Abstract 2940: Somatic hypermutation analysis of chronic lymphocytic leukemia research samples by IGH chain next-generation sequencing
Authors: Toro Michelle , Pickle Loni , Sarda Shrutii , Marshall Robyn , Pieterse Bonnie , Venter Tania , Yang Chenchen , Gerdener Theo , Lowman Geoffrey Marc 
Published: June 2022

Abstract
Chronic lymphocytic leukemia (CLL) is a common form of leukemia characterized by clonal expansion of neoplastic B cells and a heterogenous disease course. Somatic hypermutation (SHM) status of the IGHV gene in CLL clinical research samples is important as SHM frequency is an established prognostic biomarker. Conventionally, SHM analysis is performed via Sanger sequencing which is limited by the inability to evaluate more than one rearrangement due to mutations, multiplex constraints, and template input requirements.
Ion AmpliSeq™ next generation sequencing (NGS) assays for research in IGH chain SHM are evaluated using multiple cohorts of CLL research samples. These NGS assays employ multiplex primers that target the Leader or FR1 regions of the IGHV gene and the IGHJ gene in either RNA or DNA templates. The robustness of these assays was demonstrated by an evaluation across multiple labs, including comparisons between DNA and RNA input and correlation to orthogonal NGS testing.
Ion AmpliSeq™ multiplex primer sets were designed to target the Leader or Framework 1 (FR1) regions of the IGHV gene with joining gene (J) or constant region (C) primers. The Oncomine™ IGHV Leader-J Assay uses Leader primers, while the RNA-input Oncomine™ IGH-LR Assay, and the DNA-input Oncomine™ IGH FR1-J Assay use FR1 variable gene primers in combination with C and J region primers, respectively. Primer panels were designed to amplify all variable gene families, including all individual alleles, listed in the IMGT database. To evaluate accuracy, each assay was used to measure SHM from a panel of gDNA and RNA rearrangements extracted from CLL research samples for clonality testing and SHM analysis. To test the Leader-J and FR1-J DNA input assays, 24 orthogonally characterized CLL samples were evaluated on the Ion Torrent S5 platform. Stereotypy and confirmation of individual software findings assessed by placing FASTA file results into the online ARResT tool.
Both RNA and DNA input assays correctly determined the SHM status of CLL rearrangements, with IGHV SHM values concordant between both RNA and DNA approaches. SHM values derived from FR1 variable gene primers delivered concordant results compared to Leader variable gene primers across a range of SHM frequencies, with FR1 primer coverage of the IGHV gene leading to an expected over-estimation of SHM percentage. SHM status was concordant between NGS assays evaluated in over 90% (21/24) of CLL samples tested. Mutational frequency showed excellent concordance, with an R2=0.97, and V-gene usage was 100% concordant.
These results support the robustness of long-read NGS assays to quantify SHM in either DNA or RNA samples. Concordant results were shown between FR1 and Leader-targeting primers using DNA input showing the utility in both priming locations. Orthogonal testing of the Leader-J assay showed excellent concordance for mutation rate, SHM status, and stereotypy.
Citation Format: Michelle Toro, Loni Pickle, Shrutii Sarda, Robyn Marshall, Bonnie Pieterse, Tania Venter, Chenchen Yang, Theo Gerdener, Geoffrey Marc Lowman. Somatic hypermutation analysis of chronic lymphocytic leukemia research samples by IGH chain next-generation sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2940.