Comparative analysis of targeted next-generation sequencing panels for the detection of gene mutations in chronic lymphocytic leukemia: an ERIC multi-center study

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Authors	SUTTON L.A. LJUNGSTROM V. ENJUANES A. CORTESE D. SKAFTASON A. TAUSCH E. STAŇO KOZUBÍK Kateřina NADEU F. ARMAND M. MALČÍKOVÁ Jitka PANDZIC T. FORSTER J. DAVIS Z. OSCIER D. ROSSI D. GHIA P. STREFFORD J.C. POSPÍŠILOVÁ Šárka STILGENBAUER S. DAVI F. CAMPO E. STAMATOPOULOS K. ROSENQUIST R.
Year of publication	2021
Type	Article in Periodical
Magazine / Source	haematologica
MU Faculty or unit	Central European Institute of Technology
Citation
Web	https://haematologica.org/article/view/9711
Doi	http://dx.doi.org/10.3324/haematol.2019.234716
Keywords	CLINICAL IMPACTRECURRENT MUTATIONSCLONAL EVOLUTIONLABORATORY STANDARDSTP53NOTCH1SF3B1CLLBIRC3PROGRESSION
Description	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 94.299.8%. 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) 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 molecular identifiers may be necessary to reach a higher sensitivity and ensure consistent and accurate detection of low-frequency variants.
Related projects:	Alternative mechanisms of deregulation of the p53 pathway in chronic lymphocytic leukemia Comprehensive prognostic and predictive panel for chronic lymphocytic leukemia: a next-generation sequencing tool suitable for clinical practice and study of genetic architecture behind the disease progress