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Targeted next-generation sequencing in chronic lymphocytic leukemia: a high-throughput yet tailored approach will facilitate implementation within a clinical setting

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SUTTON Lesley-Ann LJUNGSTRÖM Viktor MANSOURI Larry YOUNG Emma CORTESE Diego NAVRKALOVÁ Veronika MALČÍKOVÁ Jitka MUGGEN Alice F. TRBUŠEK Martin PANAGIOTIDIS Panagiotis DAVI Frederic BELESSI Chrysoula LANGERAK Anton W. GHIA Paolo POSPÍŠILOVÁ Šárka STAMATOPOULOS Kostas ROSENQUIST Richard

Rok publikování 2015
Druh Článek v odborném periodiku
Časopis / Zdroj Haematologica
Fakulta / Pracoviště MU

Středoevropský technologický institut

Citace
www http://www.haematologica.org/content/100/3/370.full-text.pdf+html
Doi http://dx.doi.org/10.3324/haematol.2014.109777
Obor Onkologie a hematologie
Klíčová slova Chronic Lymphocytic Leukemia; Cytogenetics and Molecular Genetics; Next-generation sequencing; Prognosis; Targeted resequencing
Přiložené soubory
Popis Next-generation sequencing has revealed novel recurrent mutations in chronic lymphocytic leukemia, particularly in patients with aggressive disease. Here, we explored targeted re-sequencing as a novel strategy to assess the mutation status of genes with prognostic potential. To this end, we utilized the HaloPlex targeted enrichment technology and designed a panel including nine genes: ATM, BIRC3, MYD88, NOTCH1, SF3B1 and TP53, that have been linked to chronic lymphocytic leukemia prognosis, and KLHL6, POT1 and XPO1, that are less characterized but were found recurrently mutated in various sequencing studies. A total of 188 chronic lymphocytic leukemia patients with poor-prognostic features (unmutated IGHV, n=137; IGHV3-21 subset #2, n=51) were sequenced on the HiSeq 2000 and data were analyzed using well-established bioinformatics tools. Using a conservative cutoff of 10% for the mutant allele, we found that 114/180 (63%) patients carried at least one mutation, with mutations in ATM, BIRC3, NOTCH1, SF3B1 and TP53 accounting for 149/177 (84%) of all mutations. We selected 155 mutations for Sanger validation (variant allele frequency, 10-99%) and 93% (144/155) of mutations were confirmed; notably, all 11 discordant variants had a variant allele frequency between 11-27%, hence at the detection limit of conventional Sanger sequencing. Technical precision was assessed by repeating the entire Haloplex procedure for 63 patients with 77/82 (94%) mutations demonstrating concordance. In summary, this study demonstrates targeted next-generation sequencing as an accurate and reproducible technique potentially suitable for routine screening, eventually as a stand-alone test without the need for confirmation by Sanger sequencing.
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