Neuroendocrine differentiation in prostate cancer correlates with overall prognosis and disease progression after androgen-deprivation therapy, although its specific mechanisms are currently poorly understood. A role of Notch pathway has been reported in determining neuroendocrine phenotype of normal and neoplastic tissues. The aim of this study was to analyze whether this pathway might affect neuroendocrine differentiation in prostate cancer. Human achaete-scute homolog 1 (hASH1), a pivotal member of the Notch pathway, was investigated in 80 prostate cancers selected and grouped according to chromogranin A immunohistochemistry, as follows: prostate cancers without neuroendocrine differentiation, untreated (25 cases); prostate cancers with neuroendocrine differentiation, untreated (40 cases); prostate cancers with previous androgen-deprivation therapy, all having neuroendocrine differentiation (15 cases). Human ASH1 protein was analyzed by immunohistochemistry, whereas the presence of hASH1 mRNA transcripts was investigated on paraffin material by real-time PCR. By immunohistochemistry, hASH1 was colocalized with chromogranin A in neuroendocrine cells of normal prostatic gland. It was absent in all but one prostate cancers without neuroendocrine differentiation, whereas it was positive in 25% of prostate cancers with neuroendocrine differentiation/ untreated, with a significant correlation with the extent of neuroendocrine features (P ¼ 0.02). Moreover, comparing untreated and treated prostate cancers with neuroendocrine differentiation, a positive association with androgen-deprivation therapy was observed (P ¼ 0.01). In prostate cancers with neuroendocrine differentiation, RNA analysis confirmed the association of higher transcript levels in androgen deprivationtreated compared with untreated patients (P ¼ 0.01). In addition, hASH1 mRNA analysis in microdissected chromogranin A-positive and chromogranin A-negative areas within the same tumor demonstrated a two-to sevenfold increase of hASH1 mRNA expression in chromogranin A-positive tumor cell populations.

Purpose:The predictive role of the quantification of thymidylate synthase (TS) in tumors treated with antifolate drugs, such as 5-fluorouracil (5-FU), has been extensively reported in a variety of human tumors. Neuroendocrine tumors (NET) represent potential targets of antifolate agents, but no data onTS expression level in these tumors are currently available. Experimental Design: A series of 116 NETs were collected, including 58 gastroenteropancreatic (GEP) and 58 lung NETs. In 24 well-differentiated GEP neuroendocrine carcinomas (WD-NEC), a 5-FU^based treatment was given. Total RNA was extracted from microdissected paraffin blocks. TS mRNA quantification was done by real-time PCR, whereas protein expression was evaluated by immunohistochemistry. Results: By means of both quantification by real-time PCR and immunohistochemistry, a higher TS expression in pulmonary small cell lung cancer and large cell NEC compared with typical and atypical carcinoids was observed (P < 0.01). Similarly, in GEP tumors, a higher TS expression in poorly differentiated carcinomas than both WD-NEC and benign tumors (P < 0.01) was found. In patients withWD-NEC treated with 5-FU, highTS mRNA levels were associated with shorter time to progression (P = 0.002) and overall survival (P = 0.04). This negative prognostic role was confirmed in multivariate analysis adjusting for major prognostic variables (P = 0.01). No association betweenTS mRNA and survival was observed in WD-NEC patients not receiving 5-FU. Conclusions: This study, for the first time, (a) reports the differential TS expression in the spectrum of NETs and (b) indicatesTS as a possible predictive marker of treatment efficacy inWD-NEC patients treated with 5-FU.

The hormone obestatin, which is derived from the same precursor as ghrelin and whose receptor(s) is still unrecognized, possesses a variety of metabolic/modulatory functions mostly related to food intake suppression and reduction of gastrointestinal motility. The distribution of obestatin in normal and neoplastic human tissues is poorly understood. We report that in fetal tissue samples, obestatin peptide was detected in the thyroid, pituitary, lung, pancreas and gastrointestinal tract, usually being co-localized with chromogranin A. In adult tissues, obestatin protein expression was restricted to pituitary, lung, pancreas and gastrointestinal tract and was co-localized strictly with ghrelin. By contrast, in endocrine tumours obestatin was expressed in a small fraction of thyroid, parathyroid, gastrointestinal and pancreatic neoplasms, in most cases with a focal immunoreactivity and co-localized with ghrelin. Messenger RNA levels of the specific fragments of ghrelin and obestatin were comparable in both normal and tumour samples, confirming that post-translational mechanisms rather than alternative splicing events lead to ghrelin/obestatin production. Finally, in TT and BON-1 cell lines obestatin induced antiproliferative effects at pharmacological doses, opposite to those observed with ghrelin. In summary, our data demonstrate that obestatin is produced by the same endocrine cells that express ghrelin in normal tissues from fetal to adult life, whereas, as compared to ghrelin, in neoplastic conditions it is down-regulated by post-translational modulation and shows potential antiproliferative properties in vitro.

Somatostatin (SS) and its synthetic analogs have a role in the treatment of neuroendocrine tumours both in terms of symptoms control and antiproliferative activities. These effects are mediated by five SS receptors, widely expressed in both human neuroendocrine and nonneuroendocrine tumours, which were demonstrated to be diagnostically and therapeutically valuable targets . Cortistatin (CST), a brain cortex peptide, partially homologous to SS and having similar functions is also expressed in peripheral tissues and tumours. CST binds all SS receptors, and, differently from SS, also the ghrelin receptor GHSR1a and the CST specific receptor MrgX2. The expression profile of CST is mostly restricted to neuroendocrine tumours (gastrointestinal, pancreas, lung, parathyroid, thyroid, adrenal). In these tumours, CST probably acts via the SS or ghrelin receptor, the MrgX2 receptor being absent. Thus, in comparison to SS analogs, CST synthetic analogs may represent additional diagnostic/therapeutic tools in those tumors expressing the receptors for SS, for ghrelin or for both peptides.

The differential diagnosis of adrenocortical carcinoma from adrenocortical adenoma is based on different pathological parameters, usually incorporated in scoring systems, which unfortunately lack a 100% sensitivity and specificity. Little is known on the molecular mechanisms leading to the malignant phenotype in adrenocortical tumors. Among other molecules, metalloproteinases were demonstrated to be implicated in malignant progression and metastatization of solid tumors, including endocrine ones. Therefore, we aimed to investigate metalloproteinases and their inhibitors expression in a series of 50 adrenocortical carcinomas and 50 control adrenocortical adenomas, diagnosed according to the Weiss histological criteria. Immunohistochemical results were scored by semiquantitative analysis and compared with clinicopathological parameters and outcome. Metalloproteinase type 2 gave the most significant result, being detected in neoplastic cells in 1/50 adrenocortical adenomas (2%) and 37/50 adrenocortical carcinomas (74%) (Po0.001), with a focal (score 1, o20% of positive cells-two-thirds of cases) or diffuse (score 2, 420% of positive cells-one-third of cases) pattern. In addition, diffuse (score 2) metalloproteinase type 2 protein expression, as compared to focal or negative immunostaining, correlated with shorter survival (Po0.02) and disease-free interval (P ¼ 0.05). No correlation was found comparing metalloproteinase type 2 expression and any clinicopathological parameter. Our data indicate that metalloproteinase type 2 immunohistochemical localization in tumor cells is significantly restricted to malignant adrenocortical tumors, with high specificity but low sensitivity. In addition, a strong metalloproteinase type 2 expression in adrenocortical carcinoma was for the first time recognized as an unfavorable prognostic factor. Modern Pathology (2006Pathology ( ) 19, 1563Pathology ( -1569Pathology ( . doi:10.1038 published online 15 September 2006 Keywords: adrenal cortex; carcinoma; differential diagnosis; immunohistochemistry; MMP-2 Adrenocortical carcinoma is a rare tumor of the adrenal cortex, which accounts for no more than 0.2% of all malignancies. Its incidence is approximately one per million with females affected more frequently than males. Two peaks of incidence are observed in early childhood and adults over age of 60 years. Up to two-thirds of cases have distant metastases at the time of diagnosis and the mortality exceeds 90% with a mean survival of less than 30 months.