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

Abstract

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.