Vascularization of carcinomas of the esophagus and its correlation with tumor proliferation.

Vascularization and tumor cell proliferation were analyzed in 33 resected human squamous cell carcinomas of the esophagus using the endothelium-specific antibody BW 200 and the proliferation-associated antibody Ki-67. Vascular parameters (relative capillary volume, relative total vessel volume, vascular surface area, and vascular length) as well as the percentage of proliferating tumor cells (Ki-67 index) were evaluated on frozen sections by a morphometric method. Vascular parameters of the normal mucosa exceeded those of tumors significantly, by a factor of 1.4-2.3. The mean distance between tumor capillaries and the onset of necrosis was 92 +/- 34 microns. Global vascular density did not correlate with TNM stage, tumor diameter, or overall tumor proliferation (mean Ki-67 index, 35.1%; range, 14.2-64.1%). However, a significant negative correlation existed between the percentage of proliferating tumor cells per tumor cord and the intercapillary distance between capillaries located at the edges of these cords. This observation points to the fact that the esophageal cancers were composed of multiple tumor cords and that each of these cords possessed its own supply capillaries at the base of the cord. The sum of these "supply units" thus constitutes an esophageal cancer. The intercapillary distance may reflect the oxygenation status of tumor cells, which cannot be predicted on the basis of tumor staging or grading.

Monoclonal antibody 2B5 defines a truncated O-glycan, GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-6 (GalNAc), on mucins from deep gastric and duodenal glands as well as metaplasia and neoplasia of gastric differentiation.

Monoclonal antibody (MAb) 2B5 previously generated in BALB/c mice using gastric mucosa of the corpus as immunogen was characterized with regard to its binding epitope. Binding assays on structurally defined neoglycolipids from mucin glycans revealed that MAb 2B5 recognizes a carbohydrate epitope on a neutral O-glycan sequence from gastric mucins. This oligosaccharide chain has been characterized by mass spectrometry and methylation analysis and sequential exoglycosidase treatment of the derived neoglycolipid as GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-6OY (where 6OY corresponds to the GalNAc-ol fragment--O--(CH2)2 conjugated to dipalmitoylphosphatidylethanolamine). The selective binding of MAb 2B5 to mucus from deep gastric and duodenal glands, to gastric metaplasia or neoplasia with gastric differentiation may imply that mucin glycosylation in the respective cells is incomplete resulting in the accumulation of truncated blood group precursor chains. MAb 2B5 is the first monoclonal antibody which defines an epitope on M2-type antigens and may substitute, accordingly, for the inconvenient "paradoxical concanavalin A-horseradish peroxidase method" in histochemistry.

Cyclooxygenase-2 expression in human esophageal carcinoma.

On the basis of epidemiological observations that nonsteroidal antiinflammatory drugs reduce the risk of esophageal carcinoma, we studied the expression of cyclooxygenase-2 (COX-2) in esophageal squamous cell carcinomas (SCCs; n = 172) and in esophageal adenocarcinomas (ADCs; n = 27). Using immunohistochemistry, we observed COX-2 expression in 91% of the SCCs and in 78% of the ADCs. Western blot analysis showed enhanced expression of the COX-2 protein in some tumors as compared with normal esophageal squamous epithelium, whereas similar amounts of the COX-1 protein were found in normal and cancerous tissues. COX expression was also studied in two esophageal cancer cell lines (OSC-1 and OSC-2) to evaluate the functional relevance of COX-2-derived prostaglandins (PGs). OSC-2 cells expressed COX-2 but not COX-1, whereas OSC-1 cells expressed high levels of COX-1 but showed only a very weak COX-2 expression. Accordingly, PGE2 synthesis was 600 times higher in the OSC-2 cells as compared with the OSC-1 cells. Treatment of OSC-2 cells with the selective COX-2 inhibitors flosulide and NS-398 concentration dependently suppressed PGE2 synthesis and proliferation and also induced apoptosis. In contrast, no effect of the COX-2 inhibitors was seen in OSC-1 cells. Our data demonstrate that COX-2 is expressed in the majority of esophageal SCCs and ADCs and that COX-2-derived PGs play an important role in the regulation of proliferation and apoptosis of esophageal tumor cells. It is concluded that inhibition of COX-2 may be useful in the therapy of esophageal cancer.

Hypermethylation of the p16INK4a promoter in colectomy specimens of patients with long-standing and extensive ulcerative colitis.

Functional inactivation of the p16INK4a gene has been reported to be involved in the development of a variety of human malignancies. Recent evidence shows that transcriptional silencing as a consequence of hypermethylation of CpG islands is the predominant mechanism of p16INK4a gene inactivation in sporadic colon cancer. This study sought to identify the significance of p16INK4a methylation in the colonic epithelium of patients with long-standing ulcerative colitis. A total of 89 tissue samples was retrieved from three colectomy specimens. A methylation-specific PCR assay was applied. The methylation status was compared with histological findings and the flow cytometrically determined DNA index. Hypermethylation of the p16INK4a promoter region was detected in 12.7% of samples that were negative for dysplasia. However, 70.0% of samples with dysplasia and all of the samples with carcinomatous lesions revealed hypermethylation. Hypermethylation of the p16INK4a gene promoter was detected already in 40% of specimens with lesions indefinite for dysplasia and in 13.7% of samples with exclusively diploid cell populations. These results suggest that hypermethylation of the p16INK4a promoter region is a frequent and early occurring event during the process of neoplastic progression in ulcerative colitis.

Pyloric gland adenoma-- how to diagnose?

The term "pyloric gland adenoma" reflects its etiogenesis from deep mucoid glands in the stomach. The diagnosis can be confirmed by immunohistochemistry. Typically, pyloric gland adenomas are strongly positive for Mucin 6 (deep mucoid gastric glands). These lesions express Mucin 6 over the whole lesion up to the surface often only with a small layer of columnar epithelium expressing Apomucin 5AC. The amount of mucin 5AC which is expressed on normal within the apical foveolar epithelium might vary from case to case. Combination or transdifferentiation with ordinary tubular (intestinal differentiation) adenoma can be observed. The gastric corpus mucosa of elderly female patients with autoimmune gastritis is highly affected. The frequency of pyloric gland adenoma is given in the literature being 2.7% of all gastric polyps. Therefore pyloric gland adenomas are not that rare that one might assume. Only a few publications are available which makes one think that these lesions are frequently misinterpreted. Pyloric gland adenomas can arise in gastric heterotopia and gastric metaplasia in the whole gastrointestinal tract. The clinical significance is given by a 30% rate of malignant transformation. These cases represent for the most well differentiated early adenocarcinomas which are known to have an excellent prognosis after complete polypectomy and limitation to the mucosal layer.

Adenoma with gastric differentiation (so-called pyloric gland adenoma) in a heterotopic gastric corpus mucosa in the rectum.

In a 46-year-old man, a pedunculated rectal polyp measuring 3.0x3.0x2.0 cm was diagnosed histologically as a pyloric gland-type adenoma arising in heterotopic gastric corpus mucosa. The luminal site was covered by glands of the gastric foveolar type, displaying focal marked proliferation interpreted as low-grade intraepithelial neoplasia. A bidirectional gastric differentiation was found: most lower glandular structures showed positivity for the deep gastric mucin core protein Muc 6 and superficial positivity for gastric foveolar epithelium mucin core protein Muc 5AC. Pyloric gland adenoma has so far been described in one larger series only and a few case reports of the stomach, gallbladder, pancreatic duct and within heterotopic gastric corpus mucosa of the duodenal bulb. The present case report is the first case of a pyloric gland-type adenoma within a gastric corpus heterotopia of the rectal mucosa.

Alteration of DNA ploidy status and cell proliferation induced by preoperative radiotherapy is a prognostic factor in rectal cancer.

To identify predictors of prognosis after preoperative radiotherapy, DNA ploidy and cell proliferation were investigated in 116 patients with rectal cancer. For flow cytometry, a nuclear suspension was prepared by pepsin digestion of paraffin samples of biopsies taken before preoperative radiotherapy (15 x 2 Gy) and also of the resected rectal tumors after radiotherapy. The median follow-up period was 6 years. The proportion of tumor necrosis was evaluated in histological sections before and after irradiation. There was a significant decrease (74 to 48%) in aneuploid tumors after radiation. Of 86 patients with aneuploid biopsies, 28 revealed no reduction in the proportion of aneuploid tumor cells [group AN(=/increase)], and 58 showed a reduction (mean 48.9%) or complete elimination of aneuploid tumor cells [group AN(decrease/psi)]. The incidence of local or distal failure was significantly reduced in the group AN(decrease/psi) (7.8%/20%), compared with the group AN (=/increase) (27%/54%) and the group of constant diploid tumors (n = 22; 13.6%/31.8 %; P = 0.034). There was a trend of decreased recurrence rate in diploid tumors with a reduced fraction of cells in S-phase after radiotherapy. Survival was significantly increased in group AN(decrease/psi) (P < 0.0001). In a multivariate regression analysis, variables of independent prognostic significance were increased proportion of necrosis after irradiation and DNA ploidy group and the postoperative tumor stage. These results suggest that alterations in tumor DNA ploidy and cell proliferation induced by preoperative radiotherapy might help to identify patients likely to benefit from preoperative radiation in rectal cancer.

Primitive neuroectodermal tumors of the cerebral hemispheres in two siblings with TP53 germline mutation.

AWe report on two siblings (brother and sister) who developed cerebral PNETs at the age of 5 years and 6 months, respectively. Both children were treated by operation followed by polychemotherapy. The brother also received cranio-spinal irradiation. Nevertheless, the children died about 12 months and 24 months post-operatively due to extensive cerebral tumor recurrences. Shortly after having lost both of her children, the mother developed an intra-abdominal tumor, which was resected and histologically diagnosed as ovarian carcinoma. Because of this unusual familial clustering of tumors and a positive history of brain tumors and other cancers in several maternal relatives, we analyzed DNA isolated from both PNETs and the ovarian carcinoma as well as constitutional (leukocyte) DNA from the whole family for mutation of the TP53 tumor suppressor gene. This analysis revealed that all tumors were homozygous for a missense mutation at codon 213 (CGA => TGG) resulting in an amino acid exchange from arginine to tryptophane. The same mutation was present in one TP53 allele in the constitutional DNA of the mother and the children, indicating that the mother had transmitted a TP53 germline mutation to both of her children. Analysis of loss of heterozygosity at microsatellite markers from 17p confirmed deletion of the paternal (wild-type) allele in both PNETs. Further investigation of the PNETs by comparative genomic hybridization revealed multiple chromosomal abnormalities. Interestingly, some genomic changes were common to both PNETs, while many others were not, a finding suggesting substantial genomic instability, probably as a consequence of p53 inactivation.

Frequent inactivation of CDKN2A and rare mutation of TP53 in PCNSL.

Twenty primary central nervous system lymphomas (PCNSL) from immunocompetent patients (nineteen B-cell lymphomas and one T-cell lymphoma) were investigated for genetic alterations and/or expression of the genes BCL2, CCND1, CDK4, CDKN1A, CDKN2A, MDM2, MYC, RB1, REL, and TP53. The gene found to be altered most frequently was CDKN2A. Eight tumors (40%) showed homozygous and two tumors (10%) hemizygous CDKN2A deletions. Furthermore, methylation analysis of six PCNSL without homozygous CDKN2A loss revealed methylation of the CpG island within exon 1 of CDKN2A in three instances. Reverse transcription PCR analysis of CDKN2A mRNA expression was performed for 11 tumors and showed either no or weak signals. Similarly, immunocytochemistry for the CDKN2A gene product (p16) remained either completely negative or showed expression restricted to single tumor cells. None of the PCNSL showed amplification of CDK4. Similarly, investigation of CCND1 revealed no amplification, rearrangement or overexpression. The retinoblastoma protein was strongly expressed in all tumors. Only one PCNSL showed a mutation of the TP53 gene, i.e., a missense mutation at codon 248 (CGG to TGG:Arg to Trp). No evidence of BCL2 gene rearrangement was found in 11 tumors investigated. The bcl-2 protein, however, was strongly expressed in most tumors. None of the 20 PCNSL demonstrated gene amplification of MDM2, MYC or REL. In summary, inactivation of CDKN2A by either homozygous deletion or DNA methylation represents an important molecular mechanism in PCNSL. Mutation of the TP53 gene and alterations of the other genes investigated appear to be of minor significance in these tumors.

Noradrenaline content of the heart of the adrenal-demedullated rat.

1. The noradrenaline (NA) concentration in the "heart" (atria and right ventricle) of male rats was estimated at different periods following adrenal demedullation. For 1-3 weeks after the operation there was, in all rats, a reduction in NA content of the tissue, whereas, after somewhat longer intervals, the concentrations had returned to normal in some but not in all animals, so that the range of values was very wide.2. In order to be able to test the effect of 2-aminotetralin on the cardiac NA of animals deprived of their medulla but having normal initial NA concentrations, an interval of 6-8 months was allowed to elapse between operation and injection of the drug; at this time the effect of the drug on cardiac NA was the same in intact and demedullated animals. There is thus no reason to attribute to the adrenal medulla any supporting role in the resynthesis of cardiac NA during periods of increased sympathetic activity.3. The time course of the pronounced fall in cardiac NA after adrenal demedullation resembles the time course of sodium retention shown by Gaunt, Renzi, Gisoldi & Howie (1967) to follow this operation. It is therefore suggested that it is the change in electrolyte metabolism which is responsible for the abnormality of NA storage; both phenomena occur in demedullated but not in adrenalectomized animals.

Comparative genomic hybridization analysis of chromosomal alterations in patients with long-standing ulcerative colitis.

Patients with ulcerative colitis (UC) are prone to develop colorectal cancer which is related to the duration and extent of the disease. One of the earliest events in tumor progression is the development of aneuploidy. Aneuploidy is correlated with the grade of dysplasia which serves as a common but not always reproducible marker for the prediction of UC associated formation of cancer. We analyzed 48 biopsy samples from 5 patients with long-standing ulcerative colitis by comparative genomic hybridization (CGH). The majority of these samples represented premalignant stages which are not well characterized at the molecular level as yet. We compared biopsy samples from different colon locations in regard to chromosomal alterations, dysplasia status and DNA index. Besides chromosomal changes occurring only in certain patients in restricted areas of the colon we also detected amplifications and deletions which were common in all persons throughout the colon. The stage of dysplasia seems to have no influence on the number and appearance of chromosomal changes. Amplifications in 2, 3, 6, 9, 11, 12 and 15 were found in almost all cases. In dysplastic samples chromosomal regions 3, 6 and 11 revealed gains of DNA. Deletions were detected within 8q, 15, 18q, 20p and 22q. The affected chromosomal regions may contain yet unknown oncogenes or tumor suppressor genes participating in UC associated carcinogenesis. The conspicuous regions found in the CGH experiments allow the selective and detailed characterization at a molecular level.

Possible transmission of sarcoidosis via allogeneic bone marrow transplantation.

Allogeneic bone marrow transplantation (BMT) was performed in a 34-year-old man for non-Hodgkin's lymphoma. Two years before bone marrow harvest, pulmonary sarcoidosis was diagnosed in the donor. After steroid therapy, disease of the donor was in clinical remission with only minor radiological signs at the time of BMT. On day 90 after BMT, active sarcoidosis was diagnosed in the recipient. Besides radiologic signs and increased angiotensin converting enzyme levels, diagnosis was proved by characteristic histologic changes in lung and liver biopsies. Immunosuppressive therapy was changed from high dose cyclosporine to high dose methylprednisolone and symptoms promptly resolved within 10 weeks. This case indicates the possibility of transmission of sarcoidosis by marrow transplantation.

Acute monocytic leukemia complicating combined-modality therapy for localized childhood Ewing's sarcoma.

Rapidly fatal acute monocytic leukemia occurred in an 11-year-old by 33 months after the beginning of irradiation and chemotherapy for non-metastatic pelvic Ewing's sarcoma. At autopsy, no recurrent primary disease was seen. An analysis of this case together with a review of the literature suggests therapy-related leukemogenesis. Thus, the decline in mortality rate for childhood cancer may be accompanied by an increased incidence of second neoplasms in cured children having the potential of a normal life span.

Effect of N-ethyl-N-butyl-nitrosamine on the esophageal mucosa of the rat. Histometric investigation of early tumor stages.

80 male Wistar rats received an oral ad libitum application of N-ethyl-N-butyl-nitrosamine in a concentration of 0.18 g per litre of drinking water. The changes induced in the esophageal mucosa and determined at three intervals (up to 48 days, up to 91 days, and up to 112 days after commencement of carcinogen exposure) were compared by microscopy with the results from a control group of 10 male Wistar rats of the same age. Several histomorphometric parameters were investigated with the aid of a Leitz ocular micrometer. The earliest localized changes found were an increase in the thickness of the epithelium and the horny layer, and an elongation of the papillary bodies and a widening of the parabasal cellular layer. Later--with a substantial increase in the rate of mitosis in all layers of the epithelium, there was a significant thickening of the non-papillomatous and papillomatous epithelium, an enlargement of the nuclei, especially in the middle and upper layers of the epithelium and a thickening of the horny layer, parts of the latter being parakeratotic. The papillomatous changes corresponded in some cases to moderate epithelial dysplasias. As expected, no fully-developed invasive carcinomas occurred in the early period investigated. The histometric data permit the conclusion to be drawn that the lesions described are demonstrable not only at the exophytic-papillomatous epithelium but also in multifocally localized form at the flat, non-papillomatous mucosa, and that they can definitely be regarded as the expression of an incipient field cancerification.

Pattern of gastric endocrine cells in microcarcinoidosis--an immunohistochemical study of 14 gastric biopsies.

A total of 14 gastric biopsy specimens from patients with microcarcinoidosis were analysed by immunohistochemical methods to evaluate the pattern of endocrine cell hyperplasia and dysplasia. All the patients had type A gastritis (autoimmune gastritis). Nonantral proliferations of gastric endocrine cells were classified according to Solcia et al. All 14 cases had hyperplasia and 13 (92.9%) of them, dysplasia of gastric endocrine cells; 9 (64.3%) of the 14 were found to have showed a coexisting invasive gastric carcinoid at the time of diagnosis of microcarcinoidosis. The patients with invasive carcinoids had higher degrees and more complex forms of endocrine dysplasia (precarcinoid lesions). The average size of the foci of the microcarcinoidosis in gastric biopsies was 0.14 +/- 0.09 cm in the patients without invasive carcinoid, as against to 0.5 +/- 0.24 cm in the group of patients with associated invasive carcinoid. Microcarcinoid gastric biopsies about 0.5 cm in size, are suggestive of adjacent invasive carcinoid. However, even frankly invasive ECL carcinoids seem to be clinically less dangerous than was thought until recently.

Differential p53 protein expression in stomach adenomas of gastric and intestinal phenotypes: possible sequences of p53 alteration in stomach carcinogenesis.

In a comparative study, the expression of p53 protein was investigated in intestinal- and gastric-type adenomas of the stomach. The former is a conventional type, which is well known to be a premalignant lesion of the stomach, but the latter is a rare, more recently noted entity. Of 28 intestinal-type adenomas, 17 (60.7%) contained more than 5% of p53 immunoreactive cells. In these adenomas, the extent of positivity for p53 protein was significantly higher in high-grade dysplasia than in low-grade dysplasia (P < 0.05), suggesting that p53 alteration plays a part in the dysplastic progression of intestinal-type adenomas. Among 18 gastric-type adenomas in which most of the tumour cells displayed gastric-type mucin, substantial expression of p53 protein was found only in the 3 tumours with high-grade dysplasia. Thus, the incidence of p53 expression was significantly higher in intestinal-type adenomas than in gastric-type adenomas (P < 0.01). These results suggest that p53 gene alteration is an earlier event in the gastric carcinogenetic sequence with the intestinal phenotype than in that with the gastric phenotype.

Pyloric gland adenoma: a clinico-pathological analysis of 90 cases.

BACKGROUND: Pyloric gland adenoma is a rarely described neoplasia of the gastric mucosa. Recent publications have shown that similar lesions are also found in the gallbladder, the main pancreatic duct, the duodenum and the cervix of the uterus. Apart from case reports, few clinical data are available on these patients. We therefore conducted a search of the archived material collected between 1990 and 2000 for more clinical data on patients with this rare lesion. PATIENTS AND METHODS: Between 1990 and 2000, 90 patients were diagnosed as having a pyloric gland adenoma in the stomach (77 patients), duodenal bulb (7 patients), duodenum (1 patient), bile duct (3 patients) or gallbladder (2 patients). RESULTS: Pyloric gland adenomas account for 2.7% of all gastric polyps and occur predominantly in old age (73+/-12.8 years), more frequently in women (75%) than in men. The predilection site in the stomach is the corpus mucosa (64%) and they are often found in patients suffering from autoimmune gastritis (36%). At the time of diagnosis, pyloric gland adenomas measure 16.1+/-9.1 mm in size. In 30% of gastric pyloric adenomas, transition to well-differentiated adenocarcinoma has been noted. DISCUSSION: In our material, pyloric gland adenoma is the third most common neoplastic polypoid lesion in the stomach. Since a search through the literature revealed only a few case reports on this lesion, it is possible that for some reason this lesion might be diagnosed more often than reported. CONCLUSION: Our study revealed that 30% of the gastric pyloric gland adenomas showed continuous transition to well-differentiated adenocarcinoma at the time of the initial diagnosis. This underscores the malignant potential of the lesion, and the need for polypectomy.

Sebaceous gland metaplasia in cardiac-type mucosa of the oesophago-gastric junction.

The first case of sebaceous gland metaplasia arising in cardiac-type mucosa of the oesophago-gastric junction of 71-year-old man is reported. Within cardiac glands, small nests composed of clear cells closely resembling sebaceous glands of the skin were found. Immunohistochemically, the cell nests stained positively for a monoclonal antibody 115D8 against milk-fat globule membrane (MAM-6). These cells were sometimes covered by cylindrical cells positive for foveolar-type mucin of the stomach (MI), and basal marginal cells of these nests expressed high molecular weight cytokeratins (34BE12). This study documents a new type of metaplasia of the gastric mucosa.

'Pyloric gland-type adenoma' arising in heterotopic gastric mucosa of the duodenum, with dysplastic progression of the gastric type.

'Pyloric gland-type adenoma' is a recently described and very rare entity. We report a case of a pedunculated polyp of the duodenal bulb showing the features of pyloric gland-type adenoma. Heterotopic gastric mucosa was found adjacent to the tumour. Immunohistochemically, the tumour cells at the surface of the polyp showed foveolar-type mucin (M1) while most other tumour cells showed deep gastric mucin (M2), displaying a pattern of differentiation similar to the normal gastric mucosa. The polyp also showed villous or papillary structures with disorganization of gastric differentiation and marked increase of proliferating in foci cells. This is the first case of pyloric gland-type adenoma found to arise in heterotopic gastric mucosa of the duodenum, showing dysplastic progression of the gastric type.

Histopathological diagnosis of Barrett's mucosa and associated neoplasias: results of a consensus conference of the Working Group for Gastroenterological Pathology of the German Society for Pathology on 22 September 2001 in Erlangen.

There are a number of difficulties regarding the diagnosis of Barrett's mucosa and the varying grades of neoplasia that may be associated with it. It was, therefore, the aim of a consensus conference of the Working Group for Gastroenterological Pathology within the German Society of Pathology to achieve standardisation regarding the following issues: definition and diagnostic criteria for Barrett's mucosa and its discrimination from intestinal metaplasia of the cardia, diagnostic criteria for intraepithelial neoplasia, number of biopsies necessary to establish the diagnosis, significance of additional immunohistochemical and/or molecular methods as well as importance of a second opinion in the diagnosis of intraepithelial neoplasia.