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

Michiel Van Den Brand ,Jeroen A.c.w Luijks ,Jos Rijntjes ,Anton W Langerak ,Kim C Heezen ,Hongxiang Liu ,Julia Steinhilber ,Frédéric Davi ,Michael Hummel ,Blanca Scheijen ,Michèle Y Van Der Klift ,Falko Fend ,Jakub Paweł Porc ,Leonie I Kroeze ,Hesham Eldaly ,Nikos Darzentas ,Tomáš Reigl ,Ioannis Anagnostopoulos ,Markus Möbs ,Patricia J.t.a Groenen

doi:10.1016/j.jmoldx.2021.06.005

Abstract

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.

Highlights

Hesham ElDaly,yyzz Hongxiang Liu,xx Ioannis Anagnostopoulos,y Michael Hummel,y Falko Fend,z Anton W
The comparison was restricted to analysis of those targets that were shared between GeneScan and next-generation sequencing (NGS)-based clonality
One of the ratios with the highest area under the curve was used to determine cutoff ratios to differentiate between a clonal and a non-clonal result. This resulted in sensitivities ranging from 94% to 97% and specificities ranging from 95% to 99% (Supplemental Table S4).[9]. This biological validation study of NGS-based Ig gene (IG) clonality testing shows that NGS-based IG clonality and conventional

Summary

Introduction

Hesham ElDaly,yyzz Hongxiang Liu,xx Ioannis Anagnostopoulos,y Michael Hummel,y Falko Fend,z Anton W. Langerak,x and Patricia J.T.A. Groenen* on behalf of the EuroClonality-NGS Working Group. From the Department of Pathology,* Radboud University Medical Center, Nijmegen, the Netherlands; the Institute of Pathology,y CharitéUniversitätsmedizin, Berlin, Germany; the Institute of Pathology and Neuropathology,z University Hospital Tübingen, Tübingen, Germany; the Laboratory. Medical Immunology,x Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, the Netherlands; the Molecular Medicine. University Hospitals National Health Service Trust, Coventry, United Kingdom; the Clinical Pathology Department,zz Cairo University, Cairo, Egypt; and the Haematopathology and Oncology Diagnostics Service,xx Addenbrooke's Hospital, Cambridge University Hospitals National Health Service Foundation Trust, Cambridge, United Kingdom. Address correspondence to Michiel van den Brand, M.D., Ph.D., Department of Pathology, Radboud University Medical Center, Geert Grooteplein

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