Investigation of mutations, copy number variations and translocations by capture-based Next generation sequencing in patients with chronic B-cell lymphoproliferative disorders

Abstract

The current standard investigations in suspected B- and T-lymphoproliferative disorders (LPDs) include the review of morphological and histological characteristics of peripheral blood and bone marrow samples, together with immunophenotype and genetic features. The results are integrated with the aim to assign the disease to one of the WHO entities of Haematopoietic and Lymphoid tumours to identify the most appropriate therapy approach and to provide prognostic information for the patient1. Despite the multi-disciplinary investigations, up to two-thirds of non-CLL chronic B-LPD cases received by the Haematological Diagnostic Malignancy service (HMDS) at our hospital cannot be assigned to a WHO category (Dr. S. Iyengar, service evaluation). For these patients identifying the best treatment approach is difficult and they may not be able to participate in clinical trials. By expanding our investigations into the molecular pathogenesis of these patients it may be possible to increase the proportion of cases assigned to a definitive diagnosis, improve our understanding of molecular subgroups and, potentially, identify actionable mutations. Using a capture-based Next-generation sequencing approach we will enable us to simultaneously analyse patient samples for the presence of mutations, copy-number variations and translocations arising from the Immunoglobulin- and T-cell receptor loci.

Based on our literature review, seventy genes with recurrent mutations are included in the design of the NGS panel. For translocations, capture baits cover the entire IG (IGH, IGK, IGL) and TR (TRA, TRB, TRG, TRD) loci. Importantly, in contrast to Fluorescent in-situ hybridisation (FISH) methods, no prior knowledge of the fusion partners is required as any fusion arising from the IG or TR loci will be detected whilst also identifying the fusion partner2. By sequencing sites of known single nucleotide polymorphisms (SNPs), identification of copy-number variants (CNV) is possible3. A proof-of-principle study demonstrating feasibility of this approach has been performed and published by me (Wren D, Walker B. et al.) on behalf of the EuroclonalityNGS consortium4.

Outputs

Abstract to be submitted to ASH, paper in preparation. Plan to adapt panel into routine use.