Adrenocortical carcinoma: the range of appearances on CT and MRI.

OBJECTIVE: Adrenocortical carcinoma (ACC) is a rare, aggressive tumor arising from the adrenal cortex that typically presents late with a large mass. The increased use of cross-sectional imaging for unrelated reasons has led to a greater number of ACCs being detected incidentally at an earlier stage. Recognition of the typical clinical, biochemical, and imaging findings is imperative for rapid diagnosis, prompt intervention, and early use of the appropriate therapy. CONCLUSION: Cross-sectional imaging with CT and MRI is essential for determining the extent of local and distant tumor spread. Complete surgical resection is currently the only potentially curative treatment of ACC, and the information attained from CT and MRI is important to guide surgery and further patient management.

Characterization of aryl hydrocarbon receptor interacting protein (AIP) mutations in familial isolated pituitary adenoma families.

Familial isolated pituitary adenoma (FIPA) is an autosomal dominant condition with variable genetic background and incomplete penetrance. Germline mutations of the aryl hydrocarbon receptor interacting protein (AIP) gene have been reported in 15-40% of FIPA patients. Limited data are available on the functional consequences of the mutations or regarding the regulation of the AIP gene. We describe a large cohort of FIPA families and characterize missense and silent mutations using minigene constructs, luciferase and beta-galactosidase assays, as well as in silico predictions. Patients with AIP mutations had a lower mean age at diagnosis (23.6+/-11.2 years) than AIP mutation-negative patients (40.4+/-14.5 years). A promoter mutation showed reduced in vitro activity corresponding to lower mRNA expression in patient samples. Stimulation of the protein kinase A-pathway positively regulates the AIP promoter. Silent mutations led to abnormal splicing resulting in truncated protein or reduced AIP expression. A two-hybrid assay of protein-protein interaction of all missense variants showed variable disruption of AIP-phosphodiesterase-4A5 binding. In summary, exonic, promoter, splice-site, and large deletion mutations in AIP are implicated in 31% of families in our FIPA cohort. Functional characterization of AIP changes is important to identify the functional impact of gene sequence variants.

Assessment of p27 (cyclin-dependent kinase inhibitor 1B) and aryl hydrocarbon receptor-interacting protein (AIP) genes in multiple endocrine neoplasia (MEN1) syndrome patients without any detectable MEN1 gene mutations.

OBJECTIVE: Germline mutations in the MEN1 gene predispose to the multiple endocrine neoplasia (MEN1) syndrome; however, approximately 10-20% of patients with MEN1 do not have a detectable MEN1 mutation. A rat strain with multiple endocrine tumours, a phenotypic overlap of both MEN1 and MEN2, has been reported to have a homozygous germline p27 (CDKN1B) mutation. Recently, two MEN1 mutation-negative MEN1 syndrome patients have been identified to harbour a germline CDKN1B mutation. The recently identified gene AIP can also cause familial isolated pituitary adenoma, but no other specific tumour is associated with this syndrome. The objective of this study was to evaluate the possible contribution of CDKN1B and AIP germline mutations in a cohort of MEN1 mutation-negative MEN1 syndrome patients. PATIENTS: Eighteen sporadic and three familial cases of MEN1 mutation-negative MEN1 syndrome were studied (18 pituitary adenomas, 12 hyperparathyroidism, 10 neuroendocrine tumours including 2 ACTH-secreting lesions and one adrenal nodular hyperplasia). Clinical data and genomic DNA were analysed for mutations in the CDKN1B and AIP genes. RESULTS: There were no mutations in the coding region or exon/intron junction of the CDKN1B and AIP genes in any patient. Although we have a limited number of patients in our cohort, our data is consistent with others in the literature suggesting that CDKN1B and AIP mutations are extremely rare in MEN1 syndrome. CONCLUSION: Our results suggest that mutations in the CDKN1B and AIP genes are relatively uncommon in MEN1 mutation-negative MEN1 syndrome patients.

Pituitary gland and beta-catenin signaling: from ontogeny to oncogenesis.

Although pituitary tumors are mostly benign, they share certain molecular events with more malignant neoplasia, although their precise pathogenesis is far from established. The acquisition of new functional characteristics during their evolution suggests a multistep process that leads to tumor transformation. Mutations in classical tumor suppressor genes or oncogenes are infrequently associated with pituitary tumorigenesis. However, alterations in different signaling pathways, especially those involved in pituitary gland development, have emerged as significant features in pituitary adenomas. In particular, changes in inhibitory components of the beta-catenin pathway and its relationship to the cadherin family of peptides may well play an important role in tumorigenesis. We review and assess the role of the beta-catenin signaling pathway in the pathogenesis of pituitary adenomas.

Ghrelin receptor gene polymorphisms and body size in children and adults.

BACKGROUND: The GH secretagogue receptor type 1a gene (GHSR) encodes the cognate receptor of ghrelin, a gut hormone that regulates food intake and pituitary GH secretion. Previous studies in U.S. families and a German population suggested GHSR to be a candidate quantitative locus for association with human obesity and growth. AIM: The aim of the study was to test common genetic variation in GHSR for association with body size in children and adults. METHODS: Sequencing was performed to systematically identify novel single nucleotide polymorphisms (SNPs) in GHSR. A set of three haplotype-tagging SNPs that captured all the genetic variation in GHSR was identified. These three haplotype-tagging SNPs were then genotyped in three large population-based U.K. cohort studies (two adult and one childhood cohort) comprising 5807 adults and 843 children. RESULTS: No significant genotype or haplotype associations were found with adult or childhood height, weight, or body mass index. CONCLUSION: Common variation in GHSR is not associated with body size in U.K. adults or children.

The role of the aryl hydrocarbon receptor-interacting protein gene in familial and sporadic pituitary adenomas.

CONTEXT: Mutations have been identified in the aryl hydrocarbon receptor-interacting protein (AIP) gene in familial isolated pituitary adenomas (FIPA). It is not clear, however, how this molecular chaperone is involved in tumorigenesis. OBJECTIVE: AIP sequence changes and expression were studied in FIPA and sporadic adenomas. The function of normal and mutated AIP molecules was studied on cell proliferation and protein-protein interaction. Cellular and ultrastructural AIP localization was determined in pituitary cells. PATIENTS: Twenty-six FIPA kindreds and 85 sporadic pituitary adenoma patients were included in the study. RESULTS: Nine families harbored AIP mutations. Overexpression of wild-type AIP in TIG3 and HEK293 human fibroblast and GH3 pituitary cell lines dramatically reduced cell proliferation, whereas mutant AIP lost this ability. All the mutations led to a disruption of the protein-protein interaction between AIP and phosphodiesterase-4A5. In normal pituitary, AIP colocalizes exclusively with GH and prolactin, and it is found in association with the secretory vesicle, as shown by double-immunofluorescence and electron microscopy staining. In sporadic pituitary adenomas, however, AIP is expressed in all tumor types. In addition, whereas AIP is expressed in the secretory vesicle in GH-secreting tumors, similar to normal GH-secreting cells, in lactotroph, corticotroph, and nonfunctioning adenomas, it is localized to the cytoplasm and not in the secretory vesicles. CONCLUSIONS: Our functional evaluation of AIP mutations is consistent with a tumor-suppressor role for AIP and its involvement in familial acromegaly. The abnormal expression and subcellular localization of AIP in sporadic pituitary adenomas indicate deranged regulation of this protein during tumorigenesis.

Multimodality palliative treatment of (111)In-pentetreotide negative/(123)I-MIBG positive metastatic carcinoid - a case report.

Patients with carcinoid tumours frequently present with metastatic disease. There are only a few therapeutic options for these patients, and the main goal of palliative treatment is to reduce symptoms and thus to improve quality of life. Current therapy includes surgical resection, hepatic artery embolisation, chemotherapy and somatostatin analogue treatment; however, all these options have limitations. It seems probable that therapeutic modalities based on radiopharmaceuticals may provide better therapy, not only in relation to symptom reduction but may also improve patient survival. In this case report we present a 46-year-old woman with a symptomatic carcinoid, who at the time of diagnosis had liver and abdominal lymph node metastases, the primary tumour being located in the terminal ileum. (111)In-pentetreotide scanning was negative, whereas (123)I-MIBG scanning showed high avidity in the tumour tissue. After right hemicolectomy, two courses of (131)I-MIBG treatment were given (12.95 GBq and 12 GBq, respectively). After the second dose of (131)I-MIBG temporary pancytopenia was present. Octreotide therapy was given empirically only for a short time and was stopped because of drug intolerance. The patient underwent tricuspid and pulmonary valve replacement because of her carcinoid heart disease, followed by two courses of embolisation of liver metastases. While (131)I-MIBG therapy reduced the patient's symptoms of flushing and diarrhoea, there has not yet been any effect on tumour response or 5-HIAA production. This case illustrates the multimodality and multidisciplinary approach to such patients.

Examining the candidacy of ghrelin as a gene responsible for variation in adult stature in a United Kingdom population with type 2 diabetes.

CONTEXT: Recently, a quantitative trait locus for stature was reported on chromosome 3p26 in patients with type 2 diabetes. OBJECTIVE: Given that ghrelin is a peptide involved in GH release and located on 3p26, we hypothesized that variation within its gene (GHRL) may be responsible for the quantitative trait locus on 3p26. DESIGN: The evidence for linkage around GHRL was refined with the genotyping of an additional four microsatellites (D3S4545, D3S1537, D3S1597, and D3S3611), giving a total of 27 markers, followed by multipoint variance components linkage analysis. Probands from the linkage families were typed for five common single nucleotide polymorphisms (SNPs) within GHRL and tested for association with adult stature using haplotype trend regression. RESULTS: The maximum multipoint evidence for linkage between adult stature and the 27 microsatellites yielded an LOD score of 2.58 (P = 0.0003) between D3S1297 and D3S1304. Five common (frequency of > or =5%) SNPs were typed in the probands [two promoter SNPs (rs27647 and rs26802), two exonic (rs696217 and rs4684677), and one intronic (rs35683)] capturing 80% of the total common variation in GHRL. No association was found between any SNP (or haplotypes thereof) and adult stature. CONCLUSION: Common genetic variation within GHRL is not responsible for variation in adult stature in this population.

The association of p53 mutations and p53 codon 72, Her 2 codon 655 and MTHFR C677T polymorphisms with breast cancer in Northern Greece.

The aim of this study was to explore a possible association between p53 codon 72, Her 2 codon 655 and MTHFR C677T polymorphisms and breast cancer in Northern Greece. We examined 42 women with breast cancer and 51 controls. A total of 42 women with breast cancer as well as healthy controls were investigated and results showed that p53 codon 72 polymorphism is statistically significantly associated with breast cancer (OR for Arg/Arg to non-Arg/Arg was 6.66, P = 0.0001 at 95% CI 2.63-16.9), but not Her 2 and MTHFR polymorphisms are associated with breast cancer (OR for Ile/Ile to non-Ile/Ile was 1.33, P = 0.54 at 95% CI 0.52-3.38 and OR for T/T versus non-T/T was 1.07, P = 0.89 at 95% CI 0.35-3.25). All subjects were examined for p53 exons 5-8 mutations. Three novel sequence variations in exons 7 and 8 of TP53 gene were found in three patients. One of them induces an amino acid change at Ser 241Gly, the second is a silent mutation Gly244Gly, and the third one results in a premature stop codon 294 (Glu294stop) and a truncated p53 protein.

A variation in the ghrelin gene increases weight and decreases insulin secretion in tall, obese children.

Ghrelin is a recently recognized gut-brain peptide originally derived from the gastric mucosa. It stimulates growth hormone release, increases appetite and facilitates fat storage, and may interact with glucose metabolism. We studied the ghrelin gene in a group of 70 tall and obese children (mean age 9.4 year, Z body mass index [BMI] and Z height >3 and/or BMI percentile >99%). We found 10 single nucleotide polymorphisms. One common polymorphism of the ghrelin gene, which corresponds to an amino acid change in the tail of the prepro-ghrelin molecule, was significantly associated with children with a higher BMI (P = 0.001), and with lower insulin secretion during the first part of an oral glucose tolerance test (P = 0.05) although no difference in glucose levels was noted. This might suggest increased insulin sensitivity, although this is not supported by the lack of difference in fasting and 2 hour insulin levels; alternatively, this may be indicative of impaired first phase insulin secretion. These data suggest that variations in the ghrelin gene contribute to obesity in children and may modulate glucose-induced insulin secretion.

Identification of adrenocorticotropin receptor messenger ribonucleic acid in the human pituitary and its loss of expression in pituitary adenomas.

The ACTH receptor (ACTH-R) is the second member of the melanocortin (MC-2) receptor family that includes five seven-transmembrane G protein-coupled receptors and has been shown to be predominantly expressed in the adrenal cortex. It has been postulated that ACTH may regulate its own secretion through ultra-short-loop feedback within the pituitary. ACTH-secreting adenomas are characterized by resistance to glucocorticoid feedback, and they may have dysregulated ACTH feedback. We therefore investigated the ACTH-R in normal and adenomatous human pituitary tissue. We report here the identification of ACTH-R mRNA in the human pituitary gland, which was confirmed by direct sequencing. We studied the expression of the ACTH-R in 23 normal pituitary specimens and 53 pituitary adenomas (22 ACTH-secreting, nine GH-secreting, eight prolactin-secreting, one TSH-secreting, one FSH-secreting, 10 nonfunctioning, and two silent corticotroph adenomas), using the sensitive technique of real-time quantitative PCR. Contamination of ACTH-secreting adenomas and nonfunctioning pituitary adenomas with nonadenomatous tissue was excluded by lack of Pit-1 expression. ACTH-R mRNA was detected in all normal pituitary specimens, and in situ hybridization colocalized expression to ACTH staining cells only. However, ACTH-R mRNA levels were undetectable in 16 of 22 ACTH-secreting tumors and in both silent corticotroph tumors. Diagnostic preoperative plasma ACTH levels were significantly lower in the ACTH-R positive ACTH-secreting tumors, compared with those who were ACTH-R negative (P = 0.0006). Direct sequencing of the coding region of the ACTH-R in cDNA from three ACTH-secreting tumors positively expressing the receptor showed no mutations, as did sequencing of genomic DNA in three receptor negative ACTH-secreting tumors and the two silent corticotrophs. These results provide further evidence compatible with an ACTH feedback loop in the pituitary and suggest that loss of expression of the ACTH-R in corticotroph adenomas of patients with Cushing's disease may play a role in the resistance to feedback of the pituitary-adrenal axis seen in these patients.

Cell cycle dysregulation in pituitary oncogenesis.

The cell cycle is the process by which cells grow, replicate their genome and divide. The cell cycle control system is a cyclically-operating biochemical device constructed from a set of interacting proteins that induce and coordinate proper progression through the cycle, and includes cyclins, cyclin-dependent kinases (CDK) and their inhibitors (CDKI). There are mainly two families of CDKI, the INK family (INK4a/p16; INK4b/p15; INK4c/p18 and INK4d/p19) and the WAF/KIP family (WAF1/p21; KIP1/p27; KIP2/p57). Progression through the cell cycle is mainly dependent on fluctuations in the concentration of cyclins and CDKI achieved through the programmed degradation of these proteins by proteolysis within the ubiquitin-proteasome system. There is also a transcriptional regulation of cyclin expression, probably dependent on CDK phosphorylation. The p53 family--p53, p63 and p73--function as transcription factors that play a major role in regulating the response of mammalian cells to stressors and damage, in part through the transcriptional activation of genes involved in cell cycle control (e.g. p21), DNA repair, senescence, angiogenesis and apoptosis. Essential for the maintenance of euploidy during mitosis is human securin, identical to the product of the pituitary tumour-transforming gene (PTTG). Loss of regulation at the G1/S transition appears to be a common event among virtually all types of human tumours. Aberrations of one or more components of the pRb/p16/cyclin D1/CDK4 pathway seem to be a frequent event (80%) in pituitary tumours. The role of p27 is rather that of a haploinsufficient gene. p27-/- mice show an increased growth rate, due to increased cellularity, testicular and ovarian cell hyperplasia and infertility, and hyperplasia of the pituitary intermediate lobe with nearly 100% mortality caused by such a benign pituitary tumour. Although the p27 gene was not found to be mutated in human pituitary tumours and its mRNA expression was similar in tumour samples in comparison with normal pituitaries, the load of p27 protein expression in corticotroph adenomas and pituitary carcinomas was shown to be much lower than those in normal pituitary tissue or other types of pituitary adenoma, suggesting that post-translational processing of p27 accelerates its removal from the nucleus. In respect to p27 degradation and its cellular compartmentalization, several pathways have been explored. Malignant tumours are associated with increased nuclear immunostaining for Jun-activation binding protein-1 (Jab1) which is responsible for phosphorylated p27 export from the nucleus. Corticotrophinomas are characterized by massively increased phosphorylation of p27 on Thr187, but are not associated with changes in Jab1. Macrophage inhibitory factor (MIF), which binds and inactivates Jab1, was noted to be over-expressed in tumours with abundant Jab1, suggesting that it may be part of a compensatory mechanism to moderate Jab1 activity. Proteasomal degradation of p27 requires its ubiquitylation by the SCF ubiquitin ligase, with specific addressing by the F-box protein Skp2 and its co-factor Cks1. Pituitary tumours with high p27 protein expression showed significantly less Skp2 expression than samples with low p27 immunostaining, suggesting that increased Skp2 could play at least a part in this process. No difference was observed in Cks1 mRNA levels between normal pituitaries and pituitary adenomas. The present data suggest that inhibition of growth and tumour development is sensitive not only to the absolute levels of p27 protein, but also to its cellular compartmentalization. Very recent findings from our group have established up-regulation of the serine-threonine kinase Akt in pituitary tumours compared to normal pituitary, which may cause phosphorylation of p27 on Thr157 and cytoplasmic retention of p27. PTTG protein is highly expressed in various human tumours, including pituitary tumours. While its mRNA levels are low in normal pituitary, increases in PTTG transcripts from more than 50% to more than 10-fold were recorded in the majority of a series of pituitary adenomas. Control of the cell cycle is a vital part of the cell's replication machinery. Disruption of this process is commonly seen in pituitary tumours and we are now beginning to identify regulatory elements which are likely to play a major role in pituitary oncogenesis.

Genetics of the ghrelin system.

Ghrelin was originally identified as a growth hormone secretagogue, but later studies suggested that it plays an important role in the regulation of appetite and body weight. The implication of genetic variants of the ghrelin-ghrelin receptor axis in the determination of stature, appetite, body weight, glucose metabolism and eating disorders has been the focus of numerous small and larger studies. More recently, several studies have shown some involvement of ghrelin and growth hormone secretagogue receptor (GHSR) genetic variants in some cancers, or their role in the genetics of immune diseases or addictive behaviour. The overall results suggest that the effects of common genetic variants are lacking or modest, while rare sequence alteration may lead to disease status in individual patients. In this review we summarize the available data on the genetics of ghrelin axis in humans.

Association studies on ghrelin and ghrelin receptor gene polymorphisms with obesity.

Ghrelin exerts a stimulatory effect on appetite and regulates energy homeostasis. Ghrelin gene variants have been shown to be associated with metabolic traits, although there is evidence suggesting linkage and association with obesity and the ghrelin receptor (GHSR). We hypothesized that these genes are good candidates for susceptibility to obesity. Direct sequencing identified 12 ghrelin single-nucleotide polymorphisms (SNPs) and 8 GHSR SNPs. The 10 common SNPs were genotyped in 1,275 obese subjects and in 1,059 subjects from a general population cohort of European origin. In the obesity case-control study, the GHSR SNP rs572169 was found to be associated with obesity (P = 0.007 in additive model, P = 0.001 in dominant model, odds ratio (OR) 1.73, 95% confidence interval (1.23-2.44)). The ghrelin variant, g.A265T (rs4684677), showed an association with obesity (P = 0.009, BMI adjusted for age and sex) in obese families. The ghrelin variant, g.A-604G (rs27647), showed an association with insulin levels at 2-h post-oral glucose tolerance test (OGTT) (P = 0.009) in obese families. We found an association between the eating behavior "overeating" and the GHSR SNP rs2232169 (P = 0.02) in obese subjects. However, none of these associations remained significant when corrected for multiple comparisons. Replication of the nominal associations with obesity could not be confirmed in a German genome-wide association (GWA) study for rs4684677 and rs572169 polymorphisms. Our data suggest that common polymorphisms in ghrelin and its receptor genes are not major contributors to the development of polygenic obesity, although common variants may alter body weight and eating behavior and contribute to insulin resistance, in particular in the context of early-onset obesity.

The role of ghrelin and ghrelin-receptor gene variants and promoter activity in type 2 diabetes.

BACKGROUND: Ghrelin and its receptor play an important role in glucose metabolism and energy homeostasis, and therefore they are functional candidates for genes carrying susceptibility alleles for type 2 diabetes. METHODS: We assessed common genetic variation of the ghrelin (GHRL; five single nucleotide polymorphisms (SNP)) and the ghrelin-receptor (GHSR) genes (four SNPs) in 610 Caucasian patients with type 2 diabetes and 820 controls. In addition, promoter reporter assays were conducted to model the regulatory regions of both genes. RESULTS: Neither GHRL nor GHSR gene SNPs were associated with type 2 diabetes. One of the ghrelin haplotypes showed a marginal protective role in type 2 diabetes. We observed profound differences in the regulation of the GHRL gene according to promoter sequence variants. There are three different GHRL promoter haplotypes represented in the studied cohort causing up to 45% difference in the level of gene expression, while the promoter region of GHSR gene is primarily represented by a single haplotype. CONCLUSION: The GHRL and GHSR gene variants are not associated with type 2 diabetes, although GHRL promoter variants have significantly different activities.

Ghrelin, the peripheral hunger hormone.

In the current review we summarize the available data concerning the gastric hormone ghrelin and its receptor. Ghrelin stimulates short-term food intake and long-term body weight regulation via its adipogenic and diabetogenic effects. Ghrelin stimulates gastric emptying, and these effects could be explored from a therapeutic point of view. Ghrelin levels change profoundly in anorexia, in states of insulin resistance, in obesity, and after bariatric surgery, suggesting that this is an important hormone in body weight regulation.

Ghrelin--a hormone with multiple functions.

Ghrelin is brain-gut peptide with growth hormone-releasing and appetite-inducing activities. It is mainly secreted from the stomach mucosa but it is also expressed widely in different tissues and therefore may have both endocrine and paracrine effects. Ghrelin is the endogenous ligand of the G protein-coupled growth hormone secretagogue receptor. In the current review we comprehensively summarize (i) the data available regarding the structure, expression pattern and regulation of ghrelin and its receptor; (ii) the available information regarding the effect of ghrelin on the pituitary hormone axis, appetite regulation, cardiac and gastrointestinal function, carbohydrate metabolism, adipose and reproductive tissue, cell proliferation and behavioral effects; (iii) experimental and clinical data regarding circulating ghrelin levels observed in various physiological and pathological conditions; and (iv) data on gene variations of ghrelin and its receptor. It is apparent that ghrelin is involved in many more processes than originally envisaged, and in particular appears to have relatively less relevance to growth hormone physiology and more to the regulation of energy fluxes in the organism. Increasing data link ghrelin to the overall control of energy use and flow in situations where there is a limitation of energy sources and ghrelin appears to play a pivotal role in energy homeostasis.

The expression of the F-box protein Skp2 is negatively associated with p27 expression in human pituitary tumors.

The CDK inhibitor p27 plays a pivotal role in controlling cell proliferation during development, and has been implicated in tumorigenesis. Previous studies have demonstrated changes in p27 protein expression, but not in mRNA levels, in human pituitary tumors. It seems probable that the fall in p27 is due to increased degradation through the ubiquitin-proteasome pathway. Skp2 (S-phase kinase-interacting protein) is a specific F-box protein that allows the recognition and binding of phosphorylated p27 to the ubiquitin complex. The aim of our study was thus to investigate the possible role of Skp2 in pituitary tumorigenesis. A total of 59 human pituitary samples, 7 normal and 52 adenomas, were assessed for transcriptional expression of Skp2; 51 pituitary samples were assessed for protein expression. Real-time RT-PCR was performed on cDNA of reverse-transcribed mRNA for relative quantification of the Skp2 transcript. Immunostaining was performed using mouse monoclonal anti-Skp2 antibody. Skp2 mRNA and protein was detectable in every sample studied. Our results showed no significant difference between the pituitary tumors and normal pituitary tissue in Skp2 mRNA or nuclear protein expression. Individual tumor types had similar mRNA expression and variable protein expression. However, samples with high p27 protein expression showed significantly less Skp2 expression than samples with low p27 staining. Our data suggest that increased p27 degradation through the ubiquitin-proteasome pathway could be regulated in pituitary tumors by changes in Skp2 expression, although other factors probably also play a role.