Assessment of Capture and Amplicon-Based Approaches for the Development of a Targeted Next-Generation Sequencing Pipeline to Personalize Lymphoma Management

Abstract

Targeted next-generation sequencing panels are increasingly used to assess the value of gene mutations for clinical diagnostic purposes. For assay development, amplicon-based methods have been preferentially used on the basis of short preparation time and small DNA input amounts. However, capture sequencing has emerged as an alternative approach because of high testing accuracy. We compared capture hybridization and amplicon sequencing approaches using fresh-frozen and formalin-fixed, paraffin-embedded tumor samples from eight lymphoma patients. Next, we developed a targeted sequencing pipeline using a 32-gene panel for accurate detection of actionable mutations in formalin-fixed, paraffin-embedded tumor samples of the most common lymphocytic malignancies: chronic lymphocytic leukemia, diffuse large B-cell lymphoma, and follicular lymphoma. We show that hybrid capture is superior to amplicon sequencing by providing deep more uniform coverage and yielding higher sensitivity for variant calling. Sanger sequencing of 588 variants identified specificity limits of thresholds for mutation calling, and orthogonal validation on 66 cases indicated 93% concordance with whole-genome sequencing. The developed pipeline and assay identified at least one actionable mutation in 91% of tumors from 219 lymphoma patients and revealed subtype-specific mutation patterns and frequencies consistent with the literature. This pipeline is an accurate and sensitive method for identifying actionable gene mutations in routinely acquired biopsy materials, suggesting further assessment of capture-based assays in the context of personalized lymphoma management.