sábado, 15 de septiembre de 2018

Comprehensive genetic diagnosis of acute myeloid leukemia by next generation sequencing. - PubMed - NCBI

2018 Sep 6. pii: haematol.2018.194258. doi: 10.3324/haematol.2018.194258. [Epub ahead of print]

Comprehensive genetic diagnosis of acute myeloid leukemia by next generation sequencing.

Mack EKM1, Marquardt A1, Langer D1, Ross P1, Ultsch A2, Kiehl MG3, Mack HID4, Haferlach T5, Neubauer A1, Brendel C6.

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Abstract

Differential induction therapy of all subtypes of Acute Myeloid Leukemia other than Acute Promyelocytic Leukemia is impeded by the long time required to complete complex and diverse cytogenetic and molecular genetic analyses for risk stratification or targeted treatment decisions. Here, we describe a reliable, rapid and sensitive diagnostic approach that combines karyotyping and mutational screening in a single integrated next generation sequencing assay. Numerical karyotyping was performed by low coverage whole genome sequencing followed by copy number variation analysis using a novel algorithm based on in silico-generated reference karyotypes. Translocations and DNA variants were examined by targeted resequencing of fusion transcripts and mutational hotspot regions using commercially available kits and analysis pipelines. For the identification of FLT3 internal tandem duplications and KMT2A partial tandem duplications, we adapted previously described tools. In a validation cohort including 22 primary patient samples, 9/9 numerically normal karyotypes were classified correctly and 30/31 (97%) Copy number variations reported by classical cytogenetics and FISH analysis were uncovered by our next generation sequencing-karyotyping approach. Predesigned fusion and mutation panels were validated exemplarily on leukemia cell lines and a subset of patient samples and identified all expected genomic alterations. Finally, blinded analysis of eight additional patient samples using our comprehensive assay accurately reproduced reference results. Therefore, calculated karyotyping by low coverage whole genome sequencing allows for fast and reliable detection of numerical chromosomal changes and, in combination with panel-based fusion- and mutation screening, will greatly facilitate implementation of subtype-specific induction therapies in Acute Myeloid Leukemia.