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WO 2010/065940 Al (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date 10 June 2010 (10.06.2010) WO 2010/065940 Al (51) International Patent Classification: (74) Agent: AI, Bing; Fish & Richardson P.C., P.O. Box GOlN 33/50 (2006.01) GOlN 33/574 (2006.01) 1022, Minneapolis, MN 55440-1022 (US). Cl 2Q 1/68 (2006.01) (81) Designated States (unless otherwise indicated, for every (21) International Application Number: kind of national protection available): AE, AG, AL, AM, PCT/US2009/066895 AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, (22) International Filing Date: DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, 4 December 2009 (04.12.2009) HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, (25) Filing Language: English KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, (26) Publication Language: English NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, (30) Priority Data: SE, SG, SK, SL, SM, ST, SV, SY, TJ, TM, TN, TR, TT, 61/1 19,996 4 December 2008 (04.12.2008) US TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (71) Applicant (for all designated States except US): THE (84) Designated States (unless otherwise indicated, for every REGENTS OF THE UNIVERSITY OF CALIFOR¬ kind of regional protection available): ARIPO (BW, GH, NIA [US/US]; 5th Floor, 1111 Franklin Street, Oakland, GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, CA 94607-5200 (US). ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, (72) Inventors; and ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, (75) Inventors/Applicants (for US only): MCCLELLAND, MC, MK, MT, NL, NO, PL, PT, RO, SE, SI, SK, SM, Michael [US/US]; 2706 El Camino Real Del Norte, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, Encinitas, CA 92024 (US). WANG, YiPeng [CN/US]; ML, MR, NE, SN, TD, TG). 3146 Occidental Street, San Diego, CA 92122 (US). MERCOLA, Daniel [US/US]; P.O. Box 3752, Rancho Published: Santa Fe, CA 92067 (US). — with international search report (Art. 21(3)) (54) Title: MATERIALS AND METHODS FOR DETERMINING DIAGNOSIS AND PROGNOSIS OF PROSTATE CANCER (57) Abstract: Materials and methods related to diagnosing and/or determining prognosis of prostate cancer. MATERIALS AND METHODS FOR DETERMINING DIAGNOSIS AND PROGNOSIS OF PROSTATE CANCER CROSS-REFERENCE TO RELATEDAPPLICATIONS This application claims benefit of priority from U.S. Provisional Application Serial No. 61/119,996, filed on December 4, 2008. STATEMENT AS TO FEDERALLY SPONSORED RESEARCH This invention was made with government support under grant no. CAl 14810 awarded by the National Institutes of Health. The government has certain rights in the invention. TECHNICALFIELD This document relates to materials and methods for determining gene expression in cells, and for diagnosing prostate cancer and assessing prognosis of prostate cancer patients. BACKGROUND Prostate cancer is the most common malignancy in men and is the cause of considerable morbidity and mortality (Howe et al. (2001) / . Natl. Cancer Inst. 93:824-842). It may be useful to identify genes that could be reliable early diagnostic and prognostic markers and therapeutic targets for prostate cancer, as well as other diseases and disorders. SUMMARY This document is based in part on the discovery that RNA expression changes can be identified that can distinguish normal prostate stroma from tumor-adjacent stroma in the absence of tumor cells, and that such expression changes can be used to signal the "presence of tumor." A linear regression method for the identification of cell-type specific expression of RNA from array data of prostate tumor-enriched samples was previously developed and validated (see, U.S. Publication No. 20060292572 and Stuart et al. (2004) Proc. Natl. Acad. ScL USA 101:615-620, both incorporated herein by reference in their entirety). As described herein, the approach was extended to evaluate differential expression data obtained from normal volunteer prostate biopsy samples with tumor-adjacent stroma. Over a thousand gene expression changes were observed. A subset of stroma-specific genes were used to derive a classifier of 131 probe sets that accurately identified tumor or nontumor status of a large number of independent test cases. These observations indicate that tumor-adjacent stroma exhibits a larger number of gene expression changes and that subset may be selected to reliably identify tumor in the absence of tumor cells. The classifier may be useful in the diagnosis of stroma-rich biopsies of clinical cases with equivocal pathology readings. The present disclosure includes, inter alia, the following: (1) extensive cross- validation of RNA biomarkers for prostate cancer relapse, across multiple datasets; (2) a "bi- modal" method for generating classifiers and testing them on samples that have mixed tissue; and (3) two methods for identifying genes in "reactive-stroma" that can be used as markers for the presence of cancer even when the sample does not include tumor but instead has regions of reactive stroma, near tumor. In one aspect, this document features an in vitro method for identifying a subject as having or not having prostate cancer, comprising: (a) providing a prostate tissue sample from the subject; (b) measuring the level of expression for prostate cancer signature genes in the sample; (c) comparing the measured expression levels to reference expression levels for the prostate cancer signature genes; and (d) if the measured expression levels are significantly greater or less than the reference expression levels, identifying the subject as having prostate cancer, and if the measured expression levels are not significantly greater or less than the reference expression levels, identifying the subject as not having prostate cancer. The prostate tissue sample may not include tumor cells, or the prostate tissue sample may include tumor cells and stromal cells. The prostate cancer signature genes can be selected from the genes listed in Table 3 or Table 4 herein. The method can include determining whether measured expression levels for ten or more prostate cancer signature genes are significantly greater or less than reference expression levels for the ten or more prostate cancer signature genes, and classifying the subject as having prostate cancer that is likely to relapse if the measured expression levels are significantly greater or less than the reference expression levels, or classifying the subject as having prostate cancer not likely to relapse if the measured expression levels are not significantly greater or less than the reference expression levels. The ten or more prostate cancer signature genes can be selected from the genes listed in Table 3 or Table 4 herein. The method can include determining whether measured expression levels for twenty or more prostate cancer signature genes are significantly greater or less than reference expression levels for the twenty or more prostate cancer signature genes, and classifying the subject as having prostate cancer that is likely to relapse if the measured expression levels are significantly greater or less than the reference expression levels, or classifying the subject as having prostate cancer not likely to relapse if the measured expression levels are not significantly greater or less than the reference expression levels. The twenty or more prostate cancer signature genes can be selected from the genes listed in Table 3 or Table 4 herein. In another aspect, this document features a method for determining the prognosis of a subject diagnosed as having prostate cancer, comprising: (a) providing a prostate tissue sample from the subject; (b) measuring the level of expression for prostate cancer signature genes in the sample; (c) comparing the measured expression levels to reference expression levels for the prostate cancer signature genes; and (d) if the measured expression levels are not significantly greater or less than the reference expression levels, identifying the subject as having a relatively better prognosis than if the measured expression levels are significantly greater or less than the reference expression levels, or if the measured expression levels are significantly greater or less than the reference expression levels, identifying the subject as having a relatively worse prognosis than if the measured expression levels are not significantly greater or less than the reference expression levels. The prostate tissue sample may not include tumor cells, or the prostate tissue sample may include tumor cells and stromal cells. The prostate cancer signature genes can be selected from the genes listed in Table 8A or 8B herein. In another aspect, this document features a method for identifying a subject as having or not having prostate cancer, comprising: (a) providing a prostate tissue sample from the subject, wherein the sample comprises prostate stromal cells; (b) measuring expression levels for one or more genes in the stromal cells, wherein the one or more genes are prostate cancer signature genes; (c) comparing the measured expression levels to reference expression levels for the one or more genes, wherein the reference expression levels are determined in stromal cells from non-cancerous prostate tissue; and (d) if the measured expression levels are significantly greater or less than the reference expression levels, identifying the subject as having prostate cancer, and if the measured expression levels are not significantly greater or less than the reference expression levels, identifying the subject as not having prostate cancer. The prostate tissue sample may not include tumor cells, or the prostate tissue sample may include tumor cells and stromal cells.
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