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WO2012031008A2.Pdf (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 \ r / n n n A 8 March 2012 (08.03.2012) W / (51) International Patent Classification: (72) Inventors; and CI2Q 1/68 (2006.01) (75) Inventors/Applicants (for US only): BALAJ, Leonora [IT/US]; 9 Cedar Street, Charlestown, Massachusetts (21) International Application Number: 02129 (US). NOERHOLM, Mikkel [DK/DE]; PCT/US201 1/050041 Preysingstrasse 21, 8213 1 Gauting (DE). BREAKE- (22) International Filing Date: FIELD, Xandra O . [US/US]; 127 Homer Street, Newton, 31 August 201 1 (3 1.08.201 1) Massachusetts 02459 (US). (25) Filing Language: English (74) Agents: RESNICK, David S . et al; Nixon Peabody LLP, 100 Summer St., Boston, Massachusetts 021 10-21 3 1 (26) Publication Langi English (US). (30) Priority Data: (81) Designated States (unless otherwise indicated, for every 61/378,860 31 August 2010 (3 1.08.2010) US kind of national protection available): AE, AG, AL, AM, 61/421,421 9 December 2010 (09.12.2010) US AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, 61/437,547 28 January 201 1 (28.01 .201 1) us CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, 61/438,1 99 31 January 201 1 (3 1.01 .201 1) us DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, 61/493,261 3 June 201 1 (03.06.201 1) us HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, (71) Applicant (for all designated States except US): THE KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, GENERAL HOSPITAL CORPORATION [US/US]; ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, 55 Fruit Street, Boston, Massachusetts 021 14 (US). NO, NZ, OM, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, (72) Inventor; and TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, (71) Applicant : SKOG, Johan Karl Olov [SE/US]; 400 ZM, ZW. West 63rd Street, Apt. 407, New York, NY 10069 (US). [Continued on next page] (54) Title: CANCER-RELATED BIOLOGICAL MATERIALS IN MICROVESICLES (57) Abstract: Disclosed herein are AMPLIFICATION PLOT methods for assaying a biological sample rom a subject by analyzing components of microvesicle fractions in aid of risk, diagnosis, prognosis or monitoring of, or directing treatment of the subject for, a disease or other medical condition in the subject. Also disclosed are methods of treat ment and identifying biomarkers u s ing a microvesicle fraction of a sub ject. Kits, pharmaceutical composi tions, and profiles related to the 0.01 methods are also disclosed. / / 3. 0 10 12 14 1 6 18 0 22 24 2 28 30 32 34 3 38 4 CYCLE < ∞ 1 r. o o w o 2012/031008 A2 1II III II II III III I llll 1 1II III I III II I II (84) Designated States (unless otherwise indicated, for every SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, kind of regional protection available): ARIPO (BW, GH, GW, ML, MR, NE, SN, TD, TG). GM, KE, LR, LS, MW, ML, NA, SD, SL, SZ, TZ, UG, Published: ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, without international search report and to be republished EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, upon receipt of that report (Rule 48.2(g)) LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, CANCER-RELATED BIOLOGICAL MATERIALS IN MICROVESICLES CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of 35 U.S.C. § 119(e) to U.S. Provisional Application serial numbers 61/378,860 filed August 31, 2010; 61/421,421 filed December 9, 2010; 61/437,547 filed January 28, 2011; 61/438,199 filed January 31, 2011; and 61/493,261 filed June 03, 2011, the contents of each of which are incorporated herein by reference in their entirety. GOVERNMENT SUPPORT [0002] This invention was made with Government support under grants CA86355, CA69246, CA141226, and CA141150 awarded by National Cancer Institute. The Government has certain rights in the invention. FIELD OF INVENTION [0003] The present invention relates to the fields of biomarker analysis, diagnosis, prognosis, patient monitoring, therapy selection, risk assessment, and novel therapeutic agents for human or other animal subjects, particularly the profiling of biological materials from a microvesicle fraction of a biological sample, and novel therapies related to microvesicles. BACKGROUND OF THE INVENTION [0004] Increasing knowledge of the genetic and epigenetic changes occurring in cancer cells provides an opportunity to detect, characterize, and monitor tumors by analysing tumor-related nucleic acid sequences and profiles. Cancer-related changes include specific mutations in gene sequences (Cortez and Calin, 2009; Diehl et al., 2008; Network, 2008; Parsons et al., 2008), up- and down-regulation of mRNA and miRNA expression (Cortez and Calin, 2009; Itadani et al., 2008; Novakova et al., 2009), mRNA splicing variations, changes in DNA methylation patterns (Cadieux et al., 2006; Kristensen and Hansen, 2009), amplification and deletion of genomic regions (Cowell and Lo, 2009), and aberrant expression of repeated DNA sequences (Ting et al., 2011). Various molecular diagnostic tests such as mutational analysis, methylation status of genomic DNA, and gene expression analysis may detect these changes. [0005] Research uncovering the molecular mechanisms underlying cancer improves our understanding of how to select and design optimal treatment regimes for a patient' s disease based on the molecular makeup of his or her particular cancer. Over the past few years, this has led to a significant increase in the development of therapies specifically targeting gene mutations involved in disease progression. In parallel, the use of molecular diagnostic testing for cancer diagnosis, prognosis and treatment selection has expanded, driven by the need for more cost efficient applications of expensive therapies. Current molecular diagnostics has so far almost exclusively relied on assaying cancer cells from tissue biopsy by needle aspiration or surgical resection. [0006] However, the ability to perform these tests using a blood sample is sometimes more desirable than using a tissue sample from a cancer patient because, frequently, fresh tissue samples are difficult or impossible to obtain, and archival tissue samples are often less relevant to the current status of the patient's disease. A less invasive approach using a more easily accessible biological sample, e.g., a blood sample, has wide ranging implications in terms of patient welfare, the ability to conduct longitudinal disease monitoring, and the ability to obtain expression profiles even when tissue cells are not easily accessible, e.g., in ovarian or brain cancer patients. [0007] Currently, gene expression profiling of blood samples involves the analysis of RNA extracted from peripheral blood mononuclear cells (PBMC) (Hakonarson et al., 2005) or circulating tumor cells (CTC) (Cristofanilli and Mendelsohn, 2006). [0008] Many types of cancer cells release an abundance of small membrane-bound vesicles, which have been observed on their surface in culture (Skog et al., 2008). These microvesicles are generated and released through several processes and vary in size (from about 30 nm to about 1 µ in diameter) and content (Simons and Raposo, 2009). Microvesicles can bud/bleb off the plasma membrane of cells, much like retrovirus particles (Booth et al., 2006), be released by fusion of endosomal-derived multivesicular bodies with the plasma membrane (Lakkaraju and Rodriguez-Boulan, 2008), or be formed as apoptotic bodies during programmed cell death (Halicka et al., 2000). In addition, defective (i.e., non infectious without helper-virus) retrovirus particles derived from human endogenous retroviral (HERV) elements may be found within microvesicle populations (Voisset et al., 2008). [0009] Microvesicles from various cell sources have been studied with respect to protein and lipid content (Iero et al., 2008; Thery et al., 2002; Wieckowski and Whiteside, 2006). They have also been observed to contain cellular RNAs and mitochondria DNA (Baj- Krzyworzeka et al., 2006; Guescini et al.; Skog et al., 2008; Valadi et al., 2007) and may facilitate the transfer of genetic information between cells and/or act as a "release hatch" for DNA, RNA, and/or proteins that the cell is trying to eliminate. Both mRNA and miRNA in microvesicles are observed to be functional following uptake by recipient cells (Burghoff et al., 2008; Deregibus et al., 2007; Ratajczak et al., 2006; Skog et al., 2008; Valadi et al., 2007; Yuan et al., 2009) and it has also been shown that apoptotic bodies can mediate horizontal gene transfer between cells (Bergsmedh et al., 2001). [0010] Knowing the expression profile, mutational profile, or both expression and mutational profiles of individual cancer is helpful for personalized medicine as many drugs target specific pathways affected by the genetic status of the tumors. Detection of genetic biomarkers in blood samples from tumor patients is challenging due to the need for high sensitivity against a background of normal cellular nucleic acids found circulating in blood. Microvesicles released by tumor cells into the circulation can provide a window into the genetic status of individual tumors (Skog et al., 2008). [0011] The present invention is directed to microvesicular nucleic acid profiles of microvesicle fractions obtained from a biological sample from a subject, methods for aiding in diagnosis, prognosis, patient monitoring, treatment selection, and risk assessment based on detecting the presence or absence of a genetic aberration in a nucleic acid profile, or changes in a polypeptide profile of a microvesicle fraction obtained from a biological sample from a patient, and therapeutic agents and methods of cancer treatment or prevention.
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