Validation of a Cell-Free DNA NGS Assay for Hematological Malignancies

Introduction: In recent years, next-generation sequencing (NGS) of cell-free DNA (cfDNA) has been applied to non-invasive assessment and molecular monitoring of patients with cancer. In this study, we present the validation data for MSK-ACCESS HEME (Memorial Sloan Kettering-Analysis of Circulating cfDNA to Examine Somatic Status), a unique molecular indexing ultra-deep sequencing assay for hematologic neoplasms. Full coding or select exons of 117 genes are targeted in this assay based on alterations identified in 10,000 patient samples sequenced with the MSK-IMPACT HEME assay.

Methods: To assess the accuracy of the MSK-ACCESS Heme assay we sequenced 54 patient cfDNA samples harboring 187 somatic alterations confirmed by clinically validated orthogonal methods. Seventy-five healthy donor samples were used to control for sequencing artifacts and background error. Five commercial SeraCare ctDNA Reference Material samples were sequenced and analyzed for sensitivity studies.For reproducibility and precision analyses, three of the accuracy samples were resequencedwithin or across different sequencing runs.

Results: Samples were sequenced to mean duplex and simplex/duplex coverage of 1450X and 2295X, respectively.De novo variant calling accuracy was 84.4% (160/187) and 97.5% (182/187) for tumor informed genotyping. In comparing the variant allele frequencies (VAFs) of alterations between MSK-ACCESS HEME and MSK-ACCESS solid, we calculated a coefficient of determination (R2) of 99.6. For variants detected at a VAF less than 0.05 by an orthogonal method, we calculated an R2 of 96.6%. Alterations with a VAF above 0.05 had close to perfect recall, with R2=99.4%. Precision and reproducibility analysis demonstrated very close correlation when sequencing samples within and between sequencing runs.Sensitivity analysis revealed that all variants were called down to 0.5%using stringent de-novo critria.

Conclusion: These data demonstrate that the MSK-ACCESS HEME assay is a robust and reliable tool for detecting and reporting alterations in hematological malignancies with high accuracy and sensitivity. Once approved by the New York State Department of Health, this assay will be employed to prospectively monitor patients with hematological diseases in clinical setting.

Spence:Sema4: Current Employment, Current equity holder in publicly-traded company. Tsui:PetDx: Current Employment, Current holder of stock options in a privately-held company. Salles:Roche: Other: Provision of Services; Incyte: Other: Provision of Services; Ipsen: Other: Provision of Services; Janssen Pharmaceuticals, Inc.: Other: Provision of Services; Loxo Oncology: Other: Provision of Services; Miltenyi Biotec Incorporated: Other: Provision of Services; MorphoSys AG: Other: Provision of Services; VelosBio, Inc.: Other: Provision of Services; Scientific Education Support Ltd.: Other: Provision of Services; Takeda Millennium: Other: Provision of Services; BeiGene, Ltd.: Other: Provision of Services; Novartis: Other: Provision of Services; Physicians' Education Resource: Other: Provision of Services; AbbVie: Other: Provision of Services; Asociación Colombiana de Hematología y Oncología (ACHO): Other: Provision of Services; Bayer: Other: Provision of Services; Bristol-Myers Squibb: Other: Provision of Services; Celgene: Other: Provision of Services; RAPT Therapeutics: Other: Provision of Services; Debiopharm: Other: Provision of Services; Regeneron Pharmaceuticals, Inc.: Other: Provision of Services; Epizyme: Other: Provision of Services; GenMab: Other: Provision of Services; Genentech: Other: Provision of Services; Gilead Pharmaceutical: Other: Provision of Services. Batlevi:Bristol-Myers Squibb: Other: Ownership / Equity Interests; Provision of Services; ADC Therapeutics: Other: Provision of Services; Dava Oncology: Other: Provision of Services; Autolus: Research Funding; Bayer: Research Funding; Epizyme: Research Funding; Janssen: Research Funding; Novartis: Research Funding; Roche/Genentech: Research Funding; Xynomic: Research Funding; GLG Pharma: Consultancy; Juno/Celgene: Consultancy; Kite Pharma: Consultancy; Life Sciences: Consultancy; Seattle Genetics: Consultancy. von Keudell:Pharmacyclics: Other: Provision of Services; Merck Sharp & Dohme: Other: Provision of Services; MorphoSys AG: Other: Provision of Services. Ptashkin:C2i Genomics: Current Employment. Zehir:Astrazeneca: Current Employment; Arcus Biosciences, Inc.: Other: Ownership / Equity Interests. Berger:Advances in Genome Biology and Technology (AGBT): Other: Provision of Services (uncompensated); Journal of Visualized Experiments: Other: Provision of Services (uncompensated); Journal of Molecular Diagnostics: Other: Provision of Services (uncompensated); JCO Precision Oncology: Other: Provision of Services (uncompensated); Eli Lilly and Company: Other: Provision of Services; American Society of Clinical Oncology (ASCO): Other: Provision of Services; PetDx, Inc.: Other: Provision of Services. Brannon:Johnson & Johnson: Current equity holder in private company. Arcila:PeerView Institute for Medical Education: Consultancy; Bristol-Myers Squibb: Consultancy; Biocartis US, Inc: Consultancy; AstraZeneca: Consultancy; Clinical Care Options: Consultancy; Invivoscribe: Consultancy; Janssen Global Services: Consultancy; Physicians' Education Resource: Consultancy.