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Article

A Novel Single-Tube Next Generation Sequencing Assay for B-Cell Receptor Clonality Testing

by
Landon Pastushok
1,†,
Shrutii Sarda
2,†,
Karen Mochoruk
1,
Wayne Hill
1,
Loni T. Pickle
3,
Michelle Toro
3,
Carolina Gonzalez
1,
Stephanie Ostresh
3,
Timothy J. Looney
2,
Chenchen Yang
2,
Julie Stakiw
4,
Mark J. Bosch
4,
Hadi Goubran
4,
C. Ronald Geyer
1,
Geoffrey M. Lowman
3 and
John F. DeCoteau
1,*
1
Advanced Diagnostics Research Laboratory, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5A2, Canada
2
Clinical Sequencing Division, Genetic Sciences Group, Thermo Fisher Scientific, South San Francisco, CA 94080, USA
3
Clinical Sequencing Division, Genetic Sciences Group, Thermo Fisher Scientific, Carlsbad, CA 92008, USA
4
Saskatoon Cancer Centre, Saskatoon, SK S7N 4H4, Canada
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
J. Mol. Pathol. 2024, 5(1), 45-65; https://doi.org/10.3390/jmp5010004
Submission received: 11 October 2023 / Revised: 2 December 2023 / Accepted: 29 January 2024 / Published: 2 February 2024
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Abstract

B-cell neoplasms possess clonal B-cell receptor rearrangements (BCR clonotype lineages) that can be identified by sequencing the B-cell repertoire for use in diagnostics, risk stratification, and high-sensitivity monitoring. BCR somatic hypermutation (SHM) can result in clonality detection failure from point mutations in PCR primer binding regions, often necessitating splitting samples into multiple reactions which increases test costs, turnaround times, and sample requirements. We evaluated the Oncomine BCR Pan-Clonality Assay, a novel single-tube PCR reaction that simultaneously amplifies all BCR loci for next-generation DNA sequencing, using neoplastic B-cell lines and clinical research samples from multiple myeloma (MM) patients, a plasma cell neoplasm associated with high SHM levels. The assay showed a linear detection range down to 1 ng of clonal DNA input, sensitivity to 10−6 in a polyclonal background, and high reproducibility. Clonotype lineages were identified in 42/45 (93%) MM samples. Ion Reporter software packaged with the assay permitted straightforward identification of MM subgroups. As expected, SHM was identified in 94% of MM cases, but several unexpected subgroups were identified including biased IGHV3-11 or IGHV4-34 usage in 20% of MM samples, and two cases with very low levels of SHM. Evidence of intraclonal diversity/ongoing SHM was identified in 18% of samples, suggesting a possible germinal center origin for some MM cases. The single-tube Oncomine BCR Pan-Clonality assay efficiently detects BCR clonotype lineages at rates comparable to existing multiple reaction assays and permits their characterization for cell of origin studies and lymphoma classification.
Keywords: B-cell neoplasia; multiple myeloma; B-cell receptor rearrangements; clonotypes; next generation sequencing; single-tube assay B-cell neoplasia; multiple myeloma; B-cell receptor rearrangements; clonotypes; next generation sequencing; single-tube assay

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MDPI and ACS Style

Pastushok, L.; Sarda, S.; Mochoruk, K.; Hill, W.; Pickle, L.T.; Toro, M.; Gonzalez, C.; Ostresh, S.; Looney, T.J.; Yang, C.; et al. A Novel Single-Tube Next Generation Sequencing Assay for B-Cell Receptor Clonality Testing. J. Mol. Pathol. 2024, 5, 45-65. https://doi.org/10.3390/jmp5010004

AMA Style

Pastushok L, Sarda S, Mochoruk K, Hill W, Pickle LT, Toro M, Gonzalez C, Ostresh S, Looney TJ, Yang C, et al. A Novel Single-Tube Next Generation Sequencing Assay for B-Cell Receptor Clonality Testing. Journal of Molecular Pathology. 2024; 5(1):45-65. https://doi.org/10.3390/jmp5010004

Chicago/Turabian Style

Pastushok, Landon, Shrutii Sarda, Karen Mochoruk, Wayne Hill, Loni T. Pickle, Michelle Toro, Carolina Gonzalez, Stephanie Ostresh, Timothy J. Looney, Chenchen Yang, and et al. 2024. "A Novel Single-Tube Next Generation Sequencing Assay for B-Cell Receptor Clonality Testing" Journal of Molecular Pathology 5, no. 1: 45-65. https://doi.org/10.3390/jmp5010004

APA Style

Pastushok, L., Sarda, S., Mochoruk, K., Hill, W., Pickle, L. T., Toro, M., Gonzalez, C., Ostresh, S., Looney, T. J., Yang, C., Stakiw, J., Bosch, M. J., Goubran, H., Geyer, C. R., Lowman, G. M., & DeCoteau, J. F. (2024). A Novel Single-Tube Next Generation Sequencing Assay for B-Cell Receptor Clonality Testing. Journal of Molecular Pathology, 5(1), 45-65. https://doi.org/10.3390/jmp5010004

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