Preferential MGMT hypermethylation in SDH-deficient wild-type GIST

AIMS: Wild-type gastrointestinal stromal tumours (wtGIST) are frequently caused by inherited pathogenic variants, or somatic alterations in the succinate dehydrogenase subunit genes (SDHx). Succinate dehydrogenase is a key enzyme in the citric acid cycle. SDH deficiency caused by SDHx inactivation leads to an accumulation of succinate, which inhibits DNA and histone demethylase enzymes, resulting in global hypermethylation. Epigenetic silencing of the DNA repair gene MGMT has proven utility as a positive predictor of the therapeutic efficacy of the alklyating drug temozolomide (TMZ) in tumours such as glioblastoma multiforme. The aim of this study was to examine MGMT promoter methylation status in a large cohort of GIST. METHODS: MGMT methylation analysis was performed on 65 tumour samples including 47 wtGIST (33 SDH-deficient wtGIST and 11 SDH preserved wtGIST) and 21 tyrosine kinase (TK) mutant GIST. RESULTS: MGMT promoter methylation was detected in 8 cases of SDH-deficient (dSDH) GIST but in none of the 14 SDH preserved wild-type GIST or 21 TK mutant GIST samples analysed. Mean MGMT methylation was significantly higher (p 0.0449) and MGMT expression significantly lower (p<0.0001) in dSDH wtGIST compared with TK mutant or SDH preserved GIST. No correlation was identified between SDHx subunit gene mutations or SDHC epimutation status and mean MGMT methylation levels. CONCLUSION: MGMT promoter hypermethylation occurs exclusively in a subset of dSDH wtGIST. Data from this study support testing of tumour MGMT promoter methylation in patients with dSDH wtGIST to identify those patients who may benefit from most from TMZ therapy

The role of [68 Ga]Ga-DOTATATE PET/CT in wild-type KIT/PDGFRA gastrointestinal stromal tumours (GIST).

BACKGROUND: [68 Ga]Ga-DOTATATE PET/CT is now recognised as the most sensitive functional imaging modality for the diagnosis of well-differentiated neuroendocrine tumours (NET) and can inform treatment with peptide receptor radionuclide therapy with [177Lu]Lu-DOTATATE. However, somatostatin receptor (SSTR) expression is not unique to NET, and therefore, [68 Ga]Ga-DOTATATE PET/CT may have oncological application in other tumours. Molecular profiling of gastrointestinal stromal tumours that lack activating somatic mutations in KIT or PDGFRA or so-called 'wild-type' GIST (wtGIST) has demonstrated that wtGIST and NET have overlapping molecular features and has encouraged exploration of shared therapeutic targets, due to a lack of effective therapies currently available for metastatic wtGIST. AIMS: To investigate (i) the diagnostic role of [68 Ga]Ga-DOTATATE PET/CT; and, (ii) to investigate the potential of this imaging modality to guide treatment with [177Lu]Lu-DOTATATE in patients with wtGIST. METHODS: [68 Ga]Ga-DOTATATE PET/CT was performed on 11 patients with confirmed or metastatic wtGIST and one patient with a history of wtGIST and a mediastinal mass suspicious for metastatic wtGIST, who was subsequently diagnosed with a metachronous mediastinal paraganglioma. Tumour expression of somatostatin receptor subtype 2 (SSTR2) using immunohistochemistry was performed on 54 tumour samples including samples from 8/12 (66.6%) patients who took part in the imaging study and 46 tumour samples from individuals not included in the imaging study. RESULTS: [68 Ga]Ga-DOTATATE PET/CT imaging was negative, demonstrating that liver metastases had lower uptake than background liver for nine cases (9/12 cases, 75%) and heterogeneous uptake of somatostatin tracer was noted for two cases (16.6%) of wtGIST. However, [68 Ga]Ga-DOTATATE PET/CT demonstrated intense tracer uptake in a synchronous paraganglioma in one case and a metachronous paraganglioma in another case with wtGIST. CONCLUSIONS: Our data suggest that SSTR2 is not a diagnostic or therapeutic target in wtGIST. [68 Ga]Ga-DOTATATE PET/CT may have specific diagnostic utility in differentiating wtGIST from other primary tumours such as paraganglioma in patients with sporadic and hereditary forms of wtGIST

Translating In Vivo Metabolomic Analysis of Succinate Dehydrogenase–Deficient Tumors Into Clinical Utility

Purpose Mutations in the mitochondrial enzyme succinate dehydrogenase (SDH) subunit genes are associated with a wide spectrum of tumors, including pheochromocytomas and paragangliomas, GI stromal tumors, renal cell carcinomas, and pituitary adenomas. SDH-related tumorigenesis is believed to be secondary to accumulation of the oncometabolite succinate. Our aim was to investigate the potential clinical applications of proton-1 magnetic resonance spectroscopy (1H-MRS) in a range of suspected SDH-related tumors. Patients and Methods Fifteen patients were recruited to this study. Respiratory-gated single-voxel 1H-MRS was performed at 3T to quantify the content of succinate at 2.4 ppm and choline at 3.22 ppm. Results A succinate peak was seen in six patients, all of whom had germ line SDHx mutations or loss of SDHB by immunohistochemistry. Succinate peaks were also detected in two patients with metastatic wild-type GI stromal tumors and no detectable germ line SDHx mutations but with somatic epimutations in SDHC. Three patients without tumor succinate peaks retained SDHB expression, consistent with SDH functionality. In six patients with borderline or absent peaks, technical difficulties such as motion artifact rendered 1H-MRS difficult to interpret. Sequential imaging in a patient with a metastatic abdominal paraganglioma demonstrated loss of the succinate peak after four cycles of [177Lu]DOTATATE, with a corresponding biochemical response in normetanephrine. Conclusion This study has demonstrated the translation into clinical practice of in vivo metabolomic analysis using 1H-MRS in patients with SDH-deficient tumors. Potential applications include noninvasive diagnosis and disease stratification, as well as monitoring of tumor response to targeted treatments. </jats:sec

Fixation and Spread of Somatic Mutations in Adult Human Colonic Epithelium.

We investigated the means and timing by which mutations become fixed in the human colonic epithelium by visualizing somatic clones and mathematical inference. Fixation requires two sequential steps. First, one of approximately seven active stem cells residing within each colonic crypt has to be mutated. Second, the mutated stem cell has to replace neighbors to populate the entire crypt in a process that takes several years. Subsequent clonal expansion due to crypt fission is infrequent for neutral mutations (around 0.7% of all crypts undergo fission in a single year). Pro-oncogenic mutations subvert both stem cell replacement to accelerate fixation and clonal expansion by crypt fission to achieve high mutant allele frequencies with age. The benchmarking of these behaviors allows the advantage associated with different gene-specific mutations to be compared irrespective of the cellular mechanisms by which they are conferred

5-hydroxymethylcytosine marks promoters in colon that resist DNA hypermethylation in cancer

The authors would like to acknowledge the support of The University of Cambridge, Cancer Research UK (CRUK SEB-Institute Group Award A ref10182; CRUK Senior fellowship C10112/A11388 to AEKI) and Hutchison Whampoa Limited. The Human Research Tissue Bank is supported by the NIHR Cambridge Biomedical Research Centre. FF is a ULB Professor funded by grants from the F.N.R.S. and Télévie, the IUAP P7/03 programme, the ARC (AUWB-2010-2015 ULB-No 7), the WB Health program and the Fonds Gaston Ithier. Data access: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=jpwzvsowiyuamzs&acc=GSE47592Background : The discovery of cytosine hydroxymethylation (5hmC) as a mechanism that potentially controls DNA methylation changes typical of neoplasia prompted us to investigate its behaviour in colon cancer. 5hmC is globally reduced in proliferating cells such as colon tumours and the gut crypt progenitors, from which tumours can arise. Results : Here, we show that colorectal tumours and cancer cells express Ten-Eleven-Translocation (TET) transcripts at levels similar to normal tissues. Genome-wide analyses show that promoters marked by 5hmC in normal tissue, and those identified as TET2 targets in colorectal cancer cells, are resistant to methylation gain in cancer. In vitro studies of TET2 in cancer cells confirm that these promoters are resistant to methylation gain independently of sustained TET2 expression. We also find that a considerable number of the methylation gain-resistant promoters marked by 5hmC in normal colon overlap with those that are marked with poised bivalent histone modifications in embryonic stem cells. Conclusions : Together our results indicate that promoters that acquire 5hmC upon normal colon differentiation are innately resistant to neoplastic hypermethylation by mechanisms that do not require high levels of 5hmC in tumours. Our study highlights the potential of cytosine modifications as biomarkers of cancerous cell proliferation.Publisher PDFPeer reviewe

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

The self-incompatibility (S) locus in Petunia hybrida is located on chromosome III in a region, syntenic for the Solanaceae

Seven independent transformants were previously shown to carry T-DNA inserts that are genetically linked to the self-incompatibility (S)-locus in Petunia hybrida. These T-DNAs provided reliable targets for fluorescent in situ hybridisation (FISH) localisation. The T-DNA loci were found to be distributed over the entire long arm of chromosome ill. The most tightly linked T-DNA loci were located in a subcentromeric position. Independent confirmation of this assignment of the S-locus to chromosome III was obtained by RFLP analysis of a cDNA marker linked to the S-locus of potato (CP100). Tight linkage was found between CP100 and a peroxidase isozyme locus previously mapped to chromosome ill. The co-localisation of CP100, peroxidase and the S-locus in petunia reveals synteny around the S-locus between four members of the Solanaceae family. The implications of a subcentromeric localisation of the S-locus are discussed in relation to chromosome fragments associated with self-compatible mutants