Wo 2010/070637 A2

Wo 2010/070637 A2

(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 24 June 2010 (24.06.2010) WO 2010/070637 A2 (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C12Q 1/68 (2006.01) kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, (21) International Application Number: CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, PCT/IL2009/001 181 DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (22) International Filing Date: HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, 13 December 2009 (13.12.2009) KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, (25) Filing Language: English NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, (26) Publication Language: English SE, SG, SK, SL, SM, ST, SV, SY, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (30) Priority Data: 61/122,422 15 December 2008 (15.12.2008) US (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, (71) Applicants (for all designated States except US): GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, ROSETTA GENOMICS LTD. [ILIlL]; 10 Plaut Street, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, 76706 Rehovot (IL). MOR RESEARCH APPLICA¬ TM), European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, TIONS [IL/IL]; 38 Habarzel Street, Ramat Hahyal, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, 69710 Tel Aviv (IL). MC, MK, MT, NL, NO, PL, PT, RO, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, (72) Inventors; and ML, MR, NE, SN, TD, TG). (75) Inventors/Applicants (for US only): SPECTOR, Yael [IL/IL]; Hanevihim 19, 64356 Tel Aviv (IL). FEIN- Published: MESSER, Meora [IL/IL]; 18 Eilat Street, 49925 Kfar — without international search report and to be republished Mass (IL). upon receipt of that report (Rule 48.2(g)) (74) Agent: SHOSHANI-KUPITZ, Tzipora; Rosetta Ge — with sequence listing part of description (Rule 5.2(a)) nomics Ltd., 10 Plaut Street, 76706 Rehovot (IL). (54) Title: METHOD FOR DISTINGUISHING BETWEEN ADRENAL TUMORS (57) Abstract: The present invention provides nucleic acid sequences that are used for identification, classification and diagnosis of specific types of adrenal tumors. The nucleic acid sequences can also be used for evaluation of a subject based on the expres sion pattern of a biological sample. METHOD FOR DISTINGUISHING BETWEEN ADRENAL TUMORS CROSS REFERENCE TO RELATED APPLICATIONS The present application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 61/122,422, filed December 15, 2008, which is herein incorporated by reference in its entirety. FIELD OF THE INVENTION The invention relates in general to microRNA molecules associated with specific types of adrenal tumors, as well as various nucleic acid molecules relating thereto or derived therefrom. BACKGROUND OF THE INVENTION In recent years, microRNAs (miRs) have emerged as an important novel class of regulatory RNA, and have a profound impact on a wide array of biological processes. These small (typically 18-24 nucleotides long) non-coding RNA molecules can modulate protein expression patterns by promoting RNA degradation, inhibiting mRNA translation, and also by affecting gene transcription. miRs play pivotal roles in diverse processes such as development and differentiation, control of cell proliferation, stress response and metabolism. The expression of many miRs has been found to be altered in numerous types of human cancer, and in some cases strong evidence has been put forward in support of the conjecture that such alterations may play a causative role in tumor progression. There are currently about 900 known human miRs. Classification of cancer has typically relied on the grouping of tumors based on histology, cytogenetics, immunohistochemistry, and known biological behavior. The pathologic diagnosis used to classify the tumor, taken together with the stage of the cancer, is then used to predict prognosis and direct therapy. However, current methods of cancer classification and staging are not completely reliable. Scientists strive to explore biomarkers and their possible role in the diagnosis, treatment and prognosis of specific cancers. Adrenal tumors can refer to one of several benign and malignant neoplasms of the adrenal gland, several of which are notable for their tendency to overproduce endocrine hormones. These include tumors of the medulla of the adrenal located anatomically at the center of each adrenal gland, i.e., pheochromocytomas, and adrenocortical tumors (ACTs), namely adrenocortical carcinoma (ACC) and adrenocortical adenoma (ACA). The prediction of malignant potential is usually straightforward in adrenocortical tumors, although some present with borderline features and elude a specific diagnosis. Several protocols for adrenocortical tumor analysis have been suggested. The most reproducible is that of Weiss (Am J Sug Pathol, 1984 Mar; 8(3): 163-9), who assessed nine features thought to be related to malignancy: The presence of three or more of these features in a given tumor indicates malignant potential. However, some of the features show a poor correlation amongst pathologists. Information on the molecular pathogenesis of tumors is constantly accumulating, but it has not been utilized thus far in the diagnostic setting. The clinical significance of distinguishing between types of adrenal tumors is important given that cases of adrenocortical adenoma have been detected as incidental findings with increasing frequency in recent years, due to the increasing use of CT scans and magnetic resonance imaging in a variety of medical settings. Widespread use of imaging tests is creating the clinical quandary of what to do about adrenal incidentalomas- masses of 1 cm or larger on the adrenal glands discovered serendipitously on imaging in patients with no symptoms or clinical evidence of adrenal disease. Today, most patients with masses of 4 cm or smaller do not undergo any surgery due to high risk and instead, undergo massive imaging follow-up. This can result in expensive additional testing and invasive procedures to rule out the slight possibility of an early adrenocortical carcinoma. There is an unmet need for a reliable method for distinguishing between tumors of the adrenal, specifically between adrenocortical adenoma and adrenocortical carcinoma, and also between pheochromocytomas, and adrenocortical tumors. SUMMARY OF THE INVENTION The present invention provides nucleic acid sequences for use in the identification, classification and diagnosis of specific tumors of the adrenal. The invention provides nucleic acid sequences for use in distinguishing between adrenocortical adenoma and adrenocortical carcinoma, and also between pheochromocytomas, and adrenocortical tumors.The nucleic acid sequences can also be used as prognostic markers for prognostic evaluation of a subject based on their expression pattern in a biological sample. According to one aspect, the present invention provides a method of classifying a specific tumor of the adrenal, the method comprising obtaining a biological sample from a subject, determining an expression profile of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1-17, 29, 35-51, 67, 74-107, a fragment thereof, or a sequence having at least about 80% identity thereto, and any combinations thereof, from said sample, and comparing said expression profile to a reference expression profile, wherein the results of said comparison allows for classification of said tumor of the adrenal. According to certain embodiments, the tumor of the adrenal is selected from the group consisting of pheochromocytoma and a tumor of the adrenal cortex. According to one embodiment, the tumor of the adrenal cortex is selected from the group consisting of adrenocortical carcinoma and adrenocortical adenoma. The invention further provides a method for distinguishing between adrenocortical carcinoma and adrenocortical adenoma, the method comprising obtaining a biological sample from a subject, determining an expression profile of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1-7, 79, 80, 36-41 and 95-96, a fragment thereof and a sequence having at least about 80% identity thereto, and any combinations thereof, from said sample, and comparing said expression profile to a reference expression profile, wherein the results of said comparison allows for distinguishing between adrenocortical carcinoma and adrenocortical adenoma. According to some embodiments, a relatively high expression level of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1-7, 79, 80, 36-41 and 95-96, a fragment thereof and a sequence having at least about 80% identity thereto, and any combinations thereof, compared to said reference expression profile is indicative of adrenocortical carcinoma. According to other embodiments, a relatively high expression level of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 8-17, 29, 35, 76-78, 81-85, 42-51, 67, 74, 75, 92-94 and 97-101, a fragment thereof and a sequence having at least about 80% identity thereto, and any combinations thereof, compared to said reference expression profile is indicative of adrenocortical adenoma. The invention further provides a method to distinguish between pheochromocytoma and a tumor of the adrenal cortex, the method comprising obtaining a biological sample from a subject, determining an expression profile of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 16, 18-27, 29-35, 50, 52-65, and 67-74, a fragment thereof and a sequence having at least about 80% identity thereto, and any combinations thereof, from said sample, and comparing said reference expression profile to a reference expression profile, wherein the results of said comparison allows for distinguishing between pheochromocytoma and a tumor of the adrenal cortex.

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