Fetal endocannabinoids orchestrate the organization of pancreatic islet microarchitecture.

Endocannabinoids are implicated in the control of glucose utilization and energy homeostasis by orchestrating pancreatic hormone release. Moreover, in some cell niches, endocannabinoids regulate cell proliferation, fate determination, and migration. Nevertheless, endocannabinoid contributions to the development of the endocrine pancreas remain unknown. Here, we show that α cells produce the endocannabinoid 2-arachidonoylglycerol (2-AG) in mouse fetuses and human pancreatic islets, which primes the recruitment of ß cells by CB1 cannabinoid receptor (CB1R) engagement. Using subtractive pharmacology, we extend these findings to anandamide, a promiscuous endocannabinoid/endovanilloid ligand, which impacts both the determination of islet size by cell proliferation and α/ß cell sorting by differential activation of transient receptor potential cation channel subfamily V member 1 (TRPV1) and CB1Rs. Accordingly, genetic disruption of TRPV1 channels increases islet size whereas CB1R knockout augments cellular heterogeneity and favors insulin over glucagon release. Dietary enrichment in ω-3 fatty acids during pregnancy and lactation in mice, which permanently reduces endocannabinoid levels in the offspring, phenocopies CB1R(-/-) islet microstructure and improves coordinated hormone secretion. Overall, our data mechanistically link endocannabinoids to cell proliferation and sorting during pancreatic islet formation, as well as to life-long programming of hormonal determinants of glucose homeostasis.

Neomorphic effects of recurrent somatic mutations in Yin Yang 1 in insulin-producing adenomas.

Insulinomas are pancreatic islet tumors that inappropriately secrete insulin, producing hypoglycemia. Exome and targeted sequencing revealed that 14 of 43 insulinomas harbored the identical somatic mutation in the DNA-binding zinc finger of the transcription factor Yin Yang 1 (YY1). Chromatin immunoprecipitation sequencing (ChIP-Seq) showed that this T372R substitution changes the DNA motif bound by YY1. Global analysis of gene expression demonstrated distinct clustering of tumors with and without YY1(T372R) mutations. Genes showing large increases in expression in YY1(T372R) tumors included ADCY1 (an adenylyl cyclase) and CACNA2D2 (a Ca(2+) channel); both are expressed at very low levels in normal ß-cells and show mutation-specific YY1 binding sites. Both gene products are involved in key pathways regulating insulin secretion. Expression of these genes in rat INS-1 cells demonstrated markedly increased insulin secretion. These findings indicate that YY1(T372R) mutations are neomorphic, resulting in constitutive activation of cAMP and Ca(2+) signaling pathways involved in insulin secretion.

Somatic Mutations and Genetic Heterogeneity at the CDKN1B Locus in Small Intestinal Neuroendocrine Tumors.

BACKGROUND: Until recently, the genetic landscape of small intestinal neuroendocrine tumors (SI-NETs) was limited to recurrent copy number alterations, most commonly a loss on chromosome 18. Intertumor heterogeneity with nonconcordant genotype in paired primary and metastatic lesions also is described, further contributing to the difficulty of unraveling the genetic enigma of SI-NETs. A recent study analyzing 55 SI-NET exomes nominated CDKN1B (p27) as a haploinsufficient tumor suppressor gene. METHODS: This study aimed to determine the frequency of CDKN1B inactivation and to investigate genotype-phenotype correlations. It investigated 362 tumors from 200 patients. All samples were resequenced for mutations in CDKN1B using automated Sanger sequencing. The expression of p27 was investigated in 12 CDKN1B mutant and nine wild type tumors. RESULTS: Some 8.5 % (17/200) of patients had tumors with pathogenic mutations in CDKN1B including 13 insertion deletions, four nonsense variants, and one stop-loss variant. All variants with available nontumoral DNA were classified as somatic. Inter- and intratumor heterogeneity at the CDKN1B locus was detected respectively in six of ten and two of ten patients. Patients with CDKN1B mutated tumors had both heterogeneous disease presentation and diverse prognosis. Expression of the p27 protein did not correlate with CDKN1B mutation status, and no differences in the clinical characteristics between CDKN1B mutated and CDKN1B wild type tumor carriers were found. CONCLUSION: This study corroborates the finding of CDKN1B as a potential haplo-insufficient tumor suppressor gene characterized by inter- and intratumor heterogeneity in SI-NETs.

Metastases from neuroendocrine tumors to the breast are more common than previously thought. A diagnostic pitfall?

BACKGROUND: Metastases from neuroendocrine tumors (NETs) to the breast have been described as a rare phenomenon. Presentation, imaging results, and cytopathologic findings of these tumours may closely mimic those of a mammary carcinoma. METHODS: This study was a retrospective review of 661 patients with metastatic NETs, of whom 280 were females, treated at Uppsala University Hospital, Uppsala, Sweden. Patients with pathological breast lesions were identified. Histopathological slides from available NET breast lesions were analyzed for mammary carcinoma and neuroendocrine markers. RESULTS: We have identified 20 female patients with NET metastases to the breast, 11/235 with small intestinal NETs, 8/55 with lung NETs, and 1/6 with thymic NETs. There were no male patients with NET metastatic to the breast. Four patients had their breast lesion initially diagnosed as mammary carcinoma. Retrospectively, these lesions showed negative staining for mammary carcinoma markers. CONCLUSIONS: Metastases to the breast from neuroendocrine tumors may be more common than previously thought. Patients with a lesion to the breast and symptoms typical for NET may benefit from additional histopathological investigation, because NET metastases and mammary carcinoma have different immunohistochemical profiles.

Comprehensive DNA methylation analysis of benign and malignant adrenocortical tumors.

The molecular pathogenesis of benign and malignant adrenocortical tumors (ACT) is incompletely clarified. The role of DNA methylation in adrenocortical tumorigenesis has not been analyzed in an unbiased, systematic fashion. Using the Infinium HumanMethylation27 BeadChip, the DNA methylation levels of 27,578 CpG sites were investigated in bisulfite-modified DNA from 6 normal adrenocortical tissue samples, 27 adrenocortical adenomas (ACA), and 15 adrenocortical carcinomas (ACC). Genes involved in cell cycle regulation, apoptosis, and transcriptional regulation of known or putative importance in the development of adrenal tumors showed significant and frequent hypermethylation. Such genes included CDKN2A, GATA4, BCL2, DLEC1, HDAC10, PYCARD, and SCGB3A1/HIN1. Comparing benign versus malignant ACT, a total of 212 CpG islands were identified as significantly hypermethylated in ACC. Gene expression studies of selected hypermethylated genes (CDKN2A, GATA4, DLEC1, HDAC10, PYCARD, SCGB3A1/HIN1) in 6 normal and 16 neoplastic adrenocortical tissues (10 ACA and 6 ACC), displayed reduced gene expression in benign and malignant ACT versus normal adrenocortical tissue. Treatment with 5-aza-2'-deoxycytidine of adrenocortical cancer H-295R cells increased expression of the hypermethylated genes CDKN2A, GATA4, DLEC1, HDAC10, PYCARD, and SCGB3A1/HIN1. In conclusion, the current study represents the first unbiased, quantitative, genome-wide study of adrenocortical tumor DNA methylation. Genes with altered DNA methylation patterns were identified of putative importance to benign and malignant adrenocortical tumor development.

The DNA methylome of benign and malignant parathyroid tumors.

The role of DNA methylation of CpG islands in parathyroid tumorigenesis has not been analyzed in an unbiased, systematic fashion. DNA was isolated from normal and pathologic parathyroid tissues, bisulphite modified and analyzed using the Infinium HumanMethylation27 BeadChip. Distinct hierarchical clustering of genes with altered DNA methylation profiles in normal and pathologic parathyroid tissue was evident. Comparing normal parathyroid tissue with parathyroid adenomas, 367 genes were significantly altered, while 175 genes significantly differed when comparing parathyroid carcinomas and normal parathyroid tissues. A comparison between parathyroid adenomas and parathyroid carcinomas identified 263 genes with significantly distinct methylation levels. Results were confirmed for certain genes in a validation cohort of 40 parathyroid adenomas by methylation-specific PCR. Genes of known or putative importance in the development of parathyroid tumors showed significant and frequent hypermethylation. DNA hypermethylation of CDKN2B, CDKN2A, WT1, SFRP1, SFRP2, and SFRP4 was associated with reduced gene expression in both benign and malignant parathyroid tumors. Treatment with 5-aza-2'-deoxycytidine of primary cell cultures restores expression of hypermethylated genes in benign and malignant parathyroid tumors. In conclusion, the unbiased, genome-wide study of the parathyroid tumor DNA methylome identified a number of genes with altered DNA methylation patterns of putative importance to benign and malignant parathyroid tumorigenesis.

Autoimmune polyendocrine syndrome type 1 and NALP5, a parathyroid autoantigen.

BACKGROUND: Autoimmune polyendocrine syndrome type 1 (APS-1) is a multiorgan autoimmune disorder caused by mutations in AIRE, the autoimmune regulator gene. Though recent studies concerning AIRE deficiency have begun to elucidate the molecular pathogenesis of organ-specific autoimmunity in patients with APS-1, the autoantigen responsible for hypoparathyroidism, a hallmark of APS-1 and its most common autoimmune endocrinopathy, has not yet been identified. METHODS: We performed immunoscreening of a human parathyroid complementary DNA library, using serum samples from patients with APS-1 and hypoparathyroidism, to identify patients with reactivity to the NACHT leucine-rich-repeat protein 5 (NALP5). Subsequently, serum samples from 87 patients with APS-1 and 293 controls, including patients with other autoimmune disorders, were used to determine the frequency and specificity of autoantibodies against NALP5. In addition, the expression of NALP5 was investigated in various tissues. RESULTS: NALP5-specific autoantibodies were detected in 49% of the patients with APS-1 and hypoparathyroidism but were absent in all patients with APS-1 but without hypoparathyroidism, in all patients with other autoimmune endocrine disorders, and in all healthy controls. NALP5 was predominantly expressed in the cytoplasm of parathyroid chief cells. CONCLUSIONS: NALP5 appears to be a tissue-specific autoantigen involved in hypoparathyroidism in patients with APS-1. Autoantibodies against NALP5 appear to be highly specific and may be diagnostic for this prominent component of APS-1.

4D parathyroid CT as the initial localization study for patients with de novo primary hyperparathyroidism.

BACKGROUND: Preoperative localization of parathyroid tumors of primary hyperparathyroidism (pHPT) is required for minimally invasive parathyroidectomy (MIP). Parathyroid four-dimensional computed tomography (4DCT) has mainly been used as an adjunct to other imaging modalities in the remedial setting. 4DCT was evaluated as the initial localization study in de novo patients with pHPT. MATERIALS AND METHODS: A total of 87 consecutive patients underwent parathyroidectomy for pHPT from August 2008 to November 2009. 4DCT was introduced as the preferred imaging modality instead of sestamibi with SPECT (SeS) in April 2009. Results of the imaging studies [4DCT, SeS, and ultrasonography (US)], operative and, pathologic findings, and biochemical measurements were evaluated. RESULTS: In this study, 84% of patients (73 of 87) underwent an US, 59.8% (52 of 87) a SeS, and 38.0% (33 of 87) had a 4DCT. 4DCT had improved sensitivity (85.7%) over SeS (40.4%) and US (48.0%) to localize parathyroid tumors to the correct quadrant of the neck (P < 0.005) as well as to localize (lateralize) the parathyroid lesions to one side of the neck (93.9% for 4DCT vs. 71.2% for US and 61.5% for SeS; P < 0.005). 4DCT correctly predicted multiglandular disease (MGD) in 85.7% (6 of 7) patients, whereas US and SeS were unable to detect MGD in any case. All patients achieved cure based on intraoperative parathyroid hormone (PTH) measurements and normalization of intact PTH and S-Ca during follow-up. CONCLUSIONS: 4DCT provides significantly greater sensitivity than SeS and US for precise localization of parathyroid tumors of pHPT. Additionally, it correctly predicted MGD in a majority of patients.

Clinical and histopathological characteristics of hyperparathyroidism-induced hypercalcemic crisis.

BACKGROUND: The clinical and pathological characteristics of hyperparathyroidism-induced hypercalcemic crisis (HIHC) are incompletely described. The present study was designed to elucidate the nature and effects of HIHC in patients undergoing parathyroidectomy in our unit. METHODS: A prospective database of 1,754 consecutive patients with primary hyperparathyroidism (PHPT) who underwent parathyroidectomy from 1991-2009 identified 67 (41 women) patients presenting with HIHC. Hyperparathyroidism-induced hypercalcemic crisis was defined as symptoms and signs of acute calcium intoxication with a concomitant total albumin corrected calcium level>13.5 mg/dl (range: 8.8-10.2 mg/dl). Clinical and pathological characteristics were evaluated. Data are expressed as mean±SEM. RESULTS: Mean age at presentation was 56.7±2.2 years. Twenty-four of 67 patients (35%) required preoperative in-hospital management. Of these, all were treated with saline resuscitation, whereas 20/24 (83%) were treated pharmacologically. Neurocognitive derangements and nephrolithiasis with associated hematuria were the most common presenting symptoms and signs. Preoperative serum calcium and the intact parathyroid hormone level (PTH) were 14.0±0.19 mg/dl and 393±43 pg/ml (reference range: 12-65 pg/ml), respectively. Minimally invasive parathyroidectomy under local cervical block was performed in 28/67 patients (42%); the remainder underwent standard cervical exploration. All patients had postoperative normalization of serum calcium and intact PTH. Hyperparathyroidism-induced hypercalcemic crisis was due to parathyroid carcinoma in 3/67 patients (4.5%), whereas the remainder of patients displayed a single parathyroid adenoma (n=57) or multiglandular hyperplasia (n=7). Histopathological evaluation from HIHC patients revealed a chief cell microcystic pattern in 15/21 (71.4%) of examined parathyroid tumors. CONCLUSIONS: Hyperparathyroidism-induced hypercalcemic crisis is most commonly due to a single parathyroid adenoma, often associated with a microcystic histopathological pattern. The condition is optimally managed with saline hydration and urgent parathyroidectomy.

Somatic mutations of GNA11 and GNAQ in CTNNB1-mutant aldosterone-producing adenomas presenting in puberty, pregnancy or menopause.

Most aldosterone-producing adenomas (APAs) have gain-of-function somatic mutations of ion channels or transporters. However, their frequency in aldosterone-producing cell clusters of normal adrenal gland suggests a requirement for codriver mutations in APAs. Here we identified gain-of-function mutations in both CTNNB1 and GNA11 by whole-exome sequencing of 3/41 APAs. Further sequencing of known CTNNB1-mutant APAs led to a total of 16 of 27 (59%) with a somatic p.Gln209His, p.Gln209Pro or p.Gln209Leu mutation of GNA11 or GNAQ. Solitary GNA11 mutations were found in hyperplastic zona glomerulosa adjacent to double-mutant APAs. Nine of ten patients in our UK/Irish cohort presented in puberty, pregnancy or menopause. Among multiple transcripts upregulated more than tenfold in double-mutant APAs was LHCGR, the receptor for luteinizing or pregnancy hormone (human chorionic gonadotropin). Transfections of adrenocortical cells demonstrated additive effects of GNA11 and CTNNB1 mutations on aldosterone secretion and expression of genes upregulated in double-mutant APAs. In adrenal cortex, GNA11/Q mutations appear clinically silent without a codriver mutation of CTNNB1.

Aberrant WNT/ß-catenin signaling in parathyroid carcinoma.

BACKGROUND: Parathyroid carcinoma (PC) is a very rare malignancy with a high tendency to recur locally, and recurrent disease is difficult to eradicate. In most western European countries and United States, these malignant neoplasms cause less than 1% of the cases with primary hyperparathyroidism, whereas incidence as high as 5% have been reported from Italy, Japan, and India. The molecular etiology of PC is poorly understood. RESULTS: The APC (adenomatous polyposis coli) tumor suppressor gene was inactivated by DNA methylation in five analyzed PCs, as determined by RT-PCR, Western blotting, and quantitative bisulfite pyrosequencing analyses. This was accompanied by accumulation of stabilized active nonphosphorylated ß-catenin, strongly suggesting aberrant activation of the WNT/ß-catenin signaling pathway in these tumors. Treatment of a primary PC cell culture with the DNA hypomethylating agent 5-aza-2'-deoxycytidine (decitabine, Dacogen(r)) induced APC expression, reduced active nonphosphorylated ß-catenin, inhibited cell growth, and caused apoptosis. CONCLUSION: Aberrant WNT/ß-catenin signaling by lost expression and DNA methylation of APC, and accumulation of active nonphosphorylated ß-catenin was observed in the analyzed PCs. We suggest that adjuvant epigenetic therapy should be considered as an additional option in the treatment of patients with recurrent or metastatic parathyroid carcinoma.

Parathyroid Klotho and FGF-receptor 1 expression decline with renal function in hyperparathyroid patients with chronic kidney disease and kidney transplant recipients.

Current studies suggest that short-term exposure of parathyroid glands to fibroblast growth factor 23 (FGF23) reduces parathyroid hormone secretion. However, patients with chronic kidney disease (CKD) develop secondary hyperparathyroidism despite high levels of serum FGF23, indicating a parathyroid FGF23 'resistance'. Here we analyzed the expression of the FGF23 receptors Klotho and FGF receptor 1 (FGFR1) in 88 hyperplastic parathyroid glands from 31 patients with CKD (including 21 renal allograft recipients), and their regulation in isolated bovine and human hyperplastic parathyroid cells. Glandular expression was variable, yet the Klotho and FGFR1 mRNA levels declined in parallel with the decreasing glomerular filtration rate, significantly decreasing over CKD stages. We found no association between the expression of Klotho, FGFR1, and the proliferation marker Ki67. In vitro treatment of bovine cells with FGF23 or calcium reduced the Klotho level, whereas active vitamin D(3) compounds increased its expression. Phosphate and parathyroid hormone had no effect. Treatment had less impact on Klotho in cultured human cells than in the bovine healthy cell model, whereas FGFR1 expression was induced in the hyperplastic cells. Thus parathyroid Klotho and FGFR1 decrease with declining renal function, possibly because of alterations in mineral metabolism related to the failing kidney. This could explain the observed parathyroid resistance to FGF23 in late CKD.

Molecular genetics of parathyroid disease.

BACKGROUND: Primary hyperparathyroidism (HPT) is often caused by a benign parathyroid tumor, adenoma; less commonly by multiglandular parathyroid disease/hyperplasia; and rarely by parathyroid carcinoma. Patients with multiple tumors require wider exploration to avoid recurrence and have increased risk for hereditary disease. Secondary HPT is a common complication of renal failure. Improved knowledge of the molecular background of parathyroid tumor development may help select patients for appropriate surgical treatment and can eventually provide new means of treatment. The present contribution summarizes more recent knowledge of parathyroid molecular genetics. METHODS: A literature search and review was made to evaluate the level of evidence concerning molecular biology and genetics of primary, secondary, and familial HPT according to criteria proposed by Sackett, with recommendation grading by Heinrich et al. RESULTS: Most parathyroid adenomas and hyperplastic glands are monoclonal lesions. Cyclin D1 gene (CCND1) translocation and oncogene action occur in 8% of adenomas; cyclin D1 overexpression is seen in 20% to 40% of parathyroid adenomas and in 31% of secondary hyperplastic glands. Somatic loss of one MEN1 allele is seen in 25% to 40% of sporadic parathyroid adenomas, half of which have inactivating mutation of the remaining allele. Inactivating somatic HRPT2 mutations are common in parathyroid carcinoma, often causing decreased expression of the protein parafibromin involved in cyclin D1 regulation. Aberrant regulation of Wnt/beta-catenin signaling may be important for parathyroid tumor development. CONCLUSIONS: Molecular genetic studies of parathyroid tumors are well designed basic experimental studies providing strong level III evidence, with data frequently confirmed by subsequent studies.

Aldosterone-Producing Adenomas.

Aldosterone-producing adenomas (APA) are more common than initially anticipated. APA cause primary aldosteronism (PA), which affect 3-10% of the hypertensive population. Research during recent years has led to an increased knowledge of the background dysregulation of the increased aldosterone release, where mutation in the gene encoding the potassium channel GIRK4-KCNJ5-is the most common. Moreover, the discovery of aldosterone-producing cell clusters in apparently normal adenomas has also led to increased understanding of the development of PA, and presumably also APA. A continuum ranging from low-renin hypertension to APA and overt PA is reasoned, and the secondary effects of aldosterone on especially the cardiovascular system have also become more evident. Diagnostics of PA and APA is important in order to reduce cardiovascular morbidity and mortality, but the diagnostic methods are somewhat unspecific and insensitive, indicating the need for novel methods.

RNA Sequencing Provides Novel Insights into the Transcriptome of Aldosterone Producing Adenomas.

Aldosterone producing adenomas (APAs) occur in the adrenal glands of around 30% of patients with primary aldosteronism, the most common form of secondary hypertension. Somatic mutations in KCNJ5, ATP1A1, ATP2B3, CACNA1D and CTNNB1 have been described in ~60% of these tumours. We subjected 15 aldosterone producing adenomas (13 with known mutations and two without) to RNA Sequencing and Whole Genome Sequencing (n = 2). All known mutations were detected in the RNA-Seq reads, and mutations in ATP2B3 (G123R) and CACNA1D (S410L) were discovered in the tumours without known mutations. Adenomas with CTNNB1 mutations showed a large number of differentially expressed genes (1360 compared to 106 and 75 for KCNJ5 and ATP1A1/ATP2B3 respectively) and clustered together in a hierarchical clustering analysis. RT-PCR in an extended cohort of 49 APAs confirmed higher expression of AFF3 and ISM1 in APAs with CTNNB1 mutations. Investigation of the expression of genes involved in proliferation and apoptosis revealed subtle differences between tumours with and without CTNNB1 mutations. Together our results consolidate the notion that CTNNB1 mutations characterize a distinct subgroup of APAs.

Type I membrane klotho expression is decreased and inversely correlated to serum calcium in primary hyperparathyroidism.

CONTEXT: The type I membrane protein Klotho was recently shown to mediate PTH secretion in parathyroid cells in response to low extracellular calcium. In contrast, Klotho inhibits PTH secretion indirectly through the action of fibroblast growth factor-23. Abnormal Klotho expression in parathyroid disorders remains to be elucidated. OBJECTIVE: The aim of the study was to determine: 1) Klotho expression in parathyroid adenomas from patients with primary hyperparathyroidism (pHPT) compared to normal tissue; and 2) its relation to the serum calcium and PTH levels. DESIGN: Surgically removed parathyroid glands (n = 40) and four normal parathyroid tissue specimens were analyzed for Klotho mRNA and protein levels by quantitative real-time PCR and immunohistochemistry. In vitro effects of calcium on Klotho mRNA expression were studied in bovine parathyroid cells. RESULTS: Klotho mRNA levels were significantly decreased (n = 23) or undetectable (n = 17) in parathyroid adenomas compared to normal tissues (P < 0.001). Reduced Klotho protein expression was confirmed by immunohistochemistry. Klotho mRNA levels were inversely correlated to serum calcium (r = -0.97; P < 0.0001), and calcium dose-dependently decreased Klotho mRNA expression in normal parathyroid cells in vitro (P < 0.01). Serum calcium was the only significant marker of Klotho expression in multivariate analysis with calcium, phosphate, PTH, and adenoma weight as independent variables. CONCLUSIONS: Parathyroid Klotho expression is decreased or undetectable in pHPT. We provide evidence that 1) serum calcium is strongly associated with parathyroid Klotho expression in pHPT; and 2) abnormal PTH secretion in hypercalcemic pHPT subjects is mediated by Klotho-independent mechanisms.

Stabilizing mutation of CTNNB1/beta-catenin and protein accumulation analyzed in a large series of parathyroid tumors of Swedish patients.

BACKGROUND: Aberrant accumulation of beta-catenin plays an important role in a variety of human neoplasms. We recently reported accumulation of beta-catenin in parathyroid adenomas from patients with primary hyperparathyroidism (pHPT). In CTNNB1 exon 3, we detected a stabilizing mutation (S37A) in 3 out of 20 analyzed adenomas. The aim of the present study was to determine the frequency and zygosity of mutations in CTNNB1 exon 3, and beta-catenin accumulation in a large series of parathyroid adenomas of Swedish patients. RESULTS: The mutation S37A (TCT > GCT) was detected by direct DNA sequencing of PCR fragments in 6 out of 104 sporadic parathyroid adenomas (5.8%). Taking our previous study into account, a total of 9 out of 124 (7.3%) adenomas displayed the same mutation. The mutations were homozygous by DNA sequencing, restriction enzyme cleavage, and gene copy number determination using the GeneChip 500 K Mapping Array Set. All tumors analyzed by immunohistochemistry, including those with mutation, displayed aberrant beta-catenin accumulation. Western blotting revealed a slightly higher expression level of beta-catenin and nonphosphorylated active beta-catenin in tumors with mutation compared to those without. Presence of the mutation was not related to distinct clinical characteristics. CONCLUSION: Aberrant accumulation of beta-catenin is very common in parathyroid tumors, and is caused by stabilizing homozygous mutation in 7.3% of Swedish pHPT patients.

Epigenetics of pheochromocytoma and paraganglioma.

Pheochromocytomas and paragangliomas (PPGLs) are neuroendocrine tumors arising in the medullae of the adrenal glands or in paraganglia. The knowledge of the tumor biology of these lesions has increased dramatically during the past two decades and more than a dozen recurrently mutated genes have been identified. Different clusters have been described that share epigenetic signatures. Mutations in the succinate dehydrogenase complex subunit genes play a pivotal role in reprogramming the epigenetic state of these tumors by inhibiting epigenetic regulators such as TET enzymes and histone demethylases. Another subgroup of tumors carries hypomethylated genomes, and overexpression of several micro-RNAs has been described. While much remains to be investigated regarding the epigenetics of PPGLs, it is clear that it plays an important role in PPGL biology.

Comprehensive analysis of CTNNB1 in adrenocortical carcinomas: Identification of novel mutations and correlation to survival.

The Wnt/ß-Catenin signaling pathway is one of the most frequently altered pathways in adrenocortical carcinomas (ACCs). The aim of this study was to investigate the status of Wnt/ß-Catenin signaling pathway by analyzing the expression level of ß-Catenin and the mutational status of APC, AXIN2, CTNNB1, and ZNRF3 in ACCs. Mutations in APC, CTNNB1, ZNRF3 and homozygous deletions in ZNRF3 were observed in 3.8% (2/52), 11.5% (6/52), 1.9% (1/52) and 17.3% (9/52) of the cohort respectively. Novel interstitial deletions in CTNNB1 spanning intron 1 to exon 3/intron 3 were also found in 7.7% (4/52) of the tumours. All the observed alterations were mutually exclusive. Nuclear accumulation of ß-Catenin, increased expression of Cyclin D1 and significantly higher expression of AXIN2 (p = 0.0039), ZNRF3 (p = 0.0032) and LEF1(p = 0.0090) observed in the tumours harbouring the deletion in comparison to tumours without CTNNB1 mutation demonstrates that the truncated ß-Catenin is functionally active and erroneously activates the downstream targets. Significantly lower overall survival rate in patients with tumours harbouring alterations in APC/CTNNB1/ZNRF3 in comparison to those without mutation was observed. In conclusion, the discovery of novel large deletions in addition to the point mutations in CTNNB1 infers that activation of Wnt/ß-Catenin pathway via alterations in CTNNB1 occurs frequently in ACCs. We also confirm that alterations in Wnt/ß-Catenin signaling pathway members have a negative effect on overall survival of patients.

Accumulation of nonphosphorylated beta-catenin and c-myc in primary and uremic secondary hyperparathyroid tumors.

CONTEXT: Primary hyperparathyroidism (pHPT) resulting from parathyroid tumors is a common endocrine disorder with incompletely understood etiology, affecting about 1% of the adult population, with an even higher prevalence for elderly individuals. In renal failure, secondary hyperparathyroidism (sHPT) occurs with multiple tumor development as a result of calcium and vitamin D regulatory disturbance. OBJECTIVE: Aberrant Wnt/beta-catenin signaling with accumulation of beta-catenin in the cytoplasm/nucleus is involved in the development of a variety of neoplasms. The aim of this study was to evaluate whether the Wnt/beta-catenin signaling pathway is activated in parathyroid adenomas of pHPT and in hyperplastic glands from uremic patients with sHPT. DESIGN: Immunohistochemistry, Western blotting, real-time quantitative RT-PCR, and DNA sequencing were performed. RESULTS: beta-Catenin was accumulated in all analyzed parathyroid tumors (n = 47) from patients with pHPT and from patients with HPT secondary to uremia. The accumulation included nonphosphorylated, stabilized (transcriptionally active) beta-catenin. The overexpression was not related to increased beta-catenin mRNA levels. A protein-stabilizing mutation in exon 3 of beta-catenin (S37A) was detected in three of 20 pHPT tumors (15%). No mutation was detected in secondary hyperplastic glands (n = 20), and no evidence for truncated adenomatosis polyposis coli proteins was found in adenomas and secondary hyperplastic glands. Mutations in other Wnt signaling components leading to beta-catenin accumulation, other than in beta-catenin itself, are therefore anticipated. The beta-catenin target gene c-myc was overexpressed in a substantial fraction of the parathyroid tumors. CONCLUSION: Our results strongly suggest that modifications in the Wnt/beta-catenin signaling pathway may be involved in the development of hyperparathyroidism.