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WO 2017/181183 Al 19 October 2017 (19.10.2017) P O P C T

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WO 2017/181183 Al 19 October 2017 (19.10.2017) 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 WO 2017/181183 Al 19 October 2017 (19.10.2017) P O P C T (51) International Patent Classification: (74) Agents: KARNAKIS, Jennifer A. et al; COOLEY LLP, C12Q 1/68 (2006.01) Attn: Patent Group, 1299 Pennsylvania Avenue, NW, Suite 700, Washington, District of Columbia 20004 (US). (21) International Application Number: PCT/US20 17/027944 (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, (22) International Filing Date: AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, 17 April 2017 (17.04.2017) BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, (25) Filing Language: English DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KH, KN, (26) Publication Language: English KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, (30) Priority Data: MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, 62/322,982 15 April 2016 (15.04.2016) US NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, (71) Applicant: EXOSOME DIAGNOSTICS, INC. [US/US]; TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, Riverside Technology Center, 840 Memorial Drive, Suite ZA, ZM, ZW. 3, Cambridge, Massachusetts 02139 (US). (84) Designated States (unless otherwise indicated, for every (72) Inventors: SKOG, Johan Karl Olov; c/o Exosome Dia kind of regional protection available): ARIPO (BW, GH, gnostics, Inc., 840 Memorial Drive, Suite 3, Cambridge, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, Massachusetts 02139 (US). NOERHOLM, Mikkel; c/o TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, Exosome Diagnostics GmbH, Am Klopferspitz 19a, 82152 TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, Martinsried (DE). BRINKMAN, Kay; c/o Exosome Dia DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, gnostics, Inc., 840 Memorial Drive, Suite 3, Cambridge, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, Massachusetts 02139 (US). CASTELLANOS-RIZAL- SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, DOS, Elena; c/o Exosome Diagnostics, Inc., 840 Memori GW, KM, ML, MR, NE, SN, TD, TG). al Drive, Suite 3, Cambridge, Massachusetts 021 39 (US). HURLEY, James; c/o Exosome Diagnostics, Inc., 840 Published: Memorial Drive, Suite 3, Cambridge, Massachusetts 021 39 — with international search report (Art. 21(3)) (US). (54) Title: PLASMA-BASED DETECTION OF ANAPLASTIC LYMPHOMA KINASE (ALK) NUCLEIC ACIDS AND ALK FU SION TRANSCRIPTS AND USES THEREOF IN DIAGNOSIS AND TREATMENT OF CANCER (57) Abstract: The present invention relates generally to the field of biomarker analysis, particularly determining gene expression signatures from biological samples, including plasma samples. PLASMA-BASED DETECTION OF ANAPLASTIC LYMPHOMA KINASE (ALK) NUCLEIC ACIDS AND ALK FUSION TRANSCRIPTS AND USES THEREOF IN DIAGNOSIS AND TREATMENT OF CANCER RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No. 62/322,982, filed April 15, 2016, the contents of which are incorporated herein by reference in their entirety. FIELD OF THE INVENTION [0002] The present invention relates generally to the field of biomarker analysis, particularly determining gene expression signatures from biological samples, including plasma samples. BACKGROUND [0003] Increasing knowledge of the genetic and epigenetic changes occurring in cancer cells provides an opportunity to detect, characterize, and monitor tumors by analyzing tumor-related nucleic acid sequences and profiles. These changes can be observed by detecting any of a variety of cancer-related biomarkers. Various molecular diagnostic assays are used to detect these biomarkers and produce valuable information for patients, doctors, clinicians and researchers. So far, these assays primarily have been performed on cancer cells derived from surgically removed tumor tissue or from tissue obtained by biopsy. [0004] However, the ability to perform these tests using a bodily fluid sample is oftentimes more desirable than using a patient tissue sample. A less invasive approach using a bodily fluid 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. [0005] Accordingly, there exists a need for new, noninvasive methods of reliably detecting biomarkers, for example, biomarkers in plasma microvesicles, to aid in diagnosis, prognosis, monitoring, or therapy selection for a disease or other medical condition. SUMMARY OF THE INVENTION [0006] The present invention is in the technical field of biotechnology. More particularly, the present invention is in the technical field of molecular biology. [0007] In molecular biology, molecules, such as nucleic acids, can be isolated from human sample material, such as plasma and other biofluids, and further analyzed with a wide range of methodologies. [0008] Human biofluids contain cells and also cell free sources of molecules shed by all cells of the body. Cell free sources include extracellular vesicles (EVs) and the molecules carried within (e.g. RNA, DNA, lipids, small metabolites and proteins) and also cell free DNA, which is likely to be derived from apoptotic and necrotic tissue. [0009] Since cell free nucleic acids, such as the RNA contained in exosomes and other EVs (exoRNA), DNA contained in exosomes and other EVs (exoDNA), free circulating or cell free DNA (cfDNA) are shed not only by normal somatic cells, but also aberrant cancer cells, an isolation of exosomal nucleic acids and DNA from human blood samples can reveal the existence and type of cancer cells in a patient. [00010] Non-small cell lung cancer (NSCLC) comprises -85% of all diagnosed lung cancers. Obtaining tissue biopsies from NSCLC is challenging, and as many as 30% of patients have no tissue for molecular analysis of genes, therefore monitoring the mutations in blood as a liquid biopsy have proven useful. The compositions and methods provided herein use the information derived from cellular living processes such as exosomal RNA (exoRNA) release, which leads to an extremely sensitive assay. It is understood that while the examples provided herein demonstrate the isolation of exoRNA, the methods and kits provided herein are useful for co-isolating any combination of exosomal nucleic acids, e.g., exoRNA and/or exoDNA, found in the sample. [00011] The existence and quantity of an ALK fusion transcript, e.g., an EML-ALK fusion transcript, in a patient can be used to guide or select the treatment options. [00012] Here we describe the application of a PCR-based assay on exoRNA and isolated from human biofluids that detects an ALK fusion transcript, e.g., an EML-ALK fusion transcript, with high sensitivity and specificity. [00013] The present invention is a complete workflow from sample extraction to nucleic acid analysis using exosomal RNA. State-of-the -art machine learning and data- mining techniques are applied to the qPCR data generated by the real time instrument to discriminate between positive and negative samples or to quantify the strength of positive or negative samples. [00014] The present disclosure provides methods of detecting one or more biomarkers in a biological sample to aid in diagnosis, prognosis, monitoring, or therapy selection for a disease such as, for example, cancer. The methods and kits provided herein are useful in detecting one or more biomarkers from plasma samples. The methods and kits provided herein are useful in detecting one or more biomarkers from the microvesicle fraction of plasma samples. [00015] The methods and kits provided herein are useful for detecting an anaplastic lymphoma kinase (ALK) fusion transcript in a biological sample. In some embodiments, the ALK fusion transcript is an EML-ALK fusion transcript. In some embodiments, the ALK fusion transcript is an EML4-ALK fusion transcript. In some embodiments, the EML4-ALK fusion transcript is EML4-ALK vl, EML4-ALK v2, EML4-ALK v3, and any combination thereof. [00016] The present disclosure provides methods and kits for detecting a EML4-ALK fusion transcript in a biological sample. In some embodiments, the biological sample is plasma. [00017] The present disclosure provides a reaction designed to capture and concentrate EVs, isolate the corresponding nucleic acids, and to simultaneously detect the presence of an ALK fusion transcript, e.g., an EML-ALK fusion transcript. [00018] Generally, the methods and kits of the disclosure include the following steps: 1) Isolation of exoRNA from a biofluid sample: a. Binding of microvesicles and other extracellular vesicles (EVs) to columns or beads; i. In some embodiments, the binding step is performed using the methods as described in PCT applications WO 2016/007755 and WO 2014/107571. b. Release from matrix using lysing conditions; c. Isolation of total nucleic acids from lysate using silica columns or beads i. In some embodiments, the isolating step is performed using the methods as described in PCT applications WO 2016/007755 and WO 2014/107571; 2) Detection and quantification of one or more EML-ALK fusion transcript(s); ) Analyzing the detected and quantified EML-ALK fusion transcript(s) using the following procedure: a. Step 1: Each sample is checked for passing the acceptance criteria for the Sample Integrity Control and the Sample Inhibition Control. i. In some embodiments, the Sample Integrity Control is the expression level of the housekeeping gene RPL4 tested by qPCR. ii. For RPL4 the acceptance criteria are defined by a cycle threshold (CT) value <28. iii. In some embodiments, the Sample Inhibition Control is the expression level of Qbeta RNA spiked into the reverse transcription reaction of each sample and tested by qPCR.
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