Tsutomu Daa

Genetic alterations in 102 primary gastric cancers by comparative genomic hybridization: gain of 20q and loss of 18q are associated with tumor progression.

Gastric cancer is one of the most common cancers. Molecular events in the carcinogenesis of gastric cancer remain, however, largely undefined. We investigated changes in DNA copy number in 102 gastric cancers by CGH. We found changes in DNA copy number in all cases, with frequent (≧30% of patients) gains at 20q, 8q, 20p, 7q, 17q, 5p, and 13q. Frequent (≧20%) losses were found at 19p, 18q, 5q, 21q, 4p, 4q, 15q, and 17p. The mean number of total alterations was significantly lower in grade 3 and scirrhous-type carcinomas (10.81 in grade 3 vs 13.98 in grade 1 and grade 2, 9.31 in scirrhous-type vs 13.18 in medullary- and intermediate-type). The mean number of losses and total alterations were higher in tumors at pT2, pT3 and pT4 (4.68 and 12.77 in pT2, pT3, and pT4 vs 2.55 and 9.22 in pT1). The mean number of losses was higher in carcinomas with lymph node metastasis (4.83). The mean number of gains and total alterations were higher in carcinomas with venous invasion (8.44 and 13.28). Several chromosomal alterations were linked in a statistically significant manner to specific clinicopathological parameters. Gain of 17q, 20p, and 20q and loss of 4p were associated with the pattern of the cancer–stroma relationship; loss of 18q was associated with pT category; gain of 5p was associated with pN category; loss of 4q and loss of 21q were associated with lymphatic invasion; gain of 7p and loss of 4q and 18q were associated with venous invasion; and loss of 18q was associated with pathological stage. These data suggest that gain of 20q and loss of 18q might play an important role in the development and progression of gastric cancer. Moreover, some genes on 20q and 18q might be target genes of gastric cancer.

Expression of CD10 in basal cell carcinoma.

We investigated the expression of CD10 by an immunohistochemical method in 51 basal cell carcinomas (BCCs), eight pilomatricomas, five trichoblastomas, two trichofolliculomas, three sebaceomas, five sebaceous carcinomas, ten syringomas, two spiradenomas, ten poromas, four porocarcinomas, one eccrine duct carcinoma (not otherwise specified, NOS), six mixed tumors of apocrine origin, and nine squamous cell carcinomas (SCCs). We detected strong expression of CD10 in tumor cells of BCC (86%), and found that the smaller the number of positive tumor cells, the larger the number of positive stromal cells, in particular in sclerosing BCCs. Spearmans rank correlation test revealed a significant negative correlation in BCCs between the expression of CD10 in tumor cells and that in stromal cells (P = 0.001). In all pilomatricomas (100%) and in four trichoblastomas (80%), strong expression was also detected in tumor cells. There was no detectable expression in trichofolliculomas. One sebaceoma (33%) and two sebaceous carcinomas (40%) expressed CD10 in a similar fashion to BCCs. All tumors of eccrine gland origin, including syringoma, spiradenoma, poroma, porocarcinoma, and eccrine duct carcinoma (NOS), did not express CD10. Five mixed tumors (83%) were immunopositive. In SCC, CD10 was overexpressed only in the stromal cells. These findings support the hypothesis that BCC is derived from the folliculo-sebaceous apocrine unit, especially having the same origin as trichoblastoma and pilomatricoma. CD10 might be an indicator of tumor invasiveness if it is expressed in stromal cells, while it might be a marker of follicular differentiation if it is expressed in the actual tumor cells of cutaneous epithelial neoplasms.

Expression of Polo-Like Kinase (PLK1) in non-Hodgkin's lymphomas

Polo-like kinases (PLKs) are protein serine/threonine kinases that play important roles in cell division. Expression of PLK1 might, moreover, play a role in the pathogenesis of human neoplasms. The expression of PLK1 mRNA is closely correlated with survival in patients with malignant tumors. We investigated the expression of PLK1 in non-Hodgkins lymphomas (NHLs) and analyzed the relationships between expression of PLK1, histological grade, and prognosis. We analyzed various types of NHLs from 118 patients using monoclonal antibodies against PLK1 and Ki-67. The levels of expression of PLK1 and Ki-67 were significantly lower in low-grade NHLs than in high-grade and intermediate-grade NHLs (P < 0.001). Moreover, when patients were grouped in terms of 5-year overall survival ( > 70%, group A; 50 - 70%, group B; 30 - 49%, group C; and < 30%, group D), levels of expression of PLK1 and Ki-67 were found to be significantly higher in group D than in group A and they were also significantly higher in group C than in group A (P < 0.001). Conversely, the level of expression, of Ki-67 was significantly lower in group D than in group C (P < 0.05). The labeling indices specific for PLK1 were generally higher than those specific for Ki-67. Once we divided all patients into two groups in terms of the expression levels, high-level expression group of PLK1 (PLK1 index of ⩾̸ 70%) and Ki-67 (Ki-67 indices of ⩾̸ 60%) and low-level expression, one of these markers (PLK1 index of < 70%, Ki-67 indices of < 60%) had a similar prognosis, an observation that can be explained by the fact that rapidly proliferating group is more drug-sensitive than the other. Our study demonstrates that expression of PLK1 might reflect the malignant potential of NHLs and that PLK1 might be more useful than Ki-67 for the detection of proliferative cells.

Insular component as a risk factor of thyroid carcinoma

Forty‐four thyroid carcinomas with an Insular component (JC) were reviewed from 2457 tumors diagnosed as papillary (PC) or follicular carcinoma (FC). These tumors were classified as FC with an IC (FCIC; 30 cases) and PC with an IC (PCIC; 14 cases). Both tumors were composed of solid cell nests in some areas and had a tendency toward a characteristic nuclear size: FCIC had a small nucleus and PCIC contained a nucleus of an Intermediate type or a large nucleus similar to that of PC, although there were numerous tumors with an exceptional nuclear size. The mean age and tumor diameter were the highest and largest in FCIC, respectively, followed by PCIC. Among the 44 cases, 17 patients died of the disease, two were alive with the disease and 18 were alive without the disease. From 13 clinicopathological factors, the presence of an IC, age, non‐encapsulation, tumor size, vascular invasion and necrosis were found to be independent variables for actual prognosis of FC and PC based on univariate analysis followed by multtvariate analysis. The results of the present study indicate that the presence of an IC is an independent aggressive prognostic factor for patients with PC and FC.

Clonal analysis of the epithelial component of W0arthin's tumor

The proliferation of the epithelial component of Warthins tumor is generally considered to represent a neoplastic condition. There has been much controversy about the histogenesis of this tumor, and the clonality of the epithelial component has not been clarified. We examined the clonal status of epithelial cells of Warthins tumor by using a polymerase chain reaction (PCR) method based on trinucleotide repeat polymorphism of the X chromosome-linked human androgen receptor gene (HUMARA) and on random inactivation of the gene by methylation. Total DNA was isolated from formalin-fixed, paraffin-embedded tissue from 16 women with Warthins tumor. Of the 16 cases analyzed, 7 were heterozygous for the HUMARA polymorphism and informative. The epithelial components of the tumors from the 7 cases were microdissected under the light microscope, and were subjected to extraction of DNA and HUMARA analysis. Using a permanent aqueous mounting medium during microdissection, we succeeded in reducing the rate of contamination by lymphocytes in the samples to less than 10%. All 7 cases showed patterns of polyclonal proliferation in the HUMARA analysis. Our results showed the nonclonal nature of Warthins tumor, suggesting that Warthins tumor is a non-neoplastic tumor-like condition. HUM PATHOL 31:1377-1380.

Analysis of p53 Mutations and the Expression of p53 and p21WAF1/CIP1 Protein in 15 Cases of Sebaceous Carcinoma of the Eyelid

PURPOSE The purpose of this study was to detect mutation of the p53 gene, to assess its relationship with p53 or p21(WAF1/CIP1) expression, and to evaluate the correlation between p53 mutation or p21(WAF1/CIP1) expression and clinicopathologic findings in sebaceous carcinoma of the eyelid. METHODS Fifteen conventional paraffin-embedded samples of sebaceous carcinoma of the eyelid were analyzed. Using the single-strand conformation polymorphism technique, the authors sequenced coding exons 5-8 of the p53 gene. The expression of p53 and p21(WAF1/CIP1) protein was analyzed by immunohistochemistry. RESULTS In 10 of the 15 cases (66.7%), point mutations were detected in the p53 gene. CC to TT double-base changes (tandem mutations), which are known to be induced only by UV, were not detected in any of the mutations. Correlations between p53 mutation and expression were found to be statistically significant (P = 0.007). There was no significant correlation between p53 mutation and clinicopathologic findings or p21(WAF1/CIP1) expression. However, there was a significant inverse correlation between p21(WAF1/CIP1) expression and presence of lymph node metastasis (P = 0.007). CONCLUSIONS Among human cancers, sebaceous carcinoma of the eyelid may be one of those showing most frequent mutation of the p53 gene, which may not be caused by exposure to UV. p21(WAF1/CIP1) downregulation may be associated with lymph node metastasis.

Human intestinal spirochetosis in Japan; its incidence, clinicopathologic features, and genotypic identification

Human intestinal spirochetosis is a common condition in Western countries, but is not well recognized in Japan. To demonstrate the incidence and clinicopathologic findings of human intestinal spirochetosis in Japan, we retrospectively investigated biopsy, and endoscopically or surgically resected specimens of the large intestine. Among a series of 2556 samples, 11 cases of human intestinal spirochetosis were detected (0.4%). Together with additional nine cases sporadically found, 20 cases of human intestinal spirochetosis were subjected to molecular detection of two strains of spirochetes (Brachyspira aalborgi and Brachyspira pilosicoli) by amplifying species-specific portion of 16S ribosomal RNA and NADH oxydase gene by polymerase chain reaction. B. aalborgi was detected in all cases examined, three of which revealed dual infection of both species. Our results suggest that human intestinal spirochetosis infection is relatively rare, and B. aalborgi is the most prevalent species in Japan. Most of human intestinal spirochetosis were asymptomatic, although symptomatic in exceptional cases. In addition, we emphasize a usefulness of immunostaining with anti-Treponema pallidum and anti-Mycobacterium bovis polyclonal antibodies for detecting the spirochetes.

Mutations in components of the Wnt signaling pathway in adenoid cystic carcinoma

The Wnt signaling pathway is essential for normal development and organogenesis. However, inappropriate activation of Wnt signaling, which results in the nuclear translocation of β-catenin, is associated with the development of various types of neoplasm. In this study, we investigated possible mutations in the genes for components of this pathway, namely, CTNNB1 (the gene for β-catenin), AXIN1, and APC, in adenoid cystic carcinoma, by PCR, analysis of single-strand conformational polymorphism, and sequencing. Among a total of 20 cases of adenoid cystic carcinoma, seven cases (35%) were associated with mutations in one or more of these three components. A mutation in CTNNB1 was detected in one case. Five cases, including the case with a mutation in CTNNB1, were associated with missense mutations in AXIN1. An aberration in the mutation cluster region of APC was detected in two cases. Mutations trended to be detected more frequently in adenoid cystic carcinoma with solid growth pattern than that with tubular and cribriform growth pattern. In the cases in which we detected mutations, it is possible that the presence of the abnormal products of the mutated genes resulted in the inappropriate activation of the Wnt signaling pathway to tumorigenesis and the growth of adenoid cystic carcinoma.

An Antigen Retrieval Method Using an Alkaline Solution Allows Immunoelectron Microscopic Identification of Secretory Granules in Conventional Epoxy-embedded Tissue Sections

Immunoelectron microscopy using chromogranin A-specific antibodies has been proposed as an efficient technique for identification of secretory granules (SGs) in tumor cells with evidence of apparent neuroendocrine differentiation. Using an antigen retrieval (AR) method, we succeeded in immunolabeling SGs with antibodies in ultrathin sections of routinely processed epoxy-embedded blocks of tissue. Samples of an insulinoma were fixed in 2% glutaraldehyde, postfixed in 1% OsO4, and embedded in epoxy resin. Ultrathin sections were immunostained with chromogranin A-specific antibodies and gold-conjugated second antibodies. There was no significant labeling in the absence of AR. Neither etching with sodium metaperiodate nor microwave irradiation of ultrathin sections in citrate buffer (pH 6.0) or in EDTA buffer (pH 8.0) was effective in improving the efficiency of immunolabeling. However, ultrathin epoxy-embedded sections that were microwaved in alkaline solution (pH 10) were adequately labeled (5.2 ± 0.34 particles per SG). Moreover, considerably improved efficiency of immunostaining was achieved by microwaving sections in alkaline solution (pH 10) with subsequent immunostaining at 60C (12.2 ± 0.51 particles per SG). This method can also be applied to epoxy-embedded sections obtained from formalin-fixed, paraffin-embedded blocks of tissue and was even valid for an old epoxy-embedded block of tissue prepared 15 years previously.

Mild hypothermia reduces expression of intercellular adhesion molecule‐1 (ICAM‐1) and the accumulation of neutrophils after acid‐induced lung injury in the rat

Background:  The pathophysiology of the acute phase of acid‐induced lung injury (AILI) has been elucidated. However, once acute respiratory distress syndrome (ARDS) develops, the mortality rate remains high and there is, as yet, no effective therapy. There are reports that application of mild hypothermia is an effective treatment for ARDS. In this study, we hypothesize that mild hypothermia inhibits activation of neutrophils and expression of intercellular adhesion molecule‐1 (ICAM‐1) in an injured lung. We studied the effects of mild hypothermia on the expression of ICAM‐1 and the accumulation of neutrophils after AILI in the rat.