A comprehensive characterisation of phaeochromocytoma and paraganglioma tumours through histone protein profiling, DNA methylation and transcriptomic analysis genome wide.

BACKGROUND: Phaeochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumours. Pathogenic variants have been identified in more than 15 susceptibility genes; associated tumours are grouped into three Clusters, reinforced by their transcriptional profiles. Cluster 1A PPGLs have pathogenic variants affecting enzymes of the tricarboxylic acid cycle, including succinate dehydrogenase. Within inherited PPGLs, these are the most common. PPGL tumours are known to undergo epigenetic reprograming, and here, we report on global histone post-translational modifications and DNA methylation levels, alongside clinical phenotypes. RESULTS: Out of the 25 histone post-translational modifications examined, Cluster 1A PPGLs were distinguished from other tumours by a decrease in hyper-acetylated peptides and an increase in H3K4me2. DNA methylation was compared between tumours from individuals who developed metastatic disease versus those that did not. The majority of differentially methylated sites identified tended to be completely methylated or unmethylated in non-metastatic tumours, with low inter-sample variance. Metastatic tumours by contrast consistently had an intermediate DNA methylation state, including the ephrin receptor EPHA4 and its ligand EFNA3. Gene expression analyses performed to identify genes involved in metastatic tumour behaviour pin-pointed a number of genes previously described as mis-regulated in Cluster 1A tumours, as well as highlighting the tumour suppressor RGS22 and the pituitary tumour-transforming gene PTTG1. CONCLUSIONS: Combined transcriptomic and DNA methylation analyses revealed aberrant pathways, including ones that could be implicated in metastatic phenotypes and, for the first time, we report a decrease in hyper-acetylated histone marks in Cluster 1 PPGLs.

SDHC phaeochromocytoma and paraganglioma: A UK-wide case series.

OBJECTIVE: Phaeochromocytomas and paragangliomas (PPGL) are rare, but strongly heritable tumours. Variants in succinate dehydrogenase (SDH) subunits are identified in approximately 25% of cases. However, clinical and genetic information of patients with SDHC variants are underreported. DESIGN: This retrospective case series collated data from 18 UK Genetics and Endocrinology departments. PATIENTS: Both asymptomatic and disease-affected patients with confirmed SDHC germline variants are included. MEASUREMENTS: Clinical data including tumour type and location, surveillance outcomes and interventions, SDHC genetic variant assessment, interpretation, and tumour risk calculation. RESULTS: We report 91 SDHC cases, 46 probands and 45 non-probands. Fifty-one cases were disease-affected. Median age at genetic diagnosis was 43 years (range: 11-79). Twenty-four SDHC germline variants were identified including six novel variants. Head and neck paraganglioma (HNPGL, n = 30, 65.2%), extra-adrenal paraganglioma (EAPGL, n = 13, 28.2%) and phaeochromocytomas (PCC) (n = 3, 6.5%) were present. One case had multiple PPGLs. Malignant disease was reported in 19.6% (9/46). Eight cases had non-PPGL SDHC-associated tumours, six gastrointestinal stromal tumours (GIST) and two renal cell cancers (RCC). Cumulative tumour risk (95% CI) at age 60 years was 0.94 (CI: 0.79-0.99) in probands, and 0.16 (CI: 0-0.31) in non-probands, respectively. CONCLUSIONS: This study describes the largest cohort of 91 SDHC patients worldwide. We confirm disease-affected SDHC variant cases develop isolated HNPGL disease in nearly 2/3 of patients, EAPGL and PCC in 1/3, with an increased risk of GIST and RCC. One fifth developed malignant disease, requiring comprehensive lifelong tumour screening and surveillance.

Case report of a man with multiple paragangliomas and pathogenic germline variants in both NF1 and SDHD.

We report a novel case of multiple paragangliomas in a patient who was identified with pathogenic variants in both NF1 and SDHD genes. The proband is a man with known familial NF1 disease, diagnosed clinically in childhood. Multiple head and neck paragangliomas (HNPGL) were found during investigations for acute left sided neurological symptoms, in the region of his known plexiform neurofibroma. He was referred for genetic counselling. He underwent surgery to remove a left carotid body tumor (CBT). A pheochromocytoma and paraganglioma gene panel was tested. Blood and HNPGL tumor DNA were analyzed by whole exome sequencing. In addition to the NF1 truncating variant c.5107delA, p.(Ser1703AlafsTer7), the SDHD truncating pathogenic variant c.3G > A, p.(Met1?) was found. Tumor sequencing showed no LOH of SDHD or NF1, but monoallelic loss of 11p15 and 11q12.2-q12.3 was observed. Co-occurrence of pathogenic variants in multiple cancer susceptibility genes is rare but possible, identified by the increased use of panel testing. This is the first description of a patient presenting with NF1 and SDHD dual pathology, with HNPGL development due to SDHD. This case illustrates the central role of genetic sequencing in PPGLs and the strong genotype-phenotype correlations of different genes.

Patients with triple-negative, JAK2V617F- and CALR-mutated essential thrombocythemia share a unique gene expression signature.

Approximately 10% to 15% of patients with essential thrombocythemia (ET) lack the common driver mutations, so-called "triple-negative" (TN) disease. We undertook a systematic approach to investigate for somatic mutations and delineate gene expression signatures in 46 TN patients and compared the results to those with known driver mutations and healthy volunteers. Deep, error-corrected, next-generation sequencing of peripheral blood mononuclear cells using the HaloPlexHS platform and whole-exome sequencing was performed. Using this platform, 10 (22%) of 46 patients had detectable mutations (MPL, n = 6; JAK2V617F, n = 4) with 3 of 10 cases harboring germline MPL mutations. RNA-sequencing and DNA methylation analysis were also performed by using peripheral blood mononuclear cells. Pathway analysis comparing healthy volunteers and ET patients (regardless of mutational status) identified significant enrichment for genes in the tumor necrosis factor, NFκB, and MAPK pathways and upregulation of platelet proliferative drivers such as ITGA2B and ITGB3. Correlation with DNA methylation showed a consistent pattern of hypomethylation at upregulated gene promoters. Interrogation of these promoter regions highlighted enrichment of transcriptional regulators, which were significantly upregulated in patients with ET regardless of mutation status, including CEBPß and NFκB. For "true" TN ET, patterns of gene expression and DNA methylation were similar to those in ET patients with known driver mutations. These observations suggest that the resultant ET phenotype may, at least in part and regardless of mutation type, be driven by transcriptional misregulation and may propagate downstream via the MAPK, tumor necrosis factor, and NFκB pathways with resultant JAK-STAT activation. These findings identify potential novel mechanisms of disease initiation that require further evaluation.