Andrea Renzullo

Effects of somatostatin analog SOM230 on cell proliferation, apoptosis, and catecholamine levels in cultured pheochromocytoma cells

Surgery is the primary therapy for pheochromocytoma (PHEO), a catecholamine-producing tumor. Benign and malignant PHEO could develop recurrences, and the intraoperative risk of recurrent PHEO is an important unresolved issue. Non-surgical treatments of PHEO recurrence would therefore better prepare patients for reintervention as well as provide them with palliative management. We investigated the effects of the new somatostatin analog (pasireotide) SOM230 versus octreotide (OCT) in primary PHEO cell cultures (Pheo-c). Pheo-c from six benign surgical samples were set up and characterized by immunocytochemistry. Real-time PCR, using both PHEO tissues and Pheo-c, showed different levels of somatostatin receptor(1-5) mRNA expression. Cells treated with various doses of OCT or SOM230 for 48 and 72 h were analyzed to assess their effects on cell proliferation and apoptosis and catecholamine levels. Even if reduction of cell viability was observed in Pheo-c treated for 48 h with either OCT or SOM230 and this effect increased after 72 h, a more significant inhibition of cell growth as well as a significantly higher induction of apoptosis was seen in Pheo-c treated with SOM230 versus OCT. In particular, apoptosis in Pheo-c was detected after 48 h and was associated with increased expression and activation of caspase-3 and cleaved poly(ADP-ribose) polymerase. OCT 10(-6) M and SOM230 10(-7) M significantly reduced catecholamine levels. Our results indicate that while both OCT and SOM230 modulate cell growth and apoptosis and catecholamine levels in Pheo-c through specific receptors, SOM230 is more effective. This improves our knowledge on the mechanism of SOM230 action in PHEO and supports a possible therapeutic use in benign PHEO recurrence.

Cardiovascular abnormalities in Klinefelter Syndrome

BACKGROUND Several epidemiological studies have demonstrated an increased mortality from cardiovascular causes in patients with Klinefelter Syndrome (KS). Little information is available about the nature of the underlying cardiovascular abnormalities. Aim of the study was to investigate exercise performance, left ventricular architecture and function, vascular reactivity, and carotid intima-media thickness in a group of patients with KS. MATERIALS AND METHODS Sixty-nine patients with KS and 48 age-matched controls participated in our population-controlled study. Forty-eight Klinefelter subjects were on testosterone treatment at the time of the investigation while 21 were naive and underwent a complete Doppler echocardiographic examination, a cardiopulmonary exercise test as well as a vascular study including measures of carotid intima-media thickness and endothelial function with flow-mediated dilation of the brachial artery. Patients with KS on testosterone therapy (n=48) were also matched against a population of men with treated secondary hypogonadism (n=21). RESULTS Patients with KS exhibited a wide array of cardiovascular abnormalities including left ventricular diastolic dysfunction, reduced maximal oxygen consumption (p<0.01), increased intima-media thickness (p<0.05) (-34% and +42% vs. controls, respectively) and a high prevalence of chronotropic incompetence (55% of patients, p<0.01). No significant difference was found between treated and untreated KS in variance with men treated for secondary hypogonadism. CONCLUSION Left ventricular diastolic dysfunction, impaired cardiopulmonary performance, chronotropic incompetence, and increased intima-media thickness suggest that cardiovascular abnormalities are a common finding in KS that is not reversed by testosterone replacement therapy and may represent the pathophysiological underpinnings of the increased risk of dying from heart disease.

Lessons to be learned from the clinical management of a MEN 2A patient bearing a novel 634/640/700 mutation of the RET proto‐oncogene

A cluster of germline gain-of-function mutations of the RET proto-oncogene are responsible for Multiple Endocrine Neoplasia type 2A (MEN 2A), an autosomal dominant, inherited disorder characterized by medullary thyroid carcinoma (MTC), phaeochromocytoma (Phaeo) and hyperparathyroidism. Genetic screening of MEN 2A patients has been available for the past decade and useful genotype–phenotype correlations have been established: specific RET mutations are associated with age at first diagnosis and tumour aggressiveness. Accordingly, MEN 2A patients can be stratified into three risk groups depending on the RET mutation. Management uncertainties remain regarding patients bearing uncommon RET mutations or genetic variations for which mutation-specific risk profiles and treatment recommendations are unavailable. Here we report the thirteen-year clinical and surgical follow-up of a patient with MEN 2A bearing three de novo RET mutations at codons 634, 640 and 700 (p. C634R, p.A640G and p.M700L) in exon 11: a combination of mutations which has not previously been described. In May 1998, a 26-year-old female patient was admitted to our unit because of recurrent episodes of hypertension, tachycardia and headache. She had recently been diagnosed with a MTC which had been removed surgically, with associated cervical central lymphectomy. Elevated urinary catecholamine levels and imaging examinations showing a left adrenal mass were suggestive of a Phaeo. Hyperplasia of the contralateral gland was also detected. The patient underwent left “open” adrenalectomy 3 months later; post-operative catecholamine levels decreased to normal values, while the serum calcitonin remained elevated (Fig. 1). The diagnosis of MEN 2A was confirmed by testing the patient’s DNA for RET mutations. Two heterozygous germline mutations were identified: a transition at position c.1900 replaced a T with a C and a transversion at position c.1919 replaced a C with a G, resulting in the substitution of a cysteine with an arginine and an alanine with a glycine at positions p.634 and p.640, respectively. The patient presented with MTC and Phaeo without parathyroid gland involvement, so we speculated that this clinical picture could be correlated with the two RET mutations identified and the unusual feature of calcitonin production by the adrenal tissue. No clinical symptoms suggestive of MEN 2A and no RET mutations were found in either parent or any of the available relatives tested (three sisters and one brother). In 2003, at

Female sexual dysfunction in women with thyroid disorders.

Background: Few data exist on the prevalence of female sexual dysfunction (FSD) in thyroid disorders. Aim: We evaluated FSD in women with thyroid diseases and in control age-matched healthy women to investigate the relationship between sexual function and thyroid hormones. Methods: One hundred and four women with thyroid diseases and 53 controls participated in the study. Eighteen with hyperthyroidism (Group 1), 22 hypothyroidism (Group 2), 45 Hashimoto’s thyroiditis (Group 3), 19 nodular goiter (Group 4) underwent thyroid function evaluation and sonography. The Female Sexual Function Index (FSFI) assessed sexual function. Results: The prevalence of FSD was 46.1% in thyroid diseases and 20.7% in controls. Only in Group 4, the prevalence (68.4%) was significantly higher than in controls (p<0.005). The mean total FSFI score was 20.1±7.1 in women with thyroid diseases and 25.6±4.7 in the controls (p<0.001). Compared with controls, there was a significant decrease of desire in Group 2; desire, arousal and lubrication in Group 3; desire, arousal, lubrication, orgasm and satisfaction in Group 4. In thyroid diseases the prevalence of FSD was 53% and 42%, while in the controls was 55% and 20%, in menopausal and pre-menopausal groups, respectively. We found a significant inverse correlation between TSH and FSFI (r=−0.7, p=0.01) in Group 4, which showed the lowest FSFI score (17.8±5.7) and the highest body mass index (28.4±7.1 kg/m2). Conclusions: Women with thyroid diseases present a higher prevalence of FSD than controls. Although our findings suggest a higher impairment of sexual function in Group 4 and a role for TSH in FSD, further researches are needed.

CDKN1B V109G polymorphism a new prognostic factor in sporadic medullary thyroid carcinoma

CONTEXT CDKN1B encodes the cyclin-dependent kinase inhibitor p27Kip1 and is mutated in multiple endocrine neoplasia-like syndromes. CDKN1B also harbors single nucleotide polymorphisms; the T/G transversion at nucleotide 326 (the V109G variant) has been reported to be protective in breast, hereditary prostate, and pancreatic tumors. Association of CDNK1B mutations or polymorphisms with sporadic medullary thyroid carcinoma (MTC) has not been investigated yet. OBJECTIVE AND DESIGN We screened germline DNA from 84 patients affected by sporadic MTC and 90 healthy age- and gender-matched controls for CDKN1B mutations or polymorphisms by PCR amplification and sequencing of the amplicons. We also tested all germline and 50 tumor tissue DNA for RET proto-oncogene mutations. Computed tomography, ultrasound scans, and serum calcitonin were carried out before surgery and during the follow-up and associated with CDKN1B polymorphism and disease remission. RESULTS The T/G transversion at nucleotide 326 was the only DNA variation detected. The overall frequency of the T/G and G/G alleles in combination was 46.4%. This variant (V109G) was correlated with post-operative calcitonin levels in the normal range and biochemical remission. Conversely, the wild-type (T/T) allele was associated with post-operative calcitonin levels above normal and a higher risk to develop clinical recurrence and distant metastases. Somatic RET mutations were significantly associated with a more aggressive behavior especially in wild-type allele-bearing patients. CONCLUSIONS Collectively, in sporadic MTC, the CDKN1B V109G polymorphism correlates with a more favorable disease progression than the wild-type allele and might be considered a new promising prognostic marker.

Testicular parenchymal abnormalities in Klinefelter syndrome: a question of cancer? Examination of 40 consecutive patients

Klinefelter syndrome (KS) is a hypergonadotropic hypogonadism characterized by a 47, XXY karyotype. The risk of testicular cancer in KS is of interest in relation to theories about testicular cancer etiology generally; nevertheless it seems to be low. We evaluated the need for imaging and serum tumor markers for testicular cancer screening in KS. Participants were 40 consecutive KS patients, enrolled from December 2009 to January 2013. Lactate dehydrogenase (LDH), alpha-fetoprotein (AFP), and beta-human chorionic gonadotrophin subunit (β-HCG) serum levels assays and testicular ultrasound (US) with color Doppler, were carried out at study entry, after 6 months and every year for 3 years. Abdominal magnetic resonance (MR) was performed in KS when testicular US showed micro-calcifications, testicular nodules and cysts. Nearly 62% of the KS had regular testicular echotexture, 37.5% showed an irregular echotexture and 17.5% had micro-calcifications and cysts. Eighty seven percent of KS had a regular vascular pattern, 12.5% varicocele, 12.5% nodules <1 cm, but none had nodules >1 cm. MR ruled out the diagnosis of cancer in all KS with testicular micro calcifications, nodules and cysts. No significant variations in LDH, AFP, and β-HCG levels and in US pattern have been detected during follow-up. We compared serum tumor markers and US pattern between KS with and without cryptorchidism and no statistical differences were found. We did not find testicular cancer in KS, and testicular US, tumor markers and MR were, in selected cases, useful tools for correctly discriminating benign from malignant lesions.

High circulating levels of CCL2 in patients with Klinefelter's syndrome

We would like to thank Dhindsa and colleagues for their interest and comments on our recent review article published in Clinical Endocrinology entitled ‘The Role of Obesity and Type 2 Diabetes Mellitus in the development of Male Obesity-associated Secondary Hypogonadism’. We would also like to thank the Editor for the opportunity to respond. We would like to confirm that we agree with the useful comments raised and provide further comment below. In our published review, we commented that the mechanisms implicated in the pathogenesis of secondary hypogonadism in men and its association with obesity and type 2 diabetes mellitus are multiple, complex and incompletely understood. We also stated that one of the pathogenic mechanisms implicated in the development of male obesity-associated secondary hypogonadism is increased aromatase activity within adipocytes. This results in increased peripheral conversion of testosterone into oestradiol and subsequent negative feedback on secretion of luteinizing hormone secretion from the pituitary. The suppressive effect of such a mechanism on the male hypothalamo–pituitary–gonadal axis results in a reduction in plasma testosterone levels and secondary hypogonadism. Consistent with this hypothesis, it has been reported in the literature that obese men show increased levels of oestrogens and decreased levels of bioavailable androgens within the serum. It has also been noted that use of aromatase inhibitors in men with obesity-related hypogonadism may normalize serum testosterone, again consistent with an important pathogenic role for aromatization in this condition. However, we acknowledge that there is controversy in the literature regarding the role of aromatization in the pathogenesis of male obesity-associated secondary hypogonadism. There is some inconsistency in the literature regarding relationships between serum levels of testosterone and oestradiol and the severity of obesity in this condition. We acknowledge that in some studies on obese men, serum levels of free oestradiol directly correlate with free testosterone. One explanation for this direct correlation is that with increasing obesity in men, as serum levels of testosterone fall (following suppression of the male gonadal axis), serum oestradiol levels would also be expected to fall eventually due to lack of substrate (testosterone) for the aromatase enzyme. This hypothesis has been supported by the European Male Ageing Study. We feel that it is important to emphasize that there are many potential mechanisms that underlie the complex pathogenesis of male obesity-associated secondary hypogonadism other than enhanced aromatase activity, as outlined in our published review article. These include the increasingly important roles of leptin, inflammatory mediators (TNF-a, IL-1b, CRP), the role of sleep disruption and the serotoninergic system and endogenous kisspeptin. We also outline the effects of insulin resistance at various levels including lipases, suppression of hepatic SHBG synthesis and the suppression of the hypothalamo–pituitary unit as potentially important pathogenic mechanisms. There may also be other, as yet unknown mechanisms at play. Clearly, there is a need for further focused studies in this field to develop a clear understanding of pathogenesis and to inform future novel treatment strategies.

Hashimoto's thyroiditis and entero-chromaffin-like cell hyperplasia: Early detection and somatostatin analogue Treatment

Type IIIb polyglandular autoimmune disease comprises autoimmune thyroid disease (HT) and chronic atrophic gastritis (AIG). Hypergastrinemia, secondary to AIG, predisposes to gastric enterochromaffin-like cell (ECL) hyperplasia, a preneoplastic condition. We evaluated the prevalence of AIG, hypergastrinemia and ECL hyperplasia in HT patients. A secondary end-point was to assess the efficacy of treatment with a somatostatin analogue in HT patient with ECL hyperplasia. From 2009 to 2011, 146 HT patients were enrolled in a prospective study. All cases underwent hormonal profile, and parietal cell antibody (PCA), gastrin, and chromogranin A (CgA) serum level assays. Selected patients with elevated gastrin and CgA levels underwent gastro esophageal endoscopy (EGDS). Patients positive for ECL hyperplasia received Octreotide LAR 30 mg/28 days for 12 months. Gastrin and CgA assays were repeated every three months and EGDS after one year. The results show that gastrin and CgA were significantly higher than normal in 17/115 (14.7%) patients. Seven more HT had isolated PCA positivity and in the 17 PCA positive patients histology diagnosed AIG, corpus prevalent, with mild to moderate atrophy. Diffuse ECL hyperplasia of the gastric body was present in three subjects, one of them presenting a type-1 carcinoid. Gastrin and CgA levels were significantly reduced (p<0.01) after 3 months of therapy with a somatostatin analogue and returned to normal after 1 year. ECL hyperplasia regressed in all three patients. We suggest that selected HT patients may need a more accurate surveillance for early signs of gastric EC risk. In the case of altered values of gastrin and in the presence of PCA positivity, EGDS and histology should be performed for an early diagnosis of AIG and treatment of ECL hyperplasia.

Differential expression of estrogen receptor α and β transcripts in tissues and in primary culture cells from pubertal gynecomastia

Background: Pubertal gynecomastia is a common problem occurring in up to 65% of adolescent boys. Gynecomastia comes at a time when self-image awareness is at its greatest and psychologically could be a psychologically disabling condition. Surgery is considered the mainstay of treatment for severe or persistent cases. A medical management aimed at altering the effective androgen/estrogen ratio has been suggested with inconstant results. Some promising results have been obtained by using anti-estrogens. Surprisingly there are no data on the estrogen receptor (ER) α and β RNA expression in gynecomastia. Aim: We studied ER RNA subtypes in pubertal gynecomastia. Methods: ERα and β RNA were determined by real time RT-PCR in 50 mammary samples from pubertal boys with idiopathic gynecomastia subjected to reductive mammoplasty. To study ERα and β pattern of expression, epithelial and stromal primary cell cultures were set up from fresh tissues. Results: These analyses indicated that in all stromal cells ERβ was expressed at higher level than ERα and in epithelial cells both ERα and ERβ were barely detectable. Conclusions: Our data suggest that also stromal cells are involved in the pathophysiology of pubertal gynecomastia. The high level of expression of ERβ seen in pubertal gynecomastia adds new insight on validation of ERβ as a target for candidate diseases and exploration of ERβ as a marker for clinical decision-making and treatment in pubertal gynecomastia. This could drive to search for new and selective anti-estrogen drugs for medical treatment of pubertal gynecomastia with a particular attention to the ERβ-selective ligand.