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

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.