Ig gene (IG) clonality 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 clonality 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 clonality analysis showed a high interlaboratory concordance (99%) and high concordance with conventional clonality analysis (98%) for the molecular conclusion. Detailed analysis of the individual IG heavy chain and kappa light chain targets showed that NGS-based clonality analysis was more often able to detect a clonal rearrangement or yield an interpretable result. NGS-based and conventional clonality analysis detected a clone in 96% and 95% of B-cell neoplasms, respectively, and all but one of the reactive cases were scored polyclonal. We conclude that NGS-based IG clonality analysis performs comparable to conventional clonality analysis. We provide critical parameters for interpretation and discuss a first step toward a quantitative scoring approach for NGS clonality results. Considering the advantages of NGS-based clonality analysis, including its high sensitivity and possibilities for accurate clonal comparison, this supports implementation in diagnostic practice.

Department of Pathology Radboud University Medical Center Nijmegen the Netherlands

Haematopathology and Oncology Diagnostics Service Addenbrooke's Hospital Cambridge University Hospitals National Health Service Foundation Trust Cambridge United Kingdom

Hematology Department Hospital Pitié Salpêtrière and Sorbonne University Paris France

Histopathology Department Coventry University Hospitals National Health Service Trust Coventry United Kingdom; Clinical Pathology Department Cairo University Cairo Egypt

Institute of Pathology and Neuropathology University Hospital Tübingen Tübingen Germany

Institute of Pathology Charité Universitätsmedizin Berlin Germany

Laboratory Medical Immunology Department of Immunology Erasmus MC University Medical Center Rotterdam Rotterdam the Netherlands

Molecular Medicine Program Central European Institute of Technology Brno Czech Republic

Molecular Medicine Program Central European Institute of Technology Brno Czech Republic; Department of Hematology University Hospital Schleswig Holstein Kiel Germany

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