SDHC epi-mutation testing in gastrointestinal stromal tumours and related tumours in clinical practice

Abstract

The enzyme succinate dehydrogenase (SDH) functions in the citric acid cycle and loss of function predisposes to the development of phaeochromocytoma/paraganglioma (PPGL), wild type gastrointestinal stromal tumour (wtGIST) and renal cell carcinoma. SDH-deficient tumours are most commonly associated with a germline SDH subunit gene (SDHA/B/C/D) mutation but can also be associated with epigenetic silencing of the SDHC gene. However, clinical diagnostic testing for an SDHC epimutation is not widely available. The objective of this study was to investigate the indications for and the optimum diagnostic pathways for the detection of SDHC epimutations in clinical practice. SDHC promoter methylation analysis of 32 paraffin embedded tumours (including 15 GIST and 17 PPGL) was performed using a pyrosequencing technique and correlated with SDHC gene expression. SDHC promoter methylation was identified in 6 (18.7%) tumours. All 6 SDHC epimutation cases presented with SDH deficient wtGIST and 3/6 cases had multiple primary tumours. No case of constitutional SDHC promoter hypermethylation was detected. Whole genome sequencing of germline DNA from three wtGIST cases with an SDHC epimutation, did not reveal any causative sequence anomalies. Herein, we recommend a diagnostic workflow for the detection of an SDHC epimutation in a service setting.

Figure 1

Figure (A) illustrates the distribution of methylation across the 12 individual CpG’s for the six cases demonstrated to have SDHC promoter methylation (epimutant cases), and the wt GIST, glioma and PPGL cases with no SDHC epimutation. Figure (B) demonstrates the methylation levels across the 12 individual CpG’s for the six epimutated cases (#001, #002, #003, #004, ##021, #022).

Figure 2

Figure (A) shows the difference in the mean % methylation of the SDHC promoter locus across 12 CpG’s in the tumour of the six hypermethylated cases and tumours of the non-epimutant cases and blood DNA and normal tissue of cases with and without an identified SDHC epimutation. Figure (B) shows reduced SDHC expression in the tumour versus normal tissue of 5/6 cases with an identified SDHC epimutation.

Figure 3

Figure (A) and (B) shows loss of SDHB protein expression on immunohistochemical analysis of the primary wtGIST tumour in case #001 and #003 respectively. In Figure (B) SDHB expression is preserved in adjacent normal tissue as highlighted by the red arrow. Figure (C) shows a pulmonary chondroma in case #021 as demonstrated by the white arrow and Figure (D) demonstrates the histology of a pulmonary chondroma from case #004, with evidence of normal collapsed lung tissue illustrated by the black arrow and chondrocytes in the tumor marked by the red arrow.

Figure 4

Illustrates a proposed work flow for the investigation of SDHC promoter methylation in a clinical setting for (A) PPGL and (B) wtGIST (defined as a GIST with no identified somatic mutation in KIT, PDGFRA OR BRAF) *Next generation sequencing panel for PPGL including the genes; SDHA, SDHB, SDHC, SDHD, SDHAF2, FH, TMEM127, RET, VHL, MAX and including multiplex ligation dependent probe amplification for deletions and duplication. **Next generation sequencing panel for wtGIST including the genes; SDHA, SDHB, SDHC, SDHD, KIT, PDGFRA, NF1 and including multiplex ligation dependent probe amplification for deletions and duplication.