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(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 2018/191553 A l 18 October 2018 (18.10.2018) W ! P O PCT (51) International Patent Classification: (US). BERNSTEIN, Bradley [US/US]; c/o 55 Fruit Street, G01N 33/574 (2006.01) C12Q 1/68 (2018.01) Boston, MA 021 14 (US). A61P 35/00 (2006 .01) C12Q 1/6813 (2018.01) (74) Agent: SCHER, Michael B.; Johnson, Marcou & Isaacs, A61P 35/04 (2006 .01) C12Q 1/6837 (2018.01) LLC, P.O. Box 691, Hoschton, GA 30548 (US). (21) International Application Number: (81) Designated States (unless otherwise indicated, for every PCT/US2018/027383 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, BZ, 12 April 2018 (12.04.2018) CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, (25) Filing Language: English HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, (26) Publication Langi English KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, (30) Priority Data: OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, 62/484,709 12 April 2017 (12.04.2017) US SC, SD, SE, SG, SK, SL, SM, ST, SV, SY,TH, TJ, TM, TN, 62/586,126 14 November 2017 (14. 11.2017) US TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (71) Applicants: MASSACHUSETTS EYE AND EAR (84) Designated States (unless otherwise indicated, for every INFIRMARY [US/US]; 243 Charles Street, Boston, kind of regional protection available): ARIPO (BW, GH, MA 021 14 (US). THE GENERAL HOSPITAL GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, CORPORATION [US/US]; 55 Fruit Street, Boston, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, MA 021 14 (US). THE BROAD INSTITUTE, INC. TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, [US/US]; 415 Main Street, Cambridge, MA 02142 (US). EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MASSACHUSETTS INSTITUTE O F TECHNOLO¬ MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, G Y [US/US]; 7 7 Massachusetts Avenue, Cambridge, MA TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, 02139 (US). KM, ML, MR, NE, SN, TD, TG). (72) Inventors; and (71) Applicants: PURAM, Sidharth [US/US]; c/o 243 Charles Published: Street, Boston, MA 021 14 (US). TIROSH, Itay [US/US]; — with international search report (Art. 21(3)) c/o 415 Main Street, Cambridge, MA 02142 (US). — with sequence listing part of description (Rule 5.2(a)) PARIKH, Anuraag [US/US]; c/o 243 Charles Street, Boston, MA 021 14 (US). LIN, Derrick [US/US]; c/o 243 Charles Street, Boston, MA 021 14 (US). REGEV, Aviv [US/US]; c/o 415 Main Street, Cambridge, MA 02142 (54) Title: TUMOR SIGNATURE FOR METASTASIS, COMPOSITIONS OF MATTER METHODS OF USE THEREOF FIG. 1 A primary tumors matching LNs Patients with Resect patient Dissociate isolate single Single cell RNA-Seq oral cavity HNSCC tumors tumor cells live cells (1=5942) (57) Abstract: The present invention advantageously provides for novel gene signatures, tools and methods for the treatment and prognosis of epithelial tumors. Applicants have used single cell RNA-seqto reveal novel expression programs of malignant, stromal and immune cells in the HNSCC tumor ecosystem. Malignant cells varied in expression of programs related to stress, hypoxia and epithelial differentiation. A partial EMT-like program (p-EMT) was discovered that was expressed in cells residing at the leading edge of tumors. Applicants unexpectedly linked the p-EMT state to metastasis and adverse clinical features that may be used to direct treatment of epithelial cancers (e.g., HNSCC). Applicants also show that metastases are dynamically regulated by the tumor microenvironment (TME). Finally, a computational modeling approach was developed that allows analysis of malignant cells in bulk sequencing samples. TUMOR SIGNATURE FOR METASTASIS, COMPOSITIONS OF MATTER METHODS OF USE THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application Nos. 62/484,709, filed April 12, 2017 and 62/586,126, filed November 14, 2017. The entire contents of the above-identified applications are hereby fully incorporated herein by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH [0002] This invention was made with government support under grant numbers CA216873, CA180922, CA202820 and CA14051 awarded by the National Institutes of Health. The government has certain rights in the invention. TECHNICAL FIELD [0003] The subject matter disclosed herein is generally directed to methods of using gene expression profiles representative of cell sub-types present in head and neck squamous cell carcinoma (HNSCC). Specifically, the gene signatures may be used for diagnosing, pro gnosing and/or staging of tumors and designing and selecting appropriate treatment regimens. Furthermore, novel signatures determined by single cell analysis of HNSCC are leveraged to provide for methods and systems for deconvolution of bulk sequencing data from tumors. BACKGROUND [0004] Genomic and transcriptomic studies have revealed driver mutations, identified aberrant regulatory programs, and redefined disease subtypes for major human tumors (Stratton et al., 2009; Weinberg, 2014). However, these studies relied on profiling technologies that measure the entire tumor in bulk, limiting their ability to capture intra- tumoral heterogeneity, including malignant cells in distinct genetic, epigenetic, and functional states, as well as diverse non-malignant cells such as immune cells, fibroblasts, and endothelial cells. Substantial evidence indicates that tra-tumoral heterogeneity among malignant and non-malignant cells, and their interactions within the tumor microenvironment (TME) are critical to many aspects of tumor biology, including self-renewal, immune surveillance, drug resistance and metastasis (Meacham and Morrison, 2013; Weinberg, 2014). [0005] Recent advances in single-cell genomics provide an avenue to explore genetic and functional heterogeneity at a cellular resolution (Navin, 2015; Tanay and Regev, 2017; Wagner et al., 2016). In particular, single-cell RNA-seq (scRNA-seq) studies of human tumors, circulating tumor cells and patient-derived xenografts have revealed new insights into tumor composition, cancer stem cells, and drug resistance. [0006] Despite these promising results, scRNA-seq studies have not extensively characterized epithelial tumors, in spite of their predominance. In these tumors, metastasis to nearby draining lymph nodes (locoregional metastasis) and to other organs (distant metastasis) represents a major cause of morbidity and mortality. However, lymph node (LN) and distant metastases are often treated based on molecular and pathologic features of the primary tumor, raising the question of whether metastases share the same genetics, epigenetics, and vulnerabilities (Lambert et al., 2017). The potentially different composition of primary tumors and metastases hinders the straightforward comparison of bulk tumor profiles. Single-cell expression profiling studies would, in principle, offer a compelling alternative. [0007] Epithelial-to-mesenchymal transition (EMT) has been suggested as a driver of local and distant spread of epithelial tumors (Gupta and Massague, 2006; Lambert et al., 2017). The process of EMT is fundamental to embryonic development and other physiologic processes and may be co-opted by malignant epithelial cells to facilitate invasion and dissemination (Thiery et al., 2009; Ye and Weinberg, 2015). EMT markers have been detected on circulating tumor cells (CTCs) associated with metastatic disease (Ting et al., 2014; Yu et al., 2013). However, since most EMT studies have focused on laboratory models, the nature, extent, and significance of EMT in primary human tumors and metastases remains controversial (Lambert et al., 2017; Nieto et al., 2016). For example, although mesenchymal subtypes have been identified in multiple tumor types (Cancer Genome Atlas, 2015; Cancer Genome Atlas Research, 201 1; Verhaak et al., 2010), it remains unclear whether they reflect mesenchymal cancer cells or, alternatively, contributions of non-malignant, mesenchymal cell types in the TME. [0008] Head and neck squamous cell carcinoma (HNSCC) is an epithelial tumor with strong associations to chronic alcohol and tobacco exposure (Puram and Rocco, 2015). Like many epithelial cancers, HNSCC tumors are highly heterogeneous within and between patients. Metastatic disease remains a central challenge, with patients often presenting at an advanced stage with LN metastases. Thus, there is a need for biomarkers and therapeutic targets capable of guiding treatment and predicting disease progression (e.g., metastasis) in epithelial tumors. SUMMARY [0009] The diverse malignant, stromal, and immune cells in tumors affect growth, metastasis and response to therapy. It is an objective of the present invention to understand tra-tumoral heterogeneity, invasion and metastasis in an epithelial human cancer. It is another objective of the present to provide for novel tools and methods for diagnosing, prognosing and treating tumors. Applicants investigated primary HNSCC tumors and matched lymph nodes. Specifically, Applicants profiled transcriptomes of -6,000 single cells from 18 head and neck squamous cell carcinoma (HNSCC) patients, including five matched pairs of primary
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  • Novel Targets of Apparently Idiopathic Male Infertility

    Novel Targets of Apparently Idiopathic Male Infertility

    International Journal of Molecular Sciences Review Molecular Biology of Spermatogenesis: Novel Targets of Apparently Idiopathic Male Infertility Rossella Cannarella * , Rosita A. Condorelli , Laura M. Mongioì, Sandro La Vignera * and Aldo E. Calogero Department of Clinical and Experimental Medicine, University of Catania, 95123 Catania, Italy; [email protected] (R.A.C.); [email protected] (L.M.M.); [email protected] (A.E.C.) * Correspondence: [email protected] (R.C.); [email protected] (S.L.V.) Received: 8 February 2020; Accepted: 2 March 2020; Published: 3 March 2020 Abstract: Male infertility affects half of infertile couples and, currently, a relevant percentage of cases of male infertility is considered as idiopathic. Although the male contribution to human fertilization has traditionally been restricted to sperm DNA, current evidence suggest that a relevant number of sperm transcripts and proteins are involved in acrosome reactions, sperm-oocyte fusion and, once released into the oocyte, embryo growth and development. The aim of this review is to provide updated and comprehensive insight into the molecular biology of spermatogenesis, including evidence on spermatogenetic failure and underlining the role of the sperm-carried molecular factors involved in oocyte fertilization and embryo growth. This represents the first step in the identification of new possible diagnostic and, possibly, therapeutic markers in the field of apparently idiopathic male infertility. Keywords: spermatogenetic failure; embryo growth; male infertility; spermatogenesis; recurrent pregnancy loss; sperm proteome; DNA fragmentation; sperm transcriptome 1. Introduction Infertility is a widespread condition in industrialized countries, affecting up to 15% of couples of childbearing age [1]. It is defined as the inability to achieve conception after 1–2 years of unprotected sexual intercourse [2].