Applicability of next-generation sequencing to decalcified formalin-fixed and paraffin-embedded chronic...

Decalcified formalin-fixed and paraffin-embedded (dFFPE) bone marrow trephines remain the primary source of gDNA in hematopathological diagnostics. Here, we investigated the applicability of next-generation sequencing (NGS) to dFFPE samples. Chronic myelomonocytic leukaemia (CMML) is a haematopoietic stem cell malignancy delineated by genetic heterogeneity. Recently characteristic mutations have been identified for this entity in a distinct group of genes (TET2, CBL, KRAS). We comparatively investigated DNA extracted from fresh mononuclear cells as well as dFFPE samples from four CMML patients employing a commercially available primer set covering the above mentioned and well characterized mutational hotspots in CMML followed by an amplicon based next-generation deep-sequencing (NGS) approach. As we observed high quality run data as well as complete concordance between both sample types in all cases, we further validated the potential of NGS in hematopathology on a larger cohort of CMML patients (n=39), detecting sequence variations in 84.6% of patients. Sequence analysis revealed 92 variants, including five known polymorphisms, ten silent mutations, 36 missense mutations, 14 nonsense mutations, 24 frame shift mutations and three potential splice site mutations. Our findings ultimately demonstrate the applicability of NGS to dFFPE biopsy specimen in CMML and thus allowing the pathologist to evaluate prognostically relevant mutations at a high resolution and further contribute to risk stratification for the individual patient.