P611: NEXT-CLL: A NEW NGS-BASED METHOD FOR RAPID ASSESSMENT OF IGHV MUTATIONAL STATUS IN CHRONIC LYMPHOID LEUKEMIA

Abstract

Background: Chronic Lymphocytic Leukemia (CLL) is a highly heterogeneous disease both in terms of biological landscape and clinical course. Current guidelines recommend IGHV mutation status (MS) determination prior to treatment initiation in order to guide the first-line therapeutic choice between chemoimmunotherapy and novel agents. Currently, the IGHV MS is determined in most routine laboratories by low-throughput Sanger sequencing that allows for the detection of the dominant clone in the majority of CLL cases. However, this technic remains technically challenging despite best practice guidelines, does not provide any insight into subclonal architecture and intraclonal diversity, and has a general failure rate ranging from 10 to 20%. Besides, commercially available NGS solutions have drawbacks (costs) and constraints (as they necessitate mandatorily a MiSeq sequencing machine and are not feasible on other devices) that restrict their use by any laboratory in routine practice. Aims: In this study, we aimed at providing a ready-to-use and rapid strategy to evaluate the IGHV gene MS using NGS in routine practice. We present a new method called Next-CLL, allowing rapid assessment on any NGS device available in routine diagnostic laboratories. Methods: Peripheral blood samples were obtained from 80 patients with typical CLL at diagnosis. Genomic DNA (gDNA) and complementary DNA (cDNA) were used. In order to fully cover the IGHV rearranged gene sequence, 4 mutliplex PCR reactions were designed: [LEADER-FR2R/JHc], [FR1c-JHc], [FR1-JHc] and [FR2-JHc], producing overlapping amplicons. Final products from the 4 PCR reactions were mixed and purified, subjected to A-tailing and ligated to Illumina-single indexed adapters. The library was then sequenced (2x150 bp) on a NextSeq 500 (Illumina). A reassembly of IGHV overlapping amplicons was performed using the BBTools Suite. The outputs were then uploaded on the Vidjil plateform for IGHV clonal analysis. The MS was calculate using the IMGT web interactive tool following ERIC recommendations. IgHV MS was assessed in parallel by Sanger sequencing or the commercial NGS assay IdentiClone® Assay (Invivoscribe) as reference. Gene usage, % identity to the germline and stereotype subsets were compared with the results obtained from Next-CLL. Results: Next-CLL identified a productive clone in 100% of cases (n=80), compared to PCR with Sanger sequencing (15% of failure). All cases that failed using the Sanger sequencing were subjected to a second NGS technique (Invivoscribe) that confirmed the results from Next-CLL, ruling out any possibility of false positive cases. Next-CLL showed 100% concordance with the reference techniques for IGHV gene identification and allowed assessment of the MS from the leader sequence, as recommended by the ERIC consortium. Only 2 cases showed discordant IGHV MS, close to the 98% identity cut-off, between Next-CLL and Sanger sequencing (the latter being unable to capture the leader sequences), thus providing 97% of concordance between the 2 techniques (Figure 1). Moreover, 100% concordant results were obtained between gDNA and cDNA (n=32 cases analysed for both) using Next-CLL. Image:Summary/Conclusion: Our new method Next-CLL allows assessment of the IGHV MS by NGS with an easy and rapid workflow, providing clinically useful information from the IGHV leader sequence and allowing analysis of intra-clonal diversity (not possible with the standard Sanger sequencing). Importantly, it might be used both on gDNA and cDNA and on any sequencing machine, on the contrary to existing commercially available kits.