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Comparative analysis of targeted next-generation sequencing panels for the detection of gene mutations in chronic lymphocytic leukemia: an ERIC multi-center study
LA. Sutton, V. Ljungström, A. Enjuanes, D. Cortese, A. Skaftason, E. Tausch, K. Stano Kozubik, F. Nadeu, M. Armand, J. Malcikova, T. Pandzic, J. Forster, Z. Davis, D. Oscier, D. Rossi, P. Ghia, JC. Strefford, S. Pospisilova, S. Stilgenbauer, F....
Jazyk angličtina Země Itálie
Typ dokumentu časopisecké články, multicentrická studie, práce podpořená grantem
Grantová podpora
C34999/A18087
Cancer Research UK - United Kingdom
Free Medical Journals od 1994
Freely Accessible Science Journals od 1994
PubMed Central od 2009
Europe PubMed Central od 2009
Open Access Digital Library od 1994-01-01
ROAD: Directory of Open Access Scholarly Resources od 1996
Odkazy
PubMed
32273480
DOI
10.3324/haematol.2019.234716
Knihovny.cz E-zdroje
- MeSH
- chronická lymfatická leukemie * diagnóza genetika MeSH
- lidé MeSH
- mutace MeSH
- reprodukovatelnost výsledků MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- multicentrická studie MeSH
- práce podpořená grantem MeSH
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 centers, while the remaining eight variants (7%) were undetected by a single center. Notably, 6 of 8 of these variants concerned minor subclonal mutations (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.
AP HP Hopital Pitie Salpetriere Department of Hematology Sorbonne Université Paris Franc
Cancer Sciences Faculty of Medicine University of Southampton Southampton UK
Clinical Genetics Karolinska University Laboratory Karolinska University Hospital Stockholm Swede
Department of Hematology Royal Bournemouth Hospital Bournemouth U
Department of Internal Medicine 3 Ulm University Ulm German
Department of Molecular Medicine and Surgery Karolinska Institutet Stockholm Swede
Institut d'Investigacions Biomèdiques August Pi iSunyer Barcelona Spain
Institute of Applied Biosciences Center for Research and Technology Thessaloniki Gree
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