Ighv Mutational Status By Deep Next Generation Sequencing Refines Ighv Sanger Sequencing Classification in Patients with Chronic Lymphocytic Leukaemia

Introduction: Determination of the mutational status of rearranged immunoglobulin heavy chain variable (IgHV) genes in patients with Chronic Lymphocytic Leukaemia (CLL), is considered one of the most important prognostic factors: patients with unmutated IgHV (UM; ≥98% of identity to the germline) genes have a more aggressive disease course and develop more frequently unfavourable genetic deletions or mutations than patients with mutated IgHV (M; ≤98%). Mutational status, is currently determined by Sanger sequencing (Sseq) that allows the analysis of the major clone, however, international guidelines recommend caution in assigning mutational status in cases with "Borderline" IgHV identity (97-97.9%), and cases with double rearrangements with discordant mutational status.

Objective: Analyze and determine the mutational status of the IgHV locus by High-throughput sequencing (HTS), in a cohort of CLL patients (n=51) with unclassifiable Sseq results: borderline status (n=22); double rearrangements (n=27) with discordant mutational status (n=2).

Methods: We included 51 DNA samples extracted from peripheral blood of patients diagnosed of CLL according to the National Cancer Institute Working Group guidelines in our institution between 1986 and 2019 (median absolute lymphocytes 11.4x10⁹/L [2,8-239,5x10⁹/L]).

Sseq amplification and analysis of IgHV rearrangements were performed on DNA conforming to the updated ERIC recommendations. In all the cases we were able to determinate the IGVH identity.

To switch high-throughput sequencing to the clinical practice, we assessed the reliability of different library preparation methods to sequence IGH locus in patients with CLL. Amplification was performed using the Sequencing Multiplex Kit based on IGH FR (forward primers) and consensus JH (reverse primer) multiplex. PCR products were purified using Magsi-NGS Prep magnetic beads (Magnamedics Diagnostics), normalized and pooled to create a library for sequencing using a MiSeq equipment. To simplify and make automatic the analysis of the same we developed a specific bioinformatic pipeline that covers from preprocessing to final data summarization and interpretation. The backbone of the analysis includes read preprocessing, mapping against IMGT reference sequences, consensus IgHV reads pairwise alignment to determine mutational status and read classification into rearrangements.

Results: This approach led to the identification of a dominant clone IgHV in all cases (n=51). Instead, the percentage of identity calculated by HTS analysis varies in:

- 15/22 borderline cases whose mutational status could be recalculated into 10 MM and 5 UM. The rest 7 remaining in borderline group.

- We could identify both clones in 29 double rearrangements cases, with concordant mutational status except 2/29 undetermined cases, included in UM group regarding HTS results.

Our tool led to the identification of a dominant clonotypic IgHV in all cases, and when compared the HTS sequence/mutational status for the most abundant clone with Sseq and for the IgHV status determination, 15 out of 22 (68,18%), could be reclassified. This case showed a major clone with productive rearrangement mutated by Sseq but unmutated by HTS.

Conclusions: Analyze and determine the mutational status of the IgHV locus by HTS, would potentially reveal multiple rearrangements and increase the prognostic precision of IgHV mutation analysis. IgHV-HTS classification is able to precisely classify patients with borderline status or/and multiple IgHV rearrangements for which Sseq is inconclusive. In this case, it has been possible to improved prognostication for 17 out of 24 patients. This is helping us to discover the advantages of the data obtained by HTS compared with current Sseq standard technique.

Samples were provided by the INCLIVA Biobank. Funded by Gilead Felowship 257/17