Applicability of next-generation sequencing to decalcified formalin-fixed and paraffin-embedded chronic myelomonocytic leukaemia samples

Abstract

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.

Keywords: Next-generation sequencing; chronic myelomonocytic leukaemia; decalcified; formalin-fixed; paraffin-embedded samples.

Figure 1

Combined coverage data of TET2, CBL and KRAS next-generation sequencing on dFFPE samples. Combined, forward and reverse reads for CBL and KRAS (A), overall forward and reverse TET2 reads per amplicon (B) and combined TET2 reads for all amplicons (C). Data reveal a consistent as well as high resolution coverage and summarized forward/reverse strand sequencing of TET2 illustrate a slight predominance of the A-sequencing-bead prepared forward strand.

Figure 2

TET2 Mutation types and distribution in CMML samples. Nine missense mutations within the two evolutionarily conserved regions (black boxes), 14 nonsense mutations (red boxes), 23 frameshift mutations (blue ovals) and three potential splice site mutations (orange ovals) in 27 of 39 patients (69.2%) at an average of 1.38 mutations per patient. Four of these mutations could be detected independently in multiple patients.