McCoy, Matthew

Abstract Clinical significance of somatic gene variants needs to be comprehensively characterized for their diagnostic, prognostic and/or therapeutic actionability in patient management. However, challenges remain due to discrepancies in interpretation and reporting of these somatic variants among different testing labs. Therefore, standardized curation, clinical interpretation and reporting of somatic variants in hematologic cancers is critical. To address this issue, the Hematologic Cancer Taskforce (HCT), composed of 52 multi-disciplinary experts including oncologists, molecular pathologists, lab directors, genomic scientists and biocurators, was formed in January 2020 within the ClinGen Somatic Cancer Clinical Domain Working Group (CDWG) with a goal to undertake systematic curation and evidence-based clinical interpretation of genes/somatic variants associated with hematologic malignancies. In collaboration with the Clinical Interpretation of Variants in Cancer (CIViC) (civicdb.org) knowledgebase, HCT members curate, edit, and verify Evidence Items for each variant extracted from peer-reviewed publications. Monthly discussions based on these Evidence Items lead to the preparation of variant Assertions, which summarize the state of the field consensus variant interpretation and include tiering based on the AMP/ASCO/CAP guidelines (PMID: 27993330). FMS-like tyrosine kinase 3 (FLT3) encodes a class III receptor tyrosine kinase expressed in hematopoietic cells. FLT3 mutations, including both internal tandem duplication (ITD) and mutations in the tyrosine kinase domain (TKD), are the most common mutations in acute myeloid leukemia (AML), occurring in approximately 30% of all AML cases. Implementing FLT3 tyrosine kinase inhibitors (TKIs) in different treatment regimens for FLT3 mutated AML patients has led to significantly improved overall survival. Type I FLT3 inhibitors, including midostaurin, gilteritinib, sunitinib, lestaurtinib, and crenolanib, bind to the ATP-binding site when the receptor is in active conformation. Type II FLT3 inhibitors, including sorafenib, ponatinib, and quizartinib, interact with a hydrophobic region directly adjacent to the ATP-binding domain that is only accessible when the receptor is inactive, which prevents receptor activation. Generally in AML cells, type I FLT3 inhibitors prevent activity for both ITD and TKD mutations, while Type II inhibitors target ITD but lack efficiency against TKD mutations. The development of TKD mutations in AML cells with ITD have proved to be a mechanism of acquired, or secondary resistance to Type II FLT3 inhibitors. The HCT is piloting curation assessments of FLT3 alterations, including ITD, TKD and non-TKD variants, in AML. So far, the HCT has curated 75 evidence items for FLT3 somatic variants. FLT3-ITD, as well as D835 and I836 were asserted as tier 1 level A variants based on the prediction of response to gilteritinib in relapsed/refractory AML (PMIDs: 27993330, 31665578, 28645776, 28516360, 27908881). Recent curation activities are focused on FLT3 D839G and N676K, as clinical trials using large AML patient cohorts are lacking in their ability to validate drug response/resistance associations of these two TKD variants due to their low frequency. Functional studies showed both variants result in increased proliferation and protection from apoptosis, supporting the oncogenic potential of these two variants (PMIDs: 26891877, 2468088). FLT3 D839G combined with ITD confers resistance to pexidartinib and ponatinib, both Type II FLT3 inhibitors (PMIDs: 25847190, 23430109). FLT3 N676K predicts response to the Type I FLT3 inhibitor, gilteritinib, when N676K is present alone or in combination with ITD. Interestingly, FLT3 N676K in the absence of ITD predicts response to sorafenib, a Type II FLT3 inhibitor (PMIDs: 32040554, 32984009). However, these results are mostly derived from in vitro studies. Two separate Tier II, Level D Assertions have been submitted for FLT3-ITD&D839G for its response to pexidartinib and ponatinib, and more evidence is being collected to form an Assertion for FLT3 N676K. The complexity of the prediction of response/resistance associated with FLT3 D839G and N676K supports the importance of evidence-based curation and collection for these variants in the context of the overall mutation profile, disease context and specific FLT3 TKIs to clearly define their clinical impact. Disclosures Pullarkat: Stemline Therapeutics: Honoraria.

Hematological malignancies comprise a genetically heterogeneous spectrum of diseases caused by abnormal proliferation or maturation of a variety of hematological cell lineages. Genomic abnormalities including chromosomal translocations, copy number variations and sequence level gene mutations underlie the pathogenesis of these disorders and frequently serve as important diagnostic, prognostic and/or therapeutic markers. However, the substantial discrepancy in interpretation and reporting of these genomic abnormalities among testing labs creates challenges for patient management. Therefore, standardizing the curation, clinical interpretation and reporting of somatic alterations within the context of their diagnostic, prognostic and therapeutic significance in hematological cancers is critical. In January 2020, the ClinGen Somatic Cancer Clinical Domain working group formed the Hematological Cancer Taskforce (HCT) with a goal to undertake systematic curation and evidence-based clinical interpretation of genes/somatic variants associated with hematological malignancies. The HCT has recruited 32 multi-disciplinary experts including oncologists, molecular pathologists, clinical lab directors, genomic scientists and biocurators with expertise in hematological malignancies. In collaboration with the Clinical Interpretation of Variants in Cancer (CIViC) (civicdb.org) knowledgebase, variants from peer-reviewed publications are curated with editor review for clinical utility as evidence items. Monthly discussions based on these evidence items lead to the creation of summary variant assertions using the AMP/ASCO/CAP guidelines (Li M, et al., Journal of Molecular Diagnostics, 2017). The HCT is currently focused on expert curation and clinical interpretation of somatic variants in FLT3 (internal tandem duplication, tyrosine kinase domain and non tyrosine kinase domain variants) in acute myeloid leukemia (AML). Expert curation of gene fusions in Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) in collaboration with the ClinGen somatic pediatric cancer taskforce is currently underway. To date, the HCT has curated 45 evidence items from clinical and pre-clinical studies on the aforementioned genes/variants. In addition, three AMP Tier I, level A variant assertions of FLT3-ITD, D835 and I836, which predict response to Gilteritinib, an FDA-approved drug for relapsed or refractory AML, have been curated. In the future, the HCT plans to extend its focus on curation of BCR-ABL1 kinase domain mutations in chronic myeloid leukemia (CML). Based on the initial pilot curation phase, the HCT will develop gene-specific recommendations to standardize the reporting and interpretation of somatic variants to better assist clinical decisions and apply to become official ClinGen Somatic Expert Panels in each of these gene-disease domains. Disclosures Blombery: Novartis: Consultancy; Invivoscribe: Honoraria; Amgen: Consultancy; Janssen: Honoraria.

Venetoclax is an oral, highly selective, BCL2 inhibitor approved by the FDA for use in chronic lymphocytic leukemia/small lymphocytic lymphoma and acute myeloid leukemia. Despite favorable responses, multiple biological mechanisms lead to treatment resistance. One such mechanism includes somatic mutations in the BCL2 gene. Multiple lines of evidence suggest that hot-spot mutations in BCL2 such as Gly101Val induce treatment resistance by disrupting the binding of BCL2 to the BCL2 inhibitors such as venetoclax. Further, widespread use of high-throughput NGS technologies has identified multiple BCL2 mutations and additional concurrent molecular alterations at various variant allele frequencies in patients with progression while undergoing venetoclax therapy. In order to understand and determine the clinical significance of each of these mutations, careful expert curation and integration into somatic variant annotation AMP/ASCO/CAP guidelines is needed. Further, curation of those somatic variants that may not have sufficient functional evidence in literature may benefit from additional tools such as in silico analysis. To address these issues, we have undertaken an effort to integrate the contributions of a multidisciplinary expert panel (clinical laboratory diagnosticians, oncologists, biomedical informaticians and lab-based researchers) for curation of BCL2 variants in hematological malignancies under the umbrella of ClinGen, an NIH/NHGRI funded consortium to establish standards and centralized resources for assessing the clinical significance of gene variants. Within the ClinGen Somatic Cancer Clinical Domain Working Group (CDWG) ((https://www.clinicalgenome.org/working-groups/somatic/), the somatic hematological malignancy taskforce has identified 56 peer-reviewed publications on BCL2 inhibitors (Jan 2014 to June 2020). The functional evidence contained within these publications was curated using CIViC (Clinical Interpretation of Variants in Cancer, civicdb.org), an open access, crowdsourced aggregation of expert curated evidence. Only a fraction of the somatic variants identified in BCL2 has established functional evidence on variant induced disruption to Venetoclax inhibition. Curation of these remaining variants of unknown significance (VUS) only have in silico functional assays to provide evidence on their potential resistance to Venetoclax. The current curation guidelines do not consider in silico prediction as a strong line of evidence for the interpretation of somatic sequence variants, however this recommendation is meant to interpret generalized in silico predictors and not robust computational models of specific protein function. The latter are more comparable to an experimental functional assay, and provide curators with more trustworthy computational assessments of disruption to protein specific functions. In order to assess their potential to integrate and supplement experimental evidence, the interaction of Ventoclax with several drug resistant BCL2 variants was simulated using AutoDock Vina (J Comput Chem. 2010;31(2):455-61). Facilitated by the SNP2SIM workflow (BMC Bioinformatics. 2019;20(1):171), the relative impact on binding energy was compared to the wildtype system. The in silico binding assay accurately predicted resistance (Fig 1), and demonstrates the utility of applying these methods to the large number of VUS in BCL2. In conclusion, the evidence-based expert curation of BCL2 variants provides a standardized approach for reporting and interpretation across all labs. For those variants (Tier 3) with limited published evidence, computational models that can predict specific changes to functional protein interactions can provide additional tools to the expert curators. Development and incorporation of these tools into curation guidelines requires the refinement of the predictive models through focused validation studies. Disclosures No relevant conflicts of interest to declare.

Abstract The Clinical Genome (ClinGen) Resource is a US National Human Genome Research Institute (NHGRI)-funded program dedicated to building an expert curated and freely available central resource that defines the clinical relevance of genes and variants for use in precision medicine. Teams of experts in various clinical domains come together as working groups within ClinGen to facilitate the interpretation, annotation and utilization of genes-variants for clinical application. Somatic Cancer is one such ClinGen Clinical Domain Working Group (CDWG) that consists of over 90 members worldwide, including clinicians, clinical laboratory diagnosticians, genomic scientists and bioinformaticians. Members of this CDWG identify high priority somatic variants in different cancer types that require expert curation and consensus in their clinical interpretation. In order to accurately implement practice guidelines/standards for variant interpretation, the Somatic Cancer CDWG recently established a somatic Variant Curation Expert Panel (VCEP) approval process. Based on their interest and clinical domain expertise, a subset of the CDWG members formed somatic VCEPs to perform authoritative curation on the shortlisted genes-somatic variants within the context of a disease, therapeutic indication or biological pathway. Expert curation within these VCEPs is performed by utilizing guidelines such as those recommended by AMP/ASCO/CAP (Li et al. 2017) and the Somatic Cancer CDWG (Ritter et al. 2016). Wherever necessary, the somatic VCEPs will develop: 1) gene- and disease-specific modifications to address gaps in existing variant assessment guidelines, 2) quantitative approaches for variant interpretation, and 3) implement standardized protocols for annotating somatic variants in genes for a specific disease, drug or biological pathway. Furthermore, ClinGen recently formed the Cancer Variant Interpretation (CVI) committee to provide support, review and feedback on the provisional somatic variant interpretation proposals developed by the VCEPs. The CVI provides somatic VCEPs with a preliminary approval before the final approval by the ClinGen CDWG oversight committee and ultimately ‘expert panel' status in ClinVar, an NCBI-maintained database of clinically relevant gene variants. NTRK fusions in cancer is the first Somatic VCEP going through the ClinGen Somatic Expert Panel approval process. Alterations in the FGFR pathway in GU cancers is the second somatic VCEP under consideration. The Somatic CDWG uses the CIViC (Clinical Interpretation of Variants in Cancer) platform for curation of somatic variants. To date, the CDWG has curated 268 evidence items relating to cancer variants in CIViC, 6 assertions, and 33 evidence source suggestions. The ultimate goal of the Somatic CDWG is to enhance the usability, dissemination and implementation of cancer somatic changes in the ClinGen resource and other cancer variant knowledgebases. Citation Format: Shruti Rao, Deborah Ritter, Arpad Danos, Gordana Raca, Angshumoy Roy, Kilannin Krysiak, Wan-Hsin Lin, Erica Barnell, Matthew McCoy, Beth Pitel, Dmitriy Sonkin, Jue Wang, Seyed Ali Hosseini, Shamini Selvarajah, Ian King, Rashmi Kanagal-Shamana, Xinjie Xu, Jeremy L. Warner, Funda Meric-Bernstam, Jason D. Merker, Marilyn Li, Alex H. Wagner, Malachi Griffith, Obi L. Griffith, Shashikant Kulkarni, Subha Madhavan. ClinGen somatic cancer working group: Disseminating standardized cancer molecular diagnostic data and evidence through global collaboration and expert curation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3215.

PURPOSE The cancer research community is constantly evolving to better understand tumor biology, disease etiology, risk stratification, and pathways to novel treatments. Yet the clinical cancer genomics field has been hindered by redundant efforts to meaningfully collect and interpret disparate data types from multiple high-throughput modalities and integrate into clinical care processes. Bespoke data models, knowledgebases, and one-off customized resources for data analysis often lack adequate governance and quality control needed for these resources to be clinical grade. Many informatics efforts focused on genomic interpretation resources for neoplasms are underway to support data collection, deposition, curation, harmonization, integration, and analytics to support case review and treatment planning. METHODS In this review, we evaluate and summarize the landscape of available tools, resources, and evidence used in the evaluation of somatic and germline tumor variants within the context of molecular tumor boards. RESULTS Molecular tumor boards (MTBs) are collaborative efforts of multidisciplinary cancer experts equipped with genomic interpretation resources to aid in the delivery of accurate and timely clinical interpretations of complex genomic results for each patient, within an institution or hospital network. Virtual MTBs (VMTBs) provide an online forum for collaborative governance, provenance, and information sharing between experts outside a given hospital network with the potential to enhance MTB discussions. Knowledge sharing in VMTBs and communication with guideline-developing organizations can lead to progress evidenced by data harmonization across resources, crowd-sourced and expert-curated genomic assertions, and a more informed and explainable usage of artificial intelligence. CONCLUSION Advances in cancer genomics interpretation aid in better patient and disease classification, more streamlined identification of relevant literature, and a more thorough review of available treatments and predicted patient outcomes.