Reproducibility of low-level residual myeloma immunoglobulin detection using ultra-deep sequencing

Oriane Cédile, Marcus Høy Hansen*, Sara Kamuk Dahlmann, Thomas Kielsgaard Kristensen, Niels Abildgaard, Charlotte Guldborg Nyvold

*Kontaktforfatter

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Abstract

Multiple myeloma, a mature B-cell neoplasm, is the second most common hematologic malignancy. Despite advancements in treatment, the disease remains incurable, with more than 100,000 annual deaths worldwide. As recommended by the International Myeloma Working Group, measurable residual disease (MRD) should be addressed at a 10−5 sensitivity level or beyond for practical purposes. Next-generation sequencing (NGS) has provided new opportunities with deep sequencing of clonal rearrangements of the immunoglobulin heavy chain (IGH) locus in B-cell malignancies. Although the ability to resolve one cancerous cell in a million other B cells is becoming attractive as a prognostic indicator in sustained patients who are MRD-negative, reaching consistent sensitivity levels is challenging because of sample stochasticity and the substantial amount of deoxyribonucleic acid (DNA) required for library preparation. Thus, in the presented study, we implemented ultra-deep sequencing of rearranged IGH to investigate the reproducibility and consistency aimed at the 10−5 sensitivity level. In this controlled setup, our data provided stable MRD detection of 1.2 clonal cells per 100,000 analyzed cells and longitudinal reproducibility. We also demonstrated a low false-negative rate using 4–5 replicates and 700–800 ng DNA per sequencing replicate. In conclusion, adding an internal control to the replicates enabled clonal cell normalization for MRD evaluation as a stable reference. These findings may guide MRD-level reporting and comparisons between laboratories.

OriginalsprogEngelsk
TidsskriftExperimental Hematology
Vol/bind119-120
Sider (fra-til)14-20
ISSN0301-472X
DOI
StatusUdgivet - 1. mar. 2023

Bibliografisk note

Funding Information:
This study received unrestricted funding from Celgene. The sponsor was not involved in the study design; collection, analysis, and interpretation of data; the writing of this article; or the decision to submit it for publication. We are thankful to Celgene (GRANT-DNK-134), the Danish Cancer Society (R246-A14603), and the Region of Southern Denmark (16/42083, Clinical Excellence grant to Odense Amyloidosis Center) for supporting this project financially.

Funding Information:
We thank Tobias Teken Christensen for excellent technical assistance and Birgitte Preiss for providing patient sample material for the project (both from the Department of Pathology, Odense University Hospital (OUH), Denmark). We are grateful to Kjell Titlestad (Department of Clinical Immunology, OUH, Denmark) for his help with donor inclusion, and we thank the donors for participating in the study. Also, thanks to Vickie Svane Kristensen (Research Unit for Haematology, OUH) for language assistance. This study received unrestricted funding from Celgene. The sponsor was not involved in the study design; collection, analysis, and interpretation of data; the writing of this article; or the decision to submit it for publication. We are thankful to Celgene (GRANT-DNK-134), the Danish Cancer Society (R246-A14603), and the Region of Southern Denmark (16/42083, Clinical Excellence grant to Odense Amyloidosis Center) for supporting this project financially. The project was approved by The Regional Committee on Health Research Ethics in Southern Denmark (S-20160069), and data were handled in accordance with requirements by the Danish Data Protection. We obtained a dispensation from written consent by the Regional ethical committee, as the material was collected as excess material from the diagnostic routine. Control donors provided informed consent. Contribution: OC, CN, and NA designed the study. OC and SD performed the experiments. OC, MH, and CN performed data analyses and drafted the manuscript. NA, SD, and TK discussed the results and commented on the manuscript. NA and CN obtained all funding for the project. All authors contributed to the final version and approved the manuscript.

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