Annotation of regulatory elements and identification of the transcription-related factors (TRFs) targeting these elements are key steps in understanding how cells interpret their genetic blueprint and their environment during development, and how that process goes awry in the case of disease. One goal of the modENCODE (model organism ENCyclopedia of DNA Elements) Project is to survey a diverse sampling of TRFs, both DNA-binding and non-DNA-binding factors, to provide a framework for the subsequent study of the mechanisms by which transcriptional regulators target the genome. Here we provide an updated map of the Drosophila melanogaster regulatory genome based on the location of 84 TRFs at various stages of development. This regulatory map reveals a variety of genomic targeting patterns, including factors with strong preferences toward proximal promoter binding, factors that target intergenic and intronic DNA, and factors with distinct chromatin state preferences. The data also highlight the stringency of the Polycomb regulatory network, and show association of the Trithorax-like (Trl) protein with hotspots of DNA binding throughout development. Furthermore, the data identify more than 5800 instances in which TRFs target DNA regions with demonstrated enhancer activity. Regions of high TRF co-occupancy are more likely to be associated with open enhancers used across cell types, while lower TRF occupancy regions are associated with complex enhancers that are also regulated at the epigenetic level. Together these data serve as a resource for the research community in the continued effort to dissect transcriptional regulatory mechanisms directing Drosophila development.

Background: Over 300 somatic molecular variants in hematologic diseases are either specified as diagnostic criteria in the World Health Organization (WHO) Classification of Tumors of Hematopoietic and Lymphoid Tissues, recognized as potentially actionable biomarkers in the National Comprehensive Cancer Network (NCCN) compendia, or supported by published well-powered clinical studies. Moreover, new molecular alterations with potential clinical implications in hematologic disease are continuously emerging in the scientific literature. These have critical use for a wide spectrum of clinicians, including hematopathologists who diagnose patient-specific hematologic malignancies, heme-oncologists who direct patient care, and clinical trial nurses who assist patients in finding appropriate clinical trials. Importantly, the utility of this information critically depends on the clinician's ability to interpret the significance of variants in a point-of-care setting. Therefore, there is an urgent and unmet need for a clinical decision support system that 1) distills the clinical implications associated with molecular alterations into a standardized and easily interpretable format and 2) democratizes access of this information to all members of the heme-oncology community. Methods: OncoKB is an established expert-guided precision oncology knowledge base that annotates the oncogenic effect and therapeutic implications of somatic molecular alterations (Chakravarty, D. et al., JCOPO, 2017). Previously, OncoKB was focused primarily on solid tumor mutation annotation. Recently, we expanded OncoKB to include alterations in hematologic malignancies. The heme-specific annotation efforts were guided by heme-oncology and hematopathology physician scientists at Memorial Sloan Kettering (MSK). Supplementing the previously published therapeutic levels of evidence (Fig. 1a), we further added level of evidence systems for diagnostic and prognostic implications (Fig. 1b, c). These three sets of evidence levels are consistent with the criteria set forth by the joint consensus of the ASCO/CAP/AMP guidelines (Li, MM. et al., J Mol Diagn, 2017). We assigned the newly curated heme-specific molecular alterations with diagnostic, prognostic or therapeutic levels of evidence, when applicable. Finally, we annotated and analyzed 1569 hematologic tumor samples from the AACR Project GENIE (release 6.1) with these levels of evidence. Results: In addition to alterations with both solid and heme clinical implications already curated in OncoKB, we annotated 288 unique heme-specific mutations, fusions, and copy number alterations in 156 newly curated cancer-associated genes. Based on MSK-expert consensus, the WHO and NCCN guidelines, and the scientific literature, we identified a total of 192 alterations with unique diagnostic levels of evidence, 65 alterations with unique prognostic levels of evidence and 55 alterations with unique therapeutic levels of evidence across 13 major hematologic tumor types (Fig. 2). To test the utility of OncoKB, we annotated all genomic events in 1569 heme cancer samples in 89 hematologic malignancies in the AACR GENIE cohort (V6.1) (Fig. 3a). Thirty-eight percent of samples harbored at least one potentially actionable alteration, and 8% were predictive of clinical benefit from an FDA-approved drug (Fig. 3b). Conclusions: OncoKB heme data is publicly available both through the web resource http://oncokb.org and through incorporation into the cBioPortal for Cancer Genomics. Heme-specific molecular alterations are used to make an accurate diagnosis, inform prognosis, optimize the use of stem cell transplant, and to link patients with the optimal mechanism-based therapies in the clinical trial setting and in routine clinical practice. This is the first study to annotate and analyze actionability of heme samples. In this proof-of-principle study, we demonstrate the ability to annotate clinical samples with their diagnostic, prognostic and therapeutic implications in a point-of-care setting. Disclosures Roshal: Celgene: Other: Provision of Services; Auron Therapeutics: Equity Ownership, Other: Provision of services; Physicians' Education Resource: Other: Provision of services. Ho:Invivoscribe, Inc.: Honoraria. Knorr:Fate Therapeutics: Patents & Royalties. LaFave:Epizyme: Patents & Royalties. Arcila:Invivoscribe, Inc.: Consultancy, Honoraria. Berger:Roche: Consultancy. Solit:Pfizer: Consultancy; Lilly Oncology: Honoraria; Vivideon Therapeutics: Consultancy; Loxo Oncology: Consultancy, Equity Ownership; Illumina: Consultancy. Dogan:Celgene: Consultancy; Seattle Genetics: Consultancy; Corvus Pharmaceuticals: Consultancy; Roche: Consultancy, Research Funding; Novartis: Consultancy; Takeda: Consultancy. Levine:C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Qiagen: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Loxo: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Novartis: Consultancy; Gilead: Consultancy; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Lilly: Honoraria; Prelude Therapeutics: Research Funding; Roche: Consultancy, Research Funding.

Genomic profiling of hematologic malignancies has augmented our understanding of variants that contribute to disease pathogenesis and supported development of prognostic models that inform disease management in the clinic. Tumor only sequencing assays are limited in their ability to identify definitive somatic variants, which can lead to ambiguity in clinical reporting and patient management. Here, we describe the MSK-IMPACT Heme cohort, a comprehensive data set of somatic alterations from paired tumor and normal DNA using a hybridization capture next generation sequencing platform. We highlight patterns of mutations, copy number alterations, and mutation signatures in a broad set of myeloid and lymphoid neoplasms. We also demonstrate the power of appropriate matching to make definitive somatic calls, including in patients who have undergone allogeneic stem cell transplant. We expect that this resource will further spur research into the pathobiology and clinical utility of clinical sequencing for patients with hematologic neoplasms.

PURPOSE The clinical utility of cell-free DNA (cfDNA) as a biomarker for advanced clear cell renal cell carcinoma (ccRCC) remains unclear. We evaluated the validity of cfDNA-based genomic profiling in a large cohort of patients with ccRCC with matched next-generation sequencing (NGS) from primary tumor tissues. MATERIALS AND METHODS We performed paired NGS of tumor DNA and plasma cfDNA using the MSK-IMPACT platform in 110 patients with metastatic ccRCC. Tissues were profiled for variants and copy number alterations with germline comparison. Manual cross-genotyping between cfDNA and tumor tissue was performed. Deep sequencing with a higher sensitivity platform, MSK-ACCESS, was performed on a subset of cfDNA samples. Clinical data and radiographic tumor volumes were assessed to correlate cfDNA yield with treatment response and disease burden. RESULTS Tumor tissue MSK-IMPACT testing identified 582 genomic alterations (GAs) across the cohort. Using standard thresholds for de novo variant calling in cfDNA, only 24 GAs were found by MSK-IMPACT in cfDNA in 7 of 110 patients (6%). With manual cross-genotyping, 210 GAs were detectable below thresholds in 74 patients (67%). Intrapatient concordance with tumor tissue was limited, including VHL (31.6%), PBRM1 (24.1%), and TP53 (52.9%). cfDNA profiling did not identify 3p loss because of low tumor fractions. Tumor volume was associated with cfDNA allele frequency, and VHL concordance was superior for patients with greater disease burden. CONCLUSION cfDNA-based NGS profiling yielded low detection rates in this metastatic ccRCC cohort. Concordance with tumor profiling was low, even for truncal mutations such as VHL, and some findings in peripheral blood may represent clonal hematopoiesis. Routine cfDNA panel testing is not supported, and its application in biomarker efforts must account for these limitations.

Next-generation sequencing of cell-free DNA (cfDNA) can be used to noninvasively assess and monitor patients with lymphoma. Here, we describe the preliminary validation of MSK-ACCESS Heme (Memorial Sloan Kettering-Analysis of Circulating cfDNA to Examine Somatic Status), a cfDNA assay that employs unique molecular indexing and ultra-deep sequencing to detect somatic alterations in 117 genes related to hematologic malignancies. To our knowledge, this is the first report of a clinical-grade cfDNA assay developed specifically for hematologic malignancies. Overall assay performance was assessed using 53 validation samples (26 normal samples and 27 cfDNA samples with somatic variants). Initial accuracy studies showed excellent correlation with the reference next-generation sequencing method (MSK-ACCESS Solid), detecting 32/32 (100%) of expected variants with a variant allele frequency over 1%. The limit of detection was assessed using standard samples, with detection of variants down to 0.5% variant allele frequency. Results were highly concordant in both inter- and intra-assay reproducibility studies. Overall, these data indicate that MSK-ACCESS Heme is a robust cfDNA-based assay that can be used to detect variants at low frequency with high reproducibility. Future work sequencing additional samples will be performed to further assess the performance of the panel. Citation Format: Sara E. DiNapoli, Coleman Spence, Erika Gedvilaite, Anita Bowman, Monica Diosdado, Anna Razumova, Dana Tsui, Gilles A. Salles, Connie Batlevi, Gottfried von Keudell, Ryan Ptashkin, Ahmet Zehir, Michael Berger, A Rose Brannon, Ryma Benayed, Maria Arcila. MSK-ACCESS Heme: A cell-free DNA next-generation sequencing assay to identify somatic alterations in patients with lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 56.