome). Identifying features include, but are not limited to, unique nucleic acid sequences used to encode (DNA), or express (RNA), said gene or epitopes specific to, or activities of, a protein encoded by said gene. All that is required is the identity of the gene(s) necessary to discriminate between ER+ breast cancer outcomes and an appropriate cell containing sample for use in an expression assay.

[0021] In another embodiment, the invention provides for the identification of the gene expression patterns by analyzing global, or near global, gene expression from single cells or homogenous cell populations which have been dissected away from, or otherwise isolated or purified from, contaminating cells beyond that possible by a simple biopsy. Because the expression of numerous genes fluctuate between cells from different patients as well as between cells from the same patient sample, multiple data from expression of individual genes and gene expression patterns are used as reference data to generate models which in turn permit the identification of individual gene(s), the expression of which are most highly correlated with particular ER+ breast cancer outcomes.

[0022] In additional embodiments, the invention provides physical and methodological means for detecting the expression of gene(s) identified by the models generated by individual expression patterns. These means may be directed to assaying one or more aspects of the DNA template(s) underlying the expression of the gene(s), of the RNA used as an intermediate to express the gene(s), or of the proteinaceous product expressed by the gene(s).

[0023] In a further embodiments, the gene(s) identified by a model as capable of discriminating between ER+ breast cancer outcomes may be used to identify the cellular state of an unknown sample of cell(s) from the breast. Preferably, the sample is isolated via non-invasive means. The expression of said gene(s) in said unknown sample may be determined and compared to the expression of said gene(s) in reference data of gene expression patterns correlated with ER+ breast cancer outcomes. Optionally, the comparison to reference samples may be by comparison to the model(s) constructed based on the reference samples.

[0024] One advantage provided by the present invention is that contaminating, non-breast cells (such as infiltrating lymphocytes or other immune system cells) are not present to possibly affect the genes identified or the subsequent analysis of gene expression to identify the survival outcomes of patients with breast cancer. Such contamination is present where a biopsy is used to generate gene expression profiles.

[0025] In a second aspect, the invention provides a non-subjective means based on the expression of three genes, or combinations thereof, for the identification of patients with ER+ breast cancer as likely to have a good or poor survival outcome following TAM treatment. These three genes are members of the expression patterns disclosed herein which have been found to be strongly predictive of clinical outcome following TAM treatment of ER+ breast cancer.

[0026] The present invention thus provides gene sequences identified as differentially expressed in ER+ breast cancer in correlation to TAM responsiveness. The sequences of two of the genes display increased expression in ER+ breast cells that respond to TAM treatment (and thus decreased expression in nonresponsive cases). The sequences of the third gene display decreased expression in ER+ breast cells that respond to TAM treatment (and thus increased expression in nonresponsive cases).

[0027] The first set of sequences found to be more highly expressed in TAM responsive, ER+ breast cells are those of interleukin 17 receptor B (IL17RB), which has been mapped to human chromosome 3 at 3p21.1. IL17RB is also referred to as interleukin 17B receptor (IL17BR) and sequences corresponding to it, and thus may be used in the practice of the instant invention, are identified by UniGene Cluster Hs.5470.

[0028] The second set of sequences found to be more highly expressed in TAM responsive, ER+ breast cells are those of the calcium channel, voltage-dependent, L type, alpha 1D subunit (CACNA1D), which has been mapped to human chromosome 3 at 3p14.3. Sequences corresponding to CACNA1D, and thus may be used in the practice of the instant invention, are identified by UniGene Cluster Hs.399966.

[0029] The set of sequences found to be expressed at lower levels in TAM responsive, ER+ breast cells are those of homeobox B13 (HOXB13), which has been mapped to human chromosome 17 at 17q21.2. Sequences corresponding to HOXB13, and thus may be used in the practice of the instant invention, are identified by UniGene Cluster Hs.66731.

[0030] The identified sequences may thus be used in methods of determining the responsiveness of a subject's ER+ breast cancer to TAM treatment via analysis of breast cells in a tissue or cell containing sample from a subject. The present invention provides an non-empirical means for determining TAM responsiveness in ER+ patients. This provides advantages over the use of a "wait and see" approach following treatment with TAM. The expression levels of these sequences may also be used as a means to assay small, node negative tumors that are not readily assessed by conventional means.

[0031] The expression levels of the identified sequences may be used alone or in combination with other sequences capable of determining responsiveness to TAM treatment. Preferably, the sequences of the invention are used alone or in combination with each other, such as in the format of a ratio of expression levels that can have improved predictive power over analysis based on expression of sequences corresponding to individual genes.

[0032] The present invention provides means for correlating a molecular expression phenotype with a physiological response in a subject with ER+ breast cancer. This correlation provides a way to molecularly diagnose and/or determine treatment for a breast cancer afflicted subject. Additional uses of the sequences are in the classification of cells and tissues; and determination of diagnosis and/or prognosis. Use of the sequences to identify cells of a sample as responsive, or not, to TAM treatment may be used to determine the choice, or alteration, of therapy used to treat such cells in the subject, as well as the subject itself, from which the sample originated.

[0033] An assay of the invention may utilize a means related to the expression level of the sequences disclosed herein as long as the assay reflects, quantitatively or qualitatively, expression of the sequence. Preferably, however, a quantitative assay means is preferred. The ability to determine TAM responsiveness and thus outcome of treatment therewith is provided by the recognition of the relevancy of the level of expression of the identified sequences and not by the form of the assay used to determine the actual level of expression. Identifying features of the sequences include, but are not limited to, unique nucleic acid sequences used to encode (DNA), or express (RNA), the disclosed sequences or epitopes specific to, or activities of, proteins encoded by the sequences. Alternative means include detection of nucleic acid amplification as indicative of increased expression levels (IL17RB and CACNA1D sequences) and nucleic acid inactivation, deletion, or methylation, as indicative of decreased expression levels (HOXB13 sequences). Stated differently, the invention may be practiced by assaying one or more aspect of the DNA template(s) underlying the expression of the disclosed sequence(s), of the RNA used as an intermediate to express the sequence(s), or of the proteinaceous product expressed by the sequence(s). As such, the detection of the amount of, stability of, or degradation (including rate) of, such DNA, RNA and proteinaceous molecules may be used in the practice of the invention.

[0034] The practice of the present invention is unaffected by the presence of minor mismatches between the disclosed sequences and those expressed by cells of a subject's sample. A non-limiting example of the existence of such mismatches are seen in cases of sequence polymorphisms between individuals of a species, such as individual human patients within Homo sapiens. Knowledge that expression of the disclosed sequences (and sequences that vary due to minor mismatches) is correlated with the presence of non-normal or abnormal breast cells and breast cancer is sufficient for the practice of the invention with an appropriate cell containing sample via an assay for expression.

[0035] In one embodiment, the invention provides for the identification of the expression levels of the disclosed sequences by analysis of their expression in a sample containing ER+ breast cells. In one preferred embodiment, the sample contains single cells or homogenous cell populations which have been dissected away from, or otherwise isolated or purified from, contaminating cells beyond that possible by a simple biopsy. Alternatively, undissected cells within a "section" of tissue may be used. Multiple means for such analysis are available, including detection of expression within an assay for global, or near global, gene expression in a sample (e.g. as part of a gene expression profiling analysis such as on a microarray) or by specific detection, such as quantitative PCR (Q-PCR), or real time quantitative PCR.

[0036] Preferably, the sample is isolated via non-invasive means. The expression of the disclosed sequence(s) in the sample may be determined and compared to the expression of said sequence(s) in reference data of non-normal breast cells. Alternatively, the expression level may be compared to expression levels in normal cells, preferably from the same sample or subject. In embodiments of the invention utilizing Q-PCR, the expression level may be compared to expression levels of reference genes in the same sample.

[0037] When individual breast cells are isolated in the practice of the invention, one benefit is that contaminating, non-breast cells (such as infiltrating lymphocytes or other immune system cells) are not present to possibly affect detection of expression of the disclosed sequence(s). Such contamination is present where a biopsy is used to generate gene expression profiles. However, analysis of differential gene expression and correlation to ER+ breast cancer outcomes with both isolated and non-isolated samples, as described herein, increases the confidence level of the disclosed sequences as capable of having significant predictive power with either type of sample.

[0038] While the present invention is described mainly in the context of human breast cancer, it may be practiced in the context of breast cancer of any animal known to be potentially afflicted by breast cancer. Preferred animals for the application of the present invention are mammals, particularly those important to agricultural applications (such as, but not limited to, cattle, sheep, horses, and other "farm animals"), animal models of breast cancer, and animals for human companionship (such as, but not limited to, dogs and cats).

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] FIG. 1 shows the survival curves for two groups of breast cancer patients defined by expression signatures based on 149 genes as described herein.

[0040] FIG. 2 shows survival curves for two groups of breast cancer patients defined by expression signatures based on genes sets identified for whole tissue sections (left graph) and laser microdissected cells (right graph) as described herein.

[0041] FIG. 3 shows the expression levels of IL17BR, HOXB13, and CACNA1D in whole tissue sections (top three graphs) and laser microdissected cells (bottom three graphs).

[0042] FIG. 4 shows receiver operating characteristic (ROC) analyses of IL17BR, HOXB13, and CACNA1D expression levels as predictors of breast cancer outcomes in whole tissue sections (top three graphs) and laser microdissected cells (bottom three graphs). AUC refers to area under the curve.

[0043] FIG. 5 shows Kaplan-Meier (KM) analyses of IL17BR, HOXB13, and CACNA1D expression levels as predictors of breast cancer outcomes in whole tissue sections (top three graphs) and laser microdissected cells (bottom three graphs).

[0044] FIG. 6 shows expression levels (top three graphs) and ROC (bottom three graphs) analysis of IL17BR, HOXB13, and CACNA1D as predictors of breast cancer outcomes in macrodissected formalin fixed, paraffin embedded (FFPE) samples from a cohort of 31 patients treated with tamoxifen.

[0045] FIG. 7 shows analysis and use of a ratio of HOXB13 to IL17BR expression levels as a predictor of breast cancer outcome. Plots of the ratios in whole tissue sections and macrodissected FFPE samples as well as ROC analysis are shown in the first four graphs. Survival curves based on "high" and "low" ratios (relative to 0.22, the horizontal line in the plots of the ratios) are shown in the last graph.

[0046] Modes of Practicing the Invention

[0047] Definitions of terms as used herein:

[0048] A gene expression "pattern" or "profile" or "signature" refers to the relative expression of genes correlated with responsiveness to TAM treatment of ER+ breast cancer. Responsiveness or lack thereof may be expressed as survival outcomes which are correlated with an expression "pattern" or "profile" or "signature" that is able to distinguish between, and predict, said outcomes.

[0049] A "gene" is a polynucleotide that encodes a discrete product, whether RNA or proteinaceous in nature. It is appreciated that more than one polynucleotide may be capable of encoding a discrete product. The term includes alleles and polymorphisms of a gene that encodes the same product, or a functionally associated (including gain, loss, or modulation of function) analog thereof, based upon chromosomal location and ability to recombine during normal mitosis.

[0050] A "sequence" or "gene sequence" as used herein is a nucleic acid molecule or polynucleotide composed of a discrete order of nucleotide bases. The term includes the ordering of bases that encodes a discrete product (i.e. "coding region"), whether RNA or proteinaceous in nature, as well as the ordered bases that precede or follow a "coding region". Non-limiting examples of the latter include 5' and 3' untranslated regions of a gene. It is appreciated that more than one polynucleotide may be capable of encoding a discrete product. It is also appreciated that alleles and polymorphisms of the disclosed sequences may exist and may be used in the practice of the invention to identify the expression level(s) of the disclosed sequences or the allele or polymorphism. Identification of an allele or polymorphism depends in part upon chromosomal location and ability to recombine during mitosis.

[0051] The terms "correlate" or "correlation" or equivalents thereof refer to an association between expression of one or more genes and a physiological response of a breast cancer cell and/or a breast cancer patient in comparison to the lack of the response. A gene may be expressed at higher or lower levels and still be correlated with responsiveness or breast cancer survival or outcome. The invention provides for the correlation between increases in expression of IL 17RB and CACNA1D sequences and TAM responsiveness in ER+ breast cells. Similarly, the invention provides for the correlation between decreases in expression of HOXB13 sequences and TAM responsiveness in ER+ breast cells. Increases and decreases may be readily expressed in the form of a ratio between expression in a non-normal cell and a normal cell such that a ratio of one (1) indicates no difference while ratios of two (2) and one-half indicate twice as much, and half as much, expression in the non-normal cell versus the normal cell, respectively. Expression levels can be readily determined by quantitative methods as described below.

[0052] For example, increases in IL17RB expression can be indicated by ratios of or about 1.1, of or about 1.2, of or about 1.3, of or about 1.4, of or about 1.5, of or about 1.6, of or about 1.7, of or about 1.8, of or about 1.9, of or about 2, of or about 2.5, of or about 3, of or about 3.5, of or about 4, of or about 4.5, of or about 5, of or about 5.5, of or about 6, of or about 6.5, of or about 7, of or about 7.5, of or about 8, of or about 8.5, of or about 9, of or about 9.5, of or about 10, of or about 15, of or about 20, of or about 30, of or about 40, of or about 50, of or about 60, of or about 70, of or about 80, of or about 90, of or about 100, of or about 150, of or about 200, of or about 300, of or about 400, of or about 500, of or about 600, of or about 700, of or about 800, of or about 900, or of or about 1000. A ratio of 2 is a 100% (or a two-fold) increase in expression. Similar ratios can be used with respect to increases in CACNA1D expression. Decreases in HOXB13 expression can be indicated by ratios of or about 0.9, of or about 0.8, of or about 0.7, of or about 0.6, of or about 0.5, of or about 0.4, of or about 0.3, of or about 0.2, of or about 0.1, of or about 0.05, of or about 0.01, of or about 0.005, of or about 0.001, of or about 0.0005, of or about 0.0001, of or about 0.00005, of or about 0.00001, of or about 0.000005, or of or about 0.000001.

[0053] A "polynucleotide" is a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double- and single-stranded DNA and RNA. It also includes known types of modifications including labels known in the art, methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as uncharged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), as well as unmodified forms of the polynucleotide.

[0054] The term "amplify" is used in the broad sense to mean creating an amplification product can be made enzymatically with DNA or RNA polymerases. "Amplification," as used herein, generally refers to the process of producing multiple copies of a desired sequence, particularly those of a sample. "Multiple copies" mean at least 2 copies. A "copy" does not necessarily mean perfect sequence complementarity or identity to the template sequence. Methods for amplifying mRNA are generally known in the art, and include reverse transcription PCR (RT-PCR) and those described in U.S. patent application Ser. No. 10/062,857 (filed on Oct. 25, 2001), as well as U.S. Provisional Patent Application 60/298,847 (filed Jun. 15, 2001) and 60/257,801 (filed Dec. 22, 2000), all of which are hereby incorporated by reference in their entireties as if fully set forth. Another method which may be used is quantitative PCR (or Q-PCR). Alternatively, RNA may be directly labeled as the corresponding cDNA by methods known in the art.

[0055] By "corresponding", it is meant that a nucleic acid molecule shares a substantial amount of sequence identity with another nucleic acid molecule. Substantial amount means at least 95%, usually at least 98% and more usually at least 99%, and sequence identity is determined using the BLAST algorithm, as described in Altschul et al. (1990), J. Mol. Biol. 215:403-410 (using the published default setting, i.e. parameters w=4, t=17).

[0056] A "microarray" is a linear or two-dimensional array of preferably discrete regions, each having a defined area, formed on the surface of a solid support such as, but not limited to, glass, plastic, or synthetic membrane. The density of the discrete regions on a microarray is determined by the total numbers of immobilized polynucleotides to be detected on the surface of a single solid phase support, preferably at least about 50/cm.sup.2, more preferably at least about 100/cm.sup.2, even more preferably at least about 500/cm.sup.2, but preferably below about 1,000/cm.sup.2. Preferably, the arrays contain less than about 500, about 1000, about 1500, about 2000, about 2500, or about 3000 immobilized polynucleotides in total. As used herein, a DNA microarray is an array of oligonucleotides or polynucleotides placed on a chip or other surfaces used to hybridize to amplified or cloned polynucleotides from a sample. Since the position of each particular group of primers in the array is known, the identities of a sample polynucleotides can be determined based on their binding to a particular position in the microarray.

[0057] Because the invention relies upon the identification of genes that are over- or under-expressed, one embodiment of the invention involves determining expression by hybridization of mRNA, or an amplified or cloned version thereof, of a sample cell to a polynucleotide that is unique to a particular gene sequence. Preferred polynucleotides of this type contain at least about 20, at least about 22, at least about 24, at least about 26, at least about 28, at least about 30, or at least about 32 consecutive basepairs of a gene sequence that is not found in other gene sequences. The term "about" as used in the previous sentence refers to an increase or decrease of 1 from the stated numerical value. Even more preferred are polynucleotides of at least or about 50, at least or about 100, at least about or 150, at least or about 200, at least or about 250, at least or about 300, at least or about 350, at least or about 400, at least or about 450, or at least or about 500 consecutive bases of a sequence that is not found in other gene sequences. The term "about" as used in the preceding sentence refers to an increase or decrease of 10% from the stated numerical value. Longer polynucleotides may of course contain minor mismatches (e.g. via the presence of mutations) which do not affect hybridization to the nucleic acids of a sample. Such polynucleotides may also be referred to as polynucleotide probes that are capable of hybridizing to sequences of the genes, or unique portions thereof, described herein. Such polynucleotides may be labeled to assist in their detection. Preferably, the sequences are those of mRNA encoded by the genes, the corresponding cDNA to such mRNAs, and/or amplified versions of such sequences. In preferred embodiments of the invention, the polynucleotide probes are immobilized on an array, other solid support devices, or in individual spots that localize the probes.

[0058] In another embodiment of the invention, all or part of a disclosed sequence may be amplified and detected by methods such as the polymerase chain reaction (PCR) and variations thereof, such as, but not limited to, quantitative PCR (Q-PCR), reverse transcription PCR (RT-PCR), and real-time PCR, optionally real-time RT-PCR. Such methods would utilize one or two primers that are complementary to portions of a disclosed sequence, where the primers are used to prime nucleic acid synthesis. The newly synthesized nucleic acids are optionally labeled and may be detected directly or by hybridization to a polynucleotide of the invention. The newly synthesized nucleic acids may be contacted with polynucleotides (containing sequences) of the invention under conditions which allow for their hybridization.

[0059] Alternatively, and in yet another embodiment of the invention, gene expression may be determined by analysis of expressed protein in a cell sample of interest by use of one or more antibodies specific for one or more epitopes of individual gene products (proteins) in said cell sample. Such antibodies are preferably labeled to permit their easy detection after binding to the gene product.

[0060] The term "label" refers to a composition capable of producing a detectable signal indicative of the presence of the labeled molecule. Suitable labels include radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, bioluminescent moieties, and the like. As such, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.

[0061] The term "support" refers to conventional supports such as beads, particles, dipsticks, fibers, filters, membranes and silane or silicate supports such as glass slides.

[0062] As used herein, a "breast tissue sample" or "breast cell sample" refers to a sample of breast tissue or fluid isolated from an individual suspected of being afflicted with, or at risk of developing, breast cancer. Such samples are primary isolates (in contrast to cultured cells) and may be collected by any non-invasive means, including, but not limited to, ductal lavage, fine needle aspiration, needle biopsy, the devices and methods described in U.S. Pat. No. 6,328,709, or any other suitable means recognized in the art. Alternatively, the "sample" may be collected by an invasive method, including, but not limited to, surgical biopsy.

[0063] "Expression" and "gene expression" include transcription and/or translation of nucleic acid material.

[0064] As used herein, the term "comprising" and its cognates are used in their inclusive sense; that is, equivalent to the term "including" and its corresponding cognates.

[0065] Conditions that "allow" an event to occur or conditions that are "suitable" for an event to occur, such as hybridization, strand extension, and the like, or "suitable" conditions are conditions that do not prevent such events from occurring. Thus, these conditions permit, enhance, facilitate, and/or are conducive to the event. Such conditions, known in the art and described herein, depend upon, for example, the nature of the nucleotide sequence, temperature, and buffer conditions. These conditions also depend on what event is desired, such as hybridization, cleavage, strand extension or transcription.

[0066] Sequence "mutation," as used herein, refers to any sequence alteration in the sequence of a gene disclosed herein interest in comparison to a reference sequence. A sequence mutation includes single nucleotide changes, or alterations of more than one nucleotide in a sequence, due to mechanisms such as substitution, deletion or insertion. Single nucleotide polymorphism (SNP) is also a sequence mutation as used herein. Because the present invention is based on the relative level of gene expression, mutations in non-coding regions of genes as disclosed herein may also be assayed in the practice of the invention.

[0067] "Detection" includes any means of detecting, including direct and indirect detection of gene expression and changes therein. For example, "detectably less" products may be observed directly or indirectly, and the term indicates any reduction (including the absence of detectable signal). Similarly, "detectably more" product means any increase, whether observed directly or indirectly.

[0068] Increases and decreases in expression of the disclosed sequences are defined in the following terms based upon percent or fold changes over expression in normal cells. Increases may be of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, or 200% relative to expression levels in normal cells. Alternatively, fold increases may be of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 fold over expression levels in normal cells. Decreases may be of 10, 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% relative to expression levels in normal cells.

[0069] Unless defined otherwise all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs.

[0070] Embodiments of the Invention

[0071] In a first aspect, the disclosed invention relates to the identification and use of gene expression patterns (or profiles or "signatures") which discriminate between (or are correlated with) breast cancer survival in a subject treated with tamoxifen (TAM). Such patterns may be determined by the methods of the invention by use of a number of reference cell or tissue samples, such as those reviewed by a pathologist of ordinary skill in the pathology of breast cancer, which reflect breast cancer cells as opposed to normal or other non-cancerous cells. The outcomes experienced by the subjects from whom the samples may be correlated with expression data to identify patterns that correlate with the outcomes following TAM treatment. Because the overall gene expression profile differs from person to person, cancer to cancer, and cancer cell to cancer cell, correlations between certain cells and genes expressed or underexpressed may be made as disclosed herein to identify genes that are capable of discriminating between breast cancer outcomes.

[0072] The present invention may be practiced with any number of the genes believed, or likely to be, differentially expressed with respect to breast cancer outcomes, particularly in cases of ER+ breast cancer. The identification may be made by using expression profiles of various homogenous breast cancer cell populations, which were isolated by microdissection, such as, but not limited to, laser capture microdissection (LCM) of 100-1000 cells. The expression level of each gene of the expression profile may be correlated with a particular outcome. Alternatively, the expression levels of multiple genes may be clustered to identify correlations with particular outcomes.

[0073] Genes with significant correlations to breast cancer survival when the subject is treated with tamoxifen may be used to generate models of gene expressions that would maximally discriminate between outcomes where a subject responds to tamoxifen treatment and outcomes where the tamoxifen treatment is not successful. Alternatively, genes with significant correlations may be used in combination with genes with lower correlations without significant loss of ability to discriminate between outcomes. Such models may be generated by any appropriate means recognized in the art, including, but not limited to, cluster analysis, supported vector machines, neural networks or other algorithm known in the art. The models are capable of predicting the classification of a unknown sample based upon the expression of the genes used for discrimination in the models. "Leave one out" cross-validation may be used to test the performance of various models and to help identify weights (genes) that are uninformative or detrimental to the predictive ability of the models. Cross-validation may also be used to identify genes that enhance the predictive ability of the models.

[0074] The gene(s) identified as correlated with particular breast cancer outcomes relating to tamoxifen treatment by the above models provide the ability to focus gene expression analysis to only those genes that contribute to the ability to identify a subject as likely to have a particular outcome relative to another. The expression of other genes in a breast cancer cell would be relatively unable to provide information concerning, and thus assist in the discrimination of, a breast cancer outcome.

[0075] As will be appreciated by those skilled in the art, the models are highly useful with even a small set of reference gene expression data and can become increasingly accurate with the inclusion of more reference data although the incremental increase in accuracy will likely diminish with each additional datum. The preparation of additional reference gene expression data using genes identified and disclosed herein for discriminating between different tamoxifen treatment outcomes in breast cancer is routine and may be readily performed by the skilled artisan to permit the generation of models as described above to predict the status of an unknown sample based upon the expression levels of those genes.

[0076] To determine the (increased or decreased) expression levels of genes in the practice of the present invention, any method known in the art may be utilized. In one preferred embodiment of the invention, expression based on detection of RNA which hybridizes to the genes identified and disclosed herein is used. This is readily performed by any RNA detection or amplification+detection method known or recognized as equivalent in the art such as, but not limited to, reverse transcription-PCR, the methods disclosed in U.S. patent application Ser. No. 10/062,857 (filed on Oct. 25, 2001) as well as U.S. Provisional Patent Application 60/298,847 (filed Jun. 15, 2001) and 60/257,801 (filed Dec. 22, 2000), and methods to detect the presence, or absence, of RNA stabilizing or destabilizing sequences.

[0077] Alternatively, expression based on detection of DNA status may be used. Detection of the DNA of an identified gene as methylated or deleted may be used for genes that have decreased expression in correlation with a particular breast cancer outcome. This may be readily performed by PCR based methods known in the art, including, but not limited to, Q-PCR. Conversely, detection of the DNA of an identified gene as amplified may be used for genes that have increased expression in correlation with a particular breast cancer outcome. This may be readily performed by PCR based, fluorescent in situ hybridization (FISH) and chromosome in situ hybridization (CISH) methods known in the art.

[0078] Expression based on detection of a presence, increase, or decrease in protein levels or activity may also be used. Detection may be performed by any immunohistochemistry (IHC) based, blood based (especially for secreted proteins), antibody (including autoantibodies against the protein) based, exfoliate cell (from the cancer) based, mass spectroscopy based, and image (including used of labeled ligand) based method known in the art and recognized as appropriate for the detection of the protein. Antibody and image based methods are additionally useful for the localization of tumors after determination of cancer by use of cells obtained by a non-invasive procedure (such as ductal lavage or fine needle aspiration), where the source of the cancerous cells is not known. A labeled antibody or ligand may be used to localize the carcinoma(s) within a patient.

[0079] A preferred embodiment using a nucleic acid based assay to determine expression is by immobilization of one or more sequences of the genes identified herein on a solid support, including, but not limited to, a solid substrate as an array or to beads or bead based technology as known in the art. Alternatively, solution based expression assays known in the art may also be used. The immobilized gene(s) may be in the form of polynucleotides that are unique or otherwise specific to the gene(s) such that the polynucleotide would be capable of hybridizing to a DNA or RNA corresponding to the gene(s). These polynucleotides may be the full length of the gene(s) or be short sequences of the genes (up to one nucleotide shorter than the full length sequence known in the art by deletion from the 5' or 3' end of the sequence) that are optionally minimally interrupted (such as by mismatches or inserted non-complementary basepairs) such that hybridization with a DNA or RNA corresponding to the gene(s) is not affected. Preferably, the polynucleotides used are from the 3' end of the gene, such as within about 350, about 300, about 250, about 200, about 150, about 100, or about 50 nucleotides from the polyadenylation signal or polyadenylation site of a gene or expressed sequence. Polynucleotides containing mutations relative to the sequences of the disclosed genes may also be used so long as the presence of the mutations still allows hybridization to produce a detectable signal.

[0080] The immobilized gene(s) may be used to determine the state of nucleic acid samples prepared from sample breast cell(s) for which the outcome of the sample's subject (e.g. patient from whom the sample is obtained) is not known or for confirmation of an outcome that is already assigned to the sample's subject. Without limiting the invention, such a cell may be from a patient with ER+ breast cancer. The immobilized polynucleotide(s) need only be sufficient to specifically hybridize to the corresponding nucleic acid molecules derived from the sample under suitable conditions. While even a single correlated gene sequence may to able to provide adequate accuracy in discriminating between two breast cancer outcomes, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or eleven or more of the genes identified herein may be used as a subset capable of discriminating may be used in combination to increase the accuracy of the method. The invention specifically contemplates the selection of more than one, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or eleven or more of the genes disclosed in the tables and figures herein for use as a subset in the identification of breast cancer survival outcome.

[0081] Of course 15 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 or more, 120 or more, 130 or more, 140 or more, or all the genes provided in Tables 1 and/or 2 below may be used. "Accession" as used in the context of the Tables herein as well as the present invention refers to the GenBank accession number of a sequence of each gene, the sequences of which are hereby incorporated by reference in their entireties as they are available from GenBank as accessed on the filing date of the present application. P value refers to values assigned as described in the Examples below. The indications of "E-xx" where "xx" is a two digit number refers to alternative notation for exponential figures where "E-xx" is "10.sup.-xx". Thus in combination with the numbers to the left of "E-xx", the value being represented is the numbers to the left times 10.sup.-xx. "Description" as used in the Tables provides a brief identifier of what the sequence/gene encodes.

[0082] Genes with a correlation identified by a p value below or about 0.02, below or about 0.01, below or about 0.005, or below or about 0.001 are preferred for use in the practice of the invention. The present invention includes the use of gene(s) the expression of which identify different ER+ breast cancer outcomes after TAM treatment to permit simultaneous identification of breast cancer survival outcome of a patient based upon assaying a breast cancer sample from said patient.

[0083] In a second aspect, the present invention relates to the identification and use of three sets of sequences for the determination of responsiveness to TAM treatment in ER+ breast cancer. The differential expression of these sequences in breast cancer relative to normal breast cells is used to predict TAM responsiveness in a subject. The identity of the sets of sequences were determined by use of ER+ primary breast cancers from 60 patients uniformly treated with adjuvant TAM. The cancers were analyzed using high-density oligonucleotide microarrays to identify gene expression patterns highly correlated with treatment outcome. Expression levels of IL17BR, CACNA1D, and HOXB13 were strongly predictive of clinical outcome. In contrast, a previously reported 70-gene prognosis signature was not a significant predictor of clinical outcome in these patients. Validation in an independent cohort of 31 TAM treated patients confirmed the predictive utility of these three genes.

[0084] In comparison with existing biomarkers, including ESR1, PGR, ERBB2 and EGFR, these genes are significantly more predictive of TAM response. Multivariate analysis indicated that these three genes were significant predictors of clinical outcome independent of tumor size, nodal status and tumor grade. TAM is the most effective systemic treatment for ER+ breast cancer. ER and progesterone receptor (PR) expression have been the major clinicopathological predictors for response to TAM. However, up to 40% of ER+ tumors fail to respond or develop resistance to TAM. The invention thus provides for the use of the identified biomarkers to allow better patient management by identifying patients who are more likely to benefit from TAM or other endocrine therapy and those who are likely to develop resistance and tumor recurrence.

[0085] As noted herein, the sequences(s) identified by the present invention are expressed in correlation with ER+ breast cells. For example, IL17RB, identified by I.M.A.G.E. Consortium Clusters NM.sub.--018725 and NM.sub.--172234 ("The I.M.A.G.E. Consortium: An Integrated Molecular Analysis of Genomes and their Expression," Lennon et al., 1996, Genomics 33:151-152; see also image.11n1.gov) has been found to be useful in predicting responsiveness to TAM treatment.

[0086] In preferred embodiments of the invention, any sequence, or unique portion thereof, of the IL17RB sequences of the cluster, as well as the UniGene Homo sapiens cluster Hs.5470, may be used. Similarly, any sequence encoding all or a part of the protein encoded by any IL17RB sequence disclosed herein may be used. Consensus sequences of I.M.A.G.E. Consortium clusters are as follows, with the assigned coding region (ending with a termination codon) underlined and preceded by the 5' untranslated and/or non-coding region and followed by the 3' untranslated and/or non-coding region:

1 SEQ ID NO:1 (consensus sequence for IL17RB, transcript variant 1, identified as NM_018725 or NM_018725.2) agcgcagcgt gcgggtggcc tggatcccgc gcagtggccc ggcgatgtcg ctcgtgctgc taagcctggc cgcgctgtgc aggagcgccg taccccgaga gccgaccgtt caatgtggct ctgaaactgg gccatctcca gagtggatgc tacaacatga tctaatcccc ggagacttga gggacctccg agtagaacct gttacaacta gtgttgcaac aggggactat tcaattttga tgaatgtaag ctgggtactc cgggcagatg ccagcatccg cttgttgaag gccaccaaga tttgtgtgac gggcaaaagc aacttccagt cctacagctg tgtgaggtgc aattacacag aggccttcca gactcagacc agaccctctg gtggtaaatg gacattttcc tacatcggct tccctgtaga gctgaacaca gtctatttca ttggggccca taatattcct aatgcaaata tgaatgaaga tggcccttcc atgtctgtga atttcacctc accaggctgc ctagaccaca taatgaaata taaaaaaaag tgtgtcaagg ccggaagcct gtgggatccg aacatcactg cttgtaagaa gaatgaggag acagtagaag tgaacttcac aaccactccc ctgggaaaca gatacatggc tcttatccaa cacagcacta tcatcgggtt ttctcaggtg tttgagccac accagaagaa acaaacgcga gcttcagtgg tgattccagt gactggggat agtgaaggtg ctacggtgca gctgactcca tattttccta cttgtggcag cgactgcatc cgacataaag gaacagttgt gctctgccca caaacaggcg tccctttccc tctggataac aacaaaagca agccgggagg ctggctgcct ctcctcctgc tgtctctgct ggtggccaca tgggtgctgg tggcagggat ctatctaatg tggaggcacg aaaggatcaa gaagacttcc ttttctacca ccacactact gccccccatt aaggttcttg tggtttaccc atctgaaata tgtttccatc acacaatttg ttacttcact gaatttcttc aaaaccattg cagaagtgag gtcatccttg aaaagtggca gaaaaagaaa atagcagaga tgggtccagt gcagtggctt gccactcaaa agaaggcagc agacaaagtc gtcttccttc tttccaatga cgtcaacagt gtgtgcgatg gtacctgtgg caagagcgag ggcagtccca gtgagaactc tcaagacctc ttcccccttg cctttaacct tttctgcagt gatctaagaa gccagattca tctgcacaaa tacgtggtgg tctactttag agagattgat acaaaagacg attacaatgc tctcagtgtc tgccccaagt accacctcat gaaggatgcc actgctttct gtgcagaact tctccatgtc aagcagcagg tgtcagcagg aaaaagatca caagcctgcc acgatggctg ctgctccttg tagcccaccc atgagaagca agagacctta aaggcttcct atcccaccaa ttacagggaa aaaacgtgtg atgatcctga agcttactat gcagcctaca aacagcctta gtaattaaaa cattttatac caataaaatt ttcaaatatt gctaactaat gtagcattaa ctaacgattg gaaactacat ttacaacttc aaagctgttt tatacataga aatcaattac agttttaatt gaaaactata accattttga taatgcaaca ataaagcatc ttcagccaaa catctagtct tccatagacc atgcattgca gtgtacccag aactgtttag ctaatattct atgtttaatt aatgaatact aactctaaga acccctcact gattcactca atagcatctt aagtgaaaaa ccttctatta catgcaaaaa atcattgttt ttaagataac aaaagtaggg aataaacaag ctgaacccac ttttaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa SEQ ID NO:2 (consensus sequence for IL17RB, transcript variant 2, identified as NM_172234 or NM_172234.1) agcgcagcgt gcgggtggcc tggatcccgc gcagtggccc ggcgatgtcg ctcgtgctgc taagcctggc cgcgctgtgc aggagcgccg taccccgaga gccgaccgtt caatgtggct ctgaaactgg gccatctcca gagtggatgc tacaacatga tctaatcccc ggagacttga gggacctccg agtagaacct gttacaacta gtgttgcaac aggggactat tcaattttga tgaatgtaag ctgggtactc cgggcagatg ccagcatccg cttgttgaag gccaccaaga tttgtgtgac gggcaaaagc aacttccagt cctacagctg tgtgaggtgc aattacacag aggccttcca gactcagacc agaccctctg gtggtaaatg gacattttcc tacatcggct tccctgtaga gctgaacaca gtctatttca ttggggccca taatattcct aatgcaaata tgaatgaaga tggcccttcc atgtctgtga atttcacctc accaggctgc ctagaccaca taatgaaata taaaaaaaag tgtgtcaagg ccggaagcct gtgggatccg aacatcactg cttgtaagaa gaatgaggag acagtagaag tgaacttcac aaccactccc ctgggaaaca gatacatggc tcttatccaa cacagcacta tcatcgggtt ttctcaggtg tttgagccac accagaagaa acaaacgcga gcttcagtgg tgattccagt gactggggat agtgaaggtg ctacggtgca ggtaaagttc agtgagctgc tctggggagg gaagggacat agaagactgt tccatcattc attgctttta aggatgagtt ctctcttgtc aaatgcactt ctgccagcag acaccagtta agtggcgttc atgggggctc tttcgctgca gcctccaccg tgctgaggtc aggaggccga cgtggcagtt gtggtccctt ttgcttgtat taatggctgc tgaccttcca aagcactttt tattttcatt ttctgtcaca gacactcagg gatagcagta ccattttact tccgcaagcc tttaactgca agatgaagct gcaaagggtt tgaaatggga aggtttgagt tccaggcagc gtatgaactc tggagagggg ctgccagtcc tctctgggcc gcagcggacc cagctggaac acaggaagtt ggagcagtag gtgctccttc acctctcagt atgtctcttt caactctagt ttttgaggtg gggacacagg aggtccagtg ggacacagcc actccccaaa gagtaaggag cttccatgct tcattccctg gcataaaaag tgctcaaaca caccagaggg ggcaggcacc agccagggta tgatggctac tacccttttc tggagaacca tagacttccc ttactacagg gacttgcatg tcctaaagca ctggctgaag gaagccaaga ggatcactgc tgctcctttt ttctagagga aatgtttgtc tacgtggtaa gatatgacct agccctttta ggtaagcgaa ctggtatgtt agtaacgtgt acaaagttta ggttcagacc ccgggagtct tgggcacgtg ggtctcgggt cactggtttt gactttaggg ctttgttaca gatgtgtgac caaggggaaa atgtgcatga caacactaga ggtatgggcg aagccagaaa gaagggaagt tttggctgaa gtaggagtct tggtgagatt ttgctctgat gcatggtgtg aactttctga gcctcttgtt tttcctcagc tgactccata ttttcctact tgtggcagcg actgcatccg acataaagga acagttgtgc tctgcccaca aacaggcgtc cctttccctc tggataacaa caaaagcaag ccgggaggct ggctgcctct cctcctgctg tctctgctgg tggccacatg ggtgctggtg gcagggatct atctaatgtg gaggcacgaa aggatcaaga agacttcctt ttctaccacc acactactgc cccccattaa ggttcttgtg gtttacccat ctgaaatatg tttccatcac acaatttgtt acttcactga atttcttcaa aaccattgca gaagtgaggt catccttgaa aagtggcaga aaaagaaaat agcagagatg ggtccagtgc agtggcttgc cactcaaaag aaggcagcag acaaagtcgt cttccttctt tccaatgacg tcaacagtgt gtgcgatggt acctgtggca agagcgaggg cagtcccagt gagaactctc aagacctctt cccccttgcc tttaaccttt tctgcagtga tctaagaagc cagattcatc tgcacaaata cgtggtggtc tactttagag agattgatac aaaagacgat tacaatgctc tcagtgtctg ccccaagtac cacctcatga aggatgccac tgctttctgt gcagaacttc tccatgtcaa gcagcaggtg tcagcaggaa aaagatcaca agcctgccac gatggctgct gctccttgta gcccacccat gagaagcaag agaccttaaa ggcttcctat cccaccaatt acagggaaaa aacgtgtgat gatcctgaag cttactatgc agcctacaaa cagccttagt aattaaaaca ttttatacca ataaaatttt caaatattgc taactaatgt agcattaact aacgattgga aactacattt acaacttcaa agctgtttta tacatagaaa tcaattacag ttttaattga aaactataac cattttgata atgcaacaat aaagcatctt cagccaaaca tctagtcttc catagaccat gcattgcagt gtacccagaa ctgtttagct aatattctat gtttaattaa tgaatactaa ctctaagaac ccctcactga ttcactcaat agcatcttaa gtgaaaaacc ttctattaca tgcaaaaaat cattgttttt aagataacaa aagtagggaa taaacaagct gaacccactt ttaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaa

[0087] I.M.A.G.E. Consortium Clone ID numbers and the corresponding GenBank accession numbers of sequences identified as belonging to the I.M.A.G.E. Consortium and UniGene clusters, are listed below. Also included are sequences that are not identified as having a Clone ID number but still identitied as being those of IL17RB. The sequences include those of the "sense" and complementary strands sequences corresponding to IL17RB. The sequence of each GenBank accession number is presented in the attached Appendix.

2 Clone ID numbers GenBank accession numbers 2985728 AW675096, AW673932, BC000980 5286745 BI602183 5278067 BI458542 5182255 BI823321 924000 AA514396 3566736 BF110326 3195409 BE466508 3576775 BF740045 2772915 AW299271 1368826 AA836217 1744837 AI203628 2285564 AI627783 2217709 AI744263 2103651 AI401622 2419487 AI826949 3125592 BE047352 2284721 AI911549 3643302 BF194822 1646910 AI034244 1647001 AI033911 3323709 BF064177 1419779 AA847767 2205190 AI538624 2295838 AI913613 2461335 AI942234 2130362 AI580483 2385555 AI831909 2283817 AI672344 2525596 AW025192 454687 AA677205 1285273 AA721647 3134106 BF115018 342259 W61238, W61239 1651991 AI032064 2687714 AW236941 3302808 BG057174 2544461 AW058532 122014 T98360, T98361 2139250 AI470845 2133899 AI497731 121300 T96629, T96740 162274 H25975, H25941 3446667 BE539514, BX282554 156864 R74038, R74129 4611491 BG433769 4697316 BG530489 429376 AA007528, AA007529 5112415 BI260259 701357 AA287951, AA287911 121909 T97852, T97745 268037 N40294 1307489 AA809841 1357543 AA832389 48442 H14692 1302619 AA732635 1562857 AA928257 1731938 AI184427 1896025 AI298577 2336350 AI692717 1520997 AA910922 240506 H90761 2258560 AI620122 1569921 AI793318, AA962325, AI733290 6064627 BQ226353 299018 W04890 5500181 BM455231 2484011 BI492426 4746376 BG674622 233783 BX111256 1569921 BX117618 450450 AA682806 1943085 AI202376 2250390 AI658949 4526156 BG403405 3249181 BE673417 2484395 AW021469 30515867 CF455736 2878155 AW339874 4556884 BG399724 3254505 BF475787 3650593 BF437145 233783 H64601 None (mRNA AF212365, AF208110, AF208111, AF250309, sequences) AK095091 None BM983744, CB305764, BM715988, BM670929, BI792416, BI715216, N56060, CB241389, AV660618, BX088671, CB154426, CA434589, CA412162, CA314073, BF921554, BF920093, AV685699, AV650175, BX483104, CD675121, BE081436, AW970151, AW837146, AW368264, D25960, AV709899, BX431018, AL535617, AL525465, BX453536, BX453537, AV728945, AV728939, AV727345

[0088] In one preferred embodiment, any sequence, or unique portion thereof, of the following IL17RB sequence, identified by AF208111 or AF208111.1, may be used in the practice of the invention.

3 SEQ ID NO:3 (sequence for IL17RB): CGGCGATGTCGCTCGTGCTGATAAGCCTGGCCGCGCTGTGCAGGAGCGCCGTACCCCGAG AGCCGACCGTTCAATGTGGCTCTGAAACTGGGCCATCTCCAGAGTGGATGCTACAACATG ATCTAATCCCCGGAGACTTGAGGGACCTCCGAGTAGAACCTGTTACAACTAGTGTTGCAA CAGGGGACTATTCAATTTTGATGAATGTAAGCTGGGTACTCCGGGCAGATGCCAGCATCC GCTTGTTGAAGGCCACCAAGATTTGTGTGACGGGCAAAAGCAACTTCCAGTCCTACAGCT GTGTGAGGTGCAATTACACAGAGGCCTTCCAGACTCAGACCAGACCCTCTGGTGGTAAAT GGACATTTTCCTATATCGGCTTCCCTGTAGAGCTGAACACAGTCTATTTCATTGGGGCCC ATAATATTCCTAATGCAAATATGAATGAAGATGGCCCTTCCATGTCTGTGAATTTCACCT CACCAGGCTGCCTAGACCACATAATGAAATATAAAAAAAAGTGTGTCAAGGCCGGAAGCC TGTGGGATCCGAACATCACTGCTTGTAAGAAGAATGAGGAGACAGTAGAAGTGAACTTCA CAACCACTCCCCTGGGAAACAGATACATGGCTCTTATCCAACACAGCACTATCATCGGGT TTTCTCAGGTGTTTGAGCCACACCAGAAGAAACAAACGCGAGCTTCAGTGGTGATTCCAG TGACTGGGGATAGTGAAGGTGCTACGGTGCAGGTAAAGTTCAGTGAGCTGCTCTGGGGAG GGAAGGGACATAGAAGACTGTTCCATCATTCATTGCTTTTAAGGATGAGTTCTCTCTTGT CAAATGCACTTCTGCCAGCAGACACCAGTTAAGTGGCGTTCATGGGGGTTCTTTCGCTGC AGCCTCCACCGTGCTGAGGTCAGGAGGCCGACGTGGCAGTTGTGGTCCCTTTTGCTTGTA TTAATGGCTGCTGACCTTCCAAAGCACTTTTTATTTTCATTTTCTGTCACAGACACTCAG GGATAGCAGTACCATTTTACTTCCGCAAGCCTTTAACTGCAAGATGAAGCTGCAAAGGGT TTGAAATGGGAAGGTTTGAGTTCCAGGCAGCGTATGAACTCTGGAGAGGGGCTGCCAGTC CTCTCTGGGCCGCAGCGGACCCAGCTGGAACACAGGAAGTTGGAGCAGTAGGTGCTCCTT CACCTCTCAGTATGTCTCTTTCAACTCTAGTTTTTGAAGTGGGGACACAGGAAGTCCAGT GGGGACACAGCCACTCCCCAAAGAATAAGGAACTTCCATGCTTCATTCCCTGGCATAAAA AGTGNTCAAACACACCAGAGGGGGCAGGCACCAGCCAGGGTATGATGGGTACTACCCTTT TCTGGAGAACCATAGACTTCCCTTACTACAGGGACTTGCATGTCCTAAAGCACTGGCTGA AGGAAGCCAAGAGGATCACTGCTGCTCCTTTTTTGTAGAGGAAATGTTTGTGTACGTGGT AAGATATGACCTAGCCCTTTTAGGTAAGCGAACTGGTATGTTAGTAACGTGTACAAAGTT TAGGTTCAGACCCCGGGAGTCTTGGGCATGTGGGTCTCGGGTCACTGGTTTTGACTTTAG GGCTTTGTTACAGATGTGTGACCAAGGGGAAAATGTGCATGACAACACTAGAGGTAGGGG CGAAGCCAGAAAGAAGGGAAGTTTTGGCTGAAGTAGGAGTCTTGGTGAGATTTTGCTGTG ATGCATGGTGTGAACTTTCTGAGCCTCTTGTTTTTCCTCAGCTGACTCCATATTTTCCTA CTTGTGGCAGCGACTGCATCCGACATAAAGGAACAGTTGTGCTCTGCCCACAAACAGGCG TCCCTTTCCCTCTGGATAACAACAAAAGCAAGCCGGGAGGCTGGCTGCCTCTCCTCCTGC TGTCTCTGCTGGTGGCCACATGGGTGCTGGTGGCAGGGATCTATCTAATGTGGAGGCACG AAAGGATCAAGAAGACTTCCTTTTCTACCACCACACTACTGCCCCCCATTAAGGTTCTTG TGGTTTACCCATCTGAAATATGTTTCCATCACACAATTTGTTACTTCACTGAATTTCTTC AAAACCATTGCAGAAGTGAGGTCATCCTTGAAAAGTGGCAGAAAAAGAAAATAGCAGAGA TGGGTCCAGTGCAGTGGCTTGCCACTCAAAAGAAGGCAGCAGACAAAGTCGTCTTCCTTC TTTCCAATGACGTCAACAGTGTGTGCGATGGTACCTGTGGCAAGAGCGAGGGCAGTCCCA GTGAGAACTCTCAAGACCTCTTCCCCCTTGCCTTTAACCTTTTCTGCAGTGATCTAAGAA GCCAGATTCATCTGCACAAATACGTGGTGGTCTACTTTAGAGAGATTGATACAAAAGACG ATTACAATGCTCTCAGTGTCTGCCCCAAGTACCACTTCATGAAGGATGCCACTGCTTTCT GTGCAGAACTTCTCCATGTCAAGCAGCAGGTGTCAGCAGGAAAAAGATCACAAGCCTGCC ACGATGGCTGCTGCTCCTTGTAGCCCACCCATGAGAAGCAAGAGACCTTAAAGGCTTCCT ATCCCACCAATTACAGGGAAAAAACGTGTGATGATCCTGAAGCTTACTATGCAGCCTACA AACAGCCTTAGTAATTAAAACATTTTATACCAATAAAATTTTCAAATATTACTAACTAAT GTAGCATTAACTAACGATTGGAAACTACATTTACAACTTCAAAGCTGTTTTATACATAGA AATCAATTACAGCTTTAATTGAAAACTGTAACCATTTTGATAATGCAACAATAAAGCATC TTCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0089] In another set of preferred embodiments of the invention, any sequence, or unique portion thereof, of the CACNA1D sequences of the I.M.A.G.E. Consortium cluster NM.sub.--000720, as well as the UniGene Homo sapiens cluster Hs.399966, may be used. Similarly, any sequence encoding all or a part of the protein encoded by any CACNA1D sequence disclosed herein may be used. The consensus sequence of the I.M.A.G.E. Consortium cluster is as follows, with the assigned coding region (ending with a termination codon) underlined and preceded by the 5' untranslated and/or non-coding region and followed by the 3' untranslated and/or non-coding region:

4 SEQ ID NO:4 (consensus sequence for CACNA1D, identified as NM_000720 or NM_000720.1) agaataaggg cagggaccgc ggctcctatc tcttggtgat ccccttcccc attccgcccc cgcctcaacg cccagcacag tgccctgcac acagtagtcg ctcaataaat gttcgtggat gatgatgatg atgatgatga aaaaaatgca gcatcaacgg cagcagcaag cggaccacgc gaacgaggca aactatgcaa gaggcaccag acttcctctt tctggtgaag gaccaacttc tcagccgaat agctccaagc aaactgtcct gtcttggcaa gctgcaatcg atgctgctag acaggccaag gctgcccaaa ctatgagcac ctctgcaccc ccacctgtag gatctctctc ccaaagaaaa cgtcagcaat acgccaagag caaaaaacag ggtaactcgt ccaacagccg acctgcccgc gcccttttct gtttatcact caataacccc atccgaagag cctgcattag tatagtggaa tggaaaccat ttgacatatt tatattattg gctatttttg ccaattgtgt ggccttagct atttacatcc cattccctga agatgattct aattcaacaa atcataactt ggaaaaagta gaatatgcct tcctgattat ttttacagtc gagacatttt tgaagattat agcgtatgga ttattgctac atcctaatgc ttatgttagg aatggatgga atttactgga ttttgttata gtaatagtag gattgtttag tgtaattttg gaacaattaa ccaaagaaac agaaggcggg aaccactcaa gcggcaaatc tggaggcttt gatgtcaaag ccctccgtgc ctttcgagtg ttgcgaccac ttcgactagt gtcaggggtg cccagtttac aagttgtcct gaactccatt ataaaagcca tggttcccct ccttcacata gcccttttgg tattatttgt aatcataatc tatgctatta taggattgga actttttatt ggaaaaatgc acaaaacatg tttttttgct gactcagata tcgtagctga agaggaccca gctccatgtg cgttctcagg gaatggacgc cagtgtactg ccaatggcac ggaatgtagg agtggctggg ttggcccgaa cggaggcatc accaactttg ataactttgc ctttgccatg cttactgtgt ttcagtgcat caccatggag ggctggacag acgtgctcta ctgggtaaat gatgcgatag gatgggaatg gccatgggtg tattttgtta gtctgatcat ccttggctca tttttcgtcc ttaacctggt tcttggtgtc cttagtggag aattctcaaa ggaaagagag aaggcaaaag cacggggaga tttccagaag ctccgggaga agcagcagct ggaggaggat ctaaagggct acttggattg gatcacccaa gctgaggaca tcgatccgga gaatgaggaa gaaggaggag aggaaggcaa acgaaatact agcatgccca ccagcgagac tgagtctgtg aacacagaga acgtcagcgg tgaaggcgag aaccgaggct gctgtggaag tctctggtgc tggtggagac ggagaggcgc ggccaaggcg gggccctctg ggtgtcggcg gtggggtcaa gccatctcaa aatccaaact cagccgacgc tggcgtcgct ggaaccgatt caatcgcaga agatgtaggg ccgccgtgaa gtctgtcacg ttttactggc tggttatcgt cctggtgttt ctgaacacct taaccatttc ctctgagcac tacaatcagc cagattggtt gacacagatt caagatattg ccaacaaagt cctcttggct ctgttcacct gcgagatgct ggtaaaaatg tacagcttgg gcctccaagc atatttcgtc tctcttttca accggtttga ttgcttcgtg gtgtgtggtg gaatcactga gacgatcctg gtggaactgg aaatcatgtc tcccctgggg atctctgtgt ttcggtgtgt gcgcctctta agaatcttca aagtgaccag gcactggact tccctgagca acttagtggc atccttatta aactccatga agtccatcgc ttcgctgttg cttctgcttt ttctcttcat tatcatcttt tccttgcttg ggatgcagct gtttggcggc aagtttaatt ttgatgaaac gcaaaccaag cggagcacct ttgacaattt ccctcaagca cttctcacag tgttccagat cctgacaggc gaagactgga atgctgtgat gtacgatggc atcatggctt acgggggccc atcctcttca ggaatgatcg tctgcatcta cttcatcatc ctcttcattt gtggtaacta tattctactg aatgtcttct tggccatcgc tgtagacaat ttggctgatg ctgaaagtct gaacactgct cagaaagaag aagcggaaga aaaggagagg aaaaagattg ccagaaaaga gagcctagaa aataaaaaga acaacaaacc agaagtcaac cagatagcca acagtgacaa caaggttaca attgatgact atagagaaga ggatgaagac aaggacccct atccgccttg cgatgtgcca gtaggggaag aggaagagga agaggaggag gatgaacctg aggttcctgc cggaccccgt cctcgaagga tctcggagtt gaacatgaag gaaaaaattg cccccatccc tgaagggagc gctttcttca ttcttagcaa gaccaacccg atccgcgtag gctgccacaa gctcatcaac caccacatct tcaccaacct catccttgtc ttcatcatgc tgagcagcgc tgccctggcc gcagaggacc ccatccgcag ccactccttc cggaacacga tactgggtta ctttgactat gccttcacag ccatctttac tgttgagatc ctgttgaaga tgacaacttt tggagctttc ctccacaaag gggccttctg caggaactac ttcaatttgc tggatatgct ggtggttggg gtgtctctgg tgtcatttgg gattcaatcc agtgccatct ccgttgtgaa gattctgagg gtcttaaggg tcctgcgtcc cctcagggcc atcaacagag caaaaggact taagcacgtg gtccagtgcg tcttcgtggc catccggacc atcggcaaca tcatgatcgt cactaccctc ctgcagttca tgtttgcctg tatcggggtc cagttgttca aggggaagtt ctatcgctgt acggatgaag ccaaaagtaa ccctgaagaa tgcaggggac ttttcatcct ctacaaggat ggggatgttg acagtcctgt ggtccgtgaa cggatctggc aaaacagtga tttcaacttc gacaacgtcc tctctgctat gatggcgctc ttcacagtct ccacgtttga gggctggcct gcgttgctgt ataaagccat cgactcgaat ggagagaaca tcggcccaat ctacaaccac cgcgtggaga tctccatctt cttcatcatc tacatcatca ttgtagcttt cttcatgatg aacatctttg tgggctttgt catcgttaca tttcaggaac aaggagaaaa agagtataag aactgtgagc tggacaaaaa tcagcgtcag tgtgttgaat acgccttgaa agcacgtccc ttgcggagat acatccccaa aaacccctac cagtacaagt tctggtacgt ggtgaactct tcgcctttcg aatacatgat gtttgtcctc atcatgctca acacactctg cttggccatg cagcactacg agcagtccaa gatgttcaat gatgccatgg acattctgaa catggtcttc accggggtgt tcaccgtcga gatggttttg aaagtcatcg catttaagcc taaggggtat tttagtgacg cctggaacac gtttgactcc ctcatcgtaa tcggcagcat tatagacgtg gccctcagcg aagcggaccc aactgaaagt gaaaatgtcc ctgtcccaac tgctacacct gggaactctg aagagagcaa tagaatctcc atcacctttt tccgtctttt ccgagtgatg cgattggtga agcttctcag caggggggaa ggcatccgga cattgctgtg gacttttatt aagtcctttc aggcgctccc gtatgtggcc ctcctcatag ccatgctgtt cttcatctat gcggtcattg gcatgcagat gtttgggaaa gttgccatga gagataacaa ccagatcaat aggaacaata acttccagac gtttccccag gcggtgctgc tgctcttcag gtgtgcaaca ggtgaggcct ggcaggagat catgctggcc tgtctcccag ggaagctctg tgaccctgag tcagattaca accccgggga ggagtataca tgtgggagca actttgccat tgtctatttc atcagttttt acatgctctg tgcatttctg atcatcaatc tgtttgtggc tgtcatcatg gataatttcg actatctgac ccgggactgg tctattttgg ggcctcacca tttagatgaa ttcaaaagaa tatggtcaga atatgaccct gaggcaaagg gaaggataaa acaccttgat gtggtcactc tgcttcgacg catccagcct cccctggggt ttgggaagtt atgtccacac agggtagcgt gcaagagatt agttgccatg aacatgcctc tcaacagtga cgggacagtc atgtttaatg caaccctgtt tgctttggtt cgaacggctc ttaagatcaa gaccgaaggg aacctggagc aagctaatga agaacttcgg gctgtgataa agaaaatttg gaagaaaacc agcatgaaat tacttgacca agttgtccct ccagctggtg atgatgaggt aaccgtgggg aagttctatg ccactttcct gatacaggac tactttagga aattcaagaa acggaaagaa caaggactgg tgggaaagta ccctgcgaag aacaccacaa ttgccctaca ggcgggatta aggacactgc atgacattgg gccagaaatc cggcgtgcta tatcgtgtga tttgcaagat gacgagcctg aggaaacaaa acgagaagaa gaagatgatg tgttcaaaag aaatggtgcc ctgcttggaa accatgtcaa tcatgttaat agtgatagga gagattccct tcagcagacc aataccaccc accgtcccct gcatgtccaa aggccttcaa ttccacctgc aagtgatact gagaaaccgc tgtttcctcc agcaggaaat tcggtgtgtc ataaccatca taaccataat tccataggaa agcaagttcc cacctcaaca aatgccaatc tcaataatgc caatatgtcc aaagctgccc atggaaagcg gcccagcatt gggaaccttg agcatgtgtc tgaaaatggg catcattctt cccacaagca tgaccgggag cctcagagaa ggtccagtgt gaaaagaacc cgctattatg aaacttacat taggtccgac tcaggagatg aacagctccc aactatttgc cgggaagacc cagagataca tggctatttc agggaccccc actgcttggg ggagcaggag tatttcagta gtgaggaatg ctacgaggat gacagctcgc ccacctggag caggcaaaac tatggctact acagcagata cccaggcaga aacatcgact ctgagaggcc ccgaggctac catcatcccc aaggattctt ggaggacgat gactcgcccg tttgctatga ttcacggaga tctccaagga gacgcctact acctcccacc ccagcatccc accggagatc ctccttcaac tttgagtgcc tgcgccggca gagcagccag gaagaggtcc cgtcgtctcc catcttcccc catcgcacgg ccctgcctct gcatctaatg cagcaacaga tcatggcagt tgccggccta gattcaagta aagcccagaa gtactcaccg agtcactcga cccggtcgtg ggccacccct ccagcaaccc ctccctaccg ggactggaca ccgtgctaca cccccctgat ccaagtggag cagtcagagg ccctggacca ggtgaacggc agcctgccgt ccctgcaccg cagctcctgg tacacagacg agcccgacat ctcctaccgg actttcacac cagccagcct gactgtcccc agcagcttcc ggaacaaaaa cagcgacaag cagaggagtg cggacagctt ggtggaggca gtcctgatat ccgaaggctt gggacgctat gcaagggacc caaaatttgt gtcagcaaca aaacacgaaa tcgctgatgc ctgtgacctc accatcgacg agatggagag tgcagccagc accctgctta atgggaacgt gcgtccccga gccaacgggg atgtgggccc cctctcacac cggcaggact atgagctaca ggactttggt cctggctaca gcgacgaaga gccagaccct gggagggatg aggaggacct ggcggatgaa atgatatgca tcaccacctt gtagccccca gcgaggggca gactggctct ggcctcaggt ggggcgcagg agagccaggg gaaaagtgcc tcatagttag gaaagtttag gcactagttg ggagtaatat tcaattaatt agacttttgt ataagagatg tcatgcctca agaaagccat aaacctggta ggaacaggtc ccaagcggtt gagcctggca gagtaccatg cgctcggccc cagctgcagg aaacagcagg ccccgccctc tcacagagga tgggtgagga ggccagacct gccctgcccc attgtccaga tgggcactgc tgtggagtct gcttctccca tgtaccaggg caccaggccc acccaactga aggcatggcg gcggggtgca ggggaaagtt aaaggtgatg acgatcatca cacctcgtgt cgttacctca gccatcggtc tagcatatca gtcactgggc ccaacatatc catttttaaa ccctttcccc caaatacact gcgtcctggt tcctgtttag ctgttctgaa ata

[0090] I.M.A.G.E. Consortium Clone ID numbers and the corresponding GenBank accession numbers of sequences identified as belonging to the I.M.A.G.E. Consortium and UniGene clusters, are listed below. Also included are sequences that are not identified as having a Clone ID number but still identitied as being those of CACNA1D. The sequences include those of the "sense" and complementary strands sequences corresponding to CACNA1D. The sequence of each GenBank accession number is presented in the attached Appendix.

5 Clone ID numbers GenBank accession numbers 5676430 BM128550 5197948 BI755471 6027638 BQ549084, BQ549571 2338956 AI693324 36581 R25307, R46658 49630 H29256, H29339 4798765 BG716371 2187310 AI537488 838231 AA458692 2111614 AI393327 2183482 AI520947 1851007 AI248998 1675503 AI075844 2434923 AI869807 2434924 AI869800 1845827 AI243110 2511756 AI955764 628568 AA192669, AA192157 2019331 AI361691 2337381 AI914244 2503579 AW008769 2503626 AW008794 1160989 AA877582 1653475 AI051972 1627755 AI017959 287750 N79331, N62240 1867677 AI240933 1618303 AI015031 1881344 AI290994 1408031 AA861160 1557035 AA915941 956303 AA493341 2148234 AI467998 1499899 AA885585 1647592 AI033648 2341185 AI697633 981603 AA523647 6281678 BQ710377 6278348 BQ706920 5876024 BQ016847 6608849 CA943595 5440464 BM008196 5209489 BI769856 5183025 BI758971 880540 AA468565 757337 AA437099 6608849 CA867864 461797 AA682690 434787 AA701888 6151588 BU182632 6295618 BQ898429 6300779 BQ711800 434811 AA703120 1568025 AA978315 3220210 BE550599 3214121 BE502741 3009312 AW872382 2733394 AW444663 2872156 AW341279 30514550 CF456750 2718456 AW139850 2543682 AW029633 2492730 AI963788 2545866 AI951788 2272081 AI680744 2152336 AI601252 2146429 AI459166 1274498 AA885750 2272081 BX092736 287750 BX114568 3233645 BE672659 289209 N78509, N73668 277086 N46744, N39597 3272340 BF439267 3273859 BF436153 3568401 BF110611 None (mRNA M76558, AF088004, M83566 sequences) None CB410657, BQ372430, BQ366601, BQ324528, BQ318830, AL708030, BM509161, N85902, BQ774355, CA774243, CA436347, CA389011, BU679327, BU608029, BU073743, BE175413, AW969248, AI908115, BF754485, BI015409, BG202552, BF883669, BF817590, BF807128, BF806160, BF805244, BF805235, BF805080, T27949, BE836638, BE770685, BE769065,

[0091] In one preferred embodiment, any sequence, or unique portion thereof, of the following CACNA1D sequence, identified by AI240933 or AI240933.1, may be used in the practice of the invention.

6 SEQ ID NO:5 (sequence for CACNA1D): TTTTTTTTTTTTTTTTTTTTTCTTACAAAGAAAAATTTAATATTCGATGAGAGGTTGAAC CAGGCTTAAAGCAGACATACTAGGAAATGGTGCAGCCTGTAAGAATGCCAGTTTGTAAGT ACTGACTTTGGAAAAGATCATCGCCTCTATCAGACACTTAGGGTCCTGGTCTGGCAATTT TGGCCTGATGTGATGCCACAAGACCCAACAGAGAGAGACACAGAGTCCAGGATAATGTTG ACAGTGGTGTAGCCCTTTAGGAGAAATGGCGCTCCCTGCGGCTGGTATTAGGTTACCATT GGCACCGAAGGAACCAGGAGGATAAGAATATCCATAATTTCAGAGCTGCCCTGGCACAGT ACCTGCCCCGTCGGAGGCTCTCACTGGCAAATGACAGCTCTGTGCAAGGAGCACTCCCAA GTATAAAAATTATTACACAGTTTTATTCTGAAGAACATTTTGCATTTTAATAAAAAAGGA TTTATGTCAGGAAAGAGTCATTTACAAACCTTGAAGTGTTTTTGCCTGGATCAGAGTAAG AATGTCTTAAGAAGAGGTTTGTAAGGTCTTCATAACAAAGTGGTGTTTGTTATTTACAAA AAAAAAAAAAAAAAAAATTAACAGGTTGTCTGTATACTATTAAAAATTTTGGACCAAAAA AAAAAAAAAAAAAAA

[0092] In another set of preferred embodiments of the invention, any sequence, or unique portion thereof, of the HOXB13 sequences of the I.M.A.G.E. Consortium cluster NM.sub.--006361, as well as the UniGene Homo sapiens cluster Hs.66731, may be used. Similarly, any sequence encoding all or a part of the protein encoded by any HOXB13 sequence disclosed herein may be used. The consensus sequence of the I.M.A.G.E. Consortium cluster is as follows, with the assigned coding region (ending with a termination codon) underlined and preceded by the 5' untranslated and/or non-coding region and followed by the 3' untranslated and/or non-coding region:

7 SEQ ID NO:6 (consensus sequence for HOXB13, identified as NM_006361 or NM_006361.2) cgaatgcagg cgacttgcga gctgggagcg atttaaaacg ctttggattc ccccggcctg ggtggggaga gcgagctggg tgccccctag attccccgcc cccgcacctc atgagccgac cctcggctcc atggagcccg gcaattatgc caccttggat ggagccaagg atatcgaagg cttgctggga gcgggagggg ggcggaatct ggtcgcccac tcccctctga ccagccaccc agcggcgcct acgctgatgc ctgctgtcaa ctatgccccc ttggatctgc caggctcggc ggagccgcca aagcaatgcc acccatgccc tggggtgccc caggggacgt ccccagctcc cgtgccttat ggttactttg gaggcgggta ctactcctgc cgagtgtccc ggagctcgct gaaaccctgt gcccaggcag ccaccctggc cgcgtacccc gcggagactc ccacggccgg ggaagagtac cccagtcgcc ccactgagtt tgccttctat ccgggatatc cgggaaccta ccacgctatg gccagttacc tggacgtgtc tgtggtgcag actctgggtg ctcctggaga accgcgacat gactccctgt tgcctgtgga cagttaccag tcttgggctc tcgctggtgg ctggaacagc cagatgtgtt gccagggaga acagaaccca ccaggtccct tttggaaggc agcatttgca gactccagcg ggcagcaccc tcctgacgcc tgcgcctttc gtcgcggccg caagaaacgc attccgtaca gcaaggggca gttgcgggag ctggagcggg agtatgcggc taacaagttc atcaccaagg acaagaggcg caagatctcg gcagccacca gcctctcgga gcgccagatt accatctggt ttcagaaccg ccgggtcaaa gagaagaagg ttctcgccaa ggtgaagaac agcgctaccc cttaagagat ctccttgcct gggtgggagg agcgaaagtg ggggtgtcct ggggagacca gaaacctgcc aagcccaggc tggggccaag gactctgctg agaggcccct agagacaaca cccttcccag gccactggct gctggactgt tcctcaggag cggcctgggt acccagtatg tgcagggaga cggaacccca tgtgacaggc ccactccacc agggttccca aagaacctgg cccagtcata atcattcatc ctcacagtgg caataatcac gataaccagt

[0093] I.M.A.G.E. Consortium Clone ID numbers and the corresponding GenBank accession numbers of sequences identified as belonging to the I.M.A.G.E. Consortium and Uni Gene clusters, are listed below. Also included are sequences that are not identified as having a Clone ID number but still identitied as being those of HOXB13. The sequences include those of the "sense" and complementary strands sequences corresponding to HOXB13. The sequence of each GenBank accession number is presented in the attached Appendix.

8 Clone ID numbers GenBank accession numbers 4250486 BF676461, BC007092 5518335 BM462617 4874541 BG752489 4806039 BG778198 3272315 CB050884, CB050885 4356740 BF965191 6668163 BU930208 1218366 AA807966 2437746 AI884491 1187697 AA652388 3647557 BF446158 1207949 AA657924 1047774 AA644637 3649397 BF222357 971664 AA527613 996191 AA533227 813481 AA456069, AA455572, BX117624 6256333 BQ673782 2408470 AI814453 2114743 AI417272 998548 AA535663 2116027 AI400493 3040843 AW779219 1101311 AA594847 1752062 AI150430 898712 AA494387 1218874 AA662643 2460189 AI935940 986283 AA532530 1435135 AA857572 1871750 AI261980 3915135 BE888751 2069668 AI378797 667188 AA234220, AA236353 1101561 AA588193 1170268 AI821103, AI821851, AA635855 2095067 AI420753 4432770 BG180547 783296 AA468306, AA468232 3271646 CB050115, CB050116 1219276 AA661819 30570598 CF146837 30570517 CF146763 30568921 CF144902 3099071 CF141511 3096992 CF139563 3096870 CF139372 3096623 CF139319 3096798 CF139275 30572408 CF122893 2490082 AI972423 2251055 AI918975 2419308 AI826991 2249105 AI686312 2243362 AI655923 30570697 CF146922 3255712 BF476369 3478356 BF057410 3287977 BE645544 3287746 BE645408 3621499 BE388501 30571128 CF147366 30570954 CF147143 None (mRNA BT007410, BC007092, U57052, U81599 sequences) None CB120119, CB125764, AU098628, CB126130, BI023924, BM767063, BM794275, BQ363211, BM932052, AA357646, AW609525, CB126919, AW609336, AW609244, BF855145, AU126914, CB126449, AW582404, BX641644

[0094] In one preferred embodiment, any sequence, or unique portion thereof, of the following HOXB13 sequence, identified by BC007092 or BC007092.1, may be used in the practice of the invention.

9 SEQ ID NO:7 (sequence for HOXB13): GGATTCCCCCGGCCTGGGTGGGGAGAGCGAGCTGGGTGCCCCCTAGATTCCCCGCCCCCG CACCTCATGAGCCGACCCTCGGCTCCATGGAGCCCGGCAATTATGCCACCTTGGATGGAG CCAAGGATATCGAAGGCTTGCTGGGAGCGGGAGGGGGGCGGAATCTGGTCGCCCACTCCC CTCTGACCAGCCACCCAGCGGCGCCTACGCTGATGCCTGCTGTCAACTATGCCCCCTTGG ATCTGCCAGGCTCGGCGGAGCCGCCAAAGCAATGCCACCCATGCCCTGGGGTGCCCCAGG GGACGTCCCCAGCTCCCGTGCCTTATGGTTACTTTGGAGGCGGGTACTACTCCTGCCGAG TGTCCCGGAGCTCGCTGAAACCCTGTGCCCAGGCAGCCACCCTGGCCGCGTACCCCGCGG AGACTCCCACGGCCGGGGAAGAGTACCCCAGCCGCCCCACTGAGTTTGCCTTCTATCCGG GATATCCGGGAACCTACCAGCCTATGGCCAGTTACCTGGACGTGTCTGTGGTGCAGACTC TGGGTGCTCCTGGAGAACCGCGACATGACTCCCTGTTGCCTGTGGACAGTTACCAGTCTT GGGCTCTCGCTGGTGGCTGGAACAGCCAGATGTGTTGCCAGGGAGAACAGAACCCACCAG GTCCCTTTTGGAAGGCAGCATTTGCAGACTCCAGCGGGCAGCACCCTCCTGACGCCTGCG CCTTTCGTCGCGGCCGCAAGAAACGCATTCCGTACAGCAAGGGGCAGTTGCGGGAGCTGG AGCGGGAGTATGCGGCTAACAAGTTCATCACCAAGGACAAGAGGCGCAAGATCTCGGCAG CCACCAGCCTCTCGGAGCGCCAGATTACCATCTGGTTTCAGAACCGCCGGGTCAAAGAGA AGAAGGTTCTCGCCAAGGTGAAGAACAGCGCTACCCCTTAAGAGATCTCCTTGCCTGGGT GGGAGGAGCGAAAGTGGGGGTGTCCTGGGGAGACCAGGAACCTGCCAAGCCCAGGCTGGG GCCAAGGACTCTGCTGAGAGGCCCCTAGAGACAACACCCTTCCCAGGCCACTGGCTGCTG GACTGTTCCTCAGGAGCGGCCTGGGTACCCAGTATGTGCAGGGAGACGGAACCCCATGTG ACAGCCCACTCCACCAGGGTTCCCAAAGAACCTGGCCCAGTCATAATCATTCATCCTGAC AGTGGCAATAATCACGATAACCAGTACTAGCTGCCATGATCGTTAGCCTCATATTTTCTA TCTAGAGCTCTGTAGAGCACTTTAGAAACCGCTTTCATGAATTGAGCTAATTATGAATAA ATTTGGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0095] Sequences identified by SEQ ID NO. are provided using conventional representations of a DNA strand starting from the 5' phosphate linked end to the 3' hydroxyl linked end. The assignment of coding regions is generally by comparison to available consensus sequence(s) and therefore may contain inconsistencies relative to other sequences assigned to the same cluster. These have no effect on the practice of the invention because the invention can be practiced by use of shorter segments (or combinations thereof) of sequences unique to each of the three sets described above and not affected by inconsistencies. As non-limiting examples, a segment of IL17BR, CACNA1D, or HOXB13 nucleic acid sequence composed of a 3' untranslated region sequence and/or a sequence from the 3' end of the coding region may be used as a probe for the detection of IL17BR, CACNA1D, or HOXB13 expression, respectively, without being affected by the presence of any inconsistency in the coding regions due to differences between sequences. Similarly, the use of an antibody which specifically recognizes IL17BR, CACNA1D, or HOXB13 protein to detect its expression would not be affected by the presence of any inconsistency in the representation of the coding regions provided above.

[0096] As will be appreciated by those skilled in the art, some of the above sequences include 3' poly A (or poly T on the complementary strand) stretches that do not contribute to the uniqueness of the disclosed sequences. The invention may thus be practiced with sequences lacking the 3' poly A (or poly T) stretches. The uniqueness of the disclosed sequences refers to the portions or entireties of the sequences which are found only in IL17BR, CACNA1D, or HOXB13 nucleic acids, including unique sequences found at the 3' untranslated portion of the genes. Preferred unique sequences for the practice of the invention are those which contribute to the consensus sequences for each of the three sets such that the unique sequences will be useful in detecting expression in a variety of individuals rather than being specific for a polymorphism present in some individuals. Alternatively, sequences unique to an individual or a subpopulation may be used. The preferred unique sequences are preferably of the lengths of polynucleotides of the invention as discussed herein.

[0097] To determine the (increased or decreased) expression levels of the above described sequences in the practice of the present invention, any method known in the art may be utilized. In one preferred embodiment of the invention, expression based on detection of RNA which hybridizes to polynucleotides containing the above described sequences is used. This is readily performed by any RNA detection or amplification+detection method known or recognized as equivalent in the art such as, but not limited to, reverse transcription-PCR (optionally real-time PCR), the methods disclosed in U.S. patent application Ser. No. 10/062,857 entitled "Nucleic Acid Amplification" filed on Oct. 25, 2001 as well as U.S. Provisional Patent Application 60/298,847 (filed Jun. 15, 2001) and 60/257,801 (filed Dec. 22, 2000), the methods disclosed in U.S. Pat. No. 6,291,170, and quantitative PCR. Methods to identify increased RNA stability (resulting in an observation of increased expression) or decreased RNA stability (resulting in an observation of decreased expression) may also be used. These methods include the detection of sequences that increase or decrease the stability of mRNAs containing the IL17BR, CACNA1D, or HOXB13 sequences disclosed herein. These methods also include the detection of increased mRNA degradation.

[0098] In particularly preferred embodiments of the invention, polynucleotides having sequences present in the 3' untranslated and/or non-coding regions of the above disclosed sequences are used to detect expression or non-expression of IL17BR, CACNA1 D, or HOXB13 sequences in breast cells in the practice of the invention. Such polynucleotides may optionally contain sequences found in the 3' portions of the coding regions of the above disclosed sequences. Polynucleotides containing a combination of sequences from the coding and 3' non-coding regions preferably have the sequences arranged contiguously, with no intervening heterologous sequence(s).

[0099] Alternatively, the invention may be practiced with polynucleotides having sequences present in the 5' untranslated and/or non-coding regions of IL17BR, CACNA1D, or HOXB13 sequences in breast cells to detect their levels of expression. Such polynucleotides may optionally contain sequences found in the 5' portions of the coding regions. Polynucleotides containing a combination of sequences from the coding and 5' non-coding regions preferably have the sequences arranged contiguously, with no intervening heterologous sequence(s). The invention may also be practiced with sequences present in the coding regions of IL17BR, CACNA1D, or HOXB13.

[0100] Preferred polynucleotides contain sequences from 3' or 5' untranslated and/or non-coding regions of at least about 20, at least about 22, at least about 24, at least about 26, at least about 28, at least about 30, at least about 32, at least about 34, at least about 36, at least about 38, at least about 40, at least about 42, at least about 44, or at least about 46 consecutive nucleotides. The term "about" as used in the previous sentence refers to an increase or decrease of 1 from the stated numerical value. Even more preferred are polynucleotides containing sequences of at least or about 50, at least or about 100, at least about or 150, at least or about 200, at least or about 250, at least or about 300, at least or about 350, or at least or about 400 consecutive nucleotides. The term "about" as used in the preceding sentence refers to an increase or decrease of 10% from the stated numerical value.

[0101] Sequences from the 3' or 5' end of the above described coding regions as found in polynucleotides of the invention are of the same lengths as those described above, except that they would naturally be limited by the length of the coding region. The 3' end of a coding region may include sequences up to the 3' half of the coding region. Conversely, the 5' end of a coding region may include sequences up the 5' half of the coding region. Of course the above described sequences, or the coding regions and polynucleotides containing portions thereof, may be used in their entireties.

[0102] Polynucleotides combining the sequences from a 3' untranslated and/or non-coding region and the associated 3' end of the coding region are preferably at least or about 100, at least about or 150, at least or about 200, at least or about 250, at least or about 300, at least or about 350, or at least or about 400 consecutive nucleotides. Preferably, the polynucleotides used are from the 3' end of the gene, such as within about 350, about 300, about 250, about 200, about 150, about 100, or about 50 nucleotides from the polyadenylation signal or polyadenylation site of a gene or expressed sequence. Polynucleotides containing mutations relative to the sequences of the disclosed genes may also be used so long as the presence of the mutations still allows hybridization to produce a detectable signal.

[0103] In another embodiment of the invention, polynucleotides containing deletions of nucleotides from the 5' and/or 3' end of the above disclosed sequences may be used. The deletions are preferably of 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-125, 125-150, 150-175, or 175-200 nucleotides from the 5' and/or 3' end, although the extent of the deletions would naturally be limited by the length of the disclosed sequences and the need to be able to use the polynucleotides for the detection of expression levels.

[0104] Other polynucleotides of the invention from the 3' end of the above disclosed sequences include those of primers and optional probes for quantitative PCR. Preferably, the primers and probes are those which amplify a region less than about 350, less than about 300, less than about 250, less than about 200, less than about 150, less than about 100, or less than about 50 nucleotides from the from the polyadenylation signal or polyadenylation site of a gene or expressed sequence.

[0105] In yet another embodiment of the invention, polynucleotides containing portions of the above disclosed sequences including the 3' end may be used in the practice of the invention. Such polynucleotides would contain at least or about 50, at least or about 100, at least about or 150, at least or about 200, at least or about 250, at least or about 300, at least or about 350, or at least or about 400 consecutive nucleotides from the 3' end of the disclosed sequences.

[0106] The invention thus also includes polynucleotides used to detect IL17BR, CACNA1D, or HOXB13 expression in breast cells. The polynucleotides may comprise a shorter polynucleotide consisting of sequences found in the above provided SEQ ID NOS in combination with heterologous sequences not naturally found in combination with IL17BR, CACNA1D, or HOXB13 sequences.

[0107] As non-limiting examples, a polynucleotide comprising one of the following sequences may be used in the practice of the invention.

10 SEQ ID NO:8: CAATTACAGGGAAAAAACGTGTGATGATCCTGAAGCTTACTAT- GCAGCCTACAAACAGCC SEQ ID NO:9: GCTCTCACTGGCAAATGACAGCTCTGTGCAAGGAGCACTCCCAAGTATAAAAATTATTAC SEQ ID NO:10: GATCGTTAGCCTCATATTTTCTATCTAGAGCTCTGTAGAGCACTTTAGA- AACCGCTTTCA

[0108] Stated differently, the invention may be practiced with a polynucleotide consisting of the sequence of SEQ ID NOS: 8, 9 or 10 in combination with one or more heterologous sequences that are not normally found with SEQ ID NOS: 8, 9 or 10. Alternatively, the invention may also be practiced with a polynucleotide consisting of the sequence of SEQ ID NOS: 8, 9 or 10 in combination with one or more naturally occurring sequences that are normally found with SEQ ID NOS: 8, 9 or 10.

[0109] Polynucleotides with sequences comprising SEQ ID NOS: 8 or 9, either naturally occurring or synthetic, may be used to detect nucleic acids which are over expressed in breast cancer cells that are responsive to TAM treatment. Polynucleotides with sequences comprising SEQ ID NO: 10, either naturally occurring or synthetic, may be used to detect nucleic acids which are under expressed in breast cancer cells that are responsive to TAM treatment.

[0110] Additional sequences that may be used in polynucleotides as described above for SEQ ID NOS: 8 and 9 are the following:

11 SEQ ID NO:11: TGCCTAATTTCACTCTCAGAGTGAGGCAGGTAACTGGGGCTC- CACTGGGTCACTCTGAGA SEQ ID NO:12: TTGGAAGCAGAGTCCCTCTAAAGGTAACTCTTGTGGTCACTCAATATTGTATTGGCATTT SEQ ID NO:13: ACGTTAGACTTTTGCTGGCATTCAAGTCATGGCTAGTCTGTGTATTTAA- TAAATGTGTGT SEQ ID NO:14: CTGGTCAGCCACTCTGACTTTTCTA- CCACATTAAATTCTCCATTACATCTCACTATTGGT SEQ ID NO:15: TACAACTTCTGAATGCTGCACATTCTTCCAAAATGATCCTTAGCACAATCTATTGTATGA SEQ ID NO:16: GGGATGGCCTTTAGGCCACAGTAGTGTCTGTGTTAAGTTCACTAAATGT- GTATTTAATGA SEQ ID NO:17: CTCAAAGTGCTAAAGCTATGGTTGA- CTGCTCTGGTGTTTTTATATTCATTCGTGCTTTAG

[0111] Additional sequences that may be used in polynucleotides as described above for SEQ ID NO: 10 are the following:

12 SEQ ID NO:18: CTATGGGGATGGTCCACTGTCACTGTTTCTCTGCTGTTGCAA- ATACATGGATAACACATT SEQ ID NO:19: ACTGGAAAAGCAGATGGTCTGACTGTGCTATGGCCTCATCATCAAGACTTTCAATCCTAT SEQ ID NO:20: ACGCCAAGCTCTTCAGTGAAGACACGATGTTATTAAAAGCCTGTTTTAG- GGACTGCAAAA SEQ ID NO:21: TTTTTGTAAAATCTTTAACCTTCCC- TTTGTTCTTCATGTACACGCTGAACTGCAATTCTT SEQ ID NO:22: AACCTGGGGCATTTAGGGCAGAGGACAAAAGGATGTCAGCAATTGCTTGGGCTGCTTGGC SEQ ID NO:23: CTGGAACCTCTGGACTCCCCATGCTCTAACTCCCACACTCTGCTATCAG- AAACTTAAACT SEQ ID NO:24: AACCCCAGAACCATCTAAGACATGG- GATTCAGTGATCATGTGGTTCTCCTTTTAACTTAC SEQ ID NO:25: GGCCATGTGCCATGGTATTTGGGTCCTGGGAGGGTGGGTGAAATAAAGGCATACTGTCTT SEQ ID NO:26: GTGTAGGCAGTCATGGCACCAAAGCCACCAGACTGACAAATGTGTATCA- GATGCTTTTGT SEQ ID NO:27: GAAAACCTCTTCAAAAGACAAAAAG- CTGGCACTGCATTCTCTCTCTGTAGCAGGACAGAA SEQ ID NO:28: CACATCTTTAGGGTCAGTGAACAATGGGGCACATTTGGCACTAGCTTGAGCCCAACTCTG SEQ ID NO:29: GCCTTAATTTCCTCATCTGAAAACTGGAAGGCCTGACTTGACTTGTTGA- GCTTAAGATCC SEQ ID NO:30: CTTCAGGGGAGGATCAAGCTTTGAA- CCAAAGCCAATCACTGGCTTGATTTGTGTTTTTTA SEQ ID NO:31: ACAAGTTTTCACTGAATGAGCATGGCAGTGCCACTCAAGAAAATGAATCTCCAAAGTATC

[0112] Additionally, polynucleotides containing other sequences, particularly unique sequences, present in naturally occurring nucleic acid molecules comprising SEQ ID NOS: 8-31 may be used in the practice of the invention.

[0113] Other polynucleotides for use in the practice of the invention include those that have sufficient homology to those described above to detect expression by use of hybridization techniques. Such polynucleotides preferably have about or 95%, about or 96%, about or 97%, about or 98%, or about or 99% identity with IL17BR, CACNA1D, or HOXB13 sequences as described herein. Identity is determined using the BLAST algorithm, as described above. The other polynucleotides for use in the practice of the invention may also be described on the basis of the ability to hybridize to polynucleotides of the invention under stringent conditions of about 30% v/v to about 50% formamide and from about 0.01M to about 0.15M salt for hybridization and from about 0.01M to about 0.15M salt for wash conditions at about 55 to about 65.degree. C. or higher, or conditions equivalent thereto.

[0114] In a further embodiment of the invention, a population of single stranded nucleic acid molecules comprising one or both strands of a human IL17BR or CACNA1D sequence is provided as a probe such that at least a portion of said population may be hybridized to one or both strands of a nucleic acid molecule quantitatively amplified from RNA of a breast cancer cell. The population may be only the antisense strand of a human IL17BR or CACNA1D sequence such that a sense strand of a molecule from, or amplified from, a breast cancer cell may be hybridized to a portion of said population. The population preferably comprises a sufficiently excess amount of said one or both strands of a human IL17BR or CACNA1D sequence in comparison to the amount of expressed (or amplified) nucleic acid molecules containing a complementary IL17BR or CACNA1D sequence from a normal breast cell. This condition of excess permits the increased amount of nucleic acid expression in a breast cancer cell to be readily detectable as an increase.

[0115] Alternatively, the population of single stranded molecules is equal to or in excess of all of one or both strands of the nucleic acid molecules amplified from a breast cancer cell such that the population is sufficient to hybridize to all of one or both strands. Preferred cells are those of a breast cancer patient that is ER+ or for whom tamoxifen treatment is contemplated. The single stranded molecules may of course be the denatured form of any IL17BR and/or CACNA1D sequence containing double stranded nucleic acid molecule or polynucleotide as described herein.

[0116] The population may also be described as being hybridized to IL17BR or CACNA1D sequence containing nucleic acid molecules at a level of at least twice as much as that by nucleic acid molecules of a normal breast cell. As in the embodiments described above, the nucleic acid molecules may be those quantitatively amplified from a breast cancer cell such that they reflect the amount of expression in said cell.

[0117] The population is preferably immobilized on a solid support, optionally in the form of a location on a microarray. A portion of the population is preferably hybridized to nucleic acid molecules quantitatively amplified from a non-normal or abnormal breast cell by real time PCR. The real time PCR may be practiced by use of amplified RNA from a breast cancer cell, as long as the amplification used was quantitative with respect to IL17BR or CACNA1D containing sequences.

[0118] In another embodiment of the invention, expression based on detection of DNA status may be used. Detection of the HOXB13 DNA as methylated, deleted or otherwise inactivated, may be used as an indication of decreased expression as found in non-normal breast cells. This may be readily performed by PCR based methods known in the art. The status of the promoter regions of HOXB13 may also be assayed as an indication of decreased expression of HOXB13 sequences. A non-limiting example is the methylation status of sequences found in the promoter region.

[0119] Conversely, detection of the DNA of a sequence as amplified may be used for as an indication of increased expression as found in non-normal breast cells. This may be readily performed by PCR based, fluorescent in situ hybridization (FISH) and chromosome in situ hybridization (CISH) methods known in the art.

[0120] A preferred embodiment using a nucleic acid based assay to determine expression is by immobilization of one or more of the sequences identified herein on a solid support, including, but not limited to, a solid substrate as an array or to beads or bead based technology as known in the art. Alternatively, solution based expression assays known in the art may also be used. The immobilized sequence(s) may be in the form of polynucleotides as described herein such that the polynucleotide would be capable of hybridizing to a DNA or RNA corresponding to the sequence(s).

[0121] The immobilized polynucleotide(s) may be used to determine the state of nucleic acid samples prepared from sample breast cancer cell(s), optionally as part of a method to detect ER status in said cell(s). Without limiting the invention, such a cell may be from a patient suspected of being afflicted with, or at risk of developing, breast cancer. The immobilized polynucleotide(s) need only be sufficient to specifically hybridize to the corresponding nucleic acid molecules derived from the sample (and to the exclusion of detectable or significant hybridization to other nucleic acid molecules).

[0122] In yet another embodiment of the invention, a ratio of the expression levels of two of the disclosed genes may be used to predict response to TAM treatment. Preferably, the ratio is that of two genes with opposing patterns of expression, such as an underexpressed gene to an overexpressed gene. Non-limiting examples include the ratio of HOXB13 over IL17BR or the ratio of HOXB13 over CACNA1D. This aspect of the invention is based in part on the observation that such a ratio has a stronger correlation with TAM treatment outcome than the expression level of either gene alone. For example, the ratio of HOXB13 over IL17BR has an observed classification accuracy of 77%.

[0123] Additional Embodiments of the Invention

[0124] In embodiments where only one or a few genes are to be analyzed, the nucleic acid derived from the sample breast cancer cell(s) may be preferentially amplified by use of appropriate primers such that only the genes to be analyzed are amplified to reduce contaminating background signals from other genes expressed in the breast cell. Alternatively, and where multiple genes are to be analyzed or where very few cells (or one cell) is used, the nucleic acid from the sample may be globally amplified before hybridization to the immobilized polynucleotides. Of course RNA, or the cDNA counterpart thereof may be directly labeled and used, without amplification, by methods known in the art.

[0125] Sequence expression based on detection of a presence, increase, or decrease in protein levels or activity may also be used. Detection may be performed by any immunohistochemistry (IHC) based, bodily fluid based (where a IL17BR, CACNA1D, and/or HOXB13 polypeptide is found in a bodily fluid, such as but not limited to blood), antibody (including autoantibodies against the protein where present) based, ex foliate cell (from the cancer) based, mass spectroscopy based, and image (including used of labeled ligand where available) based method known in the art and recognized as appropriate for the detection of the protein. Antibody and image based methods are additionally useful for the localization of tumors after determination of cancer by use of cells obtained by a non-invasive procedure (such as ductal lavage or fine needle aspiration), where the source of the cancerous cells is not known. A labeled antibody or ligand may be used to localize the carcinoma(s) within a patient.

[0126] Antibodies for use in such methods of detection include polyclonal antibodies, optionally isolated from naturally occurring sources where available, and monoclonal antibodies, including those prepared by use of IL17BR, CACNA1D, and/or HOXB13 polypeptides as antigens. Such antibodies, as well as fragments thereof (including but not limited to Fab fragments) function to detect or diagnose non-normal or cancerous breast cells by virtue of their ability to specifically bind IL17BR, CACNA1D, or HOXB13 polypeptides to the exclusion of other polypeptides to produce a detectable signal. Recombinant, synthetic, and hybrid antibodies with the same ability may also be used in the practice of the invention. Antibodies may be readily generated by immunization with a IL17BR, CACNA1D, or HOXB13 polypeptide, and polyclonal sera may also be used in the practice of the invention.

[0127] Antibody based detection methods are well known in the art and include sandwich and ELISA assays as well as Western blot and flow cytometry based assays as non-limiting examples. Samples for analysis in such methods include any that contain IL17BR, CACNA1D, or HOXB13 polypeptides. Non-limiting examples include those containing breast cells and cell contents as well as bodily fluids (including blood, serum, saliva, lymphatic fluid, as well as mucosal and other cellular secretions as non-limiting examples) that contain the polypeptides.

[0128] The above assay embodiments may be used in a number of different ways to identify or detect the response to TAM treatment based on gene expression in a breast cancer cell sample from a patient. In some cases, this would reflect a secondary screen for the patient, who may have already undergone mammography or physical exam as a primary screen. If positive from the primary screen, the subsequent needle biopsy, ductal lavage, fine needle aspiration, or other analogous methods may provide the sample for use in the assay embodiments before, simultaneous with, or after assaying for ER status. The present invention is particularly useful in combination with non-invasive protocols, such as ductal lavage or fine needle aspiration, to prepare a breast cell sample.

[0129] The present invention provides a more objective set of criteria, in the form of gene expression profiles of a discrete set of genes, to discriminate (or delineate) between breast cancer outcomes. In particularly preferred embodiments of the invention, the assays are used to discriminate between good and poor outcomes after tamoxifen treatment. Comparisons that discriminate between outcomes after about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, or about 150 months may be performed.

[0130] While good and poor survival outcomes may be defined relatively in comparison to each other, a "good" outcome may be viewed as a better than 50% survival rate after about 60 months post surgical intervention to remove breast cancer tumor(s). A "good" outcome may also be a better than about 60%, about 70%, about 80% or about 90% survival rate after about 60 months post surgical intervention. A "poor" outcome may be viewed as a 50% or less survival rate after about 60 months post surgical intervention to remove breast cancer tumor(s). A "poor" outcome may also be about a 70% or less survival rate after about 40 months, or about a 80% or less survival rate after about 20 months, post surgical intervention.

[0131] In another embodiment of the invention based on the expression of multiple genes in an expression pattern or profile, the isolation and analysis of a breast cancer cell sample may be performed as follows:

[0132] (1) Ductal lavage or other non-invasive procedure is performed on a patient to obtain a sample.

[0133] (2) Sample is prepared and coated onto a microscope slide. Note that ductal lavage results in clusters of cells that are cytologically examined as stated above.

[0134] (3) Pathologist or image analysis software scans the sample for the presence of non-normal and/or atypical breast cancer cells.

[0135] (4) If such cells are observed, those cells are harvested (e.g. by microdissection such as LCM).

[0136] (5) RNA is extracted from the harvested cells.

[0137] (6) RNA is purified, amplified, and labeled.

[0138] (7) Labeled nucleic acid is contacted with a microarray containing polynucleotides of the genes identified herein as correlated to discriminations between breast cancer outcomes under suitable hybridization conditions, then processed and scanned to obtain a pattern of intensities of each spot (relative to a control for general gene expression in cells) which determine the level of expression of the gene(s) in the cells.

[0139] (8) The pattern of intensities is analyzed by comparison to the expression patterns of the genes in known samples of breast cancer cells correlated with outcomes (relative to the same control).

[0140] A specific example of the above method would be performing ductal lavage following a primary screen, observing and collecting non-normal and/or atypical cells for analysis. The comparison to known expression patterns, such as that made possible by a model generated by an algorithm (such as, but not limited to nearest neighbor type analysis, SVM, or neural networks) with reference gene expression data for the different breast cancer survival outcomes, identifies the cells as being correlated with subjects with good or poor outcomes. Another example would be taking a breast tumor removed from a subject after surgical intervention, optionally converting all or part of it to an FFPE sample prior to subsequent isolation and preparation of breast cancer cells from the tumor for determination/identification of atypical, non-normal, or cancer cells, and isolation of said cells followed by steps 5 through 8 above.

[0141] Alternatively, the sample may permit the collection of both normal as well as cancer cells for analysis. The gene expression patterns for each of these two samples will be compared to each other as well as the model and the normal versus individual comparisons therein based upon the reference data set. This approach can be significantly more powerful that the cancer cells only approach because it utilizes significantly more information from the normal cells and the differences between normal and cancer cells (in both the sample and reference data sets) to determine the breast cancer outcome of the patient based on gene expression in the cancer cells from the sample.

[0142] In yet another embodiment of the invention based on the expression of a few genes, the isolation and analysis of a breast cancer cell sample may be performed as follows:

[0143] (1) Ductal lavage or other non-invasive procedure is performed on a patient to obtain a sample.

[0144] (2) Sample is prepared and coated onto a microscope slide. Note that ductal lavage results in clusters of cells that are cytologically examined as stated above.

[0145] (3) Pathologist or image analysis software scans the sample for the presence of atypical cells.

[0146] (4) If atypical cells are observed, those cells are harvested (e.g. by microdissection such as LCM).

[0147] (5) RNA is extracted from the harvested cells.

[0148] (6) RNA is assayed, directly or after conversion to cDNA or amplification therefrom, for the expression of IL17BR, CACNA1D, and/or HOXB13 sequences.

[0149] One example of the above method would be performing ductal lavage following a primary screen, observing and collecting non-normal cells (or cells suspected of being non-normal) for analysis. Alternatively, the sample may permit the collection of both normal and non-normal cells (or cells suspected of being non-normal) for analysis. The expression levels of IL17BR, CACNA1D, and/or HOXB13 sequences in each of these two populations may be compared to each other. This approach can be significantly more powerful than one using the non-normal cells only approach because it utilizes information from the normal cells and the differences between normal and non-normal cells to determine the status of the non-normal cells from the sample.

[0150] With use of the present invention, skilled physicians may prescribe or withhold TAM treatment based on prognosis determined via practice of the instant invention.

[0151] The above discussion is also applicable where a palpable lesion is detected followed by fine needle aspiration or needle biopsy of cells from the breast. The cells are plated and reviewed by a pathologist or automated imaging system which selects cells for analysis as described above.

[0152] The present invention may also be used, however, with solid tissue biopsies, including those stored as an FFPE specimen. For example, a solid biopsy may be collected and prepared for visualization followed by determination of expression of one or more genes identified herein to determine the breast cancer outcome. As another non-limiting example, a solid biopsy may be collected and prepared for visualization followed by determination of increased IL17BR and/or CACNA1D expression. One preferred means is by use of in situ hybridization with polynucleotide or protein identifying probe(s) for assaying expression of said gene(s). An analogous method may be used to detect decreased expression of HOXB13 sequences.

[0153] In an alternative method, the solid tissue biopsy may be used to extract molecules followed by analysis for expression of one or more gene(s). This provides the possibility of leaving out the need for visualization and collection of only cancer cells or cells suspected of being cancerous. This method may of course be modified such that only cells that have been positively selected are collected and used to extract molecules for analysis. This would require visualization and selection as a prerequisite to gene expression analysis. In the case of an FFPE sample, cells may be obtained followed by RNA extraction, amplification and detection as described herein.

[0154] In a further modification of the above, both normal cells and cancer cells are collected and used to extract molecules for analysis of gene expression. The approach, benefits and results are as described above using non-invasive sampling.

[0155] In a further alternative to all of the above, the sequence(s) identified herein may be used as part of a simple PCR or array based assay simply to determine the response to TAM treatment by use of a sample from a non-invasive sampling procedure. The detection of sequence expression from samples may be by use of a single microarray able to assay expression of the disclosed sequences as well as other sequences, including sequences known not to vary in expression levels between normal and non-normal breast cells, for convenience and improved accuracy.

[0156] Other uses of the present invention include providing the ability to identify breast cancer cell samples as having different responses to TAM treatment for further research or study. This provides an advance based on objective genetic/molecular criteria.

[0157] The genes identified herein also may be used to generate a model capable of predicting the breast cancer survival and recurrence outcomes of an ER+ breast cell sample based on the expression of the identified genes in the sample. Such a model may be generated by any of the algorithms described herein or otherwise known in the art as well as those recognized as equivalent in the art using gene(s) (and subsets thereof) disclosed herein for the identification of breast cancer outcomes. The model provides a means for comparing expression profiles of gene(s) of the subset from the sample against the profiles of reference data used to build the model. The model can compare the sample profile against each of the reference profiles or against a model defining delineations made based upon the reference profiles. Additionally, relative values from the sample profile may be used in comparison with the model or reference profiles.

[0158] In a preferred embodiment of the invention, breast cell samples identified as normal and cancerous from the same subject may be analyzed, optionally by use of a single microarray, for their expression profiles of the genes used to generate the model. This provides an advantageous means of identifying survival and recurrence outcomes based on relative differences from the expression profile of the normal sample. These differences can then be used in comparison to differences between normal and individual cancerous reference data which was also used to generate the model.

[0159] Articles of Manufacture

[0160] The materials and methods of the present invention are ideally suited for preparation of kits produced in accordance with well known procedures. The invention thus provides kits comprising agents (like the polynucleotides and/or antibodies described herein as non-limiting examples) for the detection of expression of the disclosed sequences. Such kits, optionally comprising the agent with an identifying description or label or instructions relating to their use in the methods of the present invention, are provided. Such a kit may comprise containers, each with one or more of the various reagents (typically in concentrated form) utilized in the methods, including, for example, pre-fabricated microarrays, buffers, the appropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP and dTTP; or rATP, rCTP, rGTP and UTP), reverse transcriptase, DNA polymerase, RNA polymerase, and one or more primer complexes of the present invention (e.g., appropriate length poly(T) or random primers linked to a promoter reactive with the RNA polymerase). A set of instructions will also typically be included.

[0161] The methods provided by the present invention may also be automated in whole or in part. All aspects of the present invention may also be practiced such that they consist essentially of a subset of the disclosed genes to the exclusion of material irrelevant to the identification of breast cancer survival outcomes via a cell containing sample.

[0162] Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.

EXAMPLES

Example 1

Gene Expression Signature Predicting TAM Treatment Outcome in Breast Cancer

[0163] A cohort of 62 estrogen receptor-positive breast cancer patients were uniformly treated with the anti-estrogen drug tamoxifen (TAM), and followed for up to 14 years. 33 patients recurred whereas 29 patients remained disease-free during the entire follow up periods. Correlating gene expression patterns with tumor recurrence/non-recurrence, a set of genes was discovered whose expression levels differ significantly between these two groups. This gene expression signature can thus be used to predict whether a patient will respond to TAM as first-line treatment based on the gene expression profile of a routine biopsy of the primary cancer.

[0164] Laser capture microdissection was performed on each tumor biopsy to procure pure populations of cancerous epithelial cells, which were then analyzed on a 22000-gene high-density oligonucleotide microarray. The top 25% genes with the greatest variances across all samples (n=5475) were selected for signature extraction. Genes showing statistically significant correlations with tumor recurrence/non-recurrence were identified using two different statistical techniques.

[0165] In the first approach, patients were divided into two groups (recurrence vs. non-recurrence), and a standard t-test was performed for each gene, which identified 149 genes with p values <0.001. The results for this analysis are shown in Table 1. Genes identified by their accession numbers correlate with non-responders when the t-statistic is less than zero while genes with a t-statistic greater than zero correlate to positive responders.

13TABLE 1 149-gene signature identified by t-test Accession p value t-statistic Description BC002595 5.49E-10 -8.186189 NDUFB7.vertline.NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 7 (18 kD, B18) BC002705 1.65E-09 -7.550191 C22orf3.vertline.chromosome 22 open reading frame 3 AL080126 1.82E-09 -7.410723 KIAA0683.vertline.KIAA0683 gene product AI767799 2.02E-09 -7.768777 BBC3.vertline.Bcl-2 binding component 3 AL021683 2.78E-09 -7.083131 SCO2.vertline.SCO cytochrome oxidase deficient homolog 2 (yeast) BC000507 4.38E-09 -7.026423 MAAT1.vertline.melanoma-associated antigen recognised by cytotoxic T lymphocytes AK027124 1.70E-08 -6.740214 FLJ23471.vertline.hypothetical protein FLJ23471 BC016737 1.99E-08 -6.742271 MPST.vertline.mercaptopyruvate sulfurtransferase BC011874 3.53E-08 -6.327036 MGC20486.vertline.hypothetical protein MGC20486 BC008832 3.86E-08 -6.388736 HMGIY.vertline.high-mobility group (nonhistone chromosomal) protein isoforms I and Y AF044959 5.20E-08 -6.222993 NDUFS6.vertline.NADH dehydrogenase (ubiquinone) Fe-S protein 6 (13 kD) (NADH-coenzyme Q reductase) BC016832 6.61E-08 -6.627917 MGC4607.vertline.hypothetical protein MGC4607 BC011680 6.61E-08 -6.427017 DKFZp434G0522.vertline.hypothe- tical protein DKFZp434G0522 AA811922 6.75E-08 -6.634444 FLJ10140.vertline.hypothetical protein FLJ10140 AW075691 1.03E-07 -6.272638 KIAA1847.vertline.hypothetical protein FLJ14972 AK024627 1.14E-07 -6.019024 FLJ20974.vertline.hypothetical protein FLJ20974 BC002389 1.15E-07 -6.05372 ATP5D.vertline.ATP synthase, H+ transporting, mitochondrial F1 complex, delta subunit AK055295 1.24E-07 -6.391213 Homo sapiens cDNA FLJ30733 fis, clone FEBRA2000129, moderately similar to PROBABLE TRNA (5-METHYLAMINOMETHYL-2- THIOURIDYLATE)-METHYLTRANSFERASE (EC 2.1.1.61) BC011621 1.54E-07 5.943998 HOOK1.vertline.hook1 protein AK023601 1.69E-07 5.919878 Homo sapiens cDNA FLJ13539 fis, clone PLACE1006640 BC013959 1.83E-07 -6.09348 GNL1.vertline.guanine nucleotide binding protein-like 1 BC018346 1.84E-07 -5.929725 LAK-4P.vertline.express- ed in activated T/LAK lymphocytes AF052052 3.46E-07 -5.920813 TFPT.vertline.TCF3 (E2A) fusion partner (in childhood Leukemia) AL136921 3.66E-07 -5.742098 DKFZp586I021.vertline.hypothetical protein DKFZp586I021 AI968598 6.33E-07 -5.685799 Homo sapiens cDNA FLJ12182 fis, clone MAMMA1000761 BC011754 7.93E-07 -5.671882 ERP70.vertline.protein disulfide isomerase related protein (calcium-binding protein, intestinal-related) BC014270 3.58E-06 -5.155079 PRKCZ.vertline.protein kinase C, zeta NM_001130 3.82E-06 -5.120513 AES.vertline.amino-terminal enhancer of split BF116098 4.09E-06 5.101295 ESTs BC015594 5.01E-06 -5.027872 Homo sapiens mRNA for FLJ00083 protein, partial cds AK000081 5.74E-06 -4.996636 CDC2L1.vertline.cell division cycle 2-like 1 (PITSLRE proteins) NM_006278 6.23E-06 -4.968186 SIAT4C.vertline.sialyltransferase 4C (beta-galactosidase alpha-2,3-sialytransferase) BC008841 6.32E-06 -5.039493 KIAA0415.vertline.KIAA0415 gene product AI972367 7.05E-06 -4.93464 Homo sapiens cDNA FLJ32384 fis, clone SKMUS1000104, weakly similar to Homo sapiens mRNA for HEXIM1 protein, complete cds AI467849 7.34E-06 -4.933176 TBC1D1.vertline.TBC1 (tre-2/USP6, BUB2, cdc16) domain family, member 1 NM_014298 9.19E-06 -4.869139 QPRT.vertline.quinolinate phosphoribosyltransferase (nicotinate-nucleotide pyrophosphorylase (carboxylating)) H19223 1.15E-05 4.786877 ESTs, Weakly similar to JC5238 galactosylceramide-like protein, GCP [H. sapiens] AI638324 1.22E-05 4.783615 Homo sapiens cDNA FLJ30332 fis, clone BRACE2007254 AF208111 1.30E-05 4.761353 IL17BR.vertline.interleuki- n 17B receptor NM_020978 1.34E-05 4.803041 AMY2B.vertline.amylase, alpha 2B; pancreatic BC015497 1.59E-05 -4.722392 TEAD4.vertline.TEA domain family member 4 AI561249 1.69E-05 4.681189 KTN1.vertline.kinectin 1 (kinesin receptor) BC004235 1.73E-05 -4.684545 DDX38.vertline.DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 38 NM_013347 1.89E-05 4.67568 HSU24186.vertline.replic- ation protein A complex 34 kd subunit homolog Rpa4 AL117616 1.90E-05 4.645713 SRI.vertline.sorcin AL117478 2.00E-05 -4.634086 AGS3.vertline.likely ortholog of rat activator of G-protein signaling 3 NM_006304 2.28E-05 4.59794 DSS1.vertline.Deleted in split-hand/split-foot 1 region BC009507 2.29E-05 -4.59323 ISG15.vertline.interferon-stimulated protein, 15 kDa AK025141 2.89E-05 4.529022 Homo sapiens cDNA: FLJ21488 fis, clone COL05445 AA581602 4.04E-05 4.43179 ESTs BC006499 4.22E-05 -4.422009 HRAS.vertline.v-Ha-ras Harvey rat sarcoma viral oncogene homolog BC007066 5.23E-05 4.379391 CDA11.vertline.CDA11 protein BC009869 5.35E-05 4.352129 SERF2.vertline.small EDRK-rich factor 2 AA206609 5.68E-05 -4.339494 Homo sapiens cDNA FLJ30002 fis, clone 3NB691000085 AI682928 5.76E-05 4.350598 EST BC006284 7.29E-05 -4.359234 Homo sapiens, clone IMAGE: 3957135, mRNA, partial cds AI871458 7.41E-05 -4.303954 ESTs AF068918 7.50E-05 -4.284961 BIN1.vertline.bridging integrator 1 NM_018936 7.50E-05 -4.254075 PCDHB2.vertline.protocad- herin beta 2 AI469557 7.83E-05 -4.248879 EPHB3.vertline.EphB3 AL137521 8.02E-05 -4.27827 Homo sapiens mRNA; cDNA DKFZp434D0218 (from clone DKFZp434D0218); partial cds AI268007 8.04E-05 4.245279 Homo sapiens cDNA FLJ30137 fis, clone BRACE2000078 AW070918 8.56E-05 -4.21829 ESTs, Weakly similar to T2D3_HUMAN TRANSCRIPTION INITIATION FACTOR TFIID 135 KDA SUBUNIT [H. sapiens] AK025862 8.75E-05 4.237223 Homo sapiens cDNA: FLJ22209 fis, clone HRC01496 AI264644 9.54E-05 -4.240955 KIAA0775.vertline.KIAA0775 gene product BF438928 9.75E-05 4.180144 ESTs BC001403 9.83E-05 -4.17366 CPSF5.vertline.cleavage and polyadenylation specific factor 5, 25 kD subunit AI270018 1.01E-04 -4.167464 ECE1.vertline.endothelin converting enzyme 1 AL133427 1.04E-04 4.19331 Homo sapiens mRNA full length insert cDNA clone EUROIMAGE 261172 AI400775 1.12E-04 -4.148062 RABL2B.vertline.RAB, member of RAS oncogene family-like 2B AW016075 1.21E-04 4.132864 ESTs, Weakly similar to ALUA_HUMAN !!!! ALU CLASS A WARNING ENTRY !!! [H. sapiens] AI033912 1.26E-04 4.100849 RLN2.vertline.relaxin 2 (H2) AA668884 1.28E-04 4.104243 ESTs AL133661 1.38E-04 4.085685 DKFZp434C0328.vertline.hypothetical protein DKFZp434C0328 BC009874 1.40E-04 -4.074407 JUN.vertline.v-jun sarcoma virus 17 oncogene homolog (avian) AI357434 1.52E-04 4.055067 HSP105B.vertline.heat shock 105 kD AF119871 1.54E-04 4.081889 PRO2268.vertline.hypothetical protein PRO2268 AK024715 1.54E-04 4.043172 FLJ21062.vertline.hypothetical protein FLJ21062 X62534 1.58E-04 4.048006 HMG2.vertline.high-mobility group (nonhistone chromosomal) protein 2 BI793002 1.60E-04 4.039819 OSBPL8.vertline.oxysterol binding protein-like 8 L13738 1.61E-04 -4.041465 ACK1.vertline.activated p21cdc42Hs kinase AW297123 1.74E-04 4.019412 ESTs NM_020235 1.80E-04 4.011596 BBX.vertline.bobby sox homolog (Drosophila) AI686003 1.83E-04 4.035297 ESTs AK022916 1.84E-04 3.989755 ZNF281.vertline.zinc finger protein 281 AK025701 1.86E-04 -3.99009 PLXNB2.vertline.plexin B2 AA806831 1.91E-04 -4.126686 ESTs AL117396 1.93E-04 3.982093 DKFZP586M0622.vertline.DKFZP586M0622 protein AW192535 1.93E-04 3.982278 ESTs AW076080 1.94E-04 3.972626 Homo sapiens, clone IMAGE: 3463399, mRNA, partial cds AB014541 1.95E-04 -3.97255 AATK.vertline.apoptosis-associated tyrosine kinase AK024967 1.96E-04 4.008564 Homo sapiens cDNA: FLJ21314 fis, clone COL02248 BC018644 2.10E-04 -3.981862 NUDT8.vertline.nudix (nucleoside diphosphate linked moiety X)-type motif 8 AK026817 2.11E-04 3.9468 FLJ23577.vertline.hypothetical protein FLJ23577 BC000692 2.20E-04 -3.943535 HYAL2.vertline.hyaluronoglucosaminidase 2 BE967259 2.26E-04 3.927279 BCL2.vertline.B-cell CLL/lymphoma 2 NM_004038 2.29E-04 3.946754 AMY1A.vertline.amylase, alpha 1A; salivary AF052110 2.34E-04 -3.915428 DAF.vertline.decay accelerating factor for complement (CD55, Cromer blood group system) AW069725 2.38E-04 3.914238 CRYZ.vertline.crystallin, zeta (quinone reductase) BM127867 2.44E-04 3.908237 MDM1.vertline.nuclear protein double minute 1 AL050227 2.50E-04 3.894782 Homo sapiens mRNA; cDNA DKFZp586M0723 (from clone DKFZp586M0723) BC005377 2.61E-04 3.949255 ACADM.vertline.acyl-Coenzyme A dehydrogenase, C-4 to C-12 straight chain BC006437 2.66E-04 -3.880036 C321D2.4.vertline.hypothetical protein C321D2.4 AF153330 2.73E-04 3.871579 SLC19A2.vertline.solute carrier family 19 (thiamine transporter), member 2 AA635853 2.86E-04 3.856068 EST AK021798 2.92E-04 3.858723 FLJ11736.vertline.hypothetical protein FLJ11736 BE675157 3.06E-04 3.882041 ESTs T52873 3.08E-04 3.831368 ESTs, Moderately similar to G02075 transcription repressor zinc finger protein 85 [H. sapiens] BE645958 3.30E-04 3.812843 ESTs BF589163 3.37E-04 3.857405 ESTs AA040945 3.44E-04 -3.797113 ESTs AK001783 3.74E-04 3.771144 FLJ10921.vertline.hypothetical protein FLJ10921 R43003 4.06E-04 3.80021 ESTs, Highly similar to COBW-like protein [H. sapiens] AW135596 4.10E-04 3.742774 FLJ10058.vertline.hypothetical protein FLJ10058 NM_003489 4.20E-04 3.736095 NRIP1.vertline.nuclear receptor interacting protein 1 AL136663 4.25E-04 -3.748587 DKFZp564A176.vertline.hypothetical protein DKFZp564A176 AI376433 4.47E-04 3.774197 KIAA1912.vertline.KIAA1912 protein BC015792 4.49E-04 -3.725478 Homo sapiens, clone MGC: 23665 IMAGE: 4866941, mRNA, complete cds AI478784 4.63E-04 3.705085 FLJ11267.vertline.hypothet- ical protein FLJ11267 U50532 4.91E-04 3.723884

CG005.vertline.hypothetical protein from BCRA2 region AI700363 4.92E-04 -3.719752 ESTs BC005956 5.22E-04 3.679274 RLN1.vertline.relaxin 1 (H1) AI240933 5.44E-04 3.657963 ESTs AF330046 5.51E-04 3.652748 PIBF1.vertline.progesterone-induced blocking factor 1 AI128331 5.55E-04 3.648721 ENDOFIN.vertline.endosome-asso- ciated FYVE-domain protein BC008381 5.63E-04 3.654514 IMPA1.vertline.inositol(myo)-1(or 4)-monophosphatase 1 AF023676 5.64E-04 -3.647402 TM7SF2.vertline.transmembrane 7 superfamily member 2 AL050179 5.73E-04 3.665736 TPM1.vertline.tropomyosin 1 (alpha) BC002355 5.73E-04 3.654105 HNRPA1.vertline.heterogeneous nuclear ribonucleoprotein A1 AK056075 5.84E-04 3.632268 Homo sapiens cDNA FLJ31513 fis, clone NT2RI1000127 AK024999 6.01E-04 3.641434 Homo sapiens cDNA: FLJ21346 fis, clone COL02705 AK000305 6.30E-04 3.666154 FLJ20298.vertline.hypothetical protein FLJ20298 AF085243 6.47E-04 3.601667 ZNF236.vertline.zinc finger protein 236 AW510501 6.56E-04 3.620023 ARHGAP5.vertline.Rho GTPase activating protein 5 AI953054 6.57E-04 -3.59919 TKT.vertline.transketolase (Wernicke-Korsakoff syndrome) BC012628 7.09E-04 -3.610827 TCAP.vertline.titin-cap (telethonin) BC007092 7.12E-04 -3.598786 HOXB13.vertline.homeo box B13 AB000520 7.40E-04 -3.558109 APS.vertline.adaptor protein with pleckstrin homology and src homology 2 domains AW150267 7.47E-04 3.566503 C21orf9.vertline.chromosome 21 open reading frame 9 AI800042 7.64E-04 3.575129 ESTs AF033199 8.01E-04 -3.541312 ZNF204.vertline.zinc finger protein 204 BC002607 8.15E-04 -3.529271 KIAA1446.vertline.KIAA1446 protein BC002480 8.43E-04 -3.525938 FLJ13352.vertline.hypothetical protein FLJ13352 AI568728 9.04E-04 -3.501174 SKI.vertline.v-ski sarcoma viral oncogene homolog (avian) AA648536 9.20E-04 -3.48714 MYO1E.vertline.myosin IE AI335002 9.28E-04 3.502278 PBEF.vertline.pre-B-cell colony-enhancing factor AW452172 9.45E-04 3.483191 ESTs AF334676 9.50E-04 3.476947 TEKT3.vertline.tektin 3 AF085233 9.77E-04 3.479809 SGKL.vertline.serum/glucocorticoid regulated kinase-like

[0166] In the second approach, the actual times of recurrence or follow-up (for those who remained disease-free) were used in a Cox proportional hazard regression model using each gene as the single predictor variable, identifying 149 genes with p values (Wald statistic)<0.001. The results for this analysis are shown in Table 2. Genes identified by their accession numbers correlate with subjects likely to suffer a reoccurrence after TAM therapy when the hazard ratio is greater than one while genes with a hazard ration of less than one correlate to individuals who are likely not to suffer a reoccurrence of breast cancer.

14TABLE 2 149-gene signature identified by Cox regression Accession p value hazard ratio Description BC002595 3.00E-08 1.9899702 NDUFB7.vertline.NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 7 (18 kD, B18) BC000507 3.66E-08 2.3494974 MAAT1.vertline.melanoma-associated antigen recognised by cytotoxic T lymphocytes BC016832 5.45E-08 2.2890356 MGC4607.vertline.hypothetical protein MGC4607 BC002705 1.52E-07 2.5669791 C22orf3.vertline.chromosome 22 open reading frame 3 AI767799 1.93E-07 2.1989649 BBC3.vertline.Bcl-2 binding component 3 BC011874 2.51E-07 2.8556338 MGC20486.vertline.hypothetical protein MGC20486 AL021683 3.74E-07 2.1946935 SCO2.vertline.SCO cytochrome oxidase deficient homolog 2 (yeast) BC008832 4.28E-07 2.3960849 HMGIY.vertline.high-mobility group (nonhistone chromosomal) protein isoforms I and Y AL080126 4.46E-07 2.1613379 KIAA0683.vertline.KIAA0683 gene product BC013959 4.68E-07 2.4974081 GNL1.vertline.guanine nucleotide binding protein-like 1 AF052052 5.29E-07 2.1949663 TFPT.vertline.TCF3 (E2A) fusion partner (in childhood Leukemia) AA811922 6.00E-07 1.9841656 FLJ10140.vertline.hypothetical protein FLJ10140 BC011680 6.96E-07 2.373463 DKFZp434G0522.vertline.hypothetical protein DKFZp434G0522 BC016737 1.06E-06 1.8482073 MPST.vertline.mercaptopyruvate sulfurtransferase AI968598 1.24E-06 2.6284635 Homo sapiens cDNA FLJ12182 fis, clone MAMMA1000761 AW075691 1.35E-06 2.0681292 KIAA1847.vertline.hypothetical protein FLJ14972 AK024627 1.53E-06 2.6015319 FLJ20974.vertline.hypothetical protein FLJ20974 AF044959 1.56E-06 2.8966077 NDUFS6.vertline.NADH dehydrogenase (ubiquinone) Fe-S protein 6 (13 kD) (NADH-coenzyme Q reductase) BC002389 1.64E-06 1.8888501 ATP5D.vertline.ATP synthase, H+ transporting, mitochondrial F1 complex, delta subunit AK055295 3.03E-06 1.8815611 Homo sapiens cDNA FLJ30733 fis, clone FEBRA2000129, moderately similar to PROBABLE TRNA (5- METHYLAMINOMETHYL-2-THIOURIDYLATE)- METHYLTRANSFERASE (EC 2.1.1.61) BC005377 3.41E-06 0.5676057 ACADM.vertline.acyl-Coenzyme A dehydrogenase, C-4 to C-12 straight chain H19223 4.47E-06 0.4802045 ESTs, Weakly similar to JC5238 galactosylceramide-like protein, GCP [H. sapiens] AK023601 4.81E-06 0.4390305 Homo sapiens cDNA FLJ13539 fis, clone PLACE1006640 NM_001130 5.72E-06 2.1351138 AES.vertline.amino-terminal enhancer of split NM_014298 6.39E-06 1.8007172 QPRT.vertline.quinolinate phosphoribosyltransferase (nicotinate- nucleotide pyrophosphorylase (carboxylating)) AK027124 7.12E-06 1.968632 FLJ23471.vertline.hypothetical protein FLJ23471 AL117396 7.58E-06 0.4156321 DKFZP586M0622.vertline.DKFZP586M0622 protein AL136921 8.27E-06 2.3643799 DKFZp586I021.vertline.hypothetical protein DKFZp586I021 U50532 8.81E-06 0.4216183 CG005.vertline.hypothetical protein from BCRA2 region BC018346 1.14E-05 1.8491373 LAK-4P.vertline.expressed in activated T/LAK lymphocytes NM_013347 1.35E-05 0.3648298 HSU24186.vertline.replication protein A complex 34 kd subunit homolog Rpa4 BC011621 1.37E-05 0.5264059 HOOK1.vertline.hook1 protein BC006284 1.48E-05 2.1550372 Homo sapiens, clone IMAGE: 3957135, mRNA, partial cds BC004235 2.01E-05 2.4910338 DDX38.vertline.DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 38 NM_006278 2.06E-05 1.9872895 SIAT4C.vertline.sialyltransf- erase 4C (beta-galactosidase alpha- 2,3-sialytransferase) AI972367 2.13E-05 2.1500078 Homo sapiens cDNA FLJ32384 fis, clone SKMUS1000104, weakly similar to Homo sapiens mRNA for HEXIM1 protein, complete cds BC012628 2.31E-05 2.0388066 TCAP.vertline.titin-cap (telethonin) AA581602 2.44E-05 0.4839842 ESTs NM_018936 2.46E-05 1.4853858 PCDHB2.vertline.protocadherin beta 2 AA746504 2.68E-05 0.667095 Homo sapiens cDNA FLJ30188 fis, clone BRACE2001267 AF220030 2.73E-05 0.4441676 TRIM6.vertline.tripartite motif-containing 6 AI682928 2.90E-05 0.4144403 EST AA206609 3.05E-05 2.0738914 Homo sapiens cDNA FLJ30002 fis, clone 3NB691000085 AL117616 3.06E-05 0.5506486 SRI.vertline.sorcin U08997 3.06E-05 0.548039 GLUD2.vertline.Glutamate dehydrogenase-2 BC009869 3.17E-05 0.4884412 SERF2.vertline.small EDRK-rich factor 2 AL137521 3.24E-05 2.4199381 Homo sapiens mRNA; cDNA DKFZp434D0218 (from clone DKFZp434D0218); partial cds AI871458 3.26E-05 2.0738428 ESTs BC008841 3.27E-05 1.8195551 KIAA0415.vertline.KIAA0415 gene product AI467849 4.07E-05 1.689976 TBC1D1.vertline.TBC1 (tre-2/USP6, BUB2, cdc16) domain family, member 1 BC011754 4.42E-05 1.6224459 ERP70.vertline.protein disulfide isomerase related protein (calcium-binding protein, intestinal-related) AL050227 4.44E-05 0.7135796 Homo sapiens mRNA; cDNA DKFZp586M0723 (from clone DKFZp586M0723) AK021798 4.56E-05 0.6377454 FLJ11736.vertline.hypothetical protein FLJ11736 AI268007 4.58E-05 0.7185686 Homo sapiens cDNA FLJ30137 fis, clone BRACE2000078 BC001403 4.70E-05 2.4561451 CPSF5.vertline.cleavage and polyadenylation specific factor 5, 25 kD subunit AK000081 5.38E-05 2.3154373 CDC2L1.vertline.cell division cycle 2-like 1 (PITSLRE proteins) BC014270 5.53E-05 2.0457284 PRKCZ.vertline.protein kinase C, zeta AL117478 5.97E-05 1.7598438 AGS3.vertline.likely ortholog of rat activator of G-protein signaling 3 BF116098 7.56E-05 0.4180467 ESTs BC006499 7.83E-05 1.8287714 HRAS.vertline.v-Ha-ras Harvey rat sarcoma viral oncogene homolog NM_003489 7.94E-05 0.4637752 NRIP1.vertline.nuclear receptor interacting protein 1 AI469557 8.50E-05 1.8599762 EPHB3.vertline.EphB3 AI561249 9.19E-05 0.4329273 KTN1.vertline.kinectin 1 (kinesin receptor) BC015497 9.45E-05 1.9287915 TEAD4.vertline.TEA domain family member 4 AL133661 1.08E-04 0.4897642 DKFZp434C0328.vertline.hypothetical protein DKFZp434C0328 BC015594 1.10E-04 2.0502453 Homo sapiens mRNA for FLJ00083 protein, partial cds AW135596 1.14E-04 0.6460164 FLJ10058.vertline.hypothetical protein FLJ10058 AI033912 1.18E-04 0.6482864 RLN2.vertline.relaxin 2 (H2) NM_020978 1.28E-04 0.598655 AMY2B.vertline.amylase, alpha 2B; pancreatic BC006437 1.49E-04 2.0560166 C321D2.4.vertline.hypothetical protein C321D2.4 AW016075 1.51E-04 0.5312489 ESTs, Weakly similar to ALUA_HUMAN !!!! ALU CLASS A WARNING ENTRY !!! [H. sapiens] NM_001354 1.52E-04 1.4085552 AKR1C2.vertline.aldo-keto reductase family 1, member C2 (dihydrodiol dehydrogenase 2; bile acid binding protein; 3- alpha hydroxysteroid dehydrogenase, type III) BC007932 1.56E-04 0.5115812 FLJ11588.vertline.hypothetical protein FLJ11588 AF319520 1.57E-04 1.4189657 ARG99.vertline.ARG99 protein AA806831 1.62E-04 1.470609 ESTs AI638324 1.64E-04 0.4669648 Homo sapiens cDNA FLJ30332 fis, clone BRACE2007254 AK025141 1.70E-04 0.6098107 Homo sapiens cDNA: FLJ21488 fis, clone COL05445 AF068918 2.11E-04 1.7571167 BIN1.vertline.bridging integrator 1 AF208111 2.18E-04 0.6637063 IL17BR.vertline.interleukin 17B receptor AK024715 2.34E-04 0.5237823 FLJ21062.vertline.hypothetical protein FLJ21062 BC007836 2.45E-04 1.8806038 MDFI.vertline.MyoD family inhibitor AW192535 2.64E-04 0.46396 ESTs AA480069 2.68E-04 1.970316 KIAA1925.vertline.KIAA1925 protein AK025862 2.84E-04 0.4739154 Homo sapiens cDNA: FLJ22209 fis, clone HRC01496 AI800042 2.92E-04 0.4939835 ESTs AA977269 3.02E-04 1.3578379 FOXD1.vertline.forkhead box D1 BC018644 3.03E-04 1.6098715 NUDT8.vertline.nudix (nucleoside diphosphate linked moiety X)- type motif 8 NM_004419 3.08E-04 0.6155024 DUSP5.vertline.dual specificity phosphatase 5 AW070918 3.10E-04 2.0916912 ESTs, Weakly similar to T2D3_HUMAN TRANSCRIPTION INITIATION FACTOR TFIID 135 KDA SUBUNIT [H. sapiens] AA040945 3.22E-04 2.2990713 ESTs AF035282 3.30E-04 0.6524492 C1orf21.vertline.chromosome 1 open reading frame 21 NM_006304 3.34E-04 0.4895086 DSS1.vertline.Deleted in split-hand/split-foot 1 region R62589 3.47E-04 0.6003814 ESTs AI400775 3.52E-04 2.2438708 RABL2B.vertline.RAB, member of RAS oncogene family-like 2B AI128331 3.60E-04 0.5099963 ENDOFIN.vertline.endoso- me-associated FYVE-domain protein AW069725 3.62E-04 0.5812922 CRYZ.vertline.crystallin, zeta (quinone reductase) AK024967 3.82E-04 0.4618762 Homo sapiens cDNA: FLJ21314 fis, clone COL02248 AK022916 3.88E-04 0.5564747 ZNF281.vertline.zinc finger protein 281 BC015484 3.92E-04 1.5502435 CALB2.vertline.calbindin 2, (29 kD, calretinin) AI953054 4.06E-04 1.9805492 TKT.vertline.transketolase (Wernicke-Korsakoff syndrome) BE675157 4.28E-04 0.6073104 ESTs AF153330 4.33E-04 0.5983906 SLC19A2.vertline.solute carrier family 19 (thiamine transporter), member 2 AL133427 4.35E-04 0.4914871 Homo sapiens mRNA full length insert cDNA clone EUROIMAGE 261172 BF438928 4.77E-04 0.5752913 ESTs NM_002428 4.77E-04 1.81811 MMP15.vertline.matrix metalloproteinase 15 (membrane-inserted) AI264644 4.82E-04 1.8613174 KIAA0775.vertline.KIAA0775 gene product BE967259 4.88E-04 0.7445998 BCL2.vertline.B-cell CLL/lymphoma 2 AW076080 4.93E-04 0.5435194 Homo sapiens, clone IMAGE: 3463399, mRNA, partial cds T52873 5.05E-04 0.5449457 ESTs, Moderately similar to G02075 transcription repressor zinc finger protein 85 [H. sapiens] AF085233 5.10E-04 0.635643 SGKL.vertline.serum/glucocorticoid regulated kinase-like BE671445 5.12E-04 0.5796479 ESTs AI356375 5.23E-04 1.7149531 CDKN2A.vertline.cyclin-dependent kinase inhibitor 2A

(melanoma, p16, inhibits CDK4) BF589163 5.28E-04 0.5585288 ESTs AA909006 5.35E-04 1.5526313 LBP-32.vertline.LBP protein 32 BC015792 5.47E-04 1.841097 Homo sapiens, clone MGC: 23665 IMAGE: 4866941, mRNA, complete cds BC000692 5.61E-04 2.0170046 HYAL2.vertline.hyaluronoglucosaminidase 2 AL050090 5.73E-04 0.7500215 DKFZP586F1018.vertline.DKFZP586F1018 protein NM_020235 5.94E-04 0.5893936 BBX.vertline.bobby sox homolog (Drosophila) BF433657 5.99E-04 1.9378811 ESTs AI692302 6.01E-04 1.899281 ESTs AK024782 6.05E-04 1.9756718 KIAA1608.vertline.KIAA1608 protein AF124735 6.12E-04 1.4649329 LHX2.vertline.LIM homeobox protein 2 BC007066 6.12E-04 0.5216856 CDA11.vertline.CDA11 protein AW135238 6.20E-04 0.4896724 ESTs AK026747 6.44E-04 0.5015784 LOC54103.vertline.hypothetical protein AA542898 6.46E-04 0.7842204 P28.vertline.dynein, axonemal, light intermediate polypeptide BC014913 6.52E-04 0.6913458 Homo sapiens, Similar to synaptotagmin-like 4, clone MGC: 17313 IMAGE: 3908307, mRNA, complete cds AI270018 6.72E-04 2.0809844 ECE1.vertline.endothelin converting enzyme 1 L13738 6.90E-04 1.6894154 ACK1.vertline.activated p21cdc42Hs kinase BC002607 7.01E-04 1.5250234 KIAA1446.vertline.KIAA1446 protein BI793002 7.18E-04 0.4917655 OSBPL8.vertline.oxysterol binding protein-like 8 BC007092 7.20E-04 1.2827239 HOXB13.vertline.homeo box B13 BC009874 7.40E-04 1.730815 JUN.vertline.v-jun sarcoma virus 17 oncogene homolog (avian) AF321193 7.41E-04 1.5356899 DSCR8.vertline.Down syndrome critical region gene 8 AK000397 7.70E-04 1.5631718 FLJ10351.vertline.likely ortholog of mouse piwi like homolog 1 (Drosophila)-like AF052110 7.76E-04 1.6400255 DAF.vertline.decay accelerating factor for complement (CD55, Cromer blood group system) AA648536 8.03E-04 1.6290887 MYO1E.vertline.myosin IE BF436400 8.31E-04 0.7911405 EST AL050179 8.59E-04 0.5180149 TPM1.vertline.tropomyosin 1 (alpha) AI700363 8.60E-04 1.3675668 ESTs NM_004038 8.72E-04 0.6247207 AMY1A.vertline.amylase, alpha 1A; salivary AF060555 8.75E-04 1.5560891 ESR2.vertline.estrogen receptor 2 (ER beta) AK026756 8.85E-04 0.6360787 KIAA1603.vertline.KIAA1603 protein AI686003 8.97E-04 0.6087104 ESTs NM_019120 9.14E-04 1.4302118 PCDHB8.vertline.protocadherin beta 8 NM_020957 9.50E-04 1.4881037 PCDHB16.vertline.protocadherin beta 16 AI921700 9.73E-04 0.522736 ITGAV.vertline.integrin, alpha V (vitronectin receptor, alpha polypeptide, antigen CD51) X62534 9.87E-04 0.5796731 HMG2.vertline.high-mobility group (nonhistone chromosomal) protein 2 BC002738 9.90E-04 1.8608522 CRIP1.vertline.cysteine-rich protein 1 (intestinal)

[0167] Between the two approaches, 114 genes were in common. At the significance level of 0.001, about 6 genes are expected by chance if there are no real differences between the patient groups, indicating that the 149 genes identified by either method are highly statistically significant.

Example 2

Kaplan-Meier Survival Curves of Patients Stratified by Cross-Validation

[0168] Kaplan-Meier analysis was performed to assess the differential survival of patients stratified by the gene expression signature. Leave-one-out-cross-validation was performed. Briefly, one of the 62 patients was left out as a test sample, and the other 61 samples were used in Cox regression to both select significant genes (p<0.001) and obtain gene-specific weights (Cox regression coefficients .beta.). A linear sum of the gene-specific weights (.beta.) times expression levels (x) across all selected genes was calculated as the overall risk score for each patient: S=sum(.beta..sub.ix.sub.i) for all selected genes. The mid-point m between the median scores for the two patient groups (recurrence/non-recurrence) in the training set was calculated: m=(median score of recurrence group+median score of non-recurrence group)/2, and the score for the test sample S was compared with m to classify the test sample to either the recurrence (S>m, TAM signature-) or non-recurrence group (S<=m, TAM signature+). This entire procedure was repeated 62 times to generate a classification for each patient. Disease-free survival curves of the two groups as assigned by the cross-validation procedure are compared. The results are shown in FIG. 1.

Example 3

Identification of Biomarker Predictors of TAM Treatment Outcome

[0169] Samples from 60 patients with ER+ primary breast cancer, and treated with adjuvant TAM, were selected tamoxifen based on treatment outcome. 28 had developed tumor recurrence with a median time of 4 years, and 32 remained disease-free with a median follow-up of 10 years (Table 3). Patients who remained disease-free during the entire follow up period were likely to represent responders to TAM, although a small subset of them might have been cured by surgery alone. Those patients who developed tumor recurrence despite TAM therapy either did not respond or developed resistance to TAM and are hereafter referred to as non-responders for brevity. To control for known prognostic factors, tumors between these two groups were matched by tumor size, lymph node status and tumor grade.

15TABLE 3 Patients and tumor characteristics Tumor Sample ID type Size Grade Nodes ER PR Age DFS Status 1389 D 1.7 2 0/1 Pos Pos 80 94 0 648 D 1.1 2 0/15 Pos ND 62 160 0 289 D 3 2 0/15 Pos ND 75 63 1 749 D 1.8 2 2/9 Pos Pos 61 137 0 420 D/L 2 3 ND Pos Pos 72 58 1 633 D 2.7 3 0/11 Pos ND 61 20 1 662 D 1 3 6/11 Pos Pos 79 27 1 849 D 2 1 0/26 Pos Neg 75 23 1 356 D 1 2 2/20 Pos ND 58 24 1 1304 D 2 3 0/14 Pos Pos 57 20 1 1419 D 2.5 2 1/8 Pos Pos 59 86.04 0 1093 D 1 3 1/14 Pos Pos 66 84.96 0 1047 D/L 2.6 2 0/18 Pos Neg 70 127.92 0 1037 D/L 1.5 2 0/4 Pos Pos 85 83.04 0 319 D 4 2 1/13 Pos ND 67 44 1 25 D 3.5 2 0/9 Neg Pos 62 75 1 180 D 1.6 2 2/19 Pos Pos 69 168.96 0 687 D 3.5 3 3/16 Pos ND 73 141.96 0 856 D 1.6 2 0/16 Pos Pos 73 87.96 0 1045 D 2.5 3 1/12 Pos Neg 73 120.96 0 1205 D 2.7 2 1/19 Pos Pos 71 87.96 0 1437 D 1.7 2 2/22 Pos Pos 67 89.04 0 1507 D 3.7 3 0/40 Pos Pos 70 69.96 0 469 D 1 1 0/19 Pos ND 66 161.04 0 829 D 1.2 2 0/9 Pos ND 69 135.96 0 868 D 3 3 0/13 Pos Pos 65 129.96 0 1206 D 4.1 3 0/15 Pos Neg 84 56 1 843 D 3.4 2 11/20 Pos Neg 76 122 1 342 D 3 2 9/21 Pos ND 62 102 1 1218 D 4.5 1 3/16 Pos Pos 62 10 1 547 D/L 1.5 2 ND Pos ND 74 129 1 1125 D 2.6 2 0/18 Pos Pos 54 123 0 1368 D 2.6 2 ND Pos Pos 82 63 0 605 D 2.2 2 6/18 Pos ND 70 110.04 0 59 L 3 2 33/38 Pos ND 70 21 1 68 D 3 2 0/17 Pos ND 53 38 1 317 D 1.2 3 1/10 Pos Pos 71 5 1 374 D 1 3 0/15 Pos Neg 57 47 1 823 D 2 2 0/6 Pos Pos 51 69 1 280 D 2.2 3 0/12 Pos ND 66 44 1 651 D 4.7 3 10/13 Pos ND 48 137 1 763 D 1.8 2 0/14 Pos Pos 63 117.96 0 1085 D 4.7 2 0/8 Pos Pos 48 101 1 1363 D 2.1 2 0/15 Pos Pos 56 114 0 295 D 3.5 2 3/21 Pos Pos 52 118 1 871 D 4 3 0/16 Pos Neg 61 6 1 1343 D 2.5 3 ND Pos Pos 79 21 1 140 L >2.0 2 18/28 Pos ND 63 43 1 260 D/L 0.9 2 1/13 Pos ND 73 42 1 297 D 0.8 2 1/16 Pos Pos 66 169 0 1260 D 3.5 2 0/14 Pos Pos 58 79 0 1405 D 1 3 ND Pos Pos 81 95.04 0 518 L 5.5 2 3/20 Pos ND 68 156 0 607 D 1.2 2 5/14 Pos Pos 76 114 0 638 D 2 2 1/24 Pos Pos 67 147.96 0 655 D 2 3 ND Pos Pos 73 143.04 0 772 D 2.5 2 0/18 Pos Pos 68 69 1 878 D/L 1.6 2 0/9 Pos Neg 76 138 0 1279 D 2 2 0/12 Pos Pos 68 102 0 1370 D 2 2 ND Pos Pos 73 60.96 0 Abbreviations: D, ductal; L, lobular; pos, positive; neg, negative; ND, not determined; ER, estrogen receptor; PR, progesterone receptor; DFS, disease-free survival; status = 1, recurred; status = 0, disease-free.

[0170] The samples were used to identify gene expression signatures correlated with outcome of TAM treatment. Each breast cancer biopsy contains a mixture of cell types including epithelial breast cancer cells, infiltrating lymphocytes, endothelial cells and stromal fibroblasts. It has been suggested that complex interactions among these cell types in the tumor microenvironment determine the biological behavior of the tumor. Therefore, to identify gene expression differences in primary tumors between TAM responders and non-responders, expression profiling of both whole tissue sections, which represent this microenvironment, and microdissected, largely pure populations of epithelial cancer cells from each tumor biopsy were conducted on a custom 22k oligonucleotide microarray.

[0171] This generated two parallel datasets corresponding to each patient: one set from whole tissue sections ("sections dataset") and another from laser capture microdissected cancer cells ("LCM dataset"). Each expression dataset was first filtered based on overall variance of each gene and the top 5475 high-variance genes (75th percentile) were selected. Using the reduced datasets, t-test on each gene between the TAM responders and non-responders were carried out. From the sections dataset, 19 genes were identified at the p value cutoff of 0.001 (Table 4). The probability of selecting this many or more differentially expressed genes by chance was 0.035 as estimated by randomly permuting the patient class with respect to treatment outcome and repeating the t-test procedure 1000 times. Among the 19 genes identified in the sections dataset, genes involved in immune response are particularly prominent.

16TABLE 4 19-gene signature identified by t-test in the Sections dataset Mean in Fold Parametric p- Mean in non- difference value responders responders of means GB acc Description 1 1.96E-05 0.759 1.317 0.576 AW006861 SCYA4.vertline.small inducible cytokine A4 2 2.43E-05 1.31 0.704 1.861 AI240933 ESTs 3 8.08E-05 0.768 1.424 0.539 X59770 IL1R2.vertline.interleukin 1 receptor, type II 4 9.57E-05 0.883 1.425 0.62 AB000520 APS.vertline.adaptor protein with pleckstrin homology and src homology 2 domains 5 9.91E-05 1.704 0.659 2.586 AF208111 IL17BR.vertline.interleukin 17B receptor 6 0.0001833 0.831 1.33 0.625 AI820604 ESTs 7 0.0001935 0.853 1.459 0.585 AI087057 DOK2.vertline.docking protein 2, 56 kD 8 0.0001959 1.29 0.641 2.012 AJ272267 CHDH.vertline.choline dehydrogenase 9 0.0002218 1.801 0.943 1.91 N30081 ESTs, Weakly similar to I38022 hypothetical protein [H. sapiens] 10 0.0004234 1.055 2.443 0.432 AI700363 ESTs 11 0.0004357 0.451 1.57 0.287 AL117406 ABCC11.vertline.ATP-binding cassette, sub- family C (CFTR/MRP), member 11 12 0.0004372 1.12 3.702 0.303 BC007092 HOXB13.vertline.homeo box B13 13 0.0005436 0.754 1.613 0.467 M92432 GUCY2D.vertline.guanylate cyclase 2D, membrane (retina-specific) 14 0.0005859 1.315 0.578 2.275 AL050227 Homo sapiens mRNA; cDNA DKFZp586M0723 (from clone DKFZp586M0723) 15 0.000635 1.382 0.576 2.399 AW613732 Homo sapiens cDNA FLJ31137 fis, clone IMR322001049 16 0.0008714 0.794 1.252 0.634 BC007783 SCYA3.vertline.small inducible cytokine A3 17 0.0008912 2.572 1.033 2.49 X81896 C11orf25.vertline.chromosome 11 open reading frame 25 18 0.0009108 0.939 1.913 0.491 BC004960 MGC10955.vertline.hypothetical protein MGC10955 19 0.0009924 1.145 0.719 1.592 AK027250 Homo sapiens cDNA: FLJ23597 fis, clone LNG15281

[0172] Repeating the same analysis on the LCM dataset yielded 9 significant genes at the cutoff of p<0.001 (Table 5); however, the probability of finding 9 or more genes by chance is 0.154 in permutation analysis (n=1000). These results established that significant differences in gene expression between the two patient groups exist, but differences were subtle.

17TABLE 5 9-gene signature identified by t-test in the LCM dataset Mean in Fold Parametric Mean in non- difference p-value responders responders of means GB acc Description 1 2.67E-05 1.101 4.891 0.225 BC007092 HOXB13.vertline.homeo box B13 2 0.0003393 1.045 2.607 0.401 AI700363 ESTs 3 0.0003736 0.64 1.414 0.453 NM 014298 QPRT.vertline.quinolinate phosphoribosyltransferase (nicotinate- nucleotide pyrophosphorylase (carboxylating)) 4 0.0003777 1.642 0.694 2.366 AF208111 IL17BR.vertline.interleukin 17B receptor 5 0.0003895 0.631 1.651 0.382 AF033199 ZNF204.vertline.zinc finger protein 204 6 0.0004524 1.97 0.576 3.42 AI688494 FLJ13189.vertline.hypothetical protein FLJ13189 7 0.0005329 1.178 0.694 1.697 AI240933 ESTs 8 0.0007403 0.99 1.671 0.592 AL57459 Homo sapiens mRNA; cDNA DKFZp434B0425 (from clone DKFZp434B0425) 9 0.0007739 0.723 1.228 0.589 BC002480 FLJ13352.vertline.hypothetical protein FLJ13352

[0173] The sequence of each GenBank accession number in Tables 4 and 5 is presented in the attached Appendix.

[0174] Due to the limited sample size (n=60), leave-one-out cross validation was used to assess the predictive significance of the gene expression signature. In each round of cross validation, significant genes were identified using the training set by t-test at p<0.001, and a compound covariate predictor was built as the linear combination oft he gene expression values over all significant genes weighted by their t-statistics. The predictor was then used to predict the left-out sample. Repeating this procedure 60 times generated an "honest" prediction on each sample.

[0175] Using the sections dataset, the overall accuracy of cross validation results are 70%, and the sensitivity, specificity, positive and negative predictive values are 60%, 78%, 71%, and 69%, respectively. The results of analyzing the LCM dataset were slightly lower, with an overall accuracy of 67%, and sensitivity, specificity, positive and negative predictive values of 57%, 75%, 67%, and 67%, respectively. Patients having the "responder signature" and those having the "non-responder signature" as predicted from cross validation demonstrate significantly different disease-free survival curves (FIG. 2).

[0176] Previously a 70-gene prognostic classifier was derived from correlating gene expression profiles with distant metastasis from node-negative breast cancer patients, most of which received no adjuvant chemotherapy or endocrine therapy. 61 of the 70 genes from the study were on the microarrays used in this example. Expression data corresponding to these 61 genes were extracted from the sections dataset because the 70-gene signature study used whole tissue sections. None of these 61 genes were significantly differentially expressed between TAM responders and non-responders at the significance level of 0.001, and only 3 genes were significant at p<0.05. Leave-one-out cross-validation analysis using either all 61 genes or only genes with p<0.05 gave overall accuracies of 52% and 53% respectively. Thus the 70-gene classifier derived from mostly untreated patients cannot predict tumor recurrence after adjuvant TAM treatment. Without being bound by theory, and offered to improve the understanding of the invention, this suggests that the treatment outcome by TAM is not simply a reflection of the aggressiveness of the primary tumor, but may directly reflect the responsiveness to TAM.

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