Targeted next-generation sequencing in chronic lymphocytic leukemia: a high-throughput yet tailored approach will facilitate implementation within a clinical setting
| Authors | |
|---|---|
| Year of publication | 2015 |
| Type | Article in Periodical |
| Magazine / Source | Haematologica |
| MU Faculty or unit | |
| Citation | |
| Web | http://www.haematologica.org/content/100/3/370.full-text.pdf+html |
| Doi | http://dx.doi.org/10.3324/haematol.2014.109777 |
| Field | Oncology and hematology |
| Keywords | Chronic Lymphocytic Leukemia; Cytogenetics and Molecular Genetics; Next-generation sequencing; Prognosis; Targeted resequencing |
| Attached files | |
| Description | 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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