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Haematologica. 2015 Mar;100(3):370-6. doi: 10.3324/haematol.2014.109777. Epub 2014 Dec 5.

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

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Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Sweden.
Central European Institute of Technology, Masaryk University, Brno, Czech Republic.
Department of Immunology, Erasmus MC, University Medical Center Rotterdam, The Netherlands.
First Department of Propaedeutic Medicine, School of Medicine, University of Athens, Greece.
Laboratory of Hematology and Universite Pierre et Marie Curie, Hopital Pitie-Salpetriere, Paris, France.
Hematology Department, Nikea General Hospital, Pireaus, Greece.
Università Vita-Salute San Raffaele, Milan, Italy Division of Molecular Oncology and Department of Onco-Hematology, IRCCS San Raffaele Scientific Institute, Milan, Italy.
Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Sweden Institute of Applied Biosciences, CERTH, Thessaloniki, Greece Hematology Department and HCT Unit, G. Papanicolaou Hospital, Thessaloniki, Greece.
Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Sweden


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 HaloPlex targeted enrichment technology and designed a panel including nine genes: ATM, BIRC3, MYD88, NOTCH1, SF3B1 and TP53, which have been linked to the prognosis of chronic lymphocytic leukemia, and KLHL6, POT1 and XPO1, which are less characterized but were found to be 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; concordance was found for 77/82 (94%) mutations. In summary, this study demonstrates that targeted next-generation sequencing is 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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