The activating TERT promoter mutation C228T is recurrent in subsets of adrenal tumors.

The telomerase reverse transcriptase gene (TERT) encodes the reverse transcriptase component of the telomerase complex, which is essential for telomere stabilization and cell immortalization. Recent studies have demonstrated a transcriptional activation role for the TERT promoter mutations C228T and C250T in many human cancers, as well as a role in aggressive disease with potential clinical applications. Although telomerase activation is known in adrenal tumors, the underlying mechanisms are not established. We assessed C228T and C250T TERT mutations by direct Sanger sequencing in tumors of the adrenal gland, and further evaluated potential associations with clinical parameters and telomerase activation. A total of 199 tumors were evaluated, including 34 adrenocortical carcinomas (ACC), 47 adrenocortical adenomas (ACA), 105 pheochromocytomas (PCC; ten malignant and 95 benign), and 13 abdominal paragangliomas (PGL; nine malignant and four benign). TERT expression levels were determined by quantitative RT-PCR. The C228T mutation was detected in 4/34 ACCs (12%), but not in any ACA (P=0.028). C228T was also observed in one benign PCC and in one metastatic PGL. The C250T mutation was not observed in any case. In the ACC and PGL groups, TERT mutation-positive cases exhibited TERT expression, indicating telomerase activation; however, since expression was also revealed in TERT WT cases, this could denote additional mechanisms of TERT activation. To conclude, the TERT promoter mutation C228T is a recurrent event associated with TERT expression in ACCs, but rarely occurs in PGL and PCC. The involvement of the TERT gene in ACC represents a novel mutated gene in this entity.

Rare germline mutations identified by targeted next-generation sequencing of susceptibility genes in pheochromocytoma and paraganglioma.

CONTEXT: Pheochromocytomas and paragangliomas have a highly diverse genetic background, with a third of the cases carrying a germline mutation in 1 of 14 identified genes. OBJECTIVE: This study aimed to evaluate next-generation sequencing for more efficient genetic testing of pheochromocytoma and paraganglioma and to establish germline and somatic mutation frequencies for all known susceptibility genes. DESIGN: A targeted next-generation sequencing approach on an Illumina MiSeq instrument was used for a mutation analysis in 86 unselected pheochromocytoma and paraganglioma tumor samples. The study included the genes EGLN1, EPAS1, KIF1Bß, MAX, MEN1, NF1, RET, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, and VHL. RESULTS were verified in tumor and constitutional DNA with Sanger sequencing. RESULTS: In all cases with clinical syndromes or known germline mutations, a mutation was detected in the expected gene. Among 68 nonfamilial tumors, 32 mutations were identified in 28 of the samples (41%), including germline mutations in EGLN1, KIF1Bß, SDHA, SDHB, and TMEM127 and somatic mutations in EPAS1, KIF1Bß, MAX, NF1, RET, and VHL, including one double monoallelic EPAS1 mutation. CONCLUSIONS: Targeted next-generation sequencing proved to be fast and cost effective for the genetic analysis of pheochromocytoma and paraganglioma. More than half of the tumors harbored mutations in the investigated genes. Notably, 7% of the apparently sporadic cases carried germline mutations, highlighting the importance of comprehensive genetic testing. KIF1Bß, which previously has not been investigated in a large cohort, appears to be an equally important tumor suppressor as MAX and TMEM127 and could be considered for genetic testing of these patients.

Frequent EPAS1/HIF2α exons 9 and 12 mutations in non-familial pheochromocytoma.

Pheochromocytomas are neuroendocrine tumors arising from the adrenal medulla. While heritable mutations are frequently described, less is known about the genetics of sporadic pheochromocytoma. Mutations in genes involved in the cellular hypoxia response have been identified in tumors, and recently EPAS1, encoding HIF2α, has been revealed to be a new gene involved in the pathogenesis of pheochromocytoma and abdominal paraganglioma. The aim of this study was to further characterize EPAS1 alterations in non-familial pheochromocytomas. Tumor DNA from 42 adrenal pheochromocytoma cases with apparently sporadic presentation, without known hereditary mutations in predisposing genes, were analyzed for mutations in EPAS1 by sequencing of exons 9 and 12, which contain the two hydroxylation sites involved in HIF2α degradation, and also exon 2. In addition, the copy number at the EPAS1 locus as well as transcriptome-wide gene expression were studied by DNA and RNA microarray analyses, respectively. We identified six missense EPAS1 mutations, three in exon 9 and three in exon 12, in five of 42 pheochromocytomas (12%). The mutations were both somatic and constitutional, and had no overlap in 11 cases (26%) with somatic mutations in NF1 or RET. One sample had two different EPAS1 mutations, shown by cloning to occur in cis, possibly indicating a novel mechanism of HIF2α stabilization through inactivation of both hydroxylation sites. One of the tumors with an EPAS1 mutation also had a gain in DNA copy number at the EPAS1 locus. All EPAS1-mutated tumors displayed a pseudo-hypoxic gene expression pattern, indicating an oncogenic role of the identified mutations.

The VHL gene is epigenetically inactivated in pheochromocytomas and abdominal paragangliomas.

Pheochromocytoma (PCC) and abdominal paraganglioma (PGL) are neuroendocrine tumors that present with clinical symptoms related to increased catecholamine levels. About a third of the cases are associated with constitutional mutations in pre-disposing genes, of which some may also be somatically mutated in sporadic cases. However, little is known about inactivating epigenetic events through promoter methylation in these very genes. Using bisulphite pyrosequencing we assessed the methylation density of 11 PCC/PGL disease genes in 96 tumors (83 PCCs and 13 PGLs) and 34 normal adrenal references. Gene expression levels were determined by quantitative RT-PCR. Both tumors and normal adrenal samples exhibited low methylation index (MetI) in the EGLN1 (PDH2), MAX, MEN1, NF1, SDHB, SDHC, SDHD, SDHAF2 (SDH5), and TMEM127 promoters, not exceeding 10% in any of the samples investigated. Aberrant RET promoter methylation was observed in two cases only. For the VHL gene we found increased MetI in tumors as compared with normal adrenals (57% vs. 27%; P<0.001), in malignant vs. benign tumors (63% vs. 55%; P<0.05), and in PGL vs. PCC (66% vs. 55%; P<0.0005). Decreased expression of the VHL gene was observed in all tumors compared with normal adrenals (P<0.001). VHL MetI and gene expressions were inversely correlated (R = -0.359, P<0.0001). Our results show that the VHL gene promoter has increased methylation compared with normal adrenals (MetI>50%) in approximately 75% of PCCs and PGLs investigated, highlighting the role of VHL in the development of these tumors.

Acquired hypermethylation of the P16INK4A promoter in abdominal paraganglioma: relation to adverse tumor phenotype and predisposing mutation.

Recurrent alterations in promoter methylation of tumor suppressor genes (TSGs) and LINE1 (L1RE1) repeat elements were previously reported in pheochromocytoma and abdominal paraganglioma. This study was undertaken to explore CpG methylation abnormalities in an extended tumor panel and assess possible relationships between metastatic disease and mutation status. CpG methylation was quantified by bisulfite pyrosequencing for selected TSG promoters and LINE1 repeats. Methylation indices above normal reference were observed for DCR2 (TNFRSF10D), CDH1, P16 (CDKN2A), RARB, and RASSF1A. Z-scores for overall TSG, and individual TSG methylation levels, but not LINE1, were significantly correlated with metastatic disease, paraganglioma, disease predisposition, or outcome. Most strikingly, P16 hypermethylation was strongly associated with SDHB mutation as opposed to RET/MEN2, VHL/VHL, or NF1-related disease. Parallel analyses of constitutional, tumor, and metastasis DNA implicate an order of events where constitutional SDHB mutations are followed by TSG hypermethylation and 1p loss in primary tumors, later transferred to metastatic tissue. In the combined material, P16 hypermethylation was prevalent in SDHB-mutated samples and was associated with short disease-related survival. The findings verify the previously reported importance of P16 and other TSG hypermethylation in an independent tumor series. Furthermore, a constitutional SDHB mutation is proposed to predispose for an epigenetic tumor phenotype occurring before the emanation of clinically recognized malignancy.

Long-term storage of endocrine tissues at - 80°C does not adversely affect RNA quality or overall histomorphology.

BACKGROUND: Today, no consensus exists regarding how human tissues are best preserved for long-term storage. Very low temperature storage in liquid nitrogen is often advocated as the superlative method for extended periods, but storage in -80 degrees Celsius (-80°C) freezers, while sometimes debated, is a possible alternative. RNA is the most easily degradable component of a biological sample in a molecular biology context and the quality can reliably be measured. AIM: To investigate to what extent long-term storage of tissues in -80°C affects the RNA quality and overall histomorphology. The tissue storage period represents nearly three decades (1986-2013). METHODS: RNA extraction from 153 tissue samples with different storage periods was performed with the mirVana kit (Invitrogen). RNA integrity was assessed using an Agilent bioanalyzer to obtain RNA integrity numbers (RIN). Further, tissue representative testing using light microscopy was performed by two pathologists to assess tissue composition and morphology. RESULTS: RIN values were measured in all samples, showing a variability that did not correlate with the storage time of the tissues. Microscopically, all samples displayed acceptable tissue morphology regardless of storage time. CONCLUSION: Long-term storage in -80°C does not adversely affect the quality of the RNA extracted from the stored tissues, and the tissue morphology is maintained to a good standard.

Prolactin receptor in primary hyperparathyroidism--expression, functionality and clinical correlations.

BACKGROUND: Primary hyperparathyroidism (PHPT) is an endocrine disorder most commonly affecting women, suggesting a role for female hormones and/or their receptors in parathyroid adenomas. We here investigated the prolactin receptor (PRLr) which is associated with tumours of the breast and other organs. METHODOLOGY/PRINCIPAL FINDINGS: PRLr expression was investigated in a panel of 37 patients with sporadic parathyroid tumours and its functionality in cultured parathyroid tumour cells. In comparison with other tissues and breast cancer cells, high levels of prolactin receptor gene (PRLR) transcripts were demonstrated in parathyroid tissues. PRLr products of 60/70 kDa were highly expressed in all parathyroid tumours. In addition varying levels of the 80 kDa PRLr isoform, with known proliferative activity, were demonstrated. In parathyroid tumours, PRLr immunoreactivity was observed in the cytoplasm (in all cases, n = 36), cytoplasmic granulae (n = 16), the plasma membrane (n = 12) or enlarged lysosomes (n = 4). In normal parathyroid rim (n = 28), PRLr was uniformly expressed in the cytoplasm and granulae. In in vitro studies of short-term cultured human parathyroid tumour cells, prolactin stimulation was associated with significant transcriptional changes in JAK/STAT, RIG-I like receptor and type II interferon signalling pathways as documented by gene expression profiling. Moreover, PRLR gene expression in parathyroid tumours was inversely correlated with the patients' plasma calcium levels. CONCLUSIONS: We demonstrate that the prolactin receptor is highly abundant in human parathyroid tissues and that PRLr isoforms expression and PRLr subcellular localisation are altered in parathyroid tumours. Responsiveness of PRLr to physiological levels of prolactin was observed in the form of increased PTH secretion and altered gene transcription with significant increase of RIG-I like receptor, JAK-STAT and Type II interferon signalling pathways. These data suggest a role of the prolactin receptor in parathyroid adenomas.

Molecular characterization of parathyroid tumors from two patients with hereditary colorectal cancer syndromes.

The tumor suppressor adenomatous polyposis coli (APC) has recently been implicated in parathyroid development. We here report clinical, histopathological and molecular investigations in parathyroid tumors arising in two patients; one familial adenomatous polyposis (FAP) syndrome patient carrying a constitutional APC mutation, and one Lynch syndrome patient demonstrating a germline MLH1 mutation as well as a non-classified, missense alteration of the APC gene. We sequenced the entire APC gene in tumor and constitutional DNA from both cases, assessed the levels of APC promoter 1A and 1B methylation by bisulfite Pyrosequencing analysis and performed immunohistochemistry for APC and parafibromin. In addition, copy number analysis regarding the APC gene on chromosome 5q21-22 was performed using qRT-PCR. Histopathological workup confirmed both tumors as parathyroid adenomas without signs of malignancy or atypia. No somatic mutations or copy number changes for the APC gene were discovered in the tumors; however, in both cases, the APC promoter 1A was hypermethylated while the APC promoter 1B was unmethylated. APC promoter 1B-specific mRNA and total APC mRNA levels were higher than in normal parathyroid samples. Immunohistochemical analyses revealed strong APC protein immunoreactivity and positive parafibromin expression in both parathyroid tumors. Absence of additional somatic APC mutations and copy number changes in addition to the positive APC immunoreactivity obtained suggest that the tumors arose without biallelic inactivation of the APC tumor suppressor gene. The finding of an unmethylated APC promoter 1B and high APC 1B mRNA levels could explain the maintained APC protein expression. Moreover, the findings of positive parafibromin and APC immunoreactivity as well as a low MIB-1 proliferation index and absence of histopathological features of malignancy/atypical adenoma indicate that the parathyroid adenomas arising in these patients did not harbor malignant potential.