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Ferrata Storti Foundation
Haematologica 2021 Volume 106(3):682-691
Chronic Lymphocytic Leukemia
Comparative analysis of targeted next-generation sequencing panels for the detection of gene mutations in chronic lymphocytic leukemia: an ERIC multi-center study
Lesley-Ann Sutton,1 Viktor Ljungström,1,2 Anna Enjuanes,3 Diego Cortese,1 Aron Skaftason,1 Eugen Tausch,4 Katerina Stano Kozubik,5 Ferran Nadeu,3 Marine Armand,6 Jitka Malcikova,5 Tatjana Pandzic,2 Jade Forster,7
Zadie Davis,8 David Oscier,8 Davide Rossi,9 Paolo Ghia,10
Jonathan C. Strefford,7 Sarka Pospisilova,5 Stephan Stilgenbauer,4
Frederic Davi,6 Elias Campo,3 Kostas Stamatopoulos1,11 and Richard Rosenquist1,12 on behalf of the European Research Initiative on CLL (ERIC)
1Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden; 2Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden; 3Institut d’Investigacions Biomèdiques August Pi iSunyer (IDIBAPS), Barcelona, Spain; Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Madrid, Spain and Hospital Clínic of Barcelona, Universitat de Barcelona, Barcelona, Spain; 4Department of Internal Medicine III, Ulm University, Ulm, Germany; 5Center of Molecular Medicine, CEITEC - Central European Institute of Technology, Masaryk University, Brno, Czech Republic; 6AP-HP, Hopital Pitie-Salpetriere, Department of Hematology, Sorbonne Université, Paris, France; 7Cancer Sciences, Faculty of Medicine, University of Southampton, Southampton, UK; 8Department of Hematology, Royal Bournemouth Hospital, Bournemouth, UK; 9Hematology Department, Oncology Institute of Southern Switzerland and Institute of Oncology Research, Bellinzona, Switzerland; 10Division of Experimental Oncology, Università Vita-Salute San Raffaele and IRCCS San Raffaele Scientific Institute, Milan, Italy; 11Institute of Applied Biosciences, Center for Research and Technology, Thessaloniki, Greece and 12Clinical Genetics, Karolinska University Laboratory, Karolinska University Hospital, Stockholm, Sweden
ABSTRACT
Next-generation sequencing (NGS) has transitioned from research to clinical routine, yet the comparability of different technologies for mutation profiling remains an open question. We performed a European multicenter (n=6) evaluation of three amplicon-based NGS assays targeting 11 genes recurrently mutated in chronic lymphocytic leukemia. Each assay was assessed by two centers using 48 pre-characterized chronic lymphocytic leukemia samples; libraries were sequenced on the Illumina MiSeq instrument and bioinformatics analyses were centralized. Across all centers the median percentage of target reads ≥100x ranged from 94.2- 99.8%. In order to rule out assay-specific technical variability, we first assessed variant calling at the individual assay level i.e., pairwise analysis of variants detected amongst partner centers. After filtering for variants present in the paired normal sample and removal of PCR/sequencing artefacts, the panels achieved 96.2% (Multiplicom), 97.7% (TruSeq) and 90% (HaloPlex) concordance at a variant allele frequency (VAF) >0.5%. Reproducibility was assessed by looking at the inter-laboratory variation in detecting mutations and 107 of 115 (93% concordance) mutations were detected by all six cen- ters, while the remaining eight variants (7%) were undetected by a single center. Notably, 6 of 8 of these variants concerned minor subclonal muta- tions (VAF <5%). We sought to investigate low-frequency mutations further by using a high-sensitivity assay containing unique molecular identifiers, which confirmed the presence of several minor subclonal mutations. Thus, while amplicon-based approaches can be adopted for somatic mutation detection with VAF >5%, after rigorous validation, the use of unique molec- ular identifiers may be necessary to reach a higher sensitivity and ensure consistent and accurate detection of low-frequency variants.
Correspondence:
RICHARD ROSENQUIST
richard.rosenquist@ki.se
Received: February 11, 2020. Accepted: March 25, 2020. Pre-published: April 9, 2020.
https://doi.org/10.3324/haematol.2019.234716
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