Background: Current next-generation sequencing (NGS) approaches for analyzing SHM commonly rely on multiplex primers targeting the framework 1 (FR1) or leader region of the IGH variable gene in combination with joining gene primers to amplify rearranged IGH chains from gDNA template. Limitations include the potential for joining gene mutations to interfere with primer binding and an inability to evaluate isotype. Here we present a method for translational research investigations of IGH chain SHM employing multiplex FR1 and isotype (constant gene) specific primers (Oncomine IGH-LR assay primers) to amplify IGH chains from RNA template. We evaluated performance by comparing SHM values obtained from NGS of RNA from 54 CLL samples amplified using Oncomine IGH-LR primers to values obtained by Sanger sequencing or NGS of RNA amplified using FR1 or leader region/J gene primers.

Methods: IGH chains from 54 CLL samples derived from two separate sequencing sites (Site 1: 24 samples, Site 2: 30 samples) were amplified from peripheral blood using Oncomine IGH-LR assay followed by sequencing via the Ion Gene Studio S5. Clonotyping, clonal lineage identification and somatic hypermutation analysis was performed by Ion Reporter via comparison to the IMGT reference database. Oncomine IGH-LR assay SHM values were compared to those obtained via NGS-based sequencing utilizing FR1/J gene primers (Site 1) or Sanger sequencing utilizing IGH-leader or FR1 and joining gene primers (Site 2).

Results: IGHV SHM values were highly concordant between NGS approaches (Spearman cor =.957, Site 1) and between Sanger sequencing and NGS approaches (Spearman cor = .849, Site 2). Sequence data obtained using Oncomine IGH-LR assay enables more in-depth clonal lineage analysis, including evaluation of isotype representation and subclonal evolution.

Conclusions: These results support the robustness and reliability of multiplex FR1 and constant gene based IGH chain amplification for the translational research characterization of somatic hypermutation in CLL and other B cell neoplasms.

Citation Format: Jayde Chang, Zadie Davis, Graeme Quest, Harriet Feilloter, Michelle Toro, Geoffrey Lowman, Loni Pickle, Fiona Hyland, Timothy Looney. Clonal lineage and somatic hypermutation analysis of chronic lymphocytic leukemia by long-amplicon IGH chain sequencing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6472.