Perspective on how to approach molecular diagnostics in acute myeloid leukemia and myelodysplastic syndromes in the era of next-generation sequencing

Abstract

Molecular mutation information became essential for biological subclassification, risk stratification and therapeutic decisions in patients with acute myeloid leukemia (AML). In myelodysplastic syndromes (MDS), a broad spectrum of molecular biomarkers such as the spliceosome mutations has been identified in recent years. The currently established combination of various polymerase chain reaction (PCR) methods with capillary Sanger sequencing for mutation analysis in AML is time-consuming and labor-intensive. The constantly increasing spectrum of molecular mutations is a tremendous challenge for hematological laboratories. The introduction of high-throughput sequencing technology, which allows the massive parallel analysis of hundreds of thousands of alleles in the shortest time, provides new options for molecular mutation analyses and for follow-up diagnostics in myeloid neoplasms. In contrast to whole-genome or exome analyses, amplicon deep-sequencing focuses on distinct genomic loci and their mutation patterns and enables a comprehensive biomarker analysis in a multitude of patients per analysis. This review summarizes thus far established common molecular diagnostic strategies and intends to outline the perspective of distinct novel amplicon deep-sequencing panels for patients with AML and MDS. It is foreseeable that clearly defined algorithms for molecular investigations will revolutionize diagnosis in patients with AML and MDS in the near future.

Validation and Implementation of a Custom Next-Generation Sequencing Clinical Assay for Hematologic Malignancies

“…2e10 Given the potential clinical implications of these mutations and diagnostic difficulties that are often presented by patients with peripheral blood cytopenias or cytoses, we established a custom NGS assay to evaluate patients with known or suspected hematologic malignancies, principally acute myeloid leukemia, myelodysplasia, and myeloproliferative neoplasms. Other strategies for integrating new molecular testing approaches into the workup of myeloid neoplasms were proposed or implemented, 11 including targeted NGS panels. 12e14 Our goal was to be comprehensive, covering all of the recurrently mutated genes, and to use a platform with the potential of a turnaround time of less than a week.…”

Diagnosing and following adult patients with acute myeloid leukaemia in the genomic age

“…Exome sequencing, moreover, does not include characterization of promoters, enhancers and splicing modifiers outside these regions. Targeted sequencing, in turn, provides an option for deep coverage and can be employed in MRD quantification during follow up of AML (Patel et al , ; Kohlmann et al , ). Expert laboratories can manage more than 100 samples per week running approximately 40 targeted amplicons in a highly centralized setting (Alexander Kohlmann, MLL Munich Leukaemia Laboratory, Munich, Germany, personal communication).…”

Mutational profiling in patients with MDS: Ready for every-day use in the clinic?

“…As amplicon deep-sequencing allows combining the relevant gene targets for MDS in one analysis [42] and as a comprehensive molecular work-up requires less than five working days, NGS is already now ready to be integrated into the diagnostic management of these patients (Fig. 1).…”