We report the simultaneous occurrence of medullary thyroid carcinoma (MTC) and papillary thyroid carcinoma (PTC), presenting as spatially distinct and well-defined tumour components, in three cases. In the first patient, histology, immunohistochemistry and electron microscopy demonstrated an MTC in the one nodule and PTC in two additional lesions. Non-neoplastic thyroid parenchyma separated the three nodules. Metastasis from PTC was diagnosed in a regional lymph node. Genetic analysis of both tumour components showed a distinctive mutational pattern: in the MTC a Cys634Arg substitution in exon 11 of the RET gene and in the two PTC foci a Val600Glu substitution in exon 15 of the BRAF gene. The other two patients are members of a large multigenerational family affected with familial MTC due to a germline mutation of the RET gene (Ala891Ser). Both patients harboured, besides medullary cancer and C-cell hyperplasia, distinct foci of papillary thyroid cancer, which was positive for Val600Glu BRAF mutation. Review of the literature disclosed 18 similar lesions reported and allowed the identification of different patterns of clinical presentation and biological behaviour. So far, the pathogenesis of these peculiar cases of thyroid malignancy has been completely unknown, but an underlying common genetic drive has been hypothesised. This is the first report in which two mutations, in the RET and BRAF genes, have been identified in three cases of MTC/PTC collision tumour, thus documenting the different genetic origin of these two coexisting carcinomas.

Previous studies indicate that nuclear factor kappaB (NF-κB) transcription factor is deregulated and overexpressed in several human neoplasias. The aim of this study was to test the hypothesis that the NF-κB pathway may be involved in parathyroid tumorigenesis. For this purpose, we determined the level of NF-κB activity, evaluated as phosphorylation of the transcription subunit p65, its modulation by specific and non-specific agents and its impact on cyclin D1 expression. Phosphorylated p65 levels present in parathyroid neoplasias (n = 13) were significantly lower than those found in normal tissues (n = 3; mean optical density (OD) 0.19 ± 0.1 vs 0.4 ± 0.1, P = 0.007), but there was no significant difference between adenomas and secondary and multiple endocrine neoplasia type 1 (MEN1)-related hyperplasia. Conversely, MEN2A (Cys634Arg)-related parathyroid samples showed extremely high levels of phosphorylated p65 that exhibited a nuclear localization at immunohistochemistry (n = 3). Phosphorylated p65 levels negatively correlated with menin expression (r ² = 0.42, P = 0.05). Tumor necrosis factor-α (TNFα) caused a significant increase in phosphorylated p65 levels (183 ± 13.8% of basal) while calcium sensing receptor (CaR) agonists exerted a significant inhibition (19.2 ± 3.3% of basal). Although TNFα was poorly effective in increasing cyclin D1 expression, NF-κB blockade by the specific inhibitor BAY11-7082 reduced FCS-stimulated cyclin D1 by about 60%. Finally, the inhibitory effects of CaR and BAY11-7082 on cyclin D1 expression were not additive – by blocking NF-κB CaR activation did not induce a further reduction in cyclin D1 levels. In conclusion, the study demonstrated that in parathyroid tumors: (1) p65 phosphorylation was dramatically increased by RET constitutive activation and was negatively correlated with menin expression, (2) p65 phosphorylation was increased and reduced by TNFα and CaR agonists respectively, and (3) blockade of the NF-κB pathway caused a significant decrease in cyclin D1 expression.

Parathyroid carcinoma (PaC) is a rare cause of primary hyperparathyroidism. Though the loss of the oncosuppressor CDC73/HRPT2 gene product, parafibromin, has been involved in the hyperparathyroidism–jaw tumor syndrome and in a consistent set of sporadic PaCs, parathyroid carcinogenesis remains obscure. MicroRNAs are a new class of small, non-coding RNAs implicated in development of cancer, since their deregulation can induce aberrant expression of several target genes. The aim of the present study was to identify differentially expressed microRNAs in parathyroid cancers compared with normal tissues. We performed a TaqMan low-density array profiling of four parathyroid cancers harboring CDC73 inactivating mutations and negative for parafibromin immunostaining. Their microRNA profiling was compared with that of two normal parathyroid biopsies. Out of 362 human microRNAs assayed, 279 (77%) were successfully amplified. Fourteen and three microRNAs were significantly down- and over-expressed in parathyroid cancers respectively. Of these, miR-296 and miR-139 were down-regulated, and miR-503 and miR-222 were over-expressed with a null false discovery rate. Carcinomas could be discriminated from parathyroid adenomas by a computed score based on the expression levels of miR-296, miR-222, and miR-503 as miR-139 was similarly down-regulated in both cancers and adenomas. Finally, miR-296 and miR-222 levels negatively correlated with mRNA levels of the hepatocyte growth factor receptor-regulated tyrosine kinase substrate and p27/kip1 levels respectively. These results suggest the existence of an altered microRNA expression pattern in PaCs together with a potential role of miR-296 as novel oncosuppressor gene in these neoplasia.

Somatostatin analogs currently used in the treatment of acromegaly and other neuroendocrine tumors inhibit hormone secretion and cell proliferation by binding to somatostatin receptor type (SST) 2 and 5. The antiproliferative pathways coupled to these receptors have been only partially characterized. The aim of this study was to evaluate the effect of octreotide and super selective SST2 (BIM23120) and SST5 (BIM23206) analogs on apoptotic activity and apoptotic gene expression in human somatotroph tumor cells. Eight somatotroph tumors expressing similar levels of SST2 and SST5 evaluated by real-time PCR and western blot analyses were included in the study. In cultured cells obtained from these tumors, octreotide induced a dose-dependent increase of caspase-3 activity (160 ± 20% vs basal at 10 nM) and cleaved cytokeratin 18 levels (172 ± 25% vs basal) at concentrations higher than 0.1 nM. This effect was due to SST2 activation since BIM23120 elicited comparable responses, while BIM23206 was ineffective. BIM23120-stimulated apoptosis was dependent on phosphatases, since it was abrogated by the inhibitor orthovanadate, and independent from the induction of apoptosis-related genes, such as p53, p63, p73, Bcl-2, Bax, BID, BIK, TNFSF8, and FADD. In somatotroph tumors, both BIM23120 and BIM2306 caused growth arrest as indicated by the increase in p27 and decrease in cyclin D1 expression. In conclusion, the present study showed that octreotide-induced apoptosis in human somatotroph tumor cells by activating SST2. This effect, together with the cytostatic action exerted by both SST2 and SST5 analogs, might account for the tumor shrinkage observed in acromegalic patients treated with long-acting somatostatin analogs.

Recently, a somatic point mutation of the B-RAF gene (V600E) has been identified as the most common genetic event in papillary thyroid carcinoma (PTC), with a prevalence variable among different series. Since discordant data on the clinico-pathologic features of B-RAF mutated PTC are present in the literature, the aim of the present co-operative study was to establish the prevalence of this genetic alteration and to perform a genotype–phenotype correlation in a large cohort of patients with PTC. To this purpose, a series of 260 sporadic PTCs with different histological variants were included in the study. The mutational analysis of the B-RAF gene was performed either by RT-PCR followed by single-stranded conformational polymorphism or by PCR and direct sequencing. Statistical analyses were obtained by means of χ²/Fisher’s exact test and t-test. Overall, a heterozygous T > A transversion at nucleotide 1799 (V600E) was found in 99 out of 260 PTCs (38%). According to the histological type of the tumor, the B-RAF ^V600E mutation was present in 48.3% of cases of classic PTCs (85 out of 176), in 17.6% (nine out of 51) of follicular variants of PTCs, in 21.7% (five out of 23) in other PTC variants and in none of the ten poorly differentiated tumors. B-RAF ^V600E was significantly associated with the classic variant of PTC (P = 0.0001) and with an older age at diagnosis (P = 0.01). No statistically significant correlation was found among the presence of B-RAF ^V600E and gender, tumor node metastasis (TNM), multicentricity of the tumor, stage at diagnosis and outcome. In conclusion, the present study reports the prevalence of B-RAF ^V600E (38%) in the largest series of sporadic PTCs, including 260 cases from three different Italian referring centers. This prevalence is similar to that calculated by pooling together all data previously reported, 39.6% (759 out of 1914 cases), thus indicating that the prevalence of this genetic event lies around 38–40%. Furthermore, B-RAF ^V600E was confirmed to be associated with the papillary growth pattern, but not with poorer differentiated PTC variants. A significant association of B-RAF mutation was also found with an older age at diagnosis, the mutation being very rare in childhood and adolescent PTCs. Finally, no correlation was found with a poorer prognosis and a worse outcome after a median follow-up of 72 months.