2. Diagnostic next generation sequencing to detect MYD88 L265P in lymphoplasmacytic lymphoma compared to ddPCR

Abstract

Lymphoplasmacytic lymphoma (LPL) is a B-cell lymphoproliferative disorder characterized by bone marrow infiltration of lymphoplasmacytic cells. Studies have identified MYD88 L265P mutation as a diagnostic marker to distinguish LPL from other small B-cell lymphomas (e.g. marginal zone lymphoma, chronic lymphocytic leukemia). However, detection rates for this mutation have varied depending on the analytic methodology, with data suggesting that routine Next Generation Sequencing (NGS) does not demonstrate the required sensitivity to reliably detect MYD88 L265P. NGS has become a work-horse of the modern genetic laboratory by basketing multiple single-gene assays into one multiplexed assay. In several routine cases, visual observation using IGV demonstrated MYD88 L265P variants in cases of suspected LPL with low tumor content. To study this, we performed droplet digital PCR (ddPCR) and our routine NGS for MYD88 L265P in a cohort of 35 cases of lymphoid neoplasms (25 LPL, 6 CLL, 4 negative bone marrow cases). Our standard NGS method achieved an average depth of ∼535 reads across this region. We utilized manual review and BAMtools to assess MYD88 L265P in NGS cases. Limit of detection for ddPCR was determined to be 0.3% variant allele frequency (VAF) with 10ng DNA input. MYD88 L265P VAF detection by NGS and ddPCR was comparable down to 0.5% VAF (R²=0.968). Setting an appropriate threshold for detection based on ddPCR results resulted in zero NGS false positives. This study demonstrates that NGS can be a sensitive and reliable method for detection of MYD88 L265P with adequate coverage and specific assessment parameters.