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A small subset of familial pancreatic endocrine tumors (PET) arises in patients with von Hippel–Lindau syndrome and these tumors may have an adverse outcome compared to other familial PET. Sporadic PET rarely harbors somatic VHL mutations, but the chromosomal location of the VHL gene is frequently deleted in sporadic PET. A subset of sporadic PET shows active hypoxia signals on mRNA and protein level. To identify the frequency of functionally relevant VHL inactivation in sporadic PET and to examine a possible prognostic significance we correlated epigenetic and genetic VHL alterations with hypoxia signals. VHL mutations were absent in all 37 PETs examined. In 2 out of 35 informative PET (6%) methylation of the VHL promoter region was detected and VHL deletion by fluorescence in situ hybridization was found in 14 out of 79 PET (18%). Hypoxia inducible factor 1α (HIF1-α), carbonic anhydrase 9 (CA-9), and glucose transporter 1 (GLUT-1) protein was expressed in 19, 27, and 30% of the 152 PETs examined. Protein expression of the HIF1-α downstream target CA-9 correlated significantly with the expression of CA-9 RNA (PPVHL deletion (PPPVHL alterations and signs of hypoxia signalling were characterized by a significantly shortened disease-free survival. We conclude that VHL gene impairment by promoter methylation and VHL deletion in nearly 25% of PET leads to the activation of the HIF-pathway. Our data suggest that VHL inactivation and consecutive hypoxia signals may be a mechanism for the development of sporadic PET with an adverse outcome.

Parathyroid carcinoma as the cause of primary hyperparathyroidism was found in 3 out of 102 patients. The diagnosis was based on the findings of markedly increased serum calcium and parathormone values, definite infiltration of the tumour into the surrounding tissue, mitoses, wide connective tissue septa in the tumour as well as rupture of the capsule and penetration of vessels. The diagnosis can only be made after close cooperation between endocrinologists, surgeons and pathologists.

The pathology of insulinoma and gastrinoma was studied in 81 patients (31 men, 50 women, mean age 48.4 years) suffering from persistent hyperinsulinaemic hypoglycaemia, and in 44 patients (28 men, 16 women, mean age 48.5 years) with Zollinger-Ellison syndrome. Insulinomas were seen in the pancreas of all patients with persistent hyperinsulinaemic hypoglycaemia. In 70 of the 81 patients the insulinoma was solitary, whereas six patients had multiple insulinomas. In five patients with multiple endocrine neoplasia type I, multiple endocrine tumours of the pancreas were visible, only one of them in each case being an insulinoma. 75% of all insulinomas were less than 2 cm in size and 15% were malignant. 18 of the 44 Zollinger-Ellison syndrome patients also had multiple endocrine neoplasia type I. Nine of these patients presented with duodenal gastrinomas which were often multiple and smaller than 0.5 cm. The gastrinoma was located in the pancreas of one of the patients with multiple endocrine neoplasia, in two patients in lymph nodes, and no gastrinoma was identified in the specimens from six patients. 33% of the duodenal gastrinomas had metastasised. Solitary gastrinomas were found in all 26 patients with sporadic Zollinger-Ellison syndrome, 14 being located in the pancreas (diameter over 2 cm in eight cases) and ten in the duodenum (diameter less than 1 cm in seven cases). 16 of these gastrinomas were malignant.

Background: Patients with a multiple endocrine neoplasia type 1 (MEN1)-associated Zollinger–Ellison syndrome (ZES) show multifocal duodenal gastrinomas and precursor lesions. Aims: To test these lesions for loss of heterozygosity (LOH) of the MEN1 gene locus on chromosome 11q13, and to investigate whether the MEN1-related endocrine cell changes also involved somatostatin cells. Material and methods: Tissue specimens from six patients with MEN1 and ZES were analysed by immunohistochemistry and immunofluorescence. LOH analysis was performed by fluorescence in situ hybridisation (FISH), using probes containing the MEN1 gene locus and the centromere 11 (C11) region. For simultaneous analysis of hormones and allelic deletions, a combined FISH/immunofluorescence protocol was established. Results: 28 of a total of 33 duodenal neuroendocrine tumours (NETs) were gastrin-producing tumours; 13/28 (46.4%) revealed LOH on 11q13 and/or C11. Five of the NETs were somatostatin-expressing tumours, two revealing LOH. Allelic loss was detected in tumours as small as 300 μm (gastrin) and 400 μm (somatostatin) in diameter. The gastrin-producing tumours showed different deletion/retention patterns. Hyperplastic somatostatin cell lesions, similar to those of the gastrin cells, were present in all patients. The hyperplastic lesions of both cell lines consistently retained both 11q13 alleles. Conclusions: Allelic deletion of the MEN1 gene may reflect a pivotal event in the development of multifocal gastrin and somatostatin cell neoplasms in the duodenum of patients with MEN1. The observation of distinct deletion patterns in small synchronous tumours supports the concept that each gastrin-producing tumour in an individual MEN1 patient arises from an independent cell clone.

Das Kapitel behandelt die Pathologie des disseminierten neuroendokrinen Zellsystems. Aufgrund seiner endokrinen Funktionen kommt es im Falle von Erkrankungen haufig zu einer inadaquaten Hormonsekretion mit entsprechender Symptomatik. Die hierfur verantwortlichen hyperplastischen und neoplastischen Veranderungen werden im Einzelnen besprochen. Daruber hinaus kommen die zahlreichen nichtsyndromatischen neuroendokrinen Neoplasien zur Sprache, wobei auf die unterschiedliche Prognose in Abhangigkeit von Primarlokalisation, Hormonproduktion, histologischer Differenzierung und Stadium eingegangen wird. Da im gastroenteropankreatischen System von neuroendokrinen Neoplasien gesprochen wird, in anderen Organen jedoch von Karzinoiden, wird explizit auf die nomenklatorischen Unterschiede bei den Tumoren hingewiesen.

Das Kapitel behandelt die multiplen endokrinen Neoplasien Typ 1 und Typ 2 mit ihren Unterformen und gibt einen kurzen Uberblick uber weitere Syndrome mit endokrinen Neoplasien. Neben Atiologie, Pathogenese und Genetik werden die typische Lokalisation sowie Morphologie der Befunde zusammen mit der klinischen Symptomatik, dem Verlauf und der Prognose beschrieben. In einem zweiten Teil werden die pluriglandulare endokrine Insuffizienz und deren zwei typische Unterformen abgehandelt.

Das Kapitel fasst die paraneoplastischen Syndrome mit endokrinem Bezug zusammen. Der Fokus liegt auf den Syndromen der Hyperkalzamie, der Hypoglykamie und der inadaquaten ADH-Produktion sowie dem Cushing- und dem Choriogonadotropin-Syndrom, wobei jeweils die Pathogenese und die in Frage kommenden Tumoren besprochen werden.

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 5178–9GA splice mutation in intron 4, which was also detectable in various non-tumorous 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 5178–9GA splice mutation in intron 4 might cause a variant of the MEN 1 phenotype. Int. J. Cancer 80:373–379, 1999. © 1999 Wiley-Liss, Inc.

Optimal treatment parameters and appropriate methods of examination for Nd:YAG laser coagulation of the human prostate in dependence of power-setting and time using the SideFocus side-firing delivery system have been determined. Transurethral free-beam laser coagulation was performed in 10 patients prior to planned radical prostatectomy or cystoprostatectomy. Thereof, 6 patients underwent transperitoneal laparoscopic lymphadenectomy and laser coagulation of the prostate between 4 and 9 days prior to open surgery. These same patients had undergone MRI examination with an endorectal coil during staging and prior to radical prostatectomy. Depth and volume of coagulated prostatic tissue of a 4-quadrant laser application were measured at power-settings of 40 W/90 s and 60 W/60 s. There was no difference in volume coagulation between the two treatment modalities. In contrast there was a significant difference in volume coagulation between those prostates removed at 4-9 days and those removed at 60-210 min after laser coagulation. In histological and MRI examination, however, there was a strong correlation with view to coagulation volume in histological and MRI examination. Using the SideFocus side-firing laser fiber, both treatment modalities showed comparable volume coagulation. Laser-induced changes are conclusively discernible by 4 days, MRI examination with an endorectal coil thus being perfectly suitable for assessment.

The pathogenesis of alcoholic chronic pancreatitis and its relationship to alcoholic acute pancreatitis are debated. According to our recent clinical long-term study, alcoholic chronic pancreatitis seems to evolve from severe acute pancreatits. The aim of this study was to correlate clinical findings to the pancreatic histopathology at early and advanced stages of the disease.Morphological changes (pseudocysts, autodigestive necrosis, calcification, and perilobular and intralobular fibrosis) were recorded in 37 surgical and 46 postmortem pancreas specimens of 73 patients from our long-term series, who progressed from clinically acute to chronic pancreatitis (mean follow-up, 12 years). Pancreatic function was monitored at yearly intervals.Surgical interventions were performed at a mean of 4.1 years from onset. Histologically, focal necrosis (49%) and mild perilobular fibrosis (54%) predominated, Pseudocysts (n = 41, mostly postnecrotic) occurred in 88% within 6 years from onset. Autopsy specimens were obtained at a mean of 12 years. These pancreata often showed severe perilobular and intralobular fibrosis (85%) and calcifications (74%), but rarely necrosis (4%). Fibrosis correlated with progressive pancreatic dysfunction (P0.001), particularly in the 10 patients with two histological assessments (mean interval between biopsy and autopsy, 8 years).The data support an evolution from severe alcoholic acute pancreatitis to chronic pancreatitis.

To evaluate reliable methods for detection of hepatitis C virus (HCV) infection in routinely processed liver biopsies we analyzed formaldehyde-fixed and paraffin-embedded liver specimens of 10 patients with serological confirmed HCV infection. We compared (1) conventional histology; (2) indirect immunofluorescence using the mAb TORDJI-22 (Clonatec, Paris, France); (3) RT-PCR using total RNA and Southern blotting with chemiluminescent detection; (4) non-radioactive in-situ hybridization (ISH) with digoxigenin-labeled oligo- and cRNA probes; (5) direct in-situ RT-PCR with incorporation of labeled nucleotides into PCR-products, and (6) indirect in-situ RT-PCR using subsequent ISH for the visualization of intracellular PCR-products. Our results indicate that: (1) using the histological criteria described by Lefkowitch et al. [Gastroenerology 1993;104:595] together with clinical data, most chronic HCV infections can be diagnosed by conventional histology, if liver biopsies specimens are adequate; (2) the commercially available mAb TORDJI-22 appears to crossreact with non-HCV epitopes, resulting in false positives; (3) molecular methods performed on routinely fixed and processed liver biopsies frequently yield false negative results due to sampling problems, low viral copy number and RNA degradation in infected cells; (4) analysis of HCV-RNA by RT-PCR of extracted total RNA is more sensitive than indirect in-situ RT-PCR or ISH; and (5) direct in-situ RT-PCR is not reliable despite the use of modifications such as DNase pretreatment and hot-start procedures. It is concluded, that several molecular methods for HCV detection must await further improvements of protocols to be suitable for routine diagnostics on paraffin-embedded liver biopsies.

The syndrome of multiple endocrine neoplasia type 1 (MEN 1) is an autosomal dominant tumour disease of the neuroendocrine system with manifestations in the parathyroids, pancreas, duodenum and pituitary gland and rarely also in the stomach and thymus. Recently, the MEN 1 gene locus has been mapped to the long arm of chromosome 11. This gene most likely belongs to the tumour suppressor genes, the allelic loss of which causes tumour development. The pancreatic and duodenal tumours may metastasize, but usually have a low malignant potential. Clinically, most MEN 1 patients present between the age of 20 and 35 with hyperparathyroidism and/or Zollinger-Ellison syndrome.

We recently reported that the gold-labeled monoclonal antibody MAb 735, reactive with a long-chain form of alpha-2,8-linked polysialic acid (polySia) found on the neural cell adhesion molecule (NCAM), is useful to immunohistochemically distinguish small-cell lung carcinomas from neuroendocrine carcinomas with higher grade of differentiation (carcinoids) and other types of lung carcinomas (Am J Pathol 1991;139:297). In this study, we tested the occurrence of polySia in various types of malignant thyroid tumors and C-cell hyperplasia to determine whether polySia is a useful adjunct for the differential diagnosis of medullary thyroid carcinoma (MTC) and other thyroid neoplasms and to distinguish primary from secondary (reactive) C-cell hyperplasia (CCH). We examined formaldehyde-fixed and paraffin-embedded sections of 79 thyroid lesions, consisting of 33 MTC (14 familial and 19 sporadic tumors), 13 follicular, 11 papillary, 16 anaplastic carcinomas, and four glands with primary and two with secondary CCH. We applied a direct and an indirect immunogold-silver technique for polySia, CT, and CEA detection, respectively. All 33 MTC showed a strong cell-surface-associated immunoreactivity for polySia, which was sensitive to endoneuraminidase digestion. The polySia immunoreactivity of nerves served as an internal control in all specimens. Immunoreactivity for CT and CEA was present in all MTC with the exception of one recurrent tumor with features of an anaplastic MTC type. All other thyroid neoplasms were nonreactive for polySia, CT, and CEA. Primary CCH associated with MTC showed a strong polySia immunostaining, which was less intense in primary CCH not combined with MTC. In normal-appearing C cells and in secondary CCH, staining for polySia was absent in the majority of cases. We conclude that polySia of NCAM is a valuable marker to distinguish medullary carcinomas from other types of thyroid carcinomas. Furthermore, it allows for the discrimination of primary from secondary C-cell hyperplasia and may be helpful to better define the normal range of C cells in unaffected members of a family with a history of multiple endocrine neoplasia (MEN)-II.

For 2 years a 72-year-old man had suffered from nonspecific upper abdominal discomfort and hepatomegaly. The gamma-glutamate transaminase concentration was increased to 121 U/l, the erythrocyte sedimentation rate was 80 mm in the first hour. Histological examination of tissue from the enlarged liver (22 cm in the midclavicular line) revealed the diagnosis of amyloidosis. The gastric mucosa, duodenum and rectum were not involved. Two years later ascites developed; six months after this he was again hospitalized in hepatic coma. Now, for the first time, a type IgA-lambda paraprotein was demonstrated by serum immunoelectrophoresis. The patient died of slowly progressing anicteric liver failure after having been ill for a total of 4 1/2 years. At autopsy there were extensive amyloid deposits throughout the liver and spleen so that the structure of these organs was hardly recognizable. The amyloid deposits in the liver were restricted to the glomerular region, while there was no amyloid in the heart. Histochemical tests showed that the deposits were strongly positive to the anti-lambda antibody. This was thus a case of primary (AL-lambda) amyloidosis of the liver and spleen which had taken an unusually prolonged course, because the heart was not involved at all and the kidneys only slightly.

Parvovirus infection of pregnant women leading to a transplacentar infection of the fetus may result in hydrops fetalis, and ultimately in intrauterine death of the fetus. In situ hybridization with a biotinylated as well as with a 35S-labeled probe for human parvovirus B19 was performed on formalin-fixed paraffin-embedded (FFPE) tissue from a fetus suffering from non-immunologic hydrops fetalis. Histology was suggestive of viral infection probably with human parvovirus. Parvovirus DNA could be detected and precisely localized mainly in the nuclei of erythroid precursors cells within fetal blood vessels of all organs examined. There was no detection of B19 nucleic acid in parenchymal cells of the placenta or the fetal organs, nor within maternal blood cells. These findings are in agreement with the well-known properties of animal parvoviruses to replicate exclusively in proliferating cells. Taking into consideration the problems in diagnosing human parvovirus infection by light microscopy, we conclude that in situ hybridization with an appropriate non-radioactive probe is a valuable, rapid and safe complementary detection method for the diagnosis and study of human parvovirus infections. The 35S-labeled probe is more sensitive than the biotinylated probe, but has the disadvantages of lower resolution of the signal, longer duration of the assay, the hazard of radioactivity and the shorter shelf-life of the probe.

We investigated the utriculus prostaticus from the fetal period up to adulthood in 148 prostates. During the second half of gestation the utriculus had a simple tubular or a cystic form and was lined with metaplastic squamous epithelium which showed immunohisto-chemical positivity for different keratins, carcinoembryonic antigen, and peanut agglutinin binding sites. After birth, alveolar outgrowths of the utriculus developed. After puberty, the utriculus had become a complicated and variable structure. The epithelium no longer differed from that of the prostate glands either morphologically or immunohistochemically. Within the epithelium numerous endocrine cells were found containing neuron-specific enolase, chromogranin, and serotonin. The utriculus and ejaculatory ducts were embedded in a fibrous stroma with, after birth, numerous plexus-like blood vessels. This fibrous zone was peripherally bordered by a layer of smooth muscle. There was no evidence for a function of the utriculus differing from that of the prostate glands. Since the epithelium of both structures is identical immunohistochemically, the epithelium of the sinus urogenitalis most likely participates in the lining of the utriculus during embryogenesis.

Despite their common origin from follicular epithelial cells, papillary and follicular thyroid carcinomas differ in their histology and clinical course. In this study the transcriptional profiles of these tumors in comparison with normal thyroid tissue were established. The aim was the development of a molecular tool providing additional information to current histopathological diagnosis and allowing further insight into tumorigenesis. Genome wide expression profiling was performed using Human Unigene Set--RZPD 2 high density cDNA macroarrays comprising 76,000 genes as probes and radioactively labeled cDNA targets retrotranscribed from the isolated RNA of three papillary and three follicular thyroid carcinomas as well as three normal thyroid tissues. 8600 genes differing in their expression between the three groups were selected and printed onto subarrays. Radioactively labeled cDNA targets obtained from 16 papillary carcinomas, 13 follicular carcinomas and 17 normal thyroid tissues were hybridized to these subarrays. 200 genes exhibited a statistically significant expression difference between the two tumor types (p0.01). In a hierarchical cluster analysis of 124 of these genes (46 known genes and 78 ESTs) the algorythm divided the tumor samples into two groups corresponding to the papillary and follicular thyroid carcinomas. The clearcut diagnostic potential of this method has to be corroborated in a prospective study. Several of the differentiallly expressed genes are known to play a role in tumor development and metastasis. Some of the genes up- or down-regulated in both tumor types are members of known oncogenic pathways in thyroid carcinomas. The complete understanding of complex genome wide expression profiles however awaits a longstanding advancement of hypothesis driven research.

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

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.

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.

Aims: To investigate whether alterations of the HER2 gene occur more frequently in histologically unfavourable subtypes of invasive breast cancer. Methods: The study was composed of nine invasive apocrine, six lipid-rich, 12 glycogen-rich, 11 micropapillary and 33 pleomorphic lobular breast carcinomas. Lymph node involvement was represented in all subgroups. HER2 status was confirmed in all cases by using immunohistochemistry (CB11, Herceptest) and fluorescent in-situ hybridization (FISH) analysis (Vysis). Results: Micropapillary and apocrine carcinomas showed the highest rate of protein overexpression (72% and 66%) and gene amplification (45% and 44%). Protein overexpression was common in poorly differentiated pleomorphic lobular carcinomas (56%); however, this subgroup failed to show an increased number of gene copies by FISH (31%). The incidence of HER2 overexpression (33% and 50%, respectively) and gene amplification (25% and 33%, respectively) among glycogen-rich and lipid-rich carcinomas was not higher than that observed in breast cancer generally. Conclusion: Our data suggest that preferential involvement of the HER2 gene in micropapillary and apocrine breast carcinomas may contribute to their aggressive behaviour.

The characterization of clinical, histopathological, immunohistochemical, and genetic features of intimal sarcomas arising in the pulmonary artery is presented in this study. Four resected lungs, one endarterectomy specimen and three biopsies from eight patients (four males and four females; median age 41 years) suffering from intimal sarcomas of the pulmonary artery using conventional stains, immunohistochemistry, and comparative genomic hybridization (CGH) were analyzed. The predominant clinical presentation was dyspnea (all eight patients) and febrile pulmonary disease (six of eight). Signs of embolic lung disease were present in all patients. One patient died postoperatively, six patients died of disease 8-35 months after presentation, and one patient was alive 6 months after surgery. Histopathological examination of the submitted material showed spindle cell, partially myxoid and pleomorphic sarcomas. Metastases were histologically confirmed in three patients (lung, pleura, and skull). Immunohistochemically, vimentin was strongly expressed in all tumors. Focal positivity was observed for alpha smooth muscle actin, CD117, CD68, p53, and bcl2. No reaction could be obtained for endothelial markers. The proliferation index Ki-67 was between 5% and 80%. Six examined tumors were positive for mdm2. In the CGH analysis, gains and amplifications in the 12q13-14 region were found in six of eight tumors (75%). Other, less consistent alterations, were losses on 3p, 3q, 4q, 9p, 11q, 13q, Xp, and Xq, gains on 7p, 17p, and 17q, and amplifications on 4q, 5p, 6p, and 11q. Intimal sarcomas of the pulmonary artery are tumors with an unfavorable prognosis and poorly differentiated morphology. A majority of tumors show a consistent genetic alteration (gains and amplifications in the 12q13-14 region) and overexpression of mdm2, implicating the mdm2/p53 pathway as a possible mechanism in the tumor pathogenesis.

CD44, a family of closely related glycoproteins generated by alternative splicing, as well as the increased beta 1,6-branching of Asn-linked oligosaccharides (beta 1,6-branches), have been implicated in tumor progression and metastasis. We have investigated the expression of CD44 standard (CD44s), various CD44 splice variants (CD44v3, -v4, -v5, -v6 and -v9), and of beta 1,6-branches in a total of 37 paraffin-embedded human primary melanomas and metastases. Out of the 28 studied primary melanomas, 27 were positive for CD44s, 21 for CD44v5 (cytoplasmic staining) and 26 for beta 1,6 branches. Furthermore, superficial spreading melanomas showed a significant (p = 0.004) stronger staining for CD44s than the thick (< 1.5 mm) nodular melanomas, whereas no significant difference was found with regard to staining for CD44v5 and beta 1,6-branches. Eight of the 9 studied melanoma metastases were positive for CD44s, 6 for CD44v5 (cytoplasmic staining) and 7 for beta 1,6-branches. No CD44v3, -v4, -v6 and -v9 could be detected in any of the tumors. On average, metastases as compared to primary tumors, exhibited a significant (p = 0.002) weaker staining for CD44s. However, metastasizing melanomas could not be distinguished from non-metastasizing ones based on CD44 immunostaining.

We analysed the value of the expression of beta 1,6 branching of asparagine-linked oligosaccharide chains and polysialic acid of the neural cell adhesion molecule (NCAM) in predicting malignant behaviour in human insulinomas, as these glycoconjugates have been associated with invasive growth and metastatic potential. Fifty-three insulinomas from patients with well-documented clinical and follow-up data were investigated. Lectin histochemical staining for beta 1,6 branches revealed that 11 (74%) of the 15 malignant insulinomas stained more strongly than normal beta cells. However, in as many as 23 (63.1%) of the 38 benign insulinomas with a disease-free follow up for 4-18 years (average 8 years), a staining intensity equivalent to that of malignant tumours was found. Two (13%) of the malignant insulinomas and 1 of the 4 liver metastases studied were unstained. None of the 53 insulinomas (and the rat RIN insulinoma) re-expressed polysialic acid as demonstrated by immunohistochemistry and Western blotting with the monoclonal antibody 735. Therefore, histochemical staining for beta 1,6 branches and immunohistochemistry for polysialic acid are unlikely to be of value as prognostic indicators for patients with insulinomas.

We investigated the possible role of the RET proto-oncogene, which has recently been identified as the susceptibility gene for multiple endocrine neoplasia type 2, in the development of sporadic neuroendocrine tumors from different locations. DNA extracted from paraffin-embedded specimens of 112 neuroendocrine tumors was screened for somatic RET point mutations in exons 10, 11, 13, 15, and 16, where recently oncogenic mutations have been described in a subset of sporadic medullary thyroid carcinomas and pheochromocytomas. Methods employed included nonisotopic PCR-based single strand conformation polymorphism (PCR-SSCP) analysis, heteroduplex gel electrophoresis, and restriction enzyme digestion. The nucleotide sequence of samples with aberrant band patterns was identified by nonisotopic direct sequencing of PCR-amplified DNA. Forty-four percent (7/16) of sporadic medullary thyroid carcinomas and 15% (3/20) of pheochromocytomas contained a somatic, heterozygous point mutation at codon 918 of exon 16 (ATG --> ACG) causing a Met --> Thr substitution. None of the remaining 4 parathyroid adenomas, 8 pituitary adenomas, 17 pancreatic neuroendocrine tumors, 11 pulmonary and 10 gastrointestinal carcinoids, 7 small cell lung carcinomas, 5 neuroblastomas, 10 malignant melanomas, or 4 schwannomas contained mutations in any of the five RET exons tested. Although the numbers of each investigated neuroendocrine tumor type are small, our data indicate that oncogenic RET proto-oncogene mutations are involved in the formation of a subset of sporadically occurring medullary thyroid carcinomas and pheochromocytomas but do not appear to be generally important in the formation of other types of sporadically occurring neuroendocrine tumors.

We studied the distribution of the large hyaluronan-binding proteoglycan versican (also known as PG-M) in human adult tissues using affinity-purified polyclonal antibodies that recognize the core protein of the prominent versican splice variants VO and V1. Versican was present in the loose connective tissues of various organs and was often associated with the elastic fiber network. Furthermore, it was localized in most smooth muscle tissues and in fibrous and elastic cartilage. Versican staining was also noted in the central and peripheral nervous system, in the basal layer of the epidermis, and on the luminal surface of some glandular epithelia. In blood vessels, versican was present in all three wall layers of veins and elastic arteries. In muscular arteries the immunoreactivity was normally restricted to the tunica adventitia. However, it appeared in the media and the split elastica interna of atherosclerotically transformed vessel walls. Our survey of the distribution of versican in normal human tissues now forms the basis for extended studies of potentially aberrant versican expression during pathogenic processes.

CD44 isoforms have been reported to be involved in tumor invasion and metastasis formation. Normal human skin expresses high levels of CD44 isoforms, but little is known about their expression in epidermal skin tumors. Expression of CD44 standard (CD44s) and variant exon (CD44v3, -v4, -v5, -v6, -v9)-encoded gene products has been studied in 74 benign, semi-malignant and malignant human epithelial skin tumors using a panel of well-characterized, variant exon-specific monoclonal antibodies (MAbs). Sensitivity and resolution of the immunohistochemical staining in paraffin sections was substantially improved by using microwave-based antigen retrieval and an optimized streptavidin-biotin-peroxidase technique. Immunostaining was evaluated semi-quantitatively and correlated with tumor type and degree of histological differentiation by non-parametric statistical tests. Furthermore, the relationship between CD44 expression and cellular proliferation rate as defined by the Ki-67 antigen was analyzed in basal cell carcinomas. We found a significant correlation between tumor type and CD44 isoform expression. Basal cell carcinomas exhibited the weakest staining and keratoacanthomas the strongest. Squamous cell carcinomas ranged in between, with a tendency to down-regulate CD44 expression upon de-differentiation. In basal cell carcinomas, an inverse relationship between CD44 expression and proliferation rate was directly demonstrated at the cellular level using double immunolabelling. Our data indicate that qualitative and quantitative changes in CD44 splicevariant expression in human skin tumors do not correlate with invasive and metastatic potential but are rather related to the degree of tumor differentiation.

We report on a patient with a rare hepatosplenic γδ T-cell lymphoma (γδ TCL) presenting clinically with B-symptoms, hepatosplenomegaly and pancytopenia. During the initial stage of the disease the sparse malignant cells could not be detected histologically. Furthermore, their identification was obscured by massive macrophage proliferation with haemophagocytosis in the spleen. Diagnosis was established by detection of a clonal T-cell receptor (TcR) rearrangement and, retrospectively, by demonstration of rare cells expressing an aberrant T-cell phenotype. The findings in this patient emphasize that minimal neoplastic T-cell infiltrates can lead to severe clinical symptoms. Initial biopsy findings may be misinterpreted as benign. γδ TCL may elaborate lymphokines that suppress haematopoiesis, leading to pancytopenia and macrophage proliferation.

Multiple endocrine neoplasia type 1 (MEN 1) is an inherited disease of the neuroendocrine cell system affecting primarily the parathyroids, pancreas, duodenum and the anterior pituitary. The pancreatic and duodenal tumours may metastasize, but generally have a low malignant potential. The diagnosis of MEN 1 is usually made in the second decade of life and based on the involvement of at least two organs and a family history. The recent discovery of the MEN 1 locus on the centromeric region of the long arm of chromosome 11 may become a further diagnostic criterion. The use of flanking DNA markers permits presymptomatic testing for MEN 1 in affected families.

In this article new classifications of the neuroendocrine tumours of the lung, pancreas and gut are proposed. These classifications use a common frame work and attempt to consider the morphological, functional as well as biological features of the tumours.

In the endocrine pancreas, chromogranins A and B as well as secretoneurin (a biologically active peptide processed endoproteolytically from secretogranin II) are most intensely expressed in alpha (glucagon) cells. We examined whether the functional status of neoplastic and nonneoplastic human alpha cells is reflected in the expression patterns of chromogranins/secretogranins. Neoplastic alpha cells were analysed immunocytochemically in six functioning glucagonomas and 37 nonfunctioning neuroendocrine tumours (29 with alpha cells) for their immunoreactivity to chromogranin A and B, as well as secretoneurin. There was no difference in the staining intensity for either peptide between glucagonomas and nonfunctioning, alpha cell containing tumours. Nonneoplastic alpha cells from patients with a functioning glucagonoma showed a decreased glucagon immunoreactivity, whereas the expression of chromogranin A (but not chromogranin B and secretoneurin) was as intense as in alpha cells not associated with glucagonoma syndrome. These results suggest that the expression of chromogranins/secretogranins in neoplastic alpha cells of the pancreas may be independently regulated from the cells' functional status. In nonneoplastic alpha cells there seems to be an association between glucagon production and chromogranin B and secretoneurin expression.

The classification of chronic hepatitis distinguishing benign chronic persistent hepatitis from severe chronic active hepatitis was constructed without knowledge of well-defined aetiological factors. Better understanding of the different hepatitis-viruses has shed new light on this subject. Chronic viral hepatitis B and C each show typical histological patterns. The validity of the conventional classification has been evaluated by a comparative study of chronic viral hepatitis B and C. 130 biopsies from 110 patients with chronic hepatitis C (CH-C) proven serologically by antibodies (second generation testing) were compared with 105 biopsies from 73 patients with chronic hepatitis B (CH-B). These were scored semi-quantatively. In CH-C, lymphoid follicles and/or aggregates were found in 88.5%, fatty degeneration in 51%, bile duct lesions in 46.2%, and Mallory body-like material in the hepatocytes in 9.2%. The portal lymphocytic infiltration generally predominated over the necro-inflammatory lesions of the parenchyma. Chronic persistent hepatitis (defined by the presence of portal hepatitis) was present exclusively in CH-C. Chronic lobular hepatitis was found exclusively in CH-B. We conclude that the histological criteria described for CH-C are highly suggestive of the diagnosis, that the artificial subdivision of chronic hepatitis into CPH and CAH is obsolete and that the histological assessment of chronic hepatitis should consist of a grading of inflammatory activity (minimal, mild, moderate, severe) and staging of fibrosis (extent of distortion of architecture). The final diagnosis should be based on the demonstration of the aetiological agent.

Non-familial human adrenocortical adenomas and carcinomas were screened for mutations in exons 5-8 of the p53 tumor suppressor gene by single-strand-conformation-polymorphism (SSCP) analysis, followed by direct sequencing of PCR-amplified DNA. Point mutations in codons 12, 13 and 61 in H-ras, K-ras and N-ras proto-oncogenes were similarly assessed by direct DNA sequencing. Three out of 15 primary adrenocortical carcinomas (20%) contained a mis-sense point mutation in the conserved regions (exons 5 and 8) of the p53 gene. Mutations were located in codon 157 (GTC-->TTC; Val-->Phe), codon 163 (TAC-->AAC; Tyr-->Asn), and codon 273 (CGT-->TGT; Arg-->Cys). The mutation in codon 157 was detected in the primary tumor as well as in brain and lymph-node metastases. Among 18 adrenocortical adenomas, there was only a single non-miscoding mutation in codon 295 (CCT-->CCC; Pro-->Pro). These data suggest that mutational inactivation of the p53 gene occurs in a minority (20%) of sporadic adrenocortical carcinomas and that these mutations constitute a late event in the multi-step process of malignant transformation. No ras mutations were detected in any of these tumors, suggesting that these genes are not involved in the development of tumors originating from the adrenal cortex.

Tissue samples of 421 HIV-positive patients have been studied for the presence and distribution of Epstein–Barr virus (EBV) subtypes A and B. This was done by PCR, EBER in situ hybridization, and immunohistochemical detection of EBV latent membrane protein (LMP) in AIDS-associated malignant lymphomas (16 cases) and lymphatic organs of patients without lymphoma (5 cases). Eleven cases were considered to be EBV-positive, with type A in four, and type B virus in four other cases. In patients with malignant lymphoma, EBV was localized in tumour tissue exclusively. One patient without lymphoma presented with multiple EBV genome type B-positive cells in all the lymphoid tissue samples examined. In HIV-positive patients, both subtypes of EBV, A and B, may play a role in the pathogenesis of lymphoproliferative lesions.

A female patient suffering from a bronchial neuroendocrine tumor with unilateral left-sided carcinoid heart disease is reported. Repeated x-ray films of the chest showed a slowly growing lung tumor in the left lower lobe. The patient refused any diagnostic or therapeutic procedure to define the type of the tumor. During the follow-up of 24 years she developed severe mitral and moderate to severe aortic insufficiency, both invasively quantified by thermodilution techniques. During surgery for double valve replacement the patient died from left ventricular heart failure. Necropsy revealed the typical pattern of a bronchial neuroendocrine tumor without metastases. Examination of the heart disclosed the characteristic deposits of fibrous tissue on the cusps of both the mitral and the aortic valves whereas the right heart showed no abnormalities. Review of the literature suggests the unilateral left-sided carcinoid heart disease to be a very rare finding, its pathogenesis remains to be elucidated.

Solid-cystic (papillary-cystic) tumours (SCT) of the pancreas are distinctive neoplasms with a predilection for young female patients. This is the first detailed report describing the occurrence of SCT in two young male patients. Except for the extapancreatic occurrence of one of the tumours (in the retroperitoneal region behind the head of the pancreas), all other clinicopathological features were identical to those characterizing the SCT in women. Immunostaining was (at least focally) positive for Lu 5 (broad spectrum keratin marker), vimentin and alpha-1-antitrypsin. The tumours were negative for neuroendocrine markers (except for neuron-specific enolase), pancreatic hormones and enzymes, pancreatic stone protein, carcinoembryonic antigen, CA 19-9 and nuclear oestrogen and progesterone receptors. This report does not support the suggested female sex hormone dependence of SCT.

Eighteen granular cell tumors from various sites were examined with antisera directed against protein S-100, neuron specific enolase (NSE), alpha-1-antichymotrypsin, and alpha-1-antitrypsin, glial fibrillary acidic protein (GFAP), lysozyme, factor VIII-related antigen, myoglobin and vimentin, as well as with a monoclonal antibody (lu-5) directed against a panepithelial marker. The immunocytochemical reaction pattern of the tumors was heterogeneous. The brain and pituitary tumors and one thyroid tumor reacted for alpha-1-antichymotrypsin and alpha-1-antitrypsin, but not for S-100 protein and NSE. However, tumors from other sites showed immunoreactions for S-100 protein and NSE and some also for vimentin. Reactions for alpha-1-antichymotrypsin and alpha-1-antitrypsin were not observed. All other reactions were similarly negative. We conclude that the morphologically homogeneous group of granular cell tumors is biologically heterogeneous.

A mouse monoclonal antibody (mAB lu-5) was prepared using a lung cancer cell line as an antigen. The selected clone produces an IgG with a gamma-1 heavy chain and a kappa-light-chain. Immunohistochemical testing of mAB lu-5 on 117 normal tissue biopsies and 474 tumours revealed reactivity with an intracytoplasmic, formaldehyderesistant antigen present in most epithelial and mesothelial cells, but absent in mesenchymal cells. The antibody can therefore be used as a first order, pan-epithelial marker. It proved also useful for fast tumour diagnosis on frozen sections.

Pituitary tumors from 376 patients were investigated, using immunocytochemical techniques at the light and electron microscopic level, and autoradiography combined with immunocytochemistry for localizing somatostatin (SRIH) receptors. Prolactinomas, growth hormone-secreting adenomas causing acromegaly, and hormonally inactive adenomas were most frequently observed (153, 86, and 90 tumors, respectively). Among the latter, we could distinguish "alpha-only adenomas," many of which were oncocytomas. At the light and electron microscopic levels, cells containing (and presumably producing) simultaneously both prolactin and growth hormone, and cells containing exclusively either prolactin or growth hormone, could be demonstrated. In addition, a highly variable number and distribution of SRIH receptors could be shown in tumors secreting prolactin, growth hormone, and in tumors not associated with symptoms caused by inappropriate hormone secretion. The systematic combination of clinical, radiological, and biological techniques has currently brought great progress in the behavior and therapeutic concepts of pituitary lesions, and promises new achievements in the near future.