Next-Generation Sequencing-Based Clonality Assessment of Ig Gene Rearrangements A Multicenter Validation Study by EuroClonality-NGS

Ig gene (IG) donality analysis has an important role in the distinction of benign and malignant B-cell lymphoid proliferations and is mostly performed with the conventional EuroClonality/BIOMED-2 multiplex PCR protocol and GeneScan fragment size analysis. Recently, the EuroClonality-NGS Working Group developed a method for next-generation sequencing (NGS)-based IG donality analysis. Herein, we report the results of an international multicenter biological validation of this novel method compared with the gold standard EuroClonality/BIOMED-2 protocol, based on 209 specimens of reactive and neoplastic lymphoproliferations. NGS-based IG donality analysis showed a high interlaboratory concordance (99%) and high concordance with conventional donality analysis (98%) for the molecular conclusion. Detailed analysis of the individual IG heavy chain and kappa light chain targets showed that NGS-based donality analysis was more often able to detect a clonal rearrangement or yield an interpretable result. NGS-based and conventional donality analysis detected a done in 96% and 95% of B-cell neoplasms, respectively, and all but one of the reactive cases were scored polydonal. We conclude that NGS-based IG donality analysis performs comparable to conventional donality analysis. We provide critical parameters for interpretation and discuss a first step toward a quantitative scoring approach for NGS donality results. Considering the advantages of NGS-based donality analysis, including its high sensitivity and possibilities for accurate clonal comparison, this supports implementation in diagnostic practice.

Automated summary

NGS-based IG clonality analysis shows high interlaboratory concordance and concordance with conventional analysis, with a higher ability to detect clonal rearrangements. NGS and conventional analysis detect clonality in 96% and 95% of B-cell neoplasms, respectively, with the majority of reactive cases being polyclonal.