Amplicon-based next-generation sequencing (NGS) of immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements for clonality assessment, marker identification and quantification of minimal residual disease (MRD) in lymphoid neoplasms has been the focus of intense research, development and application. However, standardization and validation in a scientifically controlled multicentre setting is still lacking. Therefore, IG/TR assay development and design, including bioinformatics, was performed within the EuroClonality-NGS working group and validated for MRD marker identification in acute lymphoblastic leukaemia (ALL). Five EuroMRD ALL reference laboratories performed IG/TR NGS in 50 diagnostic ALL samples, and compared results with those generated through routine IG/TR Sanger sequencing. A central polytarget quality control (cPT-QC) was used to monitor primer performance, and a central in-tube quality control (cIT-QC) was spiked into each sample as a library-specific quality control and calibrator. NGS identified 259 (average 5.2/sample, range 0-14) clonal sequences vs. Sanger-sequencing 248 (average 5.0/sample, range 0-14). NGS primers covered possible IG/TR rearrangement types more completely compared with local multiplex PCR sets and enabled sequencing of bi-allelic rearrangements and weak PCR products. The cPT-QC showed high reproducibility across all laboratories. These validated and reproducible quality-controlled EuroClonality-NGS assays can be used for standardized NGS-based identification of IG/TR markers in lymphoid malignancies.

Bristol Genetics Laboratory Southmead Hospital Bristol UK

Central European Institute of Technology Masaryk University Brno Czech Republic

Centre for Cancer Research and Cell Biology Queen's University Belfast Belfast UK

Centro Ricerca Tettamanti University of Milano Bicocca Monza Italy

CLIP Childhood Leukaemia Investigation Prague Department of Paediatric Haematology and Oncology 2nd Faculty of Medicine Charles University University Hospital Motol Prague Czech Republic

CNRS CRIStAL Université Lille Inria Lille France

Department of Hematology APHP Necker Enfants Malades and Paris Descartes University Paris France

Department of Hematology Hopital Pitié Salpêtrière Paris France

Department of Hematology University Hospital Schleswig Holstein Kiel Germany

Department of Immunohematology and Blood Transfusion Leiden University Medical Center Leiden The Netherlands

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

Department of Paediatric Haematology Great Ormond Street Hospital London UK

Department of Pathology Radboud University Medical Center Nijmegen The Netherlands

Department of Pediatric Haematology Bristol Royal Hospital for Children Bristol UK

Hospital Universitario de Salamanca IBSAL Salamanca Spain

Insititute of Pathology Charité Universitätsmedizin Berlin Berlin Germany

Institute of Applied Biosciences Thessaloniki Greece

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