Introduction: Gastrointestinal stromal tumours (GIST) are the most commonly occurring sarcomas. 15% of GIST harbour germline variants or epimutations involving genes encoding for the SDH protein complex, NF1 and other rare mutations. GISTs showing an SDH deficiency (dSDH) account for up to 7.5% of gastric GISTs and mainly affect individuals below the age of 40. Up to 20% of these dSDH GIST are related to silencing of the SDHC gene by promoter hypermethylation (SDHCmet). Diagnosis of tumours with SDHCmet has proven difficult as often only sparse FFPE material is available for testing. Here we describe our clinic experience on SDHCmet analysis in 15 patients who attended the PAWS-GIST clinic. We also propose a testing algorithm for dSDHB GIST.

Method: Fifteen patients (7f / 8m) were enrolled in a study for SDHCmet analysis. Germline analysis for SDHA-D, KIT, PDGFRA and NF1 was performed on all patients. SDHB expression was assessed by immunohistochemistry. Tumours and adjacent normal tissue were investigated for SDHCmet by pyrosequencing and for SDHC expression by qRT-PCR. Multiple ligation dependent probe analysis (MLPA) was performed on all cases with SDHC promoter hypermethylation.

Results: Ten patients showed to be dSDHB (8 f / 2m). Of these, seven had germline variants in one of the SDHx genes. Six (all f) showed a median SDHCmet of 51% in their tumours compared to less than 3% in the others. One pathogenic and one benign SDHC germline variant was detected in two of the SDHCmet tumours, whereas no pathogenic variant or copy number variation was detected by MLPA in any of the other four. SDHCmet was co-occurring with lowered SDHC mRNA levels. SDHCmet was associated with female gender (6/6), metastatic disease (5/6), young age at diagnosis (average age 24 years, range 15-27) and multiple primary tumours (3/6). No raised levels of germline SDHC promoter methylation levels were detected in any patient.

Discussion: Our work shows how SDHC promoter methylation analysis can reliably be performed on FFPE material in dSDH GIST. Our data stands in line with published array and methylation sequencing data, however our method does not require fresh frozen material. Identification of dSDH GIST harbouring epigenetic SDHC silencing will help guide genetic testing algorithms and better categorise these tumours. It may also lead the way for therapeutic options with epigenetic drugs. Clinical trials with DNA methyltransferase inhibitors are in progress (ClinicalTrials.gov: NCT03165721) to test the effect of reversing DNA hypermethylation in patients with SDH deficient cancers.