Citations

Abstract Cytogenetics has long represented a critical component in the clinical evaluation of hematologic malignancies. Chromosome banding studies provide a simultaneous snapshot of genome-wide copy number and structural variation, which have been shown to drive tumorigenesis, define diseases, and guide treatment. Technological innovations in sequencing have ushered in our present-day clinical genomics era. With recent publications highlighting novel sequencing technologies as alternatives to conventional cytogenetic approaches, we, an international consortium of laboratory geneticists, pathologists, and oncologists, describe herein the advantages and limitations of both conventional chromosome banding and novel sequencing technologies and share our considerations on crucial next steps to implement these novel technologies in the global clinical setting for a more accurate cytogenetic evaluation, which may provide improved diagnosis and treatment management. Considering the clinical, logistic, technical, and financial implications, we provide points to consider for the global evolution of cytogenetic testing.

Abstract The World Health Organization category of myeloid/lymphoid neoplasms with eosinophilia and PDGFRA rearrangements is composed of a heterogeneous group of neoplasms that can present as a myeloproliferative neoplasm, acute myeloid leukemia, myeloid sarcoma, or lymphoblastic leukemia/lymphoma. The overall outcome of these neoplasms is favorable with imatinib therapy. Herein, we describe an adult female patient with a myeloid neoplasm accompanied by eosinophilia and a novel USP25::PDGFRA gene fusion.

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.

Abstract The detection of recurrent genetic abnormalities in acute myeloid leukemia (AML), including RUNX1T1/RUNX1 gene fusion, is critical for optimal medical management. Herein, we report a 45 year old woman with newly diagnosed AML and conventional chromosome studies that revealed an apparently balanced t(8;20)(q22;p13) in all 20 metaphases analyzed. A RUNX1T1/RUNX1 dual-color dual-fusion fluorescence in situ hybridization (FISH) probe set was subsequently performed and revealed a RUNX1T1/RUNX1 gene fusion. Metaphase FISH studies performed on abnormal metaphases revealed a cryptic, complex translocation resulting in RUNX1T1/RUNX1 fusion, t(8;20;21)(q22;p13;q22). This case study shows the importance of performing FISH studies or other high-resolution genetic testing concurrently with conventional chromosome studies for the detection of cryptic recurrent gene fusions in AML, particularly a focused genetic evaluation such as RUNX1T1/RUNX1 gene fusion, when specific abnormalities involving 8q22 are identified.

Abstract Despite the well-established role of recurrent gene fusions as oncogenic drivers, current practices for characterizing and interpreting gene fusion events in clinical testing and in biomedical literature are inconsistent. From the conceptual definition of gene fusions to the salient elements that characterize these alterations, a lack of community-driven standards for the curation of gene fusions has resulted in a disparate landscape of fusion representations and supporting tools. Consequently, the evidence-based clinical evaluation of gene fusions requires extensive expert review for accurate interpretation of observed gene fusions with respect to putative evidence from biomedical literature. Furthermore, the lack of these standards inhibits the interoperability of tools, resources, and pipelines - impeding data sharing and downstream utility.To address these challenges, a cross-consortia initiative between the Variant Interpretation for Cancer Consortium and ClinGen was formed to develop a standard operating procedure (SOP) for the curation of gene fusions. The SOP is under development by an international and diverse set of experts in the representation, detection, and clinical interpretation of gene fusions. Participating stakeholders across academic, government, and industry sectors showcased challenges and solutions, and participated in community surveys and discussions to define and develop the SOP for this diverse class of alterations.An initial result of this effort was the precise molecular definition of genomic events and features constituting gene fusions. We distinguish these from similar but distinct classes of structural alterations through clinically-relevant examples. Next, we discuss our findings on community practices around the description and evaluation of gene fusions. We provide our recommendations for characterization and representation of gene fusions from these practices, and compare these recommendations to existing variant representation standards and formats (e.g. HGVS variant nomenclature). We also discuss the concurrent application of formats for standardized human- and machine-readable representations of gene fusion events.We conclude with discussion of the salient elements to enable rapid, scalable, and consistent evaluation of fusions curated from the biomedical literature. Recommendations are provided for the standardized capture of these elements to enable both intuitive and precise characterization of this diverse class of alterations in clinical reporting and literature. In summary, we provide a clinical-practice driven framework and nomenclature for gene fusions, including recommendations for human readability, computational precision, and data integrity within the SOP. This work is a substantial advancement towards standardized communication, investigation, and sharing of gene fusion data across clinical and research domains and specialties. Citation Format: Alex H. Wagner, Ioannis S. Vlachos, Dmitriy Sonkin, Panieh Terraf, Chimene Kesserwan, Andrea Sboner, Thomas Coard, Christian Reich, Deborah I. Ritter, Peter Horak, Ying S. Zou, Anna Tanska, Aaron M. Berlin, Anna Lu, Daniel Cameron, Heather E. Williams, Wan-Hsin Lin, Gokce Toruner, Arpad Danos, Jason Saliba, Huiling Xu, Xinjie Xu, Georgina Ryland, Michele Ceccarelli, Liying Zhang, Sarah Rapisardo, Catherine Rehder, Xuelu Liu, Aparna Pallavajjala, Nicole Park, Laveniya Satgunaseelan, Kristy Lee, Jie Liu, Obi Griffith, Robert R. Freimuth, Albrecht Stenzinger, Linda B. Baughn, Michael Baudis, Jennifer Lee, Marilyn Li, Angshumoy Roy, Gordana Raca. A standard operating procedure for the curation of gene fusions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 449.

Abstract Childhood cancers are driven by unique profiles of somatic genetic alterations, with a significant contribution from predisposing germline variants. Understanding the genomic landscape of pediatric cancers is complicated by their rarity, the heterogeneity of variation within a given disease, and the complex forms of structural variation they contain. Variants in childhood disease may differ from those in adult versions of the same cancer type, or may have different clinical significance. Currently, pediatric variants are underrepresented in cancer variant databases, and an urgent need exists for their publicly available expert curation. To address this, the Pediatric Cancer Taskforce (PCT) was formed within the Clinical Genome Resource (ClinGen) Somatic Cancer Clinical Domain Working Group (CDWG) (https://www.clinicalgenome.org/working-groups/somatic/). The PCT is a multi-institutional group of 39 members with broad experience in childhood cancer and variant curation, whose work consists of standardization and classification of genetic variants in pediatric cancers. The CIViC knowledgebase (www.civicdb.org) is a freely available resource for Clinical Interpretation of Variants in Cancer, which leverages public curation and expert moderation to address the problem of annotating the large volume of clinically actionable cancer variants. PCT curators work together with PCT expert members and the CIViC team on variant curation, and have submitted over 230 Evidence Items and over 10 Assertions to CIViC. To further address issues specific to pediatric curation, the PCT is working with CIViC to develop new pediatric-specific CIViC features and modifications of the data model that will aid in pediatric curation. A pediatric user interface, as well as representation of large scale structural and copy number variation are being developed for version two of CIViC, expected to be released in 1-2 years, which will enable curation of a new class of structural variants often encountered in pediatric cancer. A novel standard operating procedure for childhood cancer curation in CIViC is being developed by PCT experts, curators and the CIViC team. This SOP will cover topics including curation of structural variants, as well as pediatric-specific variant tiering guidelines which take into account the sparse nature of evidence in pediatric cases. A companion resource, CIViCmine (http://bionlp.bcgsc.ca/civicmine/), will be further developed to incorporate pediatric data. These and other joint efforts of the PCT and CIViC will significantly enhance pediatric variant representation for public use, to support the care of children with cancer. Citation Format: Arpad Danos, Wan-Hsin Lin, Jason Saliba, Angshumoy Roy, Alanna J. Church, Shruti Rao, Deborah Ritter, Kilannin Krysiak, Alex Wagner, Erica Barnell, Lana Sheta, Adam Coffman, Susanna Kiwala, Joshua F. McMichael, Laura Corson, Kevin Fisher, Heather E. Williams, Matthew Hiemenz, Katherine A. Janeway, Jianling Ji, Kesserwan A. Chimene, Laura Fuqua, Lisa Dyer, Huiling Xu, Jeffrey Jean, Laveniya Satgunaseelan, Liying Zhang, Ted W. Laetsch, Donald W. Parsons, Ryan Schmidt, Lynn M. Schriml, Kristen L. Sund, Shashikant Kulkarni, Subha Madhavan, Xinjie Xu, Rashmi Kanagal-Shamana, Marian Harris, Yasmine Akkari, Nurit Paz Yacov, Panieh Terraf, Malachi Griffith, Obi L. Griffith, Gordana Raca. Advancing knowledgebase representation of pediatric cancer variants through ClinGen/CIViC collaboration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 210.

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.

Purpose: Acute myeloid leukemia (AML) is the most common acute leukemia in adults, affecting approximately 20,000 patients annually in the United States. AML genetic subtypes, as defined by the World Health Organization (WHO), are identified through fluorescence in situ hybridization (FISH), conventional chromosome analysis, and sequencing techniques. Mate pair sequencing (MPseq) is a next generation sequencing (NGS) technology optimized to detect genome wide structural variants and copy number alterations at high resolution. Our study goal was to investigate the prognostic value of MPseq in comparison to FISH, chromosome, and sequencing studies in the evaluation of AML patients. Methods: We performed a prospective study using blood and bone marrow samples from 105 patients with a diagnosis of AML, using MPseq, along with chromosome, FISH, and NGS or PCR studies to detect small mutations. Cytogenetic and molecular genetic results were correlated with MPseq findings. We also analyzed the MPseq data for chromoplexy, chromothripsis, and progressive complexity. Junction and copy number burden, the incidence of structural variation in the genome and the percent of the genome with aberrant copy number, were evaluated. Overall survival statistics were stratified by AML subtypes and observed anomalies. Results: Although structural variants in AML were characterized at a high resolution using MPseq when compared to conventional cytogenetic methods, risk stratification using current European Leukemia Net (ELN) guidelines was not improved by MPseq. The cohorts involving 5q and/or 7q deletions exhibited high levels of genomic complexity when compared to normal karyotype AML (NK-AML). The incidence of copy number gains, losses and junctions was greatest in 5q and 7q co-deletions (5q/7q) (16.5, 25.0, 69.3) and 5q deletions (5q) (9.8, 16.7, 31.6) subtypes compared to 7q deletions (3.4, 7.0, 6.7) and NK-AML (2.6, 4.3, 3.8) (p<0.001) subtypes. Chromoplexy, chromothripsis, and progressive structural complexity were detected in most samples with 5q deletions and 5q/7q co-deletions, but absent in samples with 7q or NK-AML. Biallelic inactivation of TP53 by sequencing mutation and/or deletion was common in the 5q/7q co-deletion (14/18 cases) and 5q deletion cohorts (7/10 cases), rare in the 7q deletion cohort (1/11 cases), and absent in the NK-AML (n=44) cohort. The median OS was significantly worse for patients with 5q/7q deletions (122.5 days) and 5q deletions (248 days) compared to NK-AML (413.5 days; p<0.001 and p=0.017, respectively) and between 5q/7q deletions and 7q deletions (370.5 days; p<0.001). No significant difference was observed between 5q/7q and 5q deletion subtypes, between NK and 7q deletion subtypes and between 5q and 7q deletion subtypes. The median OS was also significantly shorter for patients with TP53 alterations compared to patients with normal TP53 status. Patients with chromoplexy, chromothripsis and/or progressive structural complexity identified by MPseq had a significantly shorter median OS compared to patients without these features. (p<0.0001) Discussion: Risk stratifications based on current guidelines using cytogenetic and sequencing results were not adjusted due to MPseq results, which is not surprising when primary abnormalities are observed by conventional cytogenetic methods. NK-AML cases did not appear to benefit from a high resolution genomic evaluation. However, MPseq added value when structural variation required additional characterization, detecting novel rearrangements, such as a KAT6A/SORBS3 fusion. Lastly, we recognized common mischaracterizations made by conventional chromosome studies - including missed TP53 deletions in 7 cases, 5q/7q deletions misinterpreted as monosomies, cryptic NUP98 rearrangements, and unappreciated genomic complexity correlating with poor OS. These mischaracterizations challenge the use of conventional chromosome studies as a gold standard without accompanying FISH or MPseq studies. MPseq, similar to other structural methodologies such as optical mapping and long read sequencing, should be considered important complements to standard cytologic techniques given the important additional genomic information obtained. The additional structural variant characterization will be critical in paving the way for genomic discovery with the overall goal of improving prognostication for patient care. Figure Disclosures Vasmatzis: WholeGenome LLC.: Other: Owner; Mayo Clinic: Membership on an entity's Board of Directors or advisory committees, Research Funding.

AbstractAcute undifferentiated leukemia (AUL) is a very rare hematologic neoplasm that expresses no markers specific for either myeloid or lymphoid lineages. While commonly observed in several acute leukemias, KMT2A rearrangements in AUL have been rarely reported in the literature. We report the third case to our knowledge of AUL harboring a KMT2A rearrangement. Furthermore, the KMT2A/GIMAP8 gene fusion identified in this case represents a novel KMT2A rearrangement.