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Endocrine pancreatic tumors (EPT) and neuroendocrine tumors (NET) occur sporadically and rarely in association with multiple endocrine neoplasia type 1 (MEN1). We analyzed the frequency of allelic deletions and mutations of the recently identified MEN1 gene in 53 sporadic tumors including 30 EPT and 23 NET (carcinoids) of different locations and types. Allelic deletion of the MEN1 locus was identified in 18/49 (36.7%) tumors (13/30, 43.3% in EPT and 5/19, 26.3% in NET) and mutations of the MEN1 gene were present in 8/52 (15.3%) tumors (4/30 (13.3%) EPT and 4/22 (18.1%) NET). The somatic mutations were clustered in the 5' region of the coding sequence and most frequently encompassed missense mutations. All tumors with mutations exhibited a loss of the other allele and a wild-type sequence of the MEN1 gene in nontumorous DNA. In one additional patient with a NET of the lung and no clinical signs or history of MEN1, a 5178-9G-->A splice donor site mutation in intron 4 was identified in both the tumor and blood DNA, indicating the presence of a thus far unknown MEN1 syndrome. In most tumor groups the frequency of allelic deletions at 11q13 was 2 to 3 times higher than the frequency of identified MEN1 gene mutations. Some tumor types, including rare forms of EPT and NET of the duodenum and small intestine, exhibited mutations more frequently than other types. Furthermore, somatic mutations were not restricted to foregut tumors but were also detectable in a midgut tumor (15.2% versus 16.6%). Our data indicate that somatic MEN1 gene mutations contribute to a subset of sporadic EPT and NET, including midgut tumors. Because the frequency of mutations varies significantly among the investigated tumor subgroups and allelic deletions are 2 to 3 times more frequently observed, factors other than MEN1 gene inactivation, including other tumor-suppressor genes on 11q13, may also be involved in the tumorigenesis of these neoplasms.

Although in situ hybridization has been used to examine the distribution of messenger RNA for somatostatin receptor subtypes (sst) in human tumors, the cellular localization of sst1 and sst2A receptors has not been reported. In this study, we describe the cellular localization of human sst1 and sst2A receptor proteins in both cryostat- and paraffin-embedded sections of 25 human tumor tissues using two recently developed polyclonal antibodies. Six somatostatin (SS) receptor (SSR) positive tumors (two gastrinomas, three carcinoids, one pheochromocytoma) and one SSR negative tumor (renal cell carcinoma), selected by positive and negative SSR autoradiography, respectively, were studied by both immunohistochemistry and Western blot analysis. The six SSR positive tumors expressed sst2A, while 4 of 5 expressed sst1 as well. The SSR negative tumor did not express either sst1 or sst2A. Western blot analysis of wheat germ agglutinin purified membrane proteins confirmed the presence of the sst1 and sst2A glycosylated receptors. The paraffin-embedded sections gave best information with respect to the subcellular localization. Sst1 immunoreactivity was observed both on the membrane and in the cytoplasm, while sst2A showed predominantly membrane-associated immunoreactivity. This subcellular distribution of sst1 or sst2A receptors was confirmed in paraffin-embedded sections of 8 additional intestinal carcinoids, 5 gastrinomas and 5 pheochromocytomas. Sst1 receptors were detected in 7 out of 8 carcinoids, in all gastrinomas, and in 4 out of 5 pheochromocytomas, while 6 out of 8 carcinoids, all gastrinomas, and 3 out of 5 pheochromocytomas expressed sst2A receptors. In conclusion, sst1 and sst2A receptors show a differential subcellular localization in human SSR positive tumors. The use of SSR subtype selective antibodies to detect the subcellular distribution of SSR subtypes in individual tumor cells is an important step forward to understand more about the pathophysiological role of the different SSR subtypes in human tumors.

To clarify the role of the MEN1 gene in the tumorigenesis of sporadic adrenocortical tumors, we performed a molecular study on 35 adrenocortical lesions including 6 hyperplasias, 19 adenomas and 10 carcinomas. Loss of heterozygosity (LOH) of the MEN1 gene was assessed by PCR using an intragenic (D11S4946) and 2 flanking microsatellite markers (D11S4936, PYGM) and/or fluorescence in situ hybridization (FISH) with a 40-kb cosmid probe containing the MEN1 gene. The complete coding sequence of the MEN1 gene was screened for mutations using non-radioactive, PCR-based single-strand conformation polymorphism (SSCP) analysis and MDE heteroduplex gel electrophoresis. PCR-LOH and FISH analyses performed in 29 tumors (PCR-LOH in 4, FISH in 17 and both in 8 tumors) revealed allelic deletion of the MEN1 locus in 8 (27.5%) and at 11q13 in 9 (31%) tumors. Furthermore, the frequency of LOH at 11q13 was significantly higher in adrenocortical carcinomas (60%) than in benign lesions (11%). Mutation analysis of tumor samples revealed 9 polymorphisms in 7 tumors (S145S, R171Q, R171Q together with L432L) but no mutations, with the exception of one adrenocortical adenoma. The latter tumor contained a somatic E109X stop codon mutation in exon 2 and a 5178-9G-->A splice mutation in intron 4, which was also detectable in various nontumorous tissues and blood indicative of a germ-line mutation. The patient, who had no clinical signs or family history of MEN1, later also developed a neuroendocrine carcinoma (atypical carcinoid) of the lung. Our findings indicate that inactivating mutations of the MEN1 tumor-suppressor gene appear not to play a prominent role in the development of sporadic hyperplastic or neoplastic lesions of the adrenal cortex and that the newly reported 5178-9G-->A splice mutation in intron 4 might cause a variant of the MEN1 phenotype.

Somatic mutations in the RET proto-oncogene are involved in the pathogenesis of an important subset (40-60%) of sporadic medullary thyroid carcinomas (MTCs) and less frequently (0-31%) in benign, sporadic phaeochromocytomas. Since limited data exist regarding the significance of somatic RET mutations in malignant phaeochromocytomas, we analysed a multicentre series of proven malignant (i.e., metastasised) phaeochromocytomas. Analogous with MTCs, where RET mutations lead to an aggressive behaviour, we hypothesised that somatic mutations would occur more frequently in malignant than in benign phaeochromocytomas. Paraffin-embedded tissue was available from 29 malignant and 27 benign phaeochromocytomas. Exons 10, 11 and 16 were analysed by non-radioactive single-strand conformation polymorphism, heteroduplex gel electrophoresis, restriction enzyme digestion and aberrant band patterns by non-isotopic sequencing. In only 1 of 29 malignant phaeochromocytomas was a mis-sense mutation found (at codon 634 of exon 11), whereas in 15% (4/27) of the benign tumours a point mutation was detected (in 3 tumours in exon 16 at codon 918 and in 1 tumour in exon 10 at codon 618). Absence of these mutations in non-tumourous DNA proved their somatic origin. Contrary to what has been reported for MTCs, oncogenic RET mutations are not associated with an aggressive tumour behaviour in sporadic phaeochromocytomas.

Fetal and placental tissues and maternal sera from a series of 273 cases of first and second trimester fetal loss were collected to detect the frequency of parvovirus B19 infection. In addition, fetal tissues were studied for the presence of congenital anomalies. Serology of maternal sera, histology of fetal tissues and placenta, polymerase chain reaction (PCR), in situ hybridization (ISH), and immunohistochemistry (IHC) were used for the detection of parvovirus B19 infection. Sera were tested for B19-specific immunoglobulin M (IgM) and/or IgG using an enzyme-linked immunosorbent assay technique. Based on serology, 149 cases not related to B19 infection were excluded from further analysis. Two of the remaining 124 cases (0.7% of all 273 cases) had parvovirus B19-specific IgM and IgG at the time of abortion, indicating a recent maternal parvovirus B19 infection. In our histological examination, 10 cases contained nuclear vacuolization in fetal erythroid progenitor cells, either in fetal tissues (n = 2) or in placental tissue (n = 8). However, this vacuolization was considered a fixation artifact and not identical to parvovirus B19-specific nuclear inclusions described in previous reports. Only 1 of these 10 cases had parvovirus B19 DNA detectable in placental tissue by PCR analysis. Neither in this case nor in any of the other cases tested was parvovirus B19 DNA or protein detectable by ISH or IHC, respectively. In none of 41 cases in which fetal tissues were available were congenital anomalies found. In conclusion, the frequency of maternal parvovirus B19 infection in this series of fetal losses is low (0.8%). This low frequency does not allow any conclusions with regard to the occurrence of congenital anomalies resulting from parvovirus B19 infection and the usage of nuclear histology for the detection of fetal parvovirus B19 infection is considered a nonspecific parameter that requires confirmation by PCR.

In the fetal development of the mouse pancreas, endocrine cells have been found that express more than one hormone simultaneously. Our objective was to evaluate the existence of such cells in the human fetal pancreas. We found cells coexpressing two of the major pancreatic hormones (insulin, glucagon, and somatostatin) in sections of eight midgestational (12-18 weeks) pancreata and in 0-7% of cells in single-cell suspensions from midgestational pancreata. By electron microscopy, using granule morphology and immunoelectron microscopic techniques, we could confirm these findings and even detect cells containing three hormones. Morphologically different granules contained different immunoreactivities, suggesting parallel regulation of hormone production and packaging. In six newborn pancreata (born after 22-40 weeks of gestation), we could not find any multiple-hormone-containing cells. Subsequently, we evaluated whether multiple-hormone-containing cells proliferate by using pancreatic fragments and single-cell preparations at the light and electron microscopic level (six pancreata). No endocrine hormone-containing cells incorporated bromodeoxyuridine during a 1-hr culture period, indicating that these cells have lost the ability to proliferate under the conditions chosen. We conclude that, as in mice, the human fetal pancreas of 12-18 weeks of gestation contains endocrine cells that express multiple hormones simultaneously. These (multiple) hormone-containing cells do not seem to proliferate under basal conditions.