Morphogenesis of minute adenomas in familial polyposis coli.

The morphogenesis of minute colonic adenomas was investigated by complete serial sectioning of human tissues from 4 individuals with familial polyposis coli. Minute adenomas, consisting of a few atypical glands, were located in the upper part of the mucosa. Single-gland adenomas, consisting of single atypical glands, also were located in the upper to superficial part of the mucosa with endophytic growth. Twelve lesions smaller than single-gland adenomas also were detected. These lesions, buds of single-gland adenomas, consisting of epithelial clusters with mild atypia, were located in the middle part of the mucosa and had sprouted from normal crypts to the lamina propria. The conclusion was that buds of single-gland adenomas originate in the proliferative zone of normal crypts, sprout out of the lamina propria mucosae, move upward with migration of the epithelial cells of the crypts, and are identified as single-gland adenomas in the upper part of the mucosa.

Genes preferentially expressed in embryo stomach are predominantly expressed in gastric cancer.

Eight complementary DNA (cDNA) clones highly expressed in fetal rat stomach but not in normal adult rat stomach were isolated after screening 2 x 10(4) independent recombinants from a subtracted cDNA library. The cDNA library was first prepared from RNAs of total stomach at 16 days gestational period, and this cDNA library was subtracted by cDNAs prepared from adult rat total stomach RNA, using a novel PCR-based cDNA subtraction method. Northern blot analysis revealed that as many as six of eight clones thus isolated were overexpressed in at least some of the human or rat gastric cancers. From analysis of partial nucleotide sequence, four cDNA clones were identified as profilin, pro-alpha 1 (1) collagen, nucleolar protein B23.2, and elongation factor 1 alpha subunit. The remaining two clones were derived from novel genes. These novel genes, L-1 and L-2, are developmentally well regulated in the stomach. The present results clearly show that genes expressed preferentially in embryo stomach are most likely to be highly expressed in gastric cancer. The method described here provides us with a rapid method for identification of genes with significantly increased expression in cancer.

Overexpression of ERK, an EPH family receptor protein tyrosine kinase, in various human tumors.

The ERK gene has been isolated as a genomic DNA encoding a part of the receptor protein-tyrosine kinase which belongs to the EPH subfamily. We previously identified a partial complementary DNA (cDNA) encompassing the catalytic domain of ERK from the expression library of human gastric cancer with an antiphosphotyrosine antibody. Using this cDNA as a probe, the cDNAs encoding mature ERK protein were isolated. The putative mature ERK protein, a total of 967 deduced amino acid residues, showed high homology with chicken Cek5 (92.5%) and mouse Nuk (99.1%). Chromosomal in situ hybridization revealed that human ERK cDNA is localized to chromosome 1p34-35. In Northern blot analysis of normal human tissues, the ERK gene was ubiquitously expressed mainly in cells of epithelial origin but not in the brain. Studies on RNAs from 76 human tumor tissues and cell lines showed that ERK is expressed at higher levels in various tumors of epithelial origin than in corresponding normal tissues, most frequently in gastric cancers (12 of 16, 75.0%). Overexpression of ERK was also detected in one osteosarcoma cell line. These findings suggest that ERK plays some significant role in carcinogenesis in the stomach and other tissues.

Isolation of complementary DNA encoding K-cadherin, a novel rat cadherin preferentially expressed in fetal kidney and kidney carcinoma.

Complementary DNA for a novel member of the cadherin family, designated K-cadherin, was isolated from a rat renal cell carcinoma complementary DNA library by screening it with a short complementary DNA probe which was initially obtained from the RNA of day 16 fetal Wistar rat stomach mucosa by the polymerase chain reaction. The deduced primary structure of K-cadherin is 789 amino acid residues, which contain five internal repeats in its extracellular domain, a single putative transmembrane domain, and a cytoplasmic tail characteristic of those of classic type cadherins. K-cadherin exhibits low homology with mature proteins of mouse N- (38%), E- (35%), and P-cadherin (32%), and high homology with a partially identified human cadherin-6 protein (95%) at the amino acid level. Northern blot analysis revealed a high level of expression of K-cadherin mRNA in fetal rat kidney and brain, and rat kidney carcinoma with two major transcripts, 4.1 and 8.0 kilobases in size, whereas there was very weak or no expression in any organ of adult rats. The level of K-cadherin expression was also elevated in some human kidney cancer tissues. In the developing kidney, in situ hybridization showed localization of K-cadherin mRNA in the nephroblastic epithelial cells of comma bodies coinciding with those in the process of polarization during glomeruloneogenesis. These results demonstrate that K-cadherin must have important functions in both the process of kidney development and tumorigenesis of some types of kidney cancer.

CYP1A1 and CYP2E1 polymorphism and lung cancer, case-control study in Rio de Janeiro, Brazil.

Msp I polymorphism and exon 7 Ile-Val polymorphism of CYP1A1, and Rsa I polymorphism of CYP2E1 were studied in lung cancer patients and controls in Rio de Janeiro, Brazil. Of the three polymorphisms studied, only the exon 7 polymorphism of CYP1A1 (Val-containing genotypes) had a distribution which was statistically significant in the patients and controls. The contribution of Val containing genotypes of CYP1A1 exon 7 was greater in the subpopulation of squamous cell carcinoma patients with a lower life-time smoking consumption (OR, 2.92 vs 1.97). This association is consistent with the previous findings by Kawajiri et al. and the first observation of the positive association of this locus with lung cancer in a Western population (Kawajiri K, Nakachi K, Imai K, Yoshii A, Shimada N, Watanabe J. FEBS Let 1990; 263, 131-133). Furthermore, together with the lack of association of Msp I polymorphism in the non-coding region of CYP1A1, the locus truly responsible for lung cancer risk among pleural polymorphisms of CYP1A1 appeared to be exon 7 Ile-Val polymorphism. In the future, investigations of multiple markers in different ethnic populations may reveal cancer risk markers common to all mankind.

Cytochrome P-450 lA1 genotype in lung cancer patients and controls in Rio de Janeiro, Brazil.

Mspl restriction fragment length polymorphism in cytochrome P-450 IA1 (CypIA1) gene, which has been associated with lung cancer susceptibility in Japanese, was studied in persons from Rio de Janeiro, in the framework of a hospital-based, age, race (black or nonblack), and gender-matched case-control study (n = 222; 110 cases and 112 controls). Contrary to the hypothesis, there was no difference in the frequency of the C genotype (Mspl site-present homozygous), even after racial breakdown. There were no significant differences between cases and controls when categorized according to tobacco consumption. The lifetime quantity of tobacco smoked was not different among lung cancer patients with three different genotypes (A, Mspl site-absent, homozygous; B, heterozygote; and C). The background frequency of the Mspl polymorphism C genotype is a little less than 10%, similar to that of the Japanese healthy population. The CyplA1 Mspl polymorphism itself does not seem to be related to susceptibility to bronchial carcinogenesis in this area.

Stage-dependent evaluation of microsatellite instability in gastric carcinoma with familial clustering.

Familial clustering of gastric cancer is probably caused by multifactorial processes, both environmental and genetic. In this report, the incidence of microsatellite instability (MSI) in 31 cases of gastric cancer in Japanese (33 lesions) with familial clustering (two or more gastric cancers within second-degree relatives) was compared to MSI in Japanese cases without a family of any cancer in age ( +/- 10 years)-, stage-, and histological subtype-matched case-control study. Although the difference noted was not significant, we noted a strong trend for MSI at any of up to seven loci of CA repeats to occur more frequently in the patients with a family history of gastric than in the control patients in early cancer (intramucosal and submucosal), whereas the prevalence of MSI was similar in both groups in more advanced cases, in which the tumor invaded beyond the proper muscle layer of the gastric wall. Because the contribution of a family history of gastric cancer to MSI apparently differs in early and advanced gastric cancer, interpretation of MSI in familial gastric cancer cases published previously require reevaluation in terms of stage and proper controls. An acquisition of CA repeat alterations in the early stage rather than in the late stage of gastric carcinogenesis may have in common etiological factors, at least in some cases, with the familial clustering of gastric cancer.

The heme-binding region polymorphism of cytochrome P450IA1 (CypIA1), rather than the RsaI polymorphism of IIE1 (CypIIE1), is associated with lung cancer in Rio de Janeiro.

Ile-Val polymorphism in exon 7 of cytochrome P450IA1 (CypIA1) and RsaI polymorphism of cytochrome P450IIE1 (CypIIE1) were examined in a case-control study of lung cancer in Rio de Janeiro, Brazil. The Val-containing genotype in exon 7 of CypIA1 was found to be associated with lung cancer in this population (odds ratio, 2.26; 95% confidence interval, 1.14-4.47 for 99 cases versus 108 controls of 123 matched pairs), whereas RsaI polymorphism in CypIIE1 was not associated with lung cancer susceptibility. In squamous cell carcinoma, the degree of association of Val-containing genotype was greater in those with fewer pack-years of smoking. The RsaI polymorphism of CypIIE1 has a different distribution from the Japanese pattern and is not associated with lung cancer. When we analyzed the association of Ile-Val polymorphism to MspI polymorphism of CypIA1, the Val/Val homozygote was found only in the subpopulation with the MspI site-present homozygote. The apparent lack of association of CypIA1 MspI polymorphism with lung cancer in this area reported in our previous study and the results of the present study indicate that the "true" responsible site for lung cancer susceptibility should be the Ile-Val polymorphism in the catalytic site of CypIA1.

Depressed adenomas of the colon in familial adenomatous polyposis. Histology, immunohistochemical detection of proliferating cell nuclear antigen (PCNA), and analysis of the background mucosa.

Depressed adenomas of the colon have been reported more often during the past several years, but there are few reports on the details of their characteristics or behavior. In this study, depressed and ordinary polypoid adenomas, especially those in an early stage, in the large intestine of 28 patients with familial adenomatous polyposis (FAP) were examined macroscopically, by dissecting microscope, and histologically. Proliferating cell nuclear antigen (PCNA) and the density of crypts in the background mucosa around depressed and polypoid adenomas were also compared. Depressed adenomas of < 1.0 mm in diameter showed horizontal growth between the normal adjacent crypts, which often left the normal crypts as islands, whereas polypoid adenomas grow expansively without including remnants of normal crypts. In depressed adenomas, PCNA shifted to the upper part of the crypts, and the background mucosa in their vicinity showed a low density of crypts. These findings indicate that a unique characteristic of growth of depressed adenomas at an early stage is superficial proliferation.

Inflammatory myoglandular polyps of the colon and rectum. A clinicopathological study of 32 pedunculated polyps, distinct from other types of polyps.

Hitherto unclassified colorectal polyps were identified in 32 patients (23 men and 9 women; mean age, 53 years). The only symptom, which was observed in less than half the patients, was passage of blood or occult blood. Endoscopic examination revealed solitary pedunculated, red polyps with a smooth surface. These polyps were found in the left colon, especially in the sigmoid. Their characteristic features were inflammatory granulation tissue in the lamina propria mucosae, proliferation of smooth muscle, and hyperplastic glands with occasional cystic dilatation. The etiology of this type of polyp is unknown, but it could involve chronic trauma from the fecal stream and from peristalsis of the bowel. These polyps can be differentiated from juvenile polyps and inflammatory polyps by the presence of abundant smooth-muscle cells in the inflamed lamina propria mucosae. They also can be differentiated from Peutz-Jeghers polyps, which appear as hamartomatous structures with tree-like proliferation of muscularis mucosae covered by colonic mucosa without inflammatory granulation tissue. Their locations and macroscopic appearance distinguish these polyps from mucosal prolapse syndrome and polyps developed after colostomy. In addition, these new polyps differ from inflammatory cap polyps in that they lack a fibrin cap. We propose the name inflammatory myoglandular polyps for these polyps, which are distinct clinicopathologically from other types of colorectal polyps.

Intrasellar neuronal choristoma associated with growth hormone-producing pituitary adenoma containing amyloid deposits.

The histological, immunocytochemical, and ultrastructural features of an intrasellar neuronal choristoma associated with pituitary growth hormone (GH)-producing adenoma are reported. Immunohistochemistry studies and electron microscopy examination showed the adenoma cells to be positive for GH but negative for prolactin, and the neurons of the choristoma to have GH-releasing factor (GRF) neurosecretory activity. The adenoma also had many amyloid deposits in its extracellular space immunoreactive to GRF. This is the first report of the tumor containing amyloid deposits.

Alteration of immunoreactivity by hydrated autoclaving, microwave treatment, and simple heating of paraffin-embedded tissue sections.

The effects of treatment in a hydrated autoclave (121 degrees C, 2 atm for 20 min), microwave oven (in water), and simple heating (60 degrees C overnight in distilled water or 90 degrees C for 10 min in ZnSO4) on the stainability of 56 antigens by commercially available antibodies in formalin-fixed paraffin-embedded tissue sections were evaluated. The detectability of nuclear antigens, glycoprotein, lymphocytic surface markers, and chromogranin A was significantly and reproducibly improved by these treatments, whereas the detectability of viral antigens and peptide hormones was attenuated or unchanged. This enhancement includes not only the distinctiveness of the positive staining, but also the number of positive cells, as revealed by comparing serial sections. Among these four heating procedures, microwave heating and autoclaving were more effective than the others on p53, c-erbB-2, and CA125, whereas simple heating was best for smooth-muscle actin (HHF35 and CGA7). Generally the effects of the heating procedures for these antigens were consistent among the cases, but the effects on GFAP varied with the case. The alterations we observed could significantly influence the interpretation of immunohistochemical staining of currently popular tumor markers such as p53 in terms of their prevalence (28% vs 64% in gastric cancer; 36% vs 82% in metastatic liver cancer) and other diagnostically important markers.

Expression of p53 and flow cytometric DNA analysis of isolated neoplastic glands of the stomach: an application of the gland isolation method.

The expression of p53 was studied immunohistochemically in combination with the DNA ploidy pattern by gland isolation in 97 alcohol-fixed gastric lesions. A polyclonal antibody, CM-1, was applied to the paraffin-embedded sections in this study. Overexpression of the p53 protein was found in 73.2% of 41 well or moderately differentiated gastric carcinomas and 52.2% of 23 cases with poor differentiation (P < 0.05). Immunoreactivity of p53 was also detected in isolated cancerous glands. No p53 immunoreactivity was detected in benign gastric lesions including adenomas, hyperplastic polyps and regions of intestinal metaplasia. In addition, flow cytometric DNA analysis was performed on isolated glandular epithelium adjacent to the portions used for immunostaining. DNA aneuploidy (DA) was detected in 85.7% of the well or moderately differentiated carcinomas and 42.9% of those with poor differentiation (P < 0.05). There was a positive correlation between DA, p53 positivity and the presence of regional lymph node metastasis, but not with other clinicopathological variables. In spite of the limited applicability of this method to poorly differentiated gastric cancer, we found that immunostaining and flow cytometry in combination with the gland isolation method facilitates analysis of gastric carcinogenesis.

Detection of minute intestinal metaplastic lesions by video microscopy.

Using video microscopy in 225 resected stomachs we detected 32 minute solitary lesions of intestinal metaplasia histologically. The magnified features of the minute lesions showed a characteristic appearance and were classified into three types: mesh-like (type A), villoid (type B), and tubular (type C). All the lesions classified as type A exhibited the incomplete type of intestinal metaplasia according to the results of histopathological and/or histochemical examination. In contrast, most lesions classified as type B exhibited the complete type of intestinal metaplasia. We concluded that intentional detection of minute lesions in resected stomachs by video microscopy is simple and useful, especially in cases of minute lesions < 5 mm in diameter. Moreover, our findings demonstrate that the minute intestinal metaplastic lesions have morphological characteristics based on which they can be classified into three types of lesions. These morphological characteristics correlate with their histopathological findings.

Histological classification of the neoplastic changes arising in ulcerative colitis: a new proposal in Japan.

Patients with total ulcerative colitis with a longstanding course of the disease have a high risk of developing colorectal carcinoma. Colonoscopic surveillance to detect precancerous tissue and/or cancer in these patients has been carried out in countries with a high incidence of ulcerative colitis. Riddell's classification has been widely used for the interpretation of biopsy specimens obtained from the colonoscopic surveillance. In Japan, however, there are problems in accepting Riddell's classification, mainly because the intramucosal carcinomas diagnosed by Japanese histopathologists are included in the category of high-grade dysplasia in Riddell's classification. Based on the results of a meticulous slide review carried out by seven histopathologists in this study, a new classification is proposed: UC-I, inflammatory change; UC-II, indefinite; UC-IIa, probably inflammatory; UC-IIb, probably neoplastic; UC-III, neoplastic but not carcinomatous; and UC-IV, carcinoma. Intramucosal carcinomas is included in the category UC-IV. We consider that the diagnosis of intramucosal carcinoma is to be made when there is a high grade of cytological and structural atypia consistent with carcinoma. Interobserver and intraobserver variability with this classification was acceptable. We believe this new classification will be widely use in cancer surveillance in ulcerative colitis in Japan.

Effects of pyrroloquinoline quinone (PQQ) and PQQ-oxazole on DNA synthesis of cultured human fibroblasts.

The effects of pyrroloquinoline quinone (PQQ) and PQQ-oxazole (PQQ-glycine adduct) on DNA synthesis were examined using cultured human fibroblasts. Confluent fibroblasts were cultured in serum-free Dulbecco's modified Eagle's media, and various concentrations of PQQ and PQQ-oxazole were added to the media. After incubation for 24 h, [3H]thymidine was added to the media as an indicator for DNA synthesis of the cells. The thymidine incorporation into the cells was significantly enhanced even in the presence of very low concentrations of PQQ (0.003-0.03 microM); it remained significantly enhanced, up to 30 microM PQQ. However, the incorporation remarkably decreased at 750-1500 microM of PQQ. In contrast to the results for PQQ, DNA synthesis was not stimulated by addition of 0.003-3 microM PQQ-oxazole, but it was slightly enhanced at concentrations 15-750 microM. In morphological examination of the cultured human fibroblasts, cell density was increased by addition of 0.003-30 microM PQQ when compared with that of the control, supporting the above biochemical data. However, there were no distinct differences in morphological effect between PQQ and PQQ-oxazole.

[Pathogenesis and progression of scirrhous carcinoma].

The aim of the present paper is to elucidate the pathogenesis of gastric scirrhous carcinoma with special reference to fibrogenesis in cancer stroma and to the mode of cancer invasions. First, there are two hypotheses concerning the origin of the marked increase of collagen fibers in this type of carcinoma. One is thought to be production by fibroblasts stimulated by cancer cells; the other is synthesis by neoplastic cells. Our studies revealed that gastric cancer cells enhanced collagen production by fibroblasts in vitro, suggesting the contribution to the formation of stromal collagen in human gastric scirrhous cancer. However, one of four gastric scirrhous cancer cell lines, NKPS, was found to slightly from collagen fibers in the brain of the nude mouse. We also calculated the number of fibroblastic cells in gastric scirrhous and normal stroma by microscopical image analysis. The results showed that fibroblastic cells in scirrhous stroma were increased four-fold over those in normal stroma. This finding suggests that the mechanism by which fibrous stroma is produced in scirrhous carcinoma may be affected by the increased fibroblastic cells which were stimulated by many kinds of growth factors produced by cancer cells. It is also well known that the infiltrative growth and dispersion of scirrhous cancer cells within the gastric wall is very fast. To assess the mode of cancerous invasion of this carcinoma, we examined the urokinase-type plasminogen activator (uPA) activity of both types of cells (cancer cells and fibroblasts) with a coculture method. Scirrhous cancer cells enhanced production of uPA by fibroblasts. However, non-scirrhous gastric cancer cells produced much uPA by themselves. This finding suggests that scirrhous cancer cells can invade the gastric wall by use of uPA produced by enhanced fibroblasts, but that non-scirrhous cancer cells synthesize much uPA by themselves and can infiltrate the gastric wall.

[Immunohistochemical distribution of p53 and Ki-67 in 2 cases of colorectal carcinoma occurring in ulcerative colitis].

We studied immunohistochemically the distribution of p53 overexpression and Ki-67 as a marker of cell proliferation, in 2 cases of advanced colorectal carcinomas occurring in UC, in comparison with histopathological features such as carcinoma and dysplasia. One is a case of 4 independent colorectal carcinomas surrounded by widespread dysplasia, and the other is a case of solitary rectal carcinoma. In these 2 cases, formalin-fixed paraffin-embedded tissues covering all of the resected specimens were examined for p53 and Ki-67 immunoreactivity using microwaving technique. p53 was negative in normal mucosa and inflammative mucosa without dysplasia. p53 was strongly positive in cancerous lesions except one lesion, and various degrees of dysplasia surrounding these lesions also showed p53 positive. The numbers of p53 and Ki-67 positive cells were higher in severe dysplasia than in mild dysplasia. Some areas of the dysplastic mucosa histologically represented "hyperplastic pattern of villous feature." In these areas p53 positive cells were aggregated in the basal part of the villous crypt, and the number of Ki-67 positive cells per gland was larger than in normal gland. Our examination covering the whole mucosa revealed: 1) p53 and Ki-67 are useful diagnostic markers for grading the degree of dysplasia. 2) Positivity of immunoreactive p53 in dysplasia seems to be consistent with that in cancers occurring in the surrounding dysplastic area in most of our cases.

[Serial spectral EEG analysis in a patient with non-Hodgkin's lymphoma complicated by leukoencephalopathy induced by high-dose methotrexate].

An autopsy case of disseminated necrotizing leukoencephalopathy in non-Hodgkin's lymphoma associated with high-dose methotrexate (HD-MTX) is reported. The patient was a 5-year-old girl, who was admitted to Hamamatsu University School of Medicine because of an abdominal mass in April, 1984. She was diagnosed as having diffuse lymphoma, medium cell type, according to the LSG classification of a biopsied specimen of the liver mass and great omental lymph nodes. Chemotherapy containing HD-MTX was given until the first bone marrow relapse occurred in September, 1985. Subsequently, the physical and CT findings were normal except for decrement of deep tendon reflex, though slight gait disturbance was apparent. In serial spectral EEG analysis, the alpha/s ratio showed slowing activity upon administration of HD-MTX, and it became irreversible. In June, 1986, second bone marrow relapse occurred, and the patient died in July, 1986. At necropsy, multifocal necroses and extensive spongiosis in the white matter of the parietal and occipital lobes were revealed in the bilateral hemispheres of the cerebrum and cerebellum. Irregular confluent demyelination and necroses were also present in the brain stem, especially the pons. Therefore, serial EEG analysis might be a means of achieving early detection of leukoencephalopathy in HD-MTX-treated patients.