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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.

SUBMITTER: Bernard V

PROVIDER: S-EPMC4014247 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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

Bernard Veronica V Gebauer Niklas N Dinh Thomas T Stegemann Judith J Feller Alfred C AC Merz Hartmut H

International journal of clinical and experimental pathology 20140315 4

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 inve ...[more]

PMID: 24817963

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Project description:BackgroundWe have developed a gene expression assay (Whole-Genome DASL), capable of generating whole-genome gene expression profiles from degraded samples such as formalin-fixed, paraffin-embedded (FFPE) specimens.Methodology/principal findingsWe demonstrated a similar level of sensitivity in gene detection between matched fresh-frozen (FF) and FFPE samples, with the number and overlap of probes detected in the FFPE samples being approximately 88% and 95% of that in the corresponding FF samples, respectively; 74% of the differentially expressed probes overlapped between the FF and FFPE pairs. The WG-DASL assay is also able to detect 1.3-1.5 and 1.5-2 -fold changes in intact and FFPE samples, respectively. The dynamic range for the assay is approximately 3 logs. Comparing the WG-DASL assay with an in vitro transcription-based labeling method yielded fold-change correlations of R(2) approximately 0.83, while fold-change comparisons with quantitative RT-PCR assays yielded R(2) approximately 0.86 and R(2) approximately 0.55 for intact and FFPE samples, respectively. Additionally, the WG-DASL assay yielded high self-correlations (R(2)>0.98) with low intact RNA inputs ranging from 1 ng to 100 ng; reproducible expression profiles were also obtained with 250 pg total RNA (R(2) approximately 0.92), with approximately 71% of the probes detected in 100 ng total RNA also detected at the 250 pg level. When FFPE samples were assayed, 1 ng total RNA yielded self-correlations of R(2) approximately 0.80, while still maintaining a correlation of R(2) approximately 0.75 with standard FFPE inputs (200 ng).Conclusions/significanceTaken together, these results show that WG-DASL assay provides a reliable platform for genome-wide expression profiling in archived materials. It also possesses utility within clinical settings where only limited quantities of samples may be available (e.g. microdissected material) or when minimally invasive procedures are performed (e.g. biopsied specimens).

Dataset Information

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

Publications

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

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