Impact of long-acting octreotide in patients with early-stage MEN1-related duodeno-pancreatic neuroendocrine tumours.

BACKGROUND: Somatostatin analogues (SSA) represent one of the main therapeutic option in patients affected with functioning well-differentiated neuroendocrine tumours (NETs). There are no studies specifically focusing on NETs associated with Multiple Endocrine Neoplasia type 1 (MEN1). AIM: To evaluate the efficacy of the long-acting SSA octreotide in MEN1 patients with early-stage duodeno-pancreatic NETs. PATIENTS AND METHODS: Forty patients with MEN1 were retrospectively evaluated. Twenty patients with evidence of one or more MEN1-related duodeno-pancreatic NETs < 20 mm in size (age range 26-61 years) were treated with octreotide long-acting octreotide (LAR) as first-line therapy. Treatment duration ranged 12-75 months. At the baseline radiological evaluation, multiple duodeno-pancreatic NETs (range 1-8, size 3-18 mm) were detected. RESULTS: An objective tumour response was observed in 10%, stable disease in 80% and progression of disease in 10% of cases. In six patients with abnormally increased CgA, gastrin and/or insulin serum concentrations, a significant clinical and hormonal response occurred in 100% of cases and was stable along the time. CONCLUSIONS: Therapy with SSA is highly safe and effective in patients with early-stage MEN1 duodeno-pancreatic NETs, resulting in long-time suppression of tumour and hormonal activity and 10% objective response. This suggests to early start therapy with SSA in patients with MEN1-related NETs.

A specific DNA methylation profile correlates with a high risk of disease progression in stage I classical (Alibert-Bazin type) mycosis fungoides.

BACKGROUND: Mycosis fungoides (MF) is the most common type of cutaneous T-cell lymphoma; in its classical presentation it evolves slowly, but it can have an aggressive course in a subset of patients. OBJECTIVES: To investigate the impact of epigenetic mechanisms on the progression of early stage MF. METHODS: We analysed DNA methylation at 12 different loci and long interspersed nucleotide elements-1 (LINE-1), as a surrogate marker of global methylation, on tissue samples from 41 patients with stage I MF followed up for at least 12 years or until disease progression. The methylation profiles were also analysed in two T-cell lymphoma cell lines and correlated with gene expression. RESULTS: The selected loci were methylated in a tumour-specific manner; concomitant hypermethylation of at least four loci was more frequent in cases progressing within 1-3 and 3-6 years than in late-progressive or non-progressive cases. LINE-1 methylation was significantly lower in rapidly progressive MF at 3 years (61%, P < 0·001) than in those at 12 years (67%). PPARG, SOCS1 and NEUROG1 methylation showed remarkable differences among the prognostic groups, but only PPARG was a significant predictor of disease progression within 6 years, after adjustment for patients' age or gender. Strikingly, a methylation profile similar to progressive cases was found in highly proliferative Sézary-derived HUT78 cells but not in MF-derived HUT102 cells. Exposure to a DNA demethylating agent restored sensitivity to apoptosis and cell cycle arrest. CONCLUSIONS: Epigenetic silencing of specific biomarkers can predict the risk of disease progression in early-stage MF, providing insights into its pathogenesis, prognosis and therapy.

Multiple endocrine neoplasia, the old and the new: a mini review.

Multiple endocrine neoplasia syndromes have since been classified as types 1 and 2, each with specific phenotypic patterns. MEN1 is usually associated with pituitary, parathyroid and paraneoplastic neuroendocrine tumours. The hallmark of MEN2 is a very high lifetime risk of developing medullary thyroid carcinoma (MTC) more than 95% in untreated patients. Three clinical subtypesdMEN2A, MEN2B, and familial MTC (FMTC) have been defined based on the risk of pheochromocytoma, hyperparathyroidism, and the presence or absence of characteristic physical features). MEN2 occurs as a result of germline activating missense mutations of the RET (REarranged during Transfection) proto-oncogene. MEN2-associated mutations are almost always located in exons 10, 11, or 13 through 16. Strong genotype-phenotype correlations exist with respect to clinical subtype, age at onset, and aggressiveness of MTC in MEN2. These are used to determine the age at which prophylactic thyroidectomy should occur and whether screening for pheochromocytoma or hyperparathyroidism is necessary. Specific RET mutations can also impact management in patients presenting with apparently sporadic MTC. Therefore, genetic testing should be performed before surgical intervention in all patients diagnosed with MTC. Recently, Pellegata et al. have reported that germline mutations in CDKN1B can predispose to the development of multiple endocrine tumours in both rats and humans and this new MEN syndrome is named MENX and MEN4, respectively. CDKN1B. A recent report showed that in sporadic MTC, CDKN1B V109G polymorphism correlates with a more favorable disease progression than the wild-type allele and might be considered a new promising prognostic marker. New insights on MEN syndrome pathogenesis and related inherited endocrine disorders are of particular interest for an adequate surgical and therapeutic approach.

A novel germline mutation in peroxisome proliferator-activated receptor gamma gene associated with large intestine polyp formation and dyslipidemia.

We report a novel PPARG germline mutation in a patient affected by colorectal cancer that replaces serine 289 with cysteine in the mature protein (S289C). The mutant has impaired transactivation potential and acts as dominant negative to the wild type receptor. In addition, it no longer restrains cell proliferation both in vitro and in vivo. Interestingly, the S289C mutant poorly activates target genes and interferes with the inflammatory pathway in tumor tissues and proximal normal mucosa. Consistently, only mutation carriers exhibit colonic lesions that can evolve to dysplastic polyps. The proband presented also dyslipidemia, hypertension and overweight, not associated to type 2 diabetes; of note, family members tested positive for the mutation and display only a dyslipidemic profile at variable penetrance with other biochemical parameters in the normal range. Finally, superimposing the mutation to the crystal structure of the ligand binding domain, the new Cys289 becomes so closely positioned to Cys285 to form an S-S bridge. This would reduce the depth of the ligand binding pocket and impede agonist positioning, explaining the biological effects and subcellular distribution of the mutant protein. This is the first PPARG germline mutation associated with dyslipidemia and colonic polyp formation that can progress to full-blown adenocarcinoma.

Mutations in the extracellular domain cause RET loss of function by a dominant negative mechanism.

The RET proto-oncogene encodes a tyrosine kinase receptor expressed in neuroectoderm-derived cells. Mutations in specific regions of the gene are responsible for the tumor syndromes multiple endocrine neoplasia types 2A and 2B (MEN 2A and 2B), while mutations along the entire gene are involved in a developmental disorder of the gastrointestinal tract, Hirschsprung's disease (HSCR disease). Two mutants in the extracellular domain of RET, one associated with HSCR disease and one carrying a flag epitope, were analyzed to investigate the impact of the mutations on RET function. Both mutants were impeded in their maturation, resulting in the lack of the 170-kDa mature form and the accumulation of the 150-kDa immature form in the endoplasmic reticulum. Although not exposed on the cell surface, the 150-kDa species formed dimers and aggregates; this was more pronounced in a double mutant bearing a MEN 2A mutation. Tyrosine phosphorylation and the transactivation potential were drastically reduced in single and double mutants. Finally, in cotransfection experiments both mutants exerted a dominant negative effect over protoRET and RET2A through the formation of a heteromeric complex that prevents their maturation and function. These results suggest that HSCR mutations in the extracellular region cause RET loss of function through a dominant negative mechanism.

DNA methylation variations are required for epithelial-to-mesenchymal transition induced by cancer-associated fibroblasts in prostate cancer cells.

Widespread genome hypo-methylation and promoter hyper-methylation of epithelium-specific genes are hallmarks of stable epithelial-to-mesenchymal transition (EMT), which in prostate cancer (PCa) correlates with castration resistance, cancer stem cells generation, chemoresistance and worst prognosis. Exploiting our consolidated 'ex-vivo' system, we show that cancer-associated fibroblasts (CAFs) released factors have pivotal roles in inducing genome methylation changes required for EMT and stemness in EMT-prone PCa cells. By global DNA methylation analysis and RNA-Seq, we provide compelling evidence that conditioned media from CAFs explanted from two unrelated patients with advanced PCa, stimulates concurrent DNA hypo- and hyper-methylation required for EMT and stemness in PC3 and DU145, but not in LN-CaP and its derivative C4-2B, PCa cells. CpG island (CGI) hyper-methylation associates with repression of genes required for epithelial maintenance and invasion antagonism, whereas activation of EMT markers and stemness genes correlate with CGI hypo-methylation. Remarkably, methylation variations and EMT-regulated transcripts almost completely reverse qualitatively and quantitatively during MET. Unsupervised clustering analysis of the PRAD TCGA data set with the differentially expressed (DE) and methylated EMT signature, identified a gene cluster of DE genes defined by a CAF+ and AR- phenotype and worst diagnosis. This gene cluster includes the relevant factors for EMT and stemness, which display DNA methylation variations in regulatory regions inversely correlated to their expression changes, thus strongly sustaining the ex-vivo data. DNMT3A-dependent methylation is essential for silencing epithelial maintenance and EMT counteracting genes, such as CDH1 and GRHL2, that is, the direct repressor of ZEB1, the key transcriptional factor for EMT and stemness. Accordingly, DNMT3A knock-down prevents EMT entry. These results shed light on the mechanisms of establishment and maintenance of coexisting DNA hypo- and hyper-methylation patterns during cancer progression, the generation of EMT and cell stemness in advanced PCa, and may pave the way to new therapeutic implications.

The miR-27a-calreticulin axis affects drug-induced immunogenic cell death in human colorectal cancer cells.

Immunogenic cell death (ICD) evoked by chemotherapeutic agents implies emission of selected damage-associated molecular patterns (DAMP) such as cell surface exposure of calreticulin, secretion of ATP and HMGB1. We sought to verify whether miR-27a is implicated in ICD, having demonstrated that it directly targets calreticulin. To this goal, we exposed colorectal cancer cell lines, genetically modified to express high or low miR-27a levels, to two bona fide ICD inducers (mitoxantrone and oxaliplatin). Low miR-27a-expressing cells displayed more ecto-calreticulin on the cell surface and increased ATP and HMGB1 secretion than high miR-27a-expressing ones in time-course experiments upon drug exposure. A calreticulin target protector counteracted the miR-27a effects while specific siRNAs mimicked them, confirming the results reported. In addition, miR-27a negatively influenced the PERK-mediated route and the late PI3K-dependent secretory step of the unfolded protein response to endoplasmic reticulum stress, suggesting that miR-27a modulates the entire ICD program. Interestingly, upon chemotherapeutic exposure, low miR-27a levels associated with an earlier and stronger induction of apoptosis and with morphological and molecular features of autophagy. Remarkably, in ex vivo setting, under the same chemotherapeutic induction, the conditioned media from high miR-27a-expressing cells impeded dendritic cell maturation while increased the secretion of specific cytokines (interleukin (IL)-4, IL-6, IL-8) and negatively influenced CD4(+) T-cell interferon γ production and proliferation, all markers of a tumor immunoevasion strategy. In conclusion, we provide the first evidence that miR-27a impairs the cell response to drug-induced ICD through the regulatory axis with calreticulin.

Proteomic screening identifies calreticulin as a miR-27a direct target repressing MHC class I cell surface exposure in colorectal cancer.

Impairment of the immune response and aberrant expression of microRNAs are emerging hallmarks of tumour initiation/progression, in addition to driver gene mutations and epigenetic modifications. We performed a preliminary survey of independent adenoma and colorectal cancer (CRC) miRnoma data sets and, among the most dysregulated miRNAs, we selected miR-27a and disclosed that it is already upregulated in adenoma and further increases during the evolution to adenocarcinoma. To identify novel genes and pathways regulated by this miRNA, we employed a differential 2DE-DIGE proteome analysis. We showed that miR-27a modulates a group of proteins involved in MHC class I cell surface exposure and, mechanistically, demonstrated that calreticulin is a miR-27a direct target responsible for most downstream effects in epistasis experiments. In vitro miR-27a affected cell proliferation and angiogenesis; mouse xenografts of human CRC cell lines expressing different miR-27a levels confirmed the protein variations and recapitulated the cell growth and apoptosis effects. In vivo miR-27a inversely correlated with MHC class I molecules and calreticulin expression, CD8(+) T cells infiltration and cytotoxic activity (LAMP-1 exposure and perforin release). Tumours with high miR-27a, low calreticulin and CD8(+) T cells' infiltration were associated with distant metastasis and poor prognosis. Our data demonstrate that miR-27a acts as an oncomiRNA, represses MHC class I expression through calreticulin downregulation and affects tumour progression. These results may pave the way for better diagnosis, patient stratification and novel therapeutic approaches.

Pro12Ala substitution in the peroxisome proliferator-activated receptor-gamma2 is not associated with type 2 diabetes.

Peroxisome proliferator-activated receptor (PPAR)-gamma is a major regulator of adipogenesis and insulin sensitivity. The PPAR-gamma gene generates two isoforms through alternative splicing, PPAR-gamma1 and -gamma2, the latter having an additional stretch of 28 amino acids at its NH2-terminus in the ligand-independent activation domain. This extension renders PPAR-gamma2 more sensitive to insulin action. Since there is a Pro12Ala substitution in this domain, we tested whether it is related to type 2 diabetes or insulin resistance. Therefore, 131 type 2 diabetic patients and 312 normoglycemic control subjects were screened for the presence of the mutation and for major clinical and metabolic features. The frequency of the mutation did not differ significantly between diabetic patients and control subjects. BMI, insulin, and other metabolic and anthropometric variables were also not associated with the mutation. Although the study was carried out on a sufficiently large sample, the conclusions do not support a major role for the Pro12Ala substitution of the PPAR-gamma gene in the etiology of type 2 diabetes.

Extinction of insulin-like growth factor II gene expression in intratypic hybrids of rat liver cells.

The expression of the developmentally regulated insulin-like growth factor II (IGF-II) gene has been studied in somatic cell hybrids derived from rat liver cells. BRL3A cells, dedifferentiated variants of rat hepatocytes, producing high levels of IGF-II, were fused to BRL30E or FAO cells of the same embryonic lineage but not expressing detectable levels of IGF-II mRNA. We report here that the IGF-II gene is subject to extinction, since its specific RNA levels are decreased both in heterokaryons and stable cell hybrids. Transcriptional analysis in isolated nuclei from parental and hybrid cells showed that the IGF-II gene is transcribed at a similar rate in all cell types. Likewise, the stability of IGF-II cytoplasmic mRNA was equivalent in the high-expressing parental cells and in the hybrids. In contrast, the distribution of IGF-II mRNA between the nuclear and the cytoplasmic compartments differed markedly in parental and hybrid cell lines. The data presented show that the expression of the IGF-II gene is subject to a dominant negative control and suggest that the phenomenon involves mechanisms that operate at the posttranscriptional level.

Retinol mobilization from cultured rat hepatic stellate cells does not require retinol binding protein synthesis and secretion.

Retinol mobilization from retinyl esters stores of hepatic stellate cells (HSCs) is a key step in the regulation of mammalian retinol homeostasis, but the precise mechanisms of such a mobilization are still poorly understood. Using primary cultures of HSCs, we first demonstrated that HSCs expressed immunoreactivity against retinol-binding-protein (RBP) when cultured in a medium containing RBP but were unable to synthesize RBP transcripts and proteins. Using pulse and chase-type experiments, we demonstrated that radioactive retinol was released in culture medium without binding proteins. Inhibition of protein secretion by brefeldin A did not modify quantitatively retinol release. This data ruled out, for the first time, the direct involvement of RBP in retinol mobilization from HSCs. Moreover, HSCs co-cultured with primary isolated hepatocytes displayed an increase of retinol transfer from HSCs to hepatocytes when they established direct physical contacts, as compared with co-cultures without contact. Based on this latter data, a mechanism of retinol mobilization from HSCs via the hepatocytes using retinol transfer through cellular membranes is proposed.

3,5-diiodo-L-thyronine regulates glucose-6-phosphate dehydrogenase activity in the rat.

Thyroid hormones influence the activity of lipogenic enzymes such as malic enzyme (ME) and glucose-6-phosphate dehydrogenase (G6PD). The effect of T3 on ME is exerted at the transcriptional level, but it is unclear if its effect on G6PD is also nuclear mediated. Furthermore, other iodothyronines that have been shown to possess biological activity (such as diiodothyronines) could contribute to this enzyme's regulation. In this study the effects of 3,5-diiodothyronine (T2) on the aforementioned enzymes were examined and compared with those of T3. Rats made hypothyroid by propylthiouracil and iopanoic acid treatment were used throughout. Enzyme activities were determined spectrophotometrically, and G6PD messenger RNA (mRNA) expression was analyzed by Northern blotting using a human G6PD complementary DNA probe. Injections of T2 to hypothyroid animals significantly enhanced the activity of both enzymes. The effect of T2 on ME was nuclear mediated and mimicked the effect of T3. The effects of T2 and T3 on G6PD differed. Injection of T3 into hypothyroid rats induced an increase in both enzyme activity and G6PD mRNA expression, indicating a nuclear-mediated effect. The effect of T2 on G6PD activity, on the other hand, was not nuclear mediated. The injection of T2 into hypothyroid animals did not change G6PD mRNA expression, and the strong increase in the enzyme's activity (from +70% to +300%) was unaffected by simultaneous injection of protein synthesis inhibitors. As the lowest dose of 1 microg T2/100 g BW affects G6PD activity 3-5 times more than the same dose of T3, these data provide the first evidence that T2 is a factor capable of regulating G6PD activity.

Loss of heterozygosity at the RET protooncogene locus in a case of multiple endocrine neoplasia type 2A.

We describe a patient affected by multiple endocrine neoplasia type 2A (MEN 2A) bearing a heterozygous germline mutation (Cys(634)Arg) in exon 11 and an additional somatic mutation of the RET protooncogene. A large intragenic deletion, spanning exon 4 to exon 16, affected the normal allele and was detected by quantitative PCR, Southern blot analysis, and screening of several polymorphic markers. This deletion causes RET loss of heterozygosity exclusively in the metastasis, thus suggesting a role for this second mutational event in tumor progression. No additional mutations were found in the other exons analyzed. We provide the first evidence that RET, a dominant oncogene, is affected by a germline mutation and by an additional somatic deletion of the wild-type allele. This unusual genetic profile may be related to the clinical course and very poor outcome.

A novel case of multiple endocrine neoplasia type 2A associated with two de novo mutations of the RET protooncogene.

We report a novel case of multiple endocrine neoplasia type 2A (MEN 2A) associated with two mutations of the protooncogene RET. One affects codon 634 and causes a cysteine to arginine substitution; the second at codon 640 causes an alanine to glycine substitution in the transmembrane region. The two mutations were present on the same RET allele and were detected in germline and tumor DNA. Both mutations were de novo, i.e. they were not found in the DNA of the parents or relatives. Immunohistochemical and RT-PCR analysis showed that the pheochromocytoma expressed calcitonin as well as both RET alleles. A cell line established from the tumor and propagated in culture sustained the expression of RET and calcitonin, as did the original pheochromocytoma. Because the patient presented with medullary thyroid carcinoma and pheochromocytoma without parathyroid gland involvement, we speculate that this clinical picture could be correlated with the two RET mutations and to the unusual calcitonin production. This is the first report of a MEN 2A case due to two mutations of the RET gene and associated with a calcitonin-producing pheochromocytoma.

A novel point mutation in the intracellular domain of the ret protooncogene in a family with medullary thyroid carcinoma.

Specific mutations in the ret protooncogene have been found associated with multiple endocrine neoplasia type 2A (MEN 2A) and type 2B (MEN 2B) and familial medullary thyroid carcinoma (FMTC). Mutations in one of five cysteine residues in the extracellular domain have been found in over 95% of families with MEN 2A and 88% of families with FMTC. In MEN 2B patients, a specific mutation at codon 918, substituting a threonine for a methionine, has been found in 95% of cases. In FMTC, in addition to the mutations of the extracellular cysteines, three intracellular base pair changes have been reported at codons 768 and 804. Here we describe a novel intracellular mutation in exon 15 of the ret gene that leads to the substitution of an alanine for a serine at codon 891 in a family with medullary thyroid carcinoma. This amino acid change may be important in determining substrate specificity or, alternatively, may play a role in ATP binding.

Frequent RET protooncogene mutations in multiple endocrine neoplasia type 2A.

The occurrence of mutations in the RET protooncogene has been investigated in 12 multiple endocrine neoplasia type 2A families and 18 cases of sporadic thyroid medullary carcinomas and pheochromocytomas. Ten of 12 families showed single base substitutions in the RET protooncogene exons 10 and 11, coding for the extracellular domain of the protein. Tumor tissues from 2 multiple endocrine neoplasia type 2A patients were analyzed at the DNA and ribonucleic acid levels and revealed the same heterozygous mutations found in the peripheral blood lymphocytes. This demonstrates that both the normal and mutant alleles are expressed. No mutations in these exons were detected in the 18 cases of sporadic tumors investigated. These data provided further evidence that the mutated RET protooncogene acts in a dominant fashion and is responsible for the pathogenesis of this syndrome.

A primary, lateral-cervical medullary thyroid carcinoma: a case report.

Medullary thyroid carcinoma (MTC) arises from the parafollicular cells of the thyroid and occurs in a sporadic or in an inherited form. We present a case of an aberrant MTC in a patient with a functioning thyroid gland. At surgical dissection, the thyroid was present in its anatomical site with a nodule in the upper one third of the right lobe. A mass was also found in a lateral-cervical position distinct from the thyroid gland. Histological examination showed the mass to be the primary MTC, whereas the thyroid nodule was a follicular adenoma. Analysis of DNA extracted from the MTC, from the adenoma, and from peripheral blood revealed a mutation within exon 16 of the RET proto-oncogene only in the DNA from the tumor. The reported case represents a sporadic MTC in an aberrant localization, probably originating from a developmental abnormality of the primordial C cells. This event might have occurred during the migration and/or differentiation of the C cells and might be related to, or caused by, the mutated RET proto-oncogene.

Changes in tissue transglutaminase activity and expression during retinoic acid-induced growth arrest and apoptosis in primary cultures of human epithelial prostate cells.

We treated primary epithelial cells from human normal prostate (NEPC) and prostate cancer (CEPC) with all-trans-retinoic acid (RA) to study whether it regulates the activity of tissue transglutaminase (tTGase), an enzyme that accumulates in cells undergoing apoptosis. tTGase activity was assessed by [14C]spermidine incorporation; tTGase, P53, Bcl-2, and p21 protein levels were evaluated by Western blotting; and RA receptors (RAR alpha, -beta, and -gamma), tTGase, retinol-binding protein (RBP), and cellular RBP type I transcripts were determined by semiquantitative RT-PCR. After 72-96 h of 10(-6) mol/L RA treatment, cell growth inhibition and apoptosis were associated with increased tTGase activity in both NEPC and CEPC, and with increased tTGase protein and messenger ribonucleic acid levels only in NEPC. Moreover, RA down-regulated RAR alpha and -beta and increased RBP messenger ribonucleic acid levels in NEPC, whereas it increased RAR beta gene expression and decreased Bcl-2 protein levels in CEPC. Our results suggest that RA induces tTGase gene expression and enzyme activity in normal prostate cells, and that RA-regulated pathways are impaired in cancer cells. Moreover, down-regulation of Bcl-2 protein and up-regulation of RAR beta suggest that retinoid may act on the genetic defect responsible for prostate cancer progression.

Fenofibrate prevents and reduces body weight gain and adiposity in diet-induced obese rats.

Fibrates are hypolipidemic drugs that activate the peroxisome proliferator-activated receptors. Since fibrates may also increase energy expenditure, we investigated whether fenofibrate (FF) had this effect in diet-induced obese rats. A 2-month administration of a high-fat palatable diet to adult rats increased body weight by 25% and white adipose mass by 163% compared with a standard diet. These effects were prevented by FF, both when administered for the 2 months of high-fat feeding and when given for only the second month. Consequently, FF-treated rats had a final body weight and white adipose tissue mass similar to untreated animals on the standard diet. FF also increased resting metabolic rate, hepatic peroxisomal and mitochondrial palmitoyl-dependent oxygen uptake and mRNA levels of acyl-CoA oxidase and lipoprotein lipase. Finally, FF lowered mRNA levels of uncoupling protein-2 and did not affect mitochondrial respiration in skeletal muscle. Therefore, FF seems to act as a weight-stabilizer mainly through its effect on liver metabolism.

Germline and somatic mutations of the RET proto-oncogene in apparently sporadic medullary thyroid carcinomas.

Medullary thyroid carcinomas (MTC) occur sporadically or as part of inherited multiple endocrine neoplasia (MEN) type 2 syndromes. To recognize misdiagnosed familial cases and to establish the frequency of somatic mutations, a series of 50 patients, clinically diagnosed with sporadic MTC, were analyzed for mutations in the RET proto-oncogene. The clinical management of the patient and of the family is different in the two cases. Germline mutations were detected in three independent cases, demonstrating that they were associated to familial MTC. The mutations affected exon 11 in two cases and exon 14 in one case. Somatic mutations were detected in eight patients (30%) and they were indicative of sporadic MTC. In seven cases the mutation affected codon 918 of exon 16 and in one case codon 634 in exon 11. No RET mutations were detected in the remaining patients. A different genetic and clinical management is proposed for individuals with a diagnosis of familial or sporadic MTC.