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

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.

Not Only Mutations Matter: Molecular Picture of Acute Myeloid Leukemia Emerging from Transcriptome Studies

“…Other integrated diagnostic platforms are being developed and validated, e.g., rapid and sensitive NGS-based assay combining karyotyping and mutational screening of AML [219]. Here, three NGS libraries are generated: two DNA-based libraries—for whole genome sequencing and selected variant identification—and one RNA-based library for fusion transcript detection.…”

Myeloid malignancies with 5q and 7q deletions are associated with extreme genomic complexity, biallelic TP53 variants, and very poor prognosis

“…In summary, we describe the use of genome-wide NGS in the characterization of genomic complexity in AML, with the potential to reframe our understanding of complex genomic events. To our knowledge, very few studies have specifically evaluated the structural complexity incorporating both CNVs and SVs of AML genomes by NGS [13][14][15] . Here we show that myeloid malignancies with deletions of 5q and 7q are associated with additional complex genomic findings not appreciated by conventional chromosome studies including increased copy number burden, chromothripsis, chromoplexy, progressive genomic complexity, and very poor overall survival.…”

Molecular methods for measurable residual disease in acute myeloid leukemia: where are we and where are we going?

“…The finding of an unrelated AML clone may have implications for clinical trial eligibility, and with the emergence of more tailored AML therapies, repeated genetic characterization of AML at follow-up and relapse will continue to increase in importance [81]. Novel NGS strategies are also amenable to the identification of recurrent fusions and other large gene aberrations, such as tandem duplications, possibly allowing for NGS assessment of MRD to encompass biomarkers that have been traditionally evaluated by RT-qPCR [93,94]. With continued technical advances, NGS platforms may allow molecular MRD testing to become closer to a one-size-fits-all approach, analogous to the current situation with MFC, rather than the more limited, individually tailored approach that is currently necessary and that is more likely to engender test ordering error and misinterpretation.…”