Abstract 6673: Simultaneous detection of IGH VDJ rearrangements and JH translocations via Ring-Seq, a novel multiplex PCR technology for targeted sequencing of translocations with novel gene partners or breakpoints
Authors: Owens Shannon , Fichter Katherine , Que Richard , Shore Sabrina , Looney Timothy 
Published: April 2023

Abstract
Introduction: Next-generation sequencing (NGS) of B cell receptor rearrangements is a leading method for clonality assessment and measurable residual disease (MRD) monitoring of B cell malignancies. Current methods rely upon multiplex PCR (mPCR) amplification of rearranged IGH, IGK or IGL loci via variable and joining gene targeting primers followed by NGS to assess rearrangements. Limitations include a need for reflex testing owing to somatic hypermutation (SHM) mediated loss of sensitivity and an inability to detect clinically relevant translocations of the IGH locus. Here we apply Ring-Seq, a novel technology for the targeted detection gene fusions with unknown partners, to simultaneously detect IGH VDJ rearrangements and translocations in a single reaction. We demonstrate detection of clinically relevant translocations and VDJ rearrangements from highly degraded gDNA.
Methods: The method begins with a highly efficient circularization of DNA fragments followed by a multiplex inverse PCR of joining genes that preferentially amplifies breakpoint junction containing templates. Amplicon libraries are sequenced on the G4 platform and analyzed to detect translocations and VDJ rearrangements. We pooled gDNA from reference cell lines harboring marker IGH VDJ rearrangements and BCL1/2-JH translocations. Pooled reference gDNA was spiked into a background of healthy donor PBL gDNA to evaluate performance over a range of marker frequencies. To simulate performance with degraded materials, gDNA input was fragmented to ~200bp by sonication prior to analysis.
Results: BCL1-JH and BCL2-JH translocations and marker IGH VDJ rearrangements were detected from 50ng pooled reference gDNA. Translocations and marker VDJ rearrangements were also detected in 50ng samples consisting of reference gDNA spiked at 10% and 1% frequency into a background of healthy donor PBL gDNA. Sequencing of healthy donor PBL gDNA yielded IGH repertoire features that were highly concordant with results obtained from BIOMED-2 mPCR analysis of the same samples.
Conclusions: We have applied Ring-Seq to enable the simultaneous detection of B cell receptor rearrangements and JH translocations relevant to the diagnosis of MCL and FL while minimizing loss of sensitivity owing to SHM. Traditional mPCR often fails to detect JH translocations owing to variation in the breakpoint region. Consequently, suspected MCL and FL diagnostic samples are assessed for translocations by FISH, while separate material is submitted for NGS-based VDJ clonality analysis via traditional mPCR. Ring-Seq may streamline this process by consolidating separate tests into a single rapid NGS assay for the G4 platform. Looking ahead, we envision broad applicability of the technology to mPCR-based variant detection.
Citation Format: Shannon Owens, Katherine Fichter, Richard Que, Sabrina Shore, Timothy Looney. Simultaneous detection of IGH VDJ rearrangements and JH translocations via Ring-Seq, a novel multiplex PCR technology for targeted sequencing of translocations with novel gene partners or breakpoints. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6673.