Kenko Cupisti

The membrane-bound bile acid receptor TGR5 is localized in the epithelium of human gallbladders.

TGR5 (Gpbar‐1) is a plasma membrane‐bound, G protein–coupled receptor for bile acids. TGR5 messenger RNA (mRNA) has been detected in many tissues, including rat cholangiocytes and mouse gallbladder. A role for TGR5 in gallstone formation has been suggested, because TGR5 knockout mice did not develop gallstones when fed a lithogenic diet. In this study, expression and localization of TGR5 was studied in human gallbladders. TGR5 mRNA and protein were detected in all 19 gallbladders. Although TGR5 mRNA was significantly elevated in the presence of gallstones, no such relation was found for TGR5 protein levels. In order to study the localization of TGR5 in human gallbladders, a novel antibody was generated. The receptor was localized in the apical membrane and the rab11‐positive recycling endosome of gallbladder epithelial cells. Furthermore, the TGR5 staining colocalized with the cyclic adenosine monophosphate–regulated chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) and the apical sodium‐dependent bile salt uptake transporter, suggesting a functional coupling of TGR5 to bile acid uptake and chloride secretion. Stimulation with bile acids significantly increased cyclic adenosine monophosphate concentration in human gallbladder tissue. Incubation of gallbladder epithelial cells with a TGR5 agonist led to a rise of N‐(ethoxycarbonylmethyl)‐6‐methoxyquinolinium bromide (MQAE)‐fluorescence, suggestive of a decrease in intracellular chloride concentration. The TGR5 agonist–dependent increase in MQAE‐fluorescence was absent in TGR5 knockout mice or in the presence of a CFTR inhibitor, indicating that TGR5 mediates chloride secretion via activation of CFTR. The presence of the receptor in both the plasma membrane and the recycling endosome indicate that TGR5 can be regulated by translocation. Conclusion: The data suggest a role for TGR5 in bile acid–induced fluid secretion in biliary epithelial cells. (HEPATOLOGY 2009.)

Adrenal involvement in multiple endocrine neoplasia type 1

Adrenal lesions belong to the spectrum of multiple endocrine neoplasia type 1 (MEN-1) syndrome. However, the prevalence of adrenal involvement, the characteristics, and the clinical management of adrenal lesions have not yet been clearly defined. A total of 66 patients with confirmed MEN1 germline mutations and 1 additional patient with typical manifestations in three organ systems were monitored in a regular screening program that included evaluation of the adrenals (median follow-up 96 months; range 12 to 300 months). Age at the diagnosis of MEN-1 and of adrenal tumors and the clinical characteristics, genotype, treatment, and follow-up of adrenal disease were analyzed. Adrenal lesions were identified in 18 of 67 (26.8%) MEN-1 patients and were diagnosed 5 years later than MEN-1. The median tumor diameter at diagnosis was 3.0 cm (range 1.2–15.0 cm), with most tumors being 3 cm or smaller. Eight patients had bilateral tumors. Ten patients had nonfunctional benign tumors, three had benign adrenal Cushing syndrome, and one patient had a pheochromocytoma. Four patients developed adrenocortical carcinomas (ACCs), three of which were functional. Nine adrenalectomies and one subtotal adrenalectomy were performed in six patients. Three patients with ACC died owing to the tumor. Patients with mutations in exons 2 and 10 developed adrenal tumors significantly more often than patients with other mutations (p <0.01). Adrenal tumors are a common feature of MEN-1 but occur later in the course of the disease. The lesions are often small and nonfunctional and can therefore be managed by close surveillance; others have significant malignant potential and should be considered for surgery when they are 3 cm or larger.

Comprehensive Re-Sequencing of Adrenal Aldosterone Producing Lesions Reveal Three Somatic Mutations near the KCNJ5 Potassium Channel Selectivity Filter

Background Aldosterone producing lesions are a common cause of hypertension, but genetic alterations for tumorigenesis have been unclear. Recently, either of two recurrent somatic missense mutations (G151R or L168R) was found in the potassium channel KCNJ5 gene in aldosterone producing adenomas. These mutations alter the channel selectivity filter and result in Na+ conductance and cell depolarization, stimulating aldosterone production and cell proliferation. Because a similar mutation occurs in a Mendelian form of primary aldosteronism, these mutations appear to be sufficient for cell proliferation and aldosterone production. The prevalence and spectrum of KCNJ5 mutations in different entities of adrenocortical lesions remain to be defined. Materials and Methods The coding region and flanking intronic segments of KCNJ5 were subjected to Sanger DNA sequencing in 351 aldosterone producing lesions, from patients with primary aldosteronism and 130 other adrenocortical lesions. The specimens had been collected from 10 different worldwide referral centers. Results G151R or L168R somatic mutations were identified in 47% of aldosterone producing adenomas, each with similar frequency. A previously unreported somatic mutation near the selectivity filter, E145Q, was observed twice. Somatic G151R or L168R mutations were also found in 40% of aldosterone producing adenomas associated with marked hyperplasia, but not in specimens with merely unilateral hyperplasia. Mutations were absent in 130 non-aldosterone secreting lesions. KCNJ5 mutations were overrepresented in aldosterone producing adenomas from female compared to male patients (63 vs. 24%). Males with KCNJ5 mutations were significantly younger than those without (45 vs. 54, respectively; p<0.005) and their APAs with KCNJ5 mutations were larger than those without (27.1 mm vs. 17.1 mm; p<0.005). Discussion Either of two somatic KCNJ5 mutations are highly prevalent and specific for aldosterone producing lesions. These findings provide new insight into the pathogenesis of primary aldosteronism.

Long-term clinical and biochemical follow-up in medullary thyroid carcinoma: a single institution's experience over 20 years.

Objective:Many patients with medullary thyroid carcinomas (MTC) have reoperative surgery in different hospitals, which makes their follow-up difficult. To comprehend these complex courses and to find relevant prognostic factors we report a 20-year single center experience of 289 patients with MTC or precursor C-cell-hyperplasias. Patients and Methods:Between April 1986 and May 2006, 289 consecutive patients with MTC or MEN2 gene carriers were treated at the Department of Surgery at the University Hospital Düsseldorf. Tumor stages were documented according to the classification of the International Union against Cancer 5th edition, 1997 (Schott. Endocr Relat Cancer. 2006;13:779–795). A system to easily comprehend operative procedures is suggested. Results:There were 159 female and 130 male patients (f/m ratio 1.22). Mean age at time of diagnosis was 32 years (4–77) in the familial cases and 53 years (23–84) years in the sporadic cases. Sixty-six patients (23%) had multifocal disease. Twelve MEN2-patients had only C-cell-hyperplasia (pT0). Tumor stage was pT1 in 86 patients, pT2 in 106 patients, pT3 in 25 patients, pT4 in 52 patients and unclear in 8 patients. In the 289 patients 648 operations were performed. One hundred seventy patients had more than 1 operation (59%). Ninety-nine patients (34%) are calcitonin-negative and 91 patients (31%) live with elevated calcitonin. Median follow-up time of the surviving 211 patients was 8.9 years (range, 0.3–30.7 years). The 5- and 10-year survival of all tumor patients was 86% and 68%, respectively. Conclusion:The chance to achieve biochemical cure in MTC is clearly dependent on the primary tumor size. The chance for long-term biochemical cure in a pT4-tumor is almost nil even after multiple and extended reoperations, whereas a pT1 tumor can be cured in up to 67% of the patients. Long-term survival, however, can be achieved even in pT4 tumor patients in almost 50%.

Long-term Biochemical Results after Operative Treatment of Primary Hyperparathyroidism Associated with Multiple Endocrine Neoplasia Types I and IIa: Is a More or Less Extended Operation Essential?

OBJECTIVE To analyse our long term results in patients operated on for primary hyperparathyroidism associated with multiple endocrine neoplasia types I and IIa. DESIGN Retrospective (data collection) and prospective (follow-up) analysis. SETTING University hospital, Germany. SUBJECTS 39 patients with MEN type I-associated and 7 patients with MEN-type-IIa-associated primary hyperparathyroidism. INTERVENTIONS Subtotal parathyroidectomy (n = 25 with MEN I and 1 with MEN IIa), total parathyroidectomy and autotransplantation (one in each group) and removal of only enlarged glands (13 with MEN type I and 5 with MEN type IIa). MAIN OUTCOME MEASUREMENTS Recurrence rate of hyperparathyroidism and permanent hypocalcaemia postoperatively. RESULTS Subtotal parathyroidectomy in patients with MEN type I gave a significantly lower recurrence rate than removal of only enlarged glands (3/25 compared with 3/13, log rank, p = 0.04). Permanent hypocalcaemia developed in 3/25 compared with 3/13, respectively. 2/5 patients with MEN type IIa developed recurrences after removal of only enlarged glands and the rate was higher than expected. CONCLUSIONS A more extensive operation is essential for patients with MEN type I; the rate of permanent hypocalcaemia is not increased, but the recurrence rate is reduced. Patients with MEN type IIa should be treated by excision of enlarged glands alone, but this may be extended to subtotal parathyroidectomy in patients with severe symptoms.

Activating mutations in CTNNB1 in aldosterone producing adenomas

Primary aldosteronism (PA) is the most common cause of secondary hypertension with a prevalence of 5–10% in unreferred hypertensive patients. Aldosterone producing adenomas (APAs) constitute a large proportion of PA cases and represent a surgically correctable form of the disease. The WNT signaling pathway is activated in APAs. In other tumors, a frequent cause of aberrant WNT signaling is mutation in the CTNNB1 gene coding for β-catenin. Our objective was to screen for CTNNB1 mutations in a well-characterized cohort of 198 APAs. Somatic CTNNB1 mutations were detected in 5.1% of the tumors, occurring mutually exclusive from mutations in KCNJ5, ATP1A1, ATP2B3 and CACNA1D. All of the observed mutations altered serine/threonine residues in the GSK3β binding domain in exon 3. The mutations were associated with stabilized β-catenin and increased AXIN2 expression, suggesting activation of WNT signaling. By CYP11B2 mRNA expression, CYP11B2 protein expression, and direct measurement of aldosterone in tumor tissue, we confirmed the ability for aldosterone production. This report provides compelling evidence that aberrant WNT signaling caused by mutations in CTNNB1 occur in APAs. This also suggests that other mechanisms that constitutively activate the WNT pathway may be important in APA formation.

Neuropsychiatric and Cognitive Changes after Surgery for Primary Hyperparathyroidism

IntroductionNeuropsychiatric symptoms and cognitive impairment are mental manifestations of primary hyperparathyroidism (pHPT). The aim of our study was to determine if surgical treatment results in a long-lasting full recovery from these symptoms.MethodsIn a prospective case-control study with matching pairs, mental changes were examined preoperatively and 6 months postoperatively in 30 patients with primary hyperparathyroidism and 30 patients with nontoxic nodular goiter using the Hamilton depression score and four cognitive tests: DEM Tect, MWT (multiple word test), ZVT, and Benton test.ResultsPatients with pHPT demonstrated significantly more cognitive changes (P < 0.0001) with significant improvement 6 months postoperatively (P < 0.0001). Patients with pHPT presented more psychopathologic symptoms than patients of the control group (P = NS), and there was a tendency towards recovery in the pHPT group postoperatively. There was no correlation between biochemistry and psychopathologic or cognitive changes in the pHPT group.ConclusionsPatients with pHPT often present with neuropsychiatric symptoms and cognitive impairment. A successful parathyroid operation improves cognitive disorders in particular.

Novel somatic mutations and distinct molecular signature in aldosterone-producing adenomas

Aldosterone-producing adenomas (APAs) are found in 1.5-3.0% of hypertensive patients in primary care and can be cured by surgery. Elucidation of genetic events may improve our understanding of these tumors and ultimately improve patient care. Approximately 40% of APAs harbor a missense mutation in the KCNJ5 gene. More recently, somatic mutations in CACNA1D, ATP1A1 and ATP2B3, also important for membrane potential/intracellular Ca(2) (+) regulation, were observed in APAs. In this study, we analyzed 165 APAs for mutations in selected regions of these genes. We then correlated mutational findings with clinical and molecular phenotype using transcriptome analysis, immunohistochemistry and semiquantitative PCR. Somatic mutations in CACNA1D in 3.0% (one novel mutation), ATP1A1 in 6.1% (six novel mutations) and ATP2B3 in 3.0% (two novel mutations) were detected. All observed mutations were located in previously described hotspot regions. Patients with tumors harboring mutations in CACNA1D, ATP1A1 and ATP2B3 were operated at an older age, were more often male and had tumors that were smaller than those in patients with KCNJ5 mutated tumors. Microarray transcriptome analysis segregated KCNJ5 mutated tumors from ATP1A1/ATP2B3 mutated tumors and those without mutation. We observed significant transcription upregulation of CYP11B2, as well as the previously described glomerulosa-specific gene NPNT, in ATP1A1/ATP2B3 mutated tumors compared to KCNJ5 mutated tumors. In summary, we describe novel somatic mutations in proteins regulating the membrane potential/intracellular Ca(2) (+) levels, and also a distinct mRNA and clinical signature, dependent on genetic alteration.

Therapy of suspected intrathoracic parathyroid adenomas

Background and aims: Ectopic mediastinal parathyroid adenoma as a cause of primary hyperparathyroidism (pHPT) can normally be resected from conventional collar incision. In rare cases with adenomas deeper in the chest, a transthoracic approach is necessary. Patients/methods: We report our experience of 19 patients with suspected mediastinal parathyroid adenomas from a total of 1035 patients with pHPT who were operated on between 1986 and 2000 using an open approach (sternotomy or thoracotomy) or video-assisted mediastinal or thoracoscopic surgery (VAMS/VATS). Results: Fourteen patients underwent an open approach with a success rate of 71% (10 of 14). Four patients remained hypercalcaemic. There were four complications in three patients: three permanent recurrent nerve palsies and one chylus fistula, requiring further surgery. VATS was successful in three of four patients with conversion to sternal splitting because of a false-negative frozen section in one patient. Another patient had parathyroid adenoma retrosternally which could not be resected by means of VAMS and had to be excised using a transsternal approach. There were no complications of minimal invasive procedures. All five patients were normocalcaemic after the operation. Conclusion: Ectopic parathyroid adenomas not resectable by means of a collar incision are rare causes of pHPT and comprise 1.25% of all patients with pHPT in our series. For these patients, VATS revealed an alternative to conventional open procedures. In questionable cases, however, the collar incision should precede the VATS procedure.

Dendritic Cell Vaccination Induces Tumor Epitope-specific Th1 Immune Response in Medullary Thyroid Carcinoma

The existence of inherited aggressive forms of medullary thyroid carcinoma (MTC) and their resistance to classical therapies make it a prime candidate for adoptive immunotherapy. Highly potent antigen-presenting cells, namely dendritic cells (DCs), may serve as an interesting tool for anticancer vaccination. Here we report on the IN VITRO findings of a vaccination trial in five MTC patients, who were treated with a new DC generation protocol consisting of granulocyte-macrophage colony-stimulating factor and interferon-alpha (IFN-DCs). These cells were pulsed with tumor-specific calcitonin and administered twice. In two patients who responded to therapy we found a large increase (in mean 2.9+/-1.9%) of antigen-specific IFN-gamma-secreting CD4+ cells as well as an increase of granzyme B positive CD8+ cells (mean 2.2+/-0.2%) in the peripheral blood. In parallel, a decrease of CD4+/CD25+/FoxP3+ regulatory T cells was seen. Importantly, IN VITRO stimulation of PBMC with 10 different 15mer calcitonin peptides resulted in the identification of two HLA class II epitope regions within the central part of full-length calcitonin. These data were in accordance with the results drawn from the computer-based algorithm epitope prediction software SYFPEITHI. Measurement of different pro- and anti-angiogenic factors did not allow for a distinct outcome of prediction of the treated patients. In summary, we have demonstrated that immunization with IFN-DCs leads to a tumor epitope-specific immune response in MTC patients and may, therefore, represent a promising tool for future vaccination trials.