Next-CLL, a New Next-Generation Sequencing–Based Method for Assessment of IGHVGene Mutational Status in Chronic Lymphoid Leukemia

Current guidelines for patients with chronic lymphocytic leukemia (CLL) recommend mutation status determination of the clonotypic IGHVgene before treatment initiation to guide the choice of first-line therapy. Currently, commercially available next-generation sequencing (NGS) solutions have technical constraints, as they necessitate at least a 2 × 300 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 IGHVgene mutation status using any NGS device (including 2 × 150 bp sequencers) in routine diagnostic laboratories. The performance of Next-CLL was validated on genomic DNA and cDNA obtained from 80 patients with CLL 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 IGHVgene identification and allowed assessment of the IGHVmutation status from the leader sequence, following international guidelines. Comparing a large retrospective series of samples, analyzed by using Sanger sequencing (n = 773) or Next-CLL (n= 352), showed no bias in IGHVusage or mutational status, further validating our strategy in the real-life setting. Next-CLL represents a straightforward workflow for IGHVanalysis in routine practice to assess clonal architecture and prognosis of patients with CLL.