AIMS:The differentiation of adrenocortical carcinomas from adenomas may be difficult based on morphology alone. Differential expression of insulin-like growth factor (IGF) II and cyclin-dependent kinase (CDK) 4 has recently been described in these tumours. The aim of this study was to investigate the diagnostic usefulness of these markers immunohistochemically.METHODS AND RESULTS:We examined 22 benign and 17 malignant adrenocortical tumours and compared IGFII and CDK4 expression with known immunohistochemical as well as morphological criteria of malignancy. Thirteen of 17 carcinomas showed immunohistochemical reactivity for IGFII, whereas all adenomas but one were negative. Intense CDK4 expression was detected in 11 of 17 carcinomas but was present in only three of 22 adenomas. The MIB1 index was >5% in 14 of 16 carcinomas and was <5% in all adenomas but one. The combination of IGFII immunohistochemistry with MIB1 index led to high sensitivity and specificity in detecting adrenocortical carcinomas.CONCLUSIONS:IGFII and MIB1 are helpful immunohistochemical markers to predict malignancy in adrenocortical neoplasms. These markers can be used in addition to clinical, gross and morphological features to establish a diagnosis in difficult cases.

BACKGROUND & AIMS:The identification of precursor lesions has a great impact on the understanding of tumorigenesis. Precursor lesions of endocrine tumors are known to occur in the setting of the MEN1 syndrome. The aim of this study was to test the hypothesis that MEN1-associated duodenal gastrinomas originate from diffuse preneoplastic gastrin cell changes. Precursor lesions may precede the development of duodenal gastrinomas because, in contrast to sporadic gastrinomas, these tumors are usually multiple.METHODS:The distribution of endocrine cells in the nontumorous duodenal tissue was analyzed qualitatively and quantitatively for 25 patients operated on for a duodenal gastrinoma. MEN1 status was assessed clinically and by polymerase chain reaction-based mutational analysis.RESULTS:Fourteen of 25 patients with gastrinoma had proliferative, hyperplastic lesions consisting of gastrin cells in the nontumorous duodenal mucosa, similar to the gastric enterochromaffin-like cell lesions observed in chronic atrophic gastritis. All patients with Zollinger-Ellison syndrome with proven MEN1 had such proliferative gastrin cell lesions, and all patients with Zollinger-Ellison syndrome without precursor lesions were MEN1 negative.CONCLUSIONS:Duodenal gastrinomas in MEN1, but not sporadic duodenal gastrinomas, are associated with proliferative gastrin cell changes within the nontumorous mucosa. It is likely that these lesions precede the development of MEN1-associated duodenal gastrinomas.

Germline mutations of the three succinate dehydrogenase subunits SDHB, SDHC and SDHD have recently been associated with familial pheochromocytoma and paraganglioma. Several reasons make these genes candidate tumor suppressor genes for medullary thyroid carcinoma (MTC): (1) SDHB lies on chromosome 1p, the region known to be deleted most frequently in MTC, (2) MTCs develop from neural crest-derived cells, as do pheochromocytomas and paragangliomas and (3) patients with germline mutations of the Ret-protooncogene develop MTCs as well as pheochromocytomas, indicating a relationship of these tumors on a genetic level. Therefore, we attempted to determine whether the tumor suppressor genes SDHB, SDHC and SDHD are involved in sporadic and familial MTC. Somatic mutations of the SDH subunits were absent in all 35 investigated MTCs. Loss of heterozygosity was found in 27% (SDHB) and 4% (SDHD) respectively. While the frequency of non-coding, intronic polymorphisms did not differ in MTC patients compared with a control population, an accumulation of amino-acid coding polymorphisms (S163P in SDHB as well as G12S and H50R in SDHD) was found among MTC patients especially patients with familial tumors, suggesting a functional connection of coding SDH polymorphisms to activating Ret mutations.

We report a family with malignant sympathetic paragangliomas (PGL) exhibiting a new type of germline mutation in the succinate dehydrogenase subunit B (SDHB) gene. Two affected brothers, presenting with symptoms at the ages of 25 and 52 yr, suffered from malignant abdominal extraadrenal sympathetic PGL. They died of their disease at ages 43 and 61 yr. Their mother had the same history of signs and symptoms, suggesting a catecholamine-producing tumor at the age of 55 yr. Analysis of the germline DNA from these three patients revealed a novel mutation in exon 4 (H132P) of the SDHB gene. This mutation was absent in 160 control chromosomes. Loss of heterozygosity analysis of the tumors showed a loss of one SDHB allele, and RT-PCR-based expression analysis confirmed the exclusive expression of the mutated allele in both tumors. A review of the published PGL families revealed malignant tumors in seven of 12 well-documented families with SDHB mutation-associated extraadrenal PGL. These findings, as well as findings of the family reported here, suggest a strong causal relationship of SDHB germline mutations with malignant extraadrenal abdominal PGL and imply the necessity of a close follow-up of affected individuals and family members.

OBJECTIVE Abnormal proinsulin processing in insulinomas may result in secretory granules containing both insulin and proinsulin, a finding not encountered in healthy beta-cells. The aim of this study was to test whether such abnormalities in the proinsulin to insulin conversion have clinical implications in patients with hypoglycaemic disorders.DESIGN Case-series.PATIENTS AND METHODS Fifteen patients with histologically confirmed insulinoma and two patients with islet cell hyperplasia were included. The immunohistochemical distribution pattern of proinsulin within the tumour cells was classified as Golgi pattern (predominantly perinuclear immunolabelling) or diffuse pattern (immunolabelling in the periphery of the cells, indicating the presence of proinsulin in secretory granules). Data obtained from the 72-h fast and arterial calcium stimulation and hepatic venous sampling (ASVS) test were related to the morphological classification.RESULTS Six insulinomas exhibited a diffuse proinsulin distribution pattern, while nine insulinomas and two islet cell hyperplasias disclosed a Golgi pattern. Median proinsulin concentrations at the termination of the fast tended to be higher in patients with the diffuse proinsulin distribution pattern than in patients with the Golgi pattern (86.9 vs. 18.8 pmol/l, P = 0.07). Higher insulin (P < 0.005) and proinsulin (P < 0.05) concentrations were significantly correlated with earlier occurrence of hypoglycaemia during the prolonged fast. During the ASVS test, tumours with the diffuse proinsulin distribution pattern exhibited a higher increase in both insulin (median, 37.3- vs. 10.5-fold, P < 0.05) and proinsulin (6.3- vs. 1.6 fold, P < 0.005) concentrations following calcium stimulation than the tumours with the Golgi pattern.CONCLUSIONS Abnormalities in the proinsulin to insulin conversion in patients with insulinomas and islet cell hyperplasia correlate with impaired regulation of both insulin and proinsulin secretion during the prolonged fast as well as the ASVS test.

BACKGROUNDAtypical fibroxanthoma (AFX) and undifferentiated high grade pleomorphic sarcoma (UpS) are histologically very similar, if not identical. However, they differ significantly in clinical outcome.MATERIALS AND METHODSWe used comparative genomic hybridization (CGH) to screen 24 AFX and 12 UpS for genomic alterations.RESULTSDNA copy number changes were observed in 20/24 AFX and in all UpS. The most frequent alterations occurring with comparable frequency in both tumors were deletions on chromosomes 9p and 13q. We also detected statistically significant differences of genetic alterations between the two tumors concerning deletions on 1q, 3p, 5q, 11p, 11q, gains on 7q, 12q and high level gains on 5p and 11q.CONCLUSIONDespite their very similar histology, AFX and UpS show clear differences in their genetic alterations. This might contribute to the different biological behavior of the two tumors. On the other hand the similarities in genetic alterations on chromosomes 9p and 13q might suggest a common pathogenetic pathway.

BACKGROUND:Distinction of Spitz nevus from malignant melanoma is sometimes difficult on the basis of conventional histology. A high rate of BRAF gene mutations in malignant melanomas (66%) and nevi (82%) has recently been reported.MATERIALS AND METHODS:We screened a series of 20 Spitz nevi for BRAF mutations in exons 11 and 15 by denaturing gradient gel electrophoresis (DGGE).RESULTS:BRAF mutations could not be identified in Spitz nevi.CONCLUSION:Our results show that mutations within the BRAF gene are useful markers for the differential diagnosis between Spitz nevus and malignant melanoma.

Plasma prolactin (PRL) is released from lactotrophs in the anterior pituitary. As plasma PRL levels rise during incubation in domestic fowl, the number of lactotrophs (PRL-immunoreactive, PRL-IR cells) increases while the number of growth hormone secreting cells, somatotrophs (GH-IR cells), declines. We measured plasma PRL levels using radioimmunoassay (RIA) and examined the distribution of lactotrophs and somatotrophs in the anterior pituitary of breeding and nonbreeding zebra finches of known ages with and without prior breeding experience using fluorescent immunohistochemistry (IHC). Plasma PRL levels were higher in breeding than in nonbreeding birds, regardless of age, sex, or previous breeding history. PRL-IR cells were localized primarily, but not exclusively, to the cephalic aspect of the anterior pituitary (AP) and along the ventral margin. Birds with prior reproductive experience had more PRL-IR cells than birds with no prior reproductive experience and breeders had slightly higher PRL-IR cell counts than did nonbreeders, but there was no correlation between the number of PRL-IR cells and plasma PRL levels. GH-IR cells were concentrated in the caudal aspect of the AP with some cells in the cephalic lobe, but numbers did not differ between any of the groups studied. An increase in PRL-IR cells corresponded with an increase in GH-IR cells. An increase in lactotroph number with reproductive experience in zebra finches may facilitate future reproductive events by allowing for more robust PRL secretion and increased reproductive success.

The tumorigenesis of sporadic endocrine tumors is still not fully understood. Activating point mutations of the serine/threonine kinase gene BRAF located on 7q34 are found in a wide range of malignancies, with the highest frequency (66%) occurring in malignant melanomas. Melanomas are tumors of neural-crest-derived cells as are medullary thyroid carcinomas, pheochromocytomas and paragangliomas. BRAF has not been examined in endocrine tumors of the diffuse neuroendocrine system or of neural-crest-derived cells. We examined 130 endocrine tumors of the pancreas, parathyroid gland, adrenal medulla, paraganglia, lung and gastrointestinal tract as well as follicular and c-cell-derived thyroid tumors. We found a high rate of V559E mutations in papillary thyroid carcinomas (47%), one V599E mutation in a well-differentiated gastric endocrine carcinoma (malignant carcinoid), but no activating BRAF mutations in all other endocrine tumors examined. These results point towards different pathways in tumorigenesis of endocrine tumors of various localizations and only rare involvement of the MAP kinase (MAPK) pathway in a subset of malignant neuroendocrine tumors.

Several chromosomal loci involved in tumorigenesis of pancreatic endocrine tumors (PET) have been identified. To date, the only gene known to be frequently altered is the MEN1 gene. Recently, DPC4 mutations and homozygous deletions have been described in 5/9 (55%) non-functioning PET, thus representing the most frequent genetic aberration described in PET. However, these data are in discordance with comparative genomic hybridization (CGH) results that rarely show genetic losses on chromosome 18. They have also been challenged by immunohistochemical data. We performed a detailed combined DPC4 mutation and deletion analysis in 34 benign and malignant PET. Mutations of the conserved C-terminal exons were not found in all examined PET and allelic loss (LOH) was found to be rare (<6%) by combined microsatellite PCR and FISH analysis. In addition, DPC4 protein expression was retained in all PET that were examined by immunohistochemistry. Therefore, DPC4 inactivation by mutation or deletion appears to be very rare in PET, which confirms the current concept of unrelated mechanisms of tumorigenesis of endocrine versus exocrine pancreatic tumors.

For several reasons, chromosome 3p is thought to be involved in the pathogenesis of sporadic endocrine pancreatic tumours (EPTs): von Hippel-Lindau's disease (VHL gene at 3p25.5) is associated with EPTs; 3p is frequently involved in solid human tumours; and comparative genomic hybridization has identified frequent losses at 3p in EPTs. This study investigated 99 benign and malignant tumours, including 20 metastases, from 82 patients, by microsatellite loss of heterozygosity (LOH) analysis and fluorescence in situ hybridization (FISH) in order to evaluate the importance of chromosome 3p deletions in the molecular pathogenesis and biological behaviour of EPTs, to elaborate a common region of deletion, and to narrow down putative tumour suppressor gene loci. Allelic losses of 3p were found in 58/99 (58.6%) of tumours in 45/82 (54.9%) patients; analysis of seven microsatellite markers (3p26-p21) revealed a common region of LOH at 3p25.3-p23. The LOH frequency was significantly higher in malignant than in benign neoplasms (70.2%, versus 28.0%; p = 0.001). In addition, a strong correlation was found between the loss of alleles on chromosome 3p and clinically metastatic disease (LOH of 73.7% in metastasizing versus 41.5% in non-metastasizing tumours; p = 0.008). EPTs from these patients showed a tendency towards losing large parts or the entire short arm of chromosome 3 with tumour progression. Furthermore, FISH analysis revealed complete loss of chromosome 3 in ten out of 37 EPTs (27%). These results indicate that a putative tumour suppressor gene at 3p25.3-p23 may play a role in the oncogenesis of sporadic EPTs and that losses of larger centromeric regions are associated with metastatic progression. Copyright (C) 2001 John Wiley & Sons, Ltd.

The malignant potential among endocrine pancreatic tumors (EPTs) varies greatly and can frequently not be predicted using histopathological parameters. Thus, molecular markers that can predict the biological behavior of EPTs are required. In a previous comparative genomic hybridization study, we observed marked genetic differences between the various EPT subtypes and a correlation between losses of 3p and 6 and gains of 14q and Xq and metastatic disease. To search for genetic alterations that play a role during early tumor development, we have studied 38 small (< or =2 cm) EPTs, including 24 insulinomas and 10 nonfunctioning EPTs. Small EPTs are usually classified as clinically benign tumors in the absence of histological signs of malignancy. Using comparative genomic hybridization, we identified chromosomal aberrations in 27 EPTs (mean, 4.1). Interestingly, the number of gains differed strongly between nonfunctioning and functioning EPTs (3.4 versus 1.5, respectively; P = 0.0526), as did the number of aberrations in the benign (n = 30) and malignant (n = 8) tumors (3 versus 8.4, respectively; P = 0.0022). In the insulinomas, 9q gain (common region of involvement: 9q34) was most common (50%) and in nonfunctioning EPTs, gain of 4p was most common (40%). Most frequent losses in insulinomas involved 1p (20.8%), 1q, 4q, 11q, Xq, and Y (all 16.7%) and in nonfunctioning EPTs, 6q. Losses of 3pq and 6q and gains of 17pq and 20q proved to be strongly associated with malignant behavior in all of the small EPTs (P < or = 0.0219). Our results demonstrate marked genetic differences between small functioning and nonfunctioning EPTs, indicating that these subtypes evolve along different genetic pathways. In addition, our study endorses the importance of chromosomes 3 and 6q losses to discriminate EPTs with a malignant behavior from benign ones.

Comparative genomic hybridization (CGH) and loss of heterozygosity (LOH) studies frequently reveal losses of 11q23 distally to the MEN-1 gene in sporadic neuroendocrine tumors of the pancreas (NEPT), lung and gastrointestinal tract (NET) as well as in pheochromocytomas (PCC) and parathyroid adenomas (PTA). The recently cloned SDHD gene, which is causative of familial paragangliomas (PGA) is located in the region of the suspected tumor Suppressor gene. Therefore, we analyzed a series of neuroendocrine tumors for mutations and allelic loss of the SDHD gene.We examined 21 neuroendocrine pancreatic tumors, 14 neuroendocrine tumors of the lung and c gastrointestinal tract, 10 parathyroid adenomas, 9 pheochromocytomas and 8 paragangliomas, using the flanking polymorphic markers D11S1347 and D11S900 for detecting allelic loss of the SDHD gene. Mutation analysis was performed using PCR/SSCP (single strand conformation polymorphism) and direct sequencing.Four of 14 neuroendocrine pancreatic tumors, four of seven neuroendocrine tumors of the lung and gastrointestinal tract, one of three pheochromocytomas, one of five paragangliomas and none of five parathyroid adenomas showed allelic loss of the 11q23 region. Mutation analysis revealed two amino acid variants of SDHD present both in the tumor and germline DNA of two patients with sporadic paragangliomas. One affected patient exhibited the combination of paratracheal paraganglioma, C-cell hyperplasia of the thyroid and a pituitary tumor.No gene alterations were identified in the other tumors. Thus SDHD is unlikely to be inactivated by somatic mutations in the examined types of sporadic neuroendocrine tumors on 11q23. As SDHD is an imprinted gene, LOH alone could lead to a complete loss of function in a subset of neuroendocrine tumors. Further expression analyses are needed to clarify this issue.

Parasympathetic paragangliomas (PGLs) represent neuroendocrine tumors arising from chief cells in branchiomeric and intravagal paraganglia, which share several histological features with their sympathetic counterpart sympathoadrenal paragangliomas. In recent years, genetic analyses of the familial form of PGL have attracted considerable interest. However, the majority of paragangliomas occurs sporadically and it remains to be determined whether the pathogenesis of sporadic paraganglioma resembles that of the familial form. Furthermore, data on comparative genetic aberrations are scarce. To provide fundamental cytogenetic data on sporadic and hereditary PGLs, we performed comparative genomic hybridization using directly fluorochrome-conjugated DNA extracted from 12 frozen and 4 paraffin-embedded tumors. The comparative genomic hybridization data were extended by loss of heterozygosity analysis of chromosome 11q. DNA copy number changes were found in 10 (63%) of 16 tumors. The most frequent chromosomal imbalance involved loss of chromosome 11. Six of seven familial tumors and two of nine sporadic tumors showed loss of 11q (86% versus 22%, P = 0.012). Deletions of 11p and 5p were found in two of nine sporadic tumors. We conclude that overall DNA copy number changes are infrequent in PGLs compared to sympathetic paragangliomas and that loss of chromosome 11 may be an important event in their tumorigenesis, particularly in familial paragangliomas.

Our previous comparative genomic hybridization study on sporadic endocrine pancreatic tumors (EPTs) revealed frequent losses on chromosomes 11q, 3p, and 6q, The aim of this study was to evaluate the importance of 6q losses in the oncogenesis of sporadic EPTs and to narrow down the smallest regions of allelic deletion. A multimodal approach com bining polymerase chain reaction-based allelotyping, double-target fluorescence in situ hybridization, and comparative genomic hybridization was used in a collection of 109 sporadic EPTs from 93 patients. Nine polymorphic microsatellite markers (6q13 to 6q25q27) were investigated, demonstrating a loss of heterozygosity (LOH) in 62.2% of the patients. A LOH was significantly more common in tumors >2 cm in diameter than below this threshold as well as in malignant than in benign tumors. We were able to narrow down the smallest regions of allelic deletion at 6q22.1 (D6S262) and 6q23-q24 (D6S310-UTRN) with LOH-frequencies of 50.0% and 41.2 to 56.3%, respectively. Several promising tumor suppressor candidates are located in these regions, Additional fluorescence in situ hybridization analysis on 46 EPTs using three locus-specific probes (6q21, 6q22, and 6q27) as well as a centromere 6-specific probe revealed complete loss of chromosome 6 especially in metastatic disease, We conclude that the two hot spots found on 6q may harbor putative tumor suppressor genes involved not only in the oncogenesis but maybe also in the malignant and metastatic progression of sporadic EPTs.

The pathogenesis of sporadic endocrine pancreatic tumors (EPTs) is still primarily unknown. Comparative genomic hybridization studies revealed loss of 10q in a significant number (nine of 31) of EPTs. The tumor suppressor gene PTEN lies on 10q23, and so, is a candidate to play some role in EPT pathogenesis. Germline PTEN mutations are found in Cowden and Bannayan-Riley-Ruvalcaba syndromes, whereas somatic mutations and deletions are found in a variety of sporadic cancers. The mutation and expression status of PTEN in EPTs has not yet been examined. Mutation analysis of the entire coding region of PTEN including splice sites was performed in 33 tumors, revealing one tumor with somatic L182F (exon 6). Loss of heterozygosity of the 10q23 region was detected in eight of 15 informative malignant (53%) and in none of seven benign EPTs. PTEN expression was assessed in 24 available EPTs by immunohistochemistry using a monoclonal anti-PTEN antibody. Of these 24, 23 tumors showed strong immunoreactivity for PTEN. Only the EPTs with PTEN mutation lacked PTEN protein expression. Although normal islet cells always exhibited predominantly nuclear PTEN immunostaining, 19 of 23 EPTs had a predominantly cytoplasmic PTEN expression pattern. Exocrine pancreatic tissue was PTEN-negative throughout. PTEN mutation is a rare event in malignant EPTs and PTEN protein is expressed in most (23 of 24) EPTs. Thus, intragenic mutation or another means of physical loss of PTEN is rarely involved in the pathogenesis of EPTs. Instead, either an impaired transport system of PTEN to the nucleus or some other means of differential compartmentalization could account for impaired PTEN function. Loss of heterozygosity of the 10q23 region is a frequent event in malignant EPTs and might suggest several hypotheses: a different tumor suppressor gene in the vicinity of PTEN might be principally involved in EPT formation; alternatively, 10q loss, including PTEN, seems to be associated with malignant transformation, but the first step toward neoplasia might involve altered subcellular localization of PTEN.

Despite several loss of heterozygosity studies, a comprehensive genomic survey of pheochromocytomas is still lacking. To identify DNA copy number changes which might be important in tumor development and progression and which may have diagnostic utility, we evaluated genetic aberrations in 29 sporadic adrenal and extra-adrenal pheochromocytomas (19 clinically benign tumors and 10 malignant lesions). Comparative genomic hybridization was performed using directly fluorochrome-conjugated DNA extracted from frozen (16) and paraffin-embedded (13) tumor tissues. The most frequently observed changes were losses of chromosomes 1p11-p32 (86%), 3q (52%), 6q (34%), 3p, 17p (31% each), 11q (28%), and gains of chromosomes 9q (38%) and 17q (31%). No amplification was identified and no difference between adrenal and extra-adrenal tumors was detected. Progression to malignant tumors was strongly associated with deletions of chromosome 6q (60% versus 21% in clinically benign lesions, P = 0.0368) and 17p (50% versus 21%). Fluorescence in situ hybridization confirmed the comparative genomic hybridization data of chromosomes 1p, 3q, and 6q, and revealed aneuploidy in some tumors. Our results suggest that the development of pheochromocytomas is associated with specific genomic aberrations, such as losses of 1p, 3q, and 6q and gains of 9q and 17q. In particular, tumor suppressor genes on chromosomes 1p and 3q may be involved in early tumorigenesis, and deletions of chromosomes 6q and 17p in progression to malignancy.