Abstract
Microscopic examination of myelodysplastic syndromes (MDS) and myelodysplastic-myeloproliferative neoplasms (MDS/MPN) may be challenging because morphological features can overlap with those of reactive states. Demonstration of clonal hematopoiesis provides a diagnostic clue and has become possible by comprehensive mutation profiling of a number of frequently mutated genes, some of them with large coding regions.To emphasize the potential benefit of NGS in hematopathology we present sequencing results from routinely processed formalin-fixed and paraffin-embedded (FFPE) bone marrow trephines (n = 192). A customized amplicon-based gene panel including 23 genes frequently mutated in myeloid neoplasms was established and implemented. Thereby, 629,691 reads per sample (range 179,847–1,460,412) and a mean coverage of 2,702 (range 707–6,327) could be obtained, which are sufficient for comprehensive mutational profiling. Seven samples failed in sequencing (3.6%). In 185 samples we found in total 269 pathogenic variants (mean 1.4 variants per patient, range 0-5), 125 Patients exhibit at least one pathogenic mutation (67.6%). Variants show allele frequencies ranging from 6.7% up to 95.7%. Most frequently mutated genes were TET2 (28.7%), SRSF2 (19.5%), ASXL1 (8.6%) and U2AF1 (8.1%). The mutation profiling increases the diagnostic precision and adds prognostic information.
Highlights
Targeted cancer therapies have been developed for many types of tumors and the necessity for precise analysis of a number of marker genes for every single patient will rise in the years [1,2,3]
In the field of hematopathology analyzing the mutational status of specific driver genes can be helpful to discriminate different disease subtypes
We systematically reviewed the scientific literature and validated quality parameters for NGS mutation profiling of formalin-fixed and paraffin-embedded (FFPE) samples in order to reduce the risk of false-positive variant reporting
Summary
Targeted cancer therapies have been developed for many types of tumors and the necessity for precise analysis of a number of marker genes for every single patient will rise in the years [1,2,3]. In the field of hematopathology analyzing the mutational status of specific driver genes can be helpful to discriminate different disease subtypes. This is especially important if morphological examination or clinical presentation remains inconclusive [4,5,6]. Myeloid neoplasms show a complex genetic basis, with 50-60 assumed driver genes for myelodysplastic syndromes alone [9]. Many important marker genes exhibit large coding sequences
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