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W O 2014/074847 a L 1 5 May 2014 (15.05.2014) P O P C T (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 W O 2014/074847 A l 1 5 May 2014 (15.05.2014) P O P C T (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C12Q 1/68 (2006.01) G01N 33/48 (2006.01) kind of national protection available): AE, AG, AL, AM, C12N 15/11 (2006.01) AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, (21) International Application Number: DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, PCT/US20 13/069 192 HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, (22) International Filing Date: KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, 8 November 2013 (08.1 1.2013) MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, (25) Filing Language: English SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, (26) Publication Language: English TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (30) Priority Data: 61/724,707 ' November 2012 (09. 11.2012) US (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, (71) Applicant: THE JOHNS HOPKINS UNIVERSITY GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, [US/US]; 3400 N. Charles Street, Baltimore, MD 21218 UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, (US). TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, (72) Inventors: SPIVAK, Jerry, L.; 4904 Roland Avenue, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, Baltimore, MD 21210 (US). OCHS, Michael; 506 Burton TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, Road, Oreland, PA 19075 (US). CONSIDINE, Michael; KM, ML, MR, NE, SN, TD, TG). 2200 Shire Court, Bel Air, MD 21015 (US). ROWLEY, Donna; 4909 Quimby Avenue, Beltsville, MD 20705 (US). Published: MOLITERNO, Alison, R.; 39 10 Cloverhill Road, Bal — with international search report (Art. 21(3)) timore, MD 21218 (US). — with sequence listing part of description (Rule 5.2(a)) (74) Agent: ERGENZINGER, Edward, R.; Ward and Smith, P.A., 1001 College Court, P.O. Box 867, New Bern, NC 28563 (US). (54) Title: A GENETIC ASSAY TO DETERMINE PROGNOSIS IN POLYCYTHEMIA VERA PATIENTS DIAGNOSIS 0 YEARS 2 YEARS 00 H-273 N= H=55 o FIG. (57) Abstract: The presently disclosed subject matter provides a genetic assay to determine the prognosis in Polycythemia Vera o (PV) patients with an indolent form of PV. This assay involves measuring certain messenger RNAs (mRNAs) in blood cells, such as white blood cells. In some embodiments, the cells are CD34+ cells. These mRNA levels are inserted into an algorithm that yields a predictive score of the risk of PV in the patient transforming from an indolent form to an aggressive form. A GENETIC ASSAY TO DETERMINE PROGNOSIS IN POLYCYTHEMIA VERA PATIENTS CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 61/724,707, filed November 9, 2012, which is incorporated herein by reference in its entirety. FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with United States government support under P01CA108671 awarded by the National Institutes of Health (NIH) and W81XWH-05- 1-0347 awarded by the Department of Defense (DOD). The U.S. government has certain rights in the invention. BACKGROUND Polycythemia vera (PV) is a hematopoietic stem cell disorder characterized by the increased production of red cells, white cells and platelets and complicated by thrombotic and hemorrhagic events, extramedullary hematopoiesis, and transformation to myelofibrosis or acute leukemia (AML), albeit at variable frequencies (FIG. 1) (Spivak, 2002), and many of these clinical features are shared in common with its companion myeloproliferative disorder, primary myelofibrosis (PMF). PV is unique since with phlebotomy alone its natural history can be measured in decades. However, not all PV patients enjoy substantial longevity or freedom from significant complications but, in contrast to PMF, no satisfactory clinical criteria exist for stratification with respect to the risk of disease transformation, and usually no cytogenetic or molecular markers predictive of disease transformation are present before the event (Gaidano et al, 1997). Although JAK2 V617F is expressed in both PV and PMF, these are stem cell disorders and hematopoietic stem cells are not dependent on this tyrosine kinase for either survival or proliferation (Spivak, 2010), nor has the extent of JAK2 V617F expression been useful for prognostic purposes (Barbui et al, 201 1). Bone marrow transplantation is the only curative therapy for PV (Kerbauy et al, 2007) and pegylated interferon is the only drug that can induce a molecular remission, although not in all patients (Kiladjian et al., 2008). Both have significant toxicities, while all chemotherapeutic drugs employed to suppress marrow and extramedullary hematopoiesis as supportive therapy can increase the rate of leukemic transformation ten-fold (Najean and Rain, 1997; Berk et al, 1981). For many malignancies, gene expression profiling (GEP) has been a useful approach for risk stratifying cancer patients with a shared clinical or histologic phenotype (Radich et al, 2006), and for developing predictors of disease behavior irrespective of the clinical phenotype (Lenz et al, 2008). SUMMARY The presently disclosed subject matter provides a genetic assay and kits to determine the prognosis in Polycythemia Vera (PV) patients with an indolent form of PV. The presently disclosed assay involves measuring certain messenger RNAs (mRNAs) in blood cells. These mRNA levels are inserted into an algorithm that yields a predictive score of the risk of PV in the patient transforming from an indolent form to an aggressive form. In one aspect, the presently disclosed subject matter provides a method for determining the likelihood of an indolent form of Polycythemia Vera (PV) transforming to an aggressive form of PV in a subject, the method comprising: (a) measuring the gene products of PCNA, IFI30, TSN, CTSA, SMC4, CDKN1A, CTTN, SON, TIAl, and MYL9 in a biological sample comprising blood cells obtained from the subject; (b) making the following comparisons of the gene product levels measured in (a) and recording a score of 1 for a true result and a score of 0 for a false result: PCNA > IFI30; TSN > CTSA; SMC4 > CDKN1A; PCNA > CTTN; SON > CTTN; TIAl > MYL9; (c) adding the scores together to obtain an added score and calculating a ratio of the added score/6 to calculate a total score; and (d) using the total score to predict if the indolent form of PV in the subject is likely to transform to an aggressive form of PV in the subject. In some embodiments, the blood cells are white blood cells. In other embodiments, the blood cells are CD34+ cells. Accordingly, in some aspects, a total score of 5/6 or 6/6 predicts that the indolent form of PV in a subject is likely to transform to an aggressive form of PV in a subject. In other aspects, a total score of less than 5/6 predicts that the indolent form of PV in a subject is not likely to transform to an aggressive form of PV in the subject. In another aspect, the presently disclosed subject matter provides a diagnostic kit for determining whether an indolent form of PV in a subject is likely to transform to an aggressive form of PV, the kit comprising a means for measuring the gene product levels of PCNA, IFI30, TSN, CTSA, SMC4, CDKN1A, CTTN, SON, TIA1, and MYL9 and a set of instructions comprising an algorithm for predicting if the indolent form of PV in the subject is likely to transform to an aggressive form of PV. Certain aspects of the presently disclosed subject matter having been stated hereinabove, which are addressed in whole or in part by the presently disclosed subject matter, other aspects will become evident as the description proceeds when taken in connection with the accompanying Examples and Figures as best described herein below. BRIEF DESCRIPTION OF THE FIGURES Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein: FIG. 1 shows the natural history of PV as shown by the evolution of PV over time with transformation to myelofibrosis and acute leukemia in 273 PV patients; FIG 2 shows quantitative real-time polymerase chain reaction (Q-RT-PCR) confirmation of PV CD34+ cell gene expression using the primers described in Table i ; FIGS. 3A-3B show representative KEGG pathway analysis for: A) male and B) female patient groups; FIG. 4 shows a representative Venn diagram of the genes differentially expressed by the 8 men and 11 women PV patients illustrating the number of genes concordantly differentially regulated by both sexes; FIG. 5 shows a representative dendrogram and heat map for the unsupervised hierarchical clustering of the 19 PV patients using the 102 genes concordantly deregulated by both sexes. The green color bar indicates the normal controls; the blue and red color bars indicate the PV patients. For the heat map, red indicates decreased gene expression and green increased gene expression; FIG. 6 shows a representative dendrogram and heat map for the supervised clustering of the 19 PV patients using 30 genes identified by top scoring pair analysis.
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