Maria Chiara Zatelli

miR-15a and miR-16-1 down-regulation in pituitary adenomas

Micro RNAs (miRs) are small noncoding RNAs, functioning as antisense regulators of other RNAs. miR‐15a and miR‐16‐1 genes are located at chromosome 13q14, a region which is frequently deleted in pituitary tumors. An inverse correlation has been shown in B cell chronic lymphocytic leukemia (B‐CLL) between miR‐15a and miR‐16‐1 expression and the expression levels of arginyl‐tRNA synthetase (RARS), an enzyme which associates with the cofactor p43 in the aminoacyl‐tRNA synthetase complex. When secreted, p43 regulates local inflammatory response and macrophage chemotaxis, and seems to have anti‐neoplastic properties in mice. We explored miR‐15a and miR‐16‐1 expression in 10 GH‐secreting and in 10 PRL‐secreting pituitary macroadenomas by Northern blot, and investigated the possible correlation with in vivo and in vitro characteristics. We found that miR‐15a and miR‐16‐1 are expressed at lower levels in pituitary adenomas as compared to normal pituitary tissue. Moreover, their expression inversely correlates with tumor diameter and with RARS expression (P < 0.05), but directly correlates with p43 secretion (P < 0.02). Therefore, miR15 and miR16 down‐regulation in pituitary adenomas correlates with a greater tumor diameter and a lower p43 secretion, suggesting that these genes may, at least in part, influence tumor growth.

Occurrence of Pituitary Dysfunction following Traumatic Brain Injury

Traumatic brain injury (TBI) may be associated with impairment of pituitary hormone secretion, which may contribute to long-term physical, cognitive, and psychological disability. We studied the occurrence and risk factors of pituitary dysfunction, including growth hormone deficiency (GHD) in 50 patients (mean age 37.6 +/- 2.4 years; 40 males, age 20-60 years; 10 females, age 23-87 years) with TBI over 5 years. Cranial or facial fractures were documented in 12 patients, and neurosurgery was performed in 14. According to the Glasgow Coma Scale (GCS), 16 patients had suffered from mild, 7 moderate, and 27 severe TBI. Glasgow Outcome Scale (GOS) indicated severe disability in 5, moderate disability in 11, and good recovery in 34 cases. Basal pituitary hormone evaluation, performed once at times variable from 12 to 64 months after TBI, showed hypogonadotrophic hypogonadism in 7 (14%), central hypothyroidism in 5 (10%), low prolactin (PRL) levels in 4 (8%), and high PRL levels in 4 (8%) cases. All subjects had normal corticotrophic and posterior pituitary function. Seven patients showed low insulin-like growth factor-I (IGF-I) levels for age and sex. Results of GHRH plus arginine testing indicated partial GHD in 10 (20%) and severe GHD in 4 (8%) cases. Patients with GHD were older (p <0.05) than patients with normal GH secretion. Magnetic resonance imaging demonstrated pituitary abnormalities in 2 patients; altogether pituitary dysfunction was observed in 27 (54%) patients. Six patients (12%) showed a combination of multiple abnormalities. Occurrence of pituitary dysfunction was 37.5%, 57.1%, and 59.3% in the patients with mild, moderate, and severe TBI, respectively. GCS scores were significantly (p <0.02) lower in patients with pituitary dysfunction compared to those with normal pituitary function (8.3 +/- 0.5 vs. 10.2 +/- 0.6). No relationship was detected between pituitary dysfunction and years since TBI, type of injury, and outcome from TBI. In conclusion, subjects with a history of TBI frequently develop pituitary dysfunction, especially GHD. Therefore, evaluation of pituitary hormone secretion, including GH, should be included in the long-term follow-up of all TBI patients so that adequate hormone replacement therapy may be administered.

Identification of differentially expressed microRNAs by microarray: A possible role for microRNA genes in pituitary adenomas

MicroRNAs (miRNAs) are small non‐coding RNAs that control gene expression by targeting mRNA. It has been demonstrated that miRNA expression is altered in many human cancers, suggesting that they may play a role in human neoplasia. To determine whether miRNA expression is altered in pituitary adenomas, we analyzed the entire miRNAome in 32 pituitary adenomas and in 6 normal pituitary samples by microarray and by Real‐Time PCR. Here, we show that 30 miRNAs are differentially expressed between normal pituitary and pituitary adenomas. Moreover, 24 miRNAs were identified as a predictive signature of pituitary adenoma and 29 miRNAs were able to predict pituitary adenoma histotype. miRNA expression could differentiate micro‐ from macro‐adenomas and treated from non‐treated patient samples. Several of the identified miRNAs are involved in cell proliferation and apoptosis, suggesting that their deregulated expression may be involved in pituitary tumorigenesis. Predictive miRNAs could be potentially useful diagnostic markers, improving the classification of pituitary adenomas. J. Cell. Physiol. 210: 370–377, 2007.

Chromogranin A as a marker of neuroendocrine neoplasia: An Italian Multicenter Study

Elevated circulating chromogranin A (CgA) levels are found in neuroendocrine tumors (NETs), but the diagnostic usefulness of this marker is still debatable. To assess the role of CgA for the diagnosis of gastroenteropancreatic (GEP) NETs and the identification of metastatic patients, an Italian multicenter observational study has been performed. CgA was evaluated in 202 GEP NET patients by IRMA and ELISA. The cutoffs for diagnosis and presence of metastases were identified by receiver-operating characteristic (ROC) curve. We found good correlation between IRMA and ELISA. The ROC analysis identified a cutoff of 53 ng/ml for IRMA and 16 U/l for ELISA as discriminating between controls and patients with active disease (sensitivity 71.3 and 84%; specificity 71 and 85% respectively). Metastases were present in 123 patients, having significantly higher CgA levels than patients without metastases. ROC analysis identified a cutoff of 146 ng/ml for IRMA and 67.3 U/l for ELISA as discriminating between patients with and without metastases (sensitivity 57 and 63.3%; specificity 55.6 and 71.4% respectively). For pancreatic NETs positive and negative predictive values were 84 and 78% respectively (90% specificity and 68% sensitivity). We found lower CgA levels in patients with extensive metastatic spread than in those with liver metastases only. These data assess the role of CgA evaluation in GEP NETs, and demonstrate that higher CgA levels associate with metastatic disease, confirming that CgA levels can provide a helpful practical biochemical marker for the clinical management of NETs, but with low sensitivity and specificity.

BRAF V600E mutation analysis increases diagnostic accuracy for papillary thyroid carcinoma in fine needle aspiration biopsies

OBJECTIVE Papillary thyroid carcinoma (PTC) represents the majority of differentiated thyroid cancers, presenting the V600E activating BRAF mutation in 29-83% of cases. The aim of our study is to analyze the influence of BRAF mutation analysis on the diagnostic accuracy of fine-needle aspiration biopsy (FNAB) in patients with suspected PTC. DESIGN AND METHODS Thyroid cytoaspirates from 469 nodules (size: 1.1+/-0.8 cm) with ultrasonographic features suspicious of malignant lesion, performed in 374 patients, were submitted to cytological evaluation and to biomolecular analysis, carried out after somatic DNA isolation, specific PCR amplification, and subsequent automated direct sequencing. All PCR fragments were also processed by specific enzyme restriction analysis. RESULTS BRAF V600E mutation was found in 48 samples, 41 of which were also cytologically diagnosed as PTC, with histologic confirmation after thyroidectomy. Total thyroidectomy was perfomed also in seven patients with negative cytology but positive BRAF mutation, with histological confirmation of PTC in all. Among the 429 BRAF-negative samples, 407 had negative cytology for PTC, while 22 were diagnosed as suspected PTC and underwent total thyroidectomy with histological diagnosis of PTC in 17 and benign lesion in five. The prevalence of BRAF V600E mutation among histologically diagnosed PTC patients was 64%. Biomolecular analysis significantly increased cytology sensitivity for PTC from 77.3 to 86.7% (P<0.01). CONCLUSIONS These data indicate that BRAF V600E mutation analysis can significantly improve FNAB diagnostic accuracy. However, biomolecular analysis is complementary to cytology, which should always be performed.

Multiple endocrine neoplasia type 2 syndromes (MEN 2): results from the ItaMEN network analysis on the prevalence of different genotypes and phenotypes

OBJECTIVE Multiple endocrine neoplasia type 2 (MEN 2) is a genetic disease characterized by medullary thyroid carcinoma (MTC) associated (MEN 2A and 2B) or not familial MTC (FMTC) with other endocrine neoplasia due to germline RET gene mutations. The prevalence of these rare genetic diseases and their corresponding RET mutations are unknown due to the small size of the study population. METHODS We collected data on germline RET mutations of 250 families with hereditary MTC followed in 20 different Italian centres. RESULTS AND CONCLUSIONS The most frequent RET amino acid substitution was Val804Met (19.6%) followed by Cys634Arg (13.6%). A total of 40 different germline RET mutations were present. Six families (2.4%) were negative for germline RET mutations. The comparison of the prevalence of RET germline mutations in the present study with those published by other European studies showed a higher prevalence of Val804Met and Ser891Ala mutations and a lower prevalence of Leu790Phe and Tyr791Phe (P<0.0001). A statistically significant higher prevalence of mutations affecting non-cysteine codons was also found (P<0.0001). Furthermore, the phenotype data collection showed an unexpected higher prevalence of FMTC (57.6%) with respect to other MEN 2 syndromes (34% MEN 2A and 6.8% of MEN 2B). In conclusion, we observed a statistically significant different pattern of RET mutations in Italian MEN 2 families with respect to other European studies and a higher prevalence of FMTC phenotype. The different ethnic origins of the patients and the particular attention given to analysing apparently sporadic MTC for RET germline mutations may explain these findings.

Virologic and Immunologic Evidence Supporting an Association between HHV-6 and Hashimoto's Thyroiditis

Hashimotos thyroiditis (HT) is the most common of all thyroid diseases and is characterized by abundant lymphocyte infiltrate and thyroid impairment, caused by various cell- and antibody-mediated immune processes. Viral infections have been suggested as possible environmental triggers, but conclusive data are not available. We analyzed the presence and transcriptional state of human herpesvirus 6 (HHV-6) in thyroid fine needle aspirates (FNA) and peripheral blood mononuclear cells (PBMCs) from 34 HT patients and 28 controls, showing that HHV-6 DNA prevalence (82% vs. 10%, p≤0.001) and viral load were significantly increased in FNA from HT patients, and thyrocytes from HT FNA displayed a 100-fold higher HHV-6 DNA load compared to infiltrating lymphocytes. In addition, while HHV-6 was strictly latent in positive samples from controls, a low grade acute infection was detected in HT samples. HHV-6 variant characterization was carried out in 10 HT FNA samples, determining that all specimens harbored HHV-6 Variant A. The tropism of HHV-6 for thyroid cells was verified by infection of Nthy-ori3-1, a thyroid follicular epithelial cell line, showing that thyrocytes are permissive to HHV-6 replication, which induces de novo expression of HLA class II antigens. Furthermore, HHV-6-infected Nthy-ori3-1 cells become targets for NK-mediated killing, NK cells from HT patients show a significantly more efficient killing of HHV-6 infected thyroid cells than healthy controls, and HT patients have increased T-cell responses to HHV-6 U94 protein, associated to viral latency. These observations suggest a potential role for HHV-6 (possibly variant A) in the development or triggering of HT.

Evaluation of Hormonal Function in a Series of Incidentally Discovered Adrenal Masses

The discovery of an asymptomatic adrenal mass (incidentaloma) during the investigation of an unrelated condition is relatively common. In this study, we report the clinical, radiologic, and endocrine evaluation of 38 patients (22 women and 16 men aged 24 to 84 years) with adrenal incidentaloma (size, 1 to 12 cm). The patients underwent basal and dynamic evaluation of the hypothalamic-pituitary-adrenal (HPA) axis, renin-angiotensin-aldosterone system, and adrenomedullary function. Moreover, computed tomograpy (CT) scan and 131I-6beta-iodomethyl-19-norcholest-5(10)-en-3beta-ol(NP-59) and/or 131I-metaiodobenzylguanidine (MIBG) scintigraphy were performed. The endocrine evaluation indicated two cases of pheochromocytoma and four cases of preclinical Cushings syndrome, three of which underwent surgery with histologic diagnosis of two adrenocortical adenomas and one carcinoma. Low levels of serum dehydroepiandrosterone sulfate (DHEA-S), associated with a markedly increased 17-hydroxyprogesterone (17-OHP) response to a corticotropin (ACTH) test, were found in patients with incidentaloma. On the basis of endocrine and morphologic data, 13 patients underwent surgical treatment: five adrenocortical adenomas (two functioning), two pheochromocytomas, two ganglioneuromas, one cortisol-secreting adrenal carcinoma, one lymphangiomatous cyst, one myelolipoma, and one hemorrhage were found. Careful diagnostic assessment of incidentally discovered adrenal masses must be performed to exclude the presence of malignant and/or functioning lesions and to verify the possibility that patients with incidentaloma have a genetic or acquired deficit of adrenal steroidogenic activity.

A novel germline CDKN1B mutation causing multiple endocrine tumors: clinical, genetic and functional characterization

Multiple endocrine neoplasia (MEN) syndromes are characterized by tumors involving two or more endocrine glands. Two MEN syndromes have long been known: MEN1 and MEN2, caused by germline mutations in MEN1 or RET, respectively. Recently, mutations in CDKN1B, encoding the cyclin‐dependent kinase (Cdk) inhibitor p27, were identified in patients having a MEN1‐like phenotype but no MEN1 gene mutations. Currently, the molecular mechanisms mediating the role of p27 in tumor predisposition are ill defined. We here report a novel germline missense variant in CDKN1B (c.678C>T, p.P69L) found in a patient with multiple endocrine tumors. We previously reported a nonsense p27 mutation (c.692G>A, p.W76X) in two patients with MEN1‐like phenotype. Functional assays were used to characterize p27P69L and p27W76X in vitro. We show that p27P69L is expressed at reduced level and is impaired in both binding to Cdk2 and inhibiting cell growth. p27W76X, which is mislocalized to the cytoplasm, can no longer efficiently bind Cyclins‐Cdks, nor inhibit cell growth or induce apoptosis. In the patients tumor tissues, p27P69L associates with reduced/absent p27 expression and in one tumor with loss‐of‐heterozygosity. Our results extend previous findings of CDKN1B mutations in patients with MEN1‐related states and support the hypothesis of a tumor suppressor role for p27 in neuroendocrine cells.

Effect of Everolimus on Cell Viability in Nonfunctioning Pituitary Adenomas

CONTEXT Pituitary adenomas can cause specific syndromes due to hormone excess and/or determine sellar mass symptoms. Pituitary cell growth can sometimes be influenced by medical therapy, such as for somatotroph adenomas treated with somatostatin analogs or prolactinomas treated with dopaminergic drugs. However, nonfunctioning pituitary adenomas (NFAs) are still orphans of medical therapy. Everolimus (RAD001), a derivative of rapamycin, is a well-known immunosuppressant drug, which has been recently shown to have antineoplastic activity in several human cancers. OBJECTIVE The objective of the study was to investigate the possible antiproliferative effects of RAD001 in human NFAs. DESIGN We collected 40 NFAs that were dispersed in primary cultures, treated without or with 1 nm to 1 microm RAD001, 10 nm cabergoline, 10 nm SOM230 (a somatostatin receptor multiligand), and/or 50 nm IGF-I. Cell viability and apoptosis were evaluated after 48 h, and vascular endothelial growth factor (VEGF) secretion was assessed after an 8-h incubation. Somatostatin and dopamine subtype 2 receptor expression was investigated by quantitative PCR. RESULTS In 28 cultures (70%), Everolimus significantly reduced cell viability (by approximately 40%; P < 0.05 vs. control), promoted apoptosis (+30%; P < 0.05 vs. control), inhibited p70S6K activity (-20%), and blocked IGF-I proliferative and antiapoptotic effects. In selected tissues cotreatment with SOM230, but not cabergoline, exerted an additive effect. Everolimus did not affect VEGF secretion but blocked the stimulatory effects of IGF-I on this parameter. CONCLUSIONS Everolimus reduced NFA cell viability by inducing apoptosis, with a mechanism likely involving IGF-I signaling but not VEGF secretion, suggesting that it might represent a possible medical treatment of invasive/recurrent NFAs.