Federica Saponaro

CDC73 mutational status and loss of parafibromin in the outcome of parathyroid cancer

Inactivating mutations of the CDC73 tumor suppressor gene have been reported in parathyroid carcinomas (PC), in association with the loss of nuclear expression of the encoded protein, parafibromin. The aim of this study was to further investigate the role of the CDC73 gene in PC and evaluate whether gene carrier status and/or the loss of parafibromin staining might have an effect on the outcome of the disease. We performed genetic and immunohistochemical studies in parathyroid tumor samples from 35 patients with sporadic PC. Nonsense or frameshift CDC73 mutations were detected in 13 samples suitable for DNA sequencing. Six of these mutations were germline. Loss of parafibromin expression was found in 17 samples. The presence of the CDC73 mutation as well as the loss of parafibromin predicted a high likelihood of subsequent recurrence and/or metastasis (92.3%, P=0.049 and 94.1%, P=0.0017 respectively), but only the latter was associated with a decreased overall 5- and 10-year survival rates (59%, P=0.107, and 23%, P=0.0026 respectively). The presence of both the CDC73 mutation and loss of parafibromin staining compared with their absence predicted a lower overall survival at 10- (18 vs 84%, P=0.016) but not at 5-year follow-up. In conclusion, loss of parafibromin staining, better than CDC73 mutation, predicts the clinical outcome and mortality rate. The added value of CDC73 mutational analysis is the possibility of identifying germline mutations, which will prompt the screening of other family members.

Cinacalcet efficacy in patients with moderately severe primary hyperparathyroidism according to the European Medicine Agency prescription labeling.

Background: Patients with primary hyperparathyroidism (PHPT) with contraindications to parathyroidectomy (PTx) or persistent PHPT have few non surgical options. Aim: The aim of the study was to investigate the efficacy of cinacalcet in reducing serum calcium in patients with PHPT, for whom PTx would be indicated according to serum calcium levels, but in whom PTx is not clinically appropriate or is contraindicated [European Medicines Agency (EMA) prescription labeling]. Subjects and methods: The study (open-label prospective, conducted in a single tertiary center) included 12 sporadic and 2 multiple endocrine neoplasia type 1 PHPT patients with serum calcium greater than 11.2 mg/dl. Cinacalcet was administered in increasing doses until normal serum calcium was reached or side effects preventing a further increase occurred. Serum calcium, PTH, phosphate, 25OHD, markers of bone turnover, 24h-urinary calcium and areal bone mineral density (BMD) were measured. Safety biochemical indices and adverse events were monitored. Results: The maintenance cinacalcet dose [median 30 mg twice daily (range 30 daily-60 mg twice daily)] was maintained constant during follow-up (median 12 months). Mean±SE baseline serum calcium was 12.2±0.3 mg/dl. Serum calcium decreased by at least 1 mg/dl in all patients and normalized in 10. Serum calcium at the last observation was 9.9±0.2 mg/dl (p<0.0001 vs baseline). PTH decreased by 17.1% compared to baseline (p=0.13), and never reached a normal value. BMD was unchanged. Adverse events occurred in 6 patients (43%) and required treatment withdrawal in 2. Conclusions: Cinacalcet reduced and often normalized serum calcium in PHPT patients who met the EMA labeling.

Cinacalcet in the management of primary hyperparathyroidism: post marketing experience of an Italian multicentre group.

To report the Italian experience on cinacalcet use following its approval by the European Medical Agency (EMA) to control hypercalcaemia in patients with primary hyperparathyroidism (PHPT).

Evaluation of formalin-fixed paraffin-embedded tissues in the proteomic analysis of parathyroid glands.

BackgroundProteomic research in the field of parathyroid tissues is limited by the very small dimension of the glands and by the low incidence of cancer lesions (1%). Formalin-fixed paraffin-embedded (FFPE) tissue specimens are a potentially valuable resource for discovering protein cancer biomarkers. In this study we have verified the applicability of a heat induced protein extraction from FFPE parathyroid adenoma tissues followed by a gel-based or gel-free proteomic approach in order to achieve protein separation and identification.ResultsThe best results for high quality MS spectra and parameters, were obtained by using a gel-free approach, and up to 163 unique proteins were identified. Similar results were obtained by applying both SDS-out and SDS-out + TCA/Acetone techniques during the gel-free method. Western blot analysis carried out with specific antibodies suggested that the antigenicity was not always preserved, while specific immunoreactions were detected for calmodulin, B box and SPRY domain-containing protein (BSPRY), peroxiredoxin 6 (PRDX 6) and parvalbumin.ConclusionsIn spite of some limitations mainly due to the extensive formalin-induced covalent cross-linking, our results essentially suggest the applicability of a proteomic approach to FFPE parathyroid specimens. From our point of view, FFPE extracts might be an alternative source, especially in the validation phase of protein biomarkers when a large cohort of samples is required and the low availability of frozen tissues might be constraining.

β-catenin activation is not involved in sporadic parathyroid carcinomas and adenomas

Aberrant accumulation of beta-catenin has been found in various types of human tumors. The aim of this study was to evaluate whether Wnt/beta-catenin signaling is activated in parathyroid carcinomas and adenomas. We studied 154 parathyroid tumors (18 carcinomas (13 with distant metastases), six atypical adenomas, and 130 adenomas). Three normal parathyroid tissues were used as control. Direct sequencing of exon 3 of the CTNNB1 gene showed absence of stabilizing mutations in all the tumors. Immunostaining of beta-catenin was performed in all carcinomas and in 66 adenomas (including three atypical). Normal parathyroid showed a homogeneous distinct outer cell membrane staining in the majority of cells and no nuclear staining. A weak cytoplasmic staining was observed in one case. All tumors showed negative nuclear staining. With the exception of one carcinoma, which had a negative membrane staining, all other samples showed a membrane staining which was similar to that of the normal parathyroid. beta-Catenin expression was heterogeneous with a range of positive cells between 5 and 80%, independently of tumor type. Our results suggest that the Wnt/beta-catenin signaling pathway is not involved in the development of parathyroid carcinomas and adenomas.

Aryl hydrocarbon receptor interacting protein (AIP) mutations occur rarely in sporadic parathyroid adenomas.

CONTEXT The molecular pathogenesis of primary hyperparathyroidism is still largely unknown. The aryl hydrocarbon receptor interacting protein (AIP) gene has a major role in the pathogenesis of familial isolated pituitary adenoma. OBJECTIVE We evaluated the involvement of the AIP gene in sporadic parathyroid adenomas. PATIENTS AND DESIGN We performed direct sequencing and multiplex ligation-dependent probe amplification analyses of the AIP gene in a large series of sporadic parathyroid adenomas. Loss of heterozygosity (LOH) at the AIP locus was studied, and aryl hydrocarbon receptor interacting protein immunostaining was also performed. In addition, alterations in the MEN1 gene were studied. RESULTS A somatic AIP mutation, substitution of arginine with glutamine at codon 304 (R304Q), was identified in 2 of 132 tumors. The mutation was germline in both cases despite the nonfamilial presentation. Heterozygous AIP large deletions were detected in 29 cases including 1 of the 2 mutated tumors, confirming a biallelic inactivation of the AIP gene. The AIP-mutated tumor with LOH showed decreased AIP immunostaining compared with normal parathyroid. LOH at the MEN1 locus, which often shared LOH at the AIP locus, was found in one third of tumors. Somatic MEN1 mutations were found in the 1 of the 2 AIP-mutated tumors and in 22 parathyroid adenomas. In addition, multiplex ligation-dependent probe amplification analysis revealed 1 large deletion of the MEN1 gene in 1 patient. CONCLUSIONS The AIP gene is rarely involved in parathyroid adenomas, but the germline nature of the mutations suggests that it might predispose to primary hyperparathyroidism. MEN1 gene alterations occur in a substantial proportion of sporadic parathyroid adenomas.

Epidemiology, pathogenesis of primary hyperparathyroidism: Current data

In the early twentieth century, primary hyperparathyroidism PHPT) was considered a rare disease, with a major bone nvolvement or osteitis fibrosa cystica. In 1934, Fuller Albright firstly reported 17 cases of PHPT, ome of them without metabolic bone disease. This discovery ed to the concept that PHPT is a more common endocrinopaty, ith variable clinical manifestations, particularly urinary and idney involvement. PHPT was increasingly identified on the asis of serum calcium measurement and screening population tudies, which included serum calcium measurement, helped efining the epidemiology the of PHPT. Variable prevalence data ere obtained, depending on the diagnostic criteria: it was estiated, according to a Norway survey, that the prevalence of HPT in Europe around 1990 ranged from 3.6 to 13.9% [1]. population-based study on the incidence in Olmsted County, N, USA, revealed that the annual incidence of PHPT in 1974 as 7.8/100,000/yr [2]. However, a year later in 1975, after the ntroduction of routine serum calcium measurement with autoated chemistry panels, the incidence in the same population, djusted for previously prevalent cases, rose to 27.7/100,000. his higher incidence was prevalently due to the detection of asymptomatic” PHPT [3]. From 1983 to 1992, despite the wider and continuous use of outinely serum calcium measurement, the incidence of PHPT eclined to an overall ageand sex-adjusted rate of 20.8/100,000, s documented from Rochester and other studies. The reason of

Normocalcemic primary hyperparathyroidism: a survey in a small village of Southern Italy

We investigated the prevalence of normocalcemic primary hyperparathyroidism (NPHPT) in the adult population living in a village in Southern Italy. All residents in 2010 (n=2045) were invited by calls and 1046 individuals accepted to participate. Medical history, calcium intake, calcium, albumin, creatinine, parathyroid hormone (PTH) and 25OHD were evaluated. NPHPT was defined by normal albumin-adjusted serum calcium, elevated plasma PTH, and exclusion of common causes of secondary hyperparathyroidism (SHPT) (serum 25OHD <30 ng/ml, estimated glomerular filtration rate (eGFR) <60 ml/min per 1.73 m2 and thiazide diuretics use), overt gastrointestinal and metabolic bone diseases. Complete data were available for 685 of 1046 subjects. Twenty subjects did not meet the inclusion criteria and 341 could not be evaluated because of thawing of plasma samples. Classical PHPT was diagnosed in four women (0.58%). For diagnosing NPHPT the upper normal limit of PTH was established in the sample of the population (n=100) who had 25OHD ≥30 ng/ml and eGFR ≥60 ml/min per 1.73 m2 and was set at the mean+3s.d. Three males (0.44%) met the diagnostic criteria of NPHPT. These subjects were younger and with lower BMI than those with classical PHPT. Our data suggest, in line with previous studies, that NPHPT might be a distinct clinical entity, being either an early phenotype of asymptomatic PHPT or a distinct variant of it. However, we cannot exclude that NPHPT might also represent an early phase of non-classical SHPT, since other variables, in addition to those currently taken into account for the diagnosis of NPHPT, might cumulate in a normocalcemic subject to increase PTH secretion.

Hypovitaminosis D in patients with heart failure: effects on functional capacity and patients’ survival

Chronic heart failure is a major cause of morbidity and mortality, but its prognosis remains poor. Vitamin D hormone has many extra-skeletal functions including a positive impact on the cardiovascular system, and has been proposed for mortality risk evaluation in heart failure patients. The aim of the present study was to evaluate vitamin D status in heart failure patients, measured by high performance liquid chromatography coupled with mass spectrometry and to correlate serum 25 hydroxy-vitamin D (25OHD) levels with functional (peak VO2%) and mortality (Metabolic Exercise Cardiac Kidney Index) heart failure parameters. We enrolled 261 consecutive patients diagnosed with heart failure; all patients underwent a comprehensive clinical and biochemical characterization, and serum 25OHD levels were measured by high performance liquid chromatography coupled with mass spectrometry. Cardiopulmonary test parameters and Metabolic Exercise Cardiac Kidney Index of mortality risk were measured in all patients. Serum 25OHD levels ranged between 2 and 45 ng/ml (mean 17 ± 9 ng/ml); most patients (87%) showed hypovitaminosis D, and 25% showed severe vitamin D deficiency (serum 25OHD < 10 ng/ml). Patients with 25OHD < 10 ng/ml had significantly lower cardiopulmonary test VO2/kg, peak VO2% and significantly higher N-terminalproBrain natriuretic peptide and Metabolic Exercise Cardiac Kidney Index, than patients with 25OHD > 10 ng/ml. Patients with peak VO2% < 50% showed significantly lower 25OHD compared to those with peak VO2% > 50%. There was a significant, positive correlation (r = 0.16, p = 0.008) between 25OHD levels and peak VO2%, and an inverse correlation with Metabolic Exercise Cardiac Kidney Index (r = −0.21, p < 0.001), even when adjusted for age, Body Mass Index, MDRD, N-terminalproBrain natriuretic peptide. In conclusion, our findings show that vitamin D levels are associated with functional and mortality heart failure prognosis parameters.

Loss of p27 expression is associated with MEN1 gene mutations in sporadic parathyroid adenomas

MEN1 is the main gene responsible for tumorigenesis of syndromic and sporadic primary hyperparathyroidism (PHPT). Germline mutations of the CDKN1B/p27Kip gene have been associated with multiple endocrine tumors in rats and humans. To evaluate the involvement of the CDKN1B gene and its relationship with MEN1 in sporadic PHPT, we carried out sequencing and loss of heterozygosity analyses of the CDKN1B gene in 147 sporadic parathyroid adenomas. p27 immunohistochemistry and genetic screening of the MEN1 gene were performed in 50 cases. Three germline CDKN1B variants (c.-80C>T, c.-29_-26delAGAG, c.397C>A) were identified in 3/147 patients. Reduction of CDKN1B gene transcription rate was demonstrated in vitro for the novel c.-80C>T and the c.-29_-26delAGAG variants. Loss of p27 expression was detected in the tumor carrying the c.-29_-26delAGAG variant. Two tumors carrying the CDKN1B variants also harbored a MEN1 mutation. Fifty-four percent of 50 CDKN1B mutation-negative tumors had a reduction of p27 nuclear staining. Somatic MEN1 mutations, identified in 15/50 samples, significantly segregated in tumors negative for nuclear and cytoplasmic p27 staining. The germline nature of the CDKN1B mutations suggests that they might predispose to PHPT. The lack of somatic CDKN1B mutations in our samples points to a rare involvement in parathyroid adenomas, despite the frequent loss of nuclear p27 expression. MEN1 biallelic inactivation seems to be directly related to down-regulation of p27 expression through the inhibition of CDKN1B gene transcription.