Long-Amplicon Sequencing of Rearranged IGH Chains Enables Simultaneous Measurement of Somatic Hypermutation and Isotype in Chronic Lymphocytic Leukemia

Background

Chronic lymphocytic leukemia (CLL) is a common form of leukemia characterized by clonal expansion of neoplastic mature B cells and a heterogenous disease course ranging from aggressive clonal expansion requiring early intervention to indolent disease that does not require treatment. Accumulating evidence suggests that the somatic hypermutation (SHM) status of the IGHV gene of the malignant clone may serve as a prognostic marker of clinical outcome, where a SHM frequency of >2% indicates a favorable outcome (SHM-M) while < 2% SHM (SHM-U) indicates comparatively poor prognosis. 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, which could potentially serve as an additional prognostic marker given the mechanistic link between somatic hypermutation and class-switch recombination. Here we present a novel method for translational research investigations of IGH chain SHM employing multiplex FR1 and isotype (constant gene) specific primers to amplify IGH chains from RNA template. We demonstrate performance of the assay via sequencing of synthesized CLL IGH chains derived from literature.

Methods

Multiplex PCR primers were designed to target all IGH variable genes and constant genes and alleles in the IMGT database (Oncomine IGH-LR assay). Primers were used to amplify IGH chains in samples consisting of 25ng healthy donor peripheral blood leukocyte (PBL) total RNA spiked with one of 7 synthesized monoclonal CLL rearrangements including three germline rearrangements (0% SHM), and four mutated rearrangements (SHM ranging from 3-9%), each expressed with a different isotype. Resultant libraries were sequenced on the Gene Studio S5 and analyzed via Ion Reporter to identify clonotypes, quantify SHM, and identify B cell clonal lineages. Automated downsampling analysis was used to confirm that libraries had been sequenced to an appropriate depth.

Results

7 of 7 synthesized rearrangements were correctly classified as SHM-M or SHM-U, and in each instance the isotype was correctly identified by the software. Clonal lineage analysis indicated that each spike in rearrangement was monoclonal, consistent with expectation. In silico analysis revealed a high correlation between SHM estimates obtained using the entire v-gene sequence and those obtained from FR1-targeting primers (Pearson's correlation >.99).

Conclusions

These results support the robustness and reliability of multiplex FR1 and constant gene based IGH chain amplification, combined with clonotyping and lineage analysis, for the translational research characterization of somatic hypermutation in CLL and other B cell neoplasms, including efforts to understand the potential prognostic value of isotype.