Next-CLL: a new next generation sequencing-based method for assessment of IGHV gene mutational status in chronic lymphoid leukemia.

Current guidelines for patients with Chronic Lymphocytic Leukemia (CLL) recommend mutation status determination of the clonotypic IGHV gene prior to treatment initiation in order to guide the choice of the first-line therapy. Currently, commercially available next-generation sequencing (NGS) solutions have technical constraints, as they necessitate at least a 2x300 bp sequencing, which restricts their use for routine practice. The cost of the commercial kits also represents an important drawback. We present a new method called Next-CLL, a ready-to-use strategy to evaluate IGHV gene mutation status using any NGS device (including 2 x 150 bp sequencers) in routine diagnostic laboratories. We validated the performance of Next-CLL on genomic DNA and complementary DNA obtained from 80 CLL patients at diagnosis. Next-CLL identified a productive clone in 100% of cases, whereas PCR with Sanger sequencing led to a 12.5% failure rate. Next-CLL had 100% concordance with the reference technique for IGHV gene identification and allowed assessment of the IGHV mutation status from the leader sequence, following international guidelines. By comparing a large retrospective series of samples, analyzed using Sanger sequencing (n=773) or Next-CLL (n=352), we showed no bias in IGHV usage or mutational status, further validating our strategy in the real-life setting. Next-CLL represents a straight-forward workflow for IGHV analysis in routine practice to assess clonal architecture and prognosis of CLL patients.