Keizo Sugimachi

Vascular endothelial growth factor-C (VEGF-C) expression in human colorectal cancer tissues

Vascular endothelial growth factor-C (VEGF-C) functions specifically to induce lymphangiogenesis. We examined the relationship between expression of VEGF-C and clinicopathological features in patients with colorectal cancer. The expression of VEGF-C in the 99 primary tumours and 18 metastatic lymph nodes from colorectal cancer patients was examined immunohistochemically. To verify VEGF-C mRNA expression, reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out. The expression of VEGF-C correlated with lymphatic involvement, lymph nodes metastasis, and depth of invasion. On the other hand, correlations were nil with regard to gender of the patients, histologic type, venous involvement, and liver metastasis. The expression of VEGF-C in metastatic lymph nodes was fairly consistent with this expression in the primary tumour. Survival time was shorter for VEGF-C positive groups than for VEGF-C negative ones, but with no statistically significant difference. RT-PCR findings revealed that the expression of VEGF-C mRNA correlated mostly with that of VEGF-C protein expression. VEGF-C may play an important role in lymphatic spread of colorectal cancer.

Altered expression of beta-catenin without genetic mutation in intrahepatic cholangiocarcinoma

β-catenin which has a role in E-cadherin mediated cell-to-cell adhesion, and is also involved in Wnt signaling pathways as a downstream signaling molecule accumulating in the cytoplasm and nucleus constitutively activates Tcf/LEF-associated transcription of oncogenic genes. We examined the expression pattern and the genetic alteration of β-catenin to determine the role of β-catenin in cancer formation and/or progression in intrahepatic cholangiocarcinoma (ICC). β-catenin expression was immunohistochemically examined in 71 surgically resected ICC samples, and correlation between the expression pattern and clinicopathologic factors was investigated. Mutation analysis of β-catenin exon 3, which included the responsible element for Wnt signaling was done in 55 samples, using PCR-SSCP and direct sequence methods. Immunohistochemical analysis revealed the reduced membranous expression of β-catenin in 58 (82%) ICCs and aberrant nuclear expression in 11 (15%) ICCs. The membranous expression was preserved in 62% of the papillary adenocarcinomas, and was frequently reduced in tumors with a poorer histological differentiation (84%), with a significant difference (P =.01). Genetic analysis showed that none of the 55 ICCs examined carried mutations in β-catenin exon 3. The present study indicates that reduced membranous expression of β-catenin is associated with non-papillary ICCs which have a more malignant behavior, and that nuclear translocation of β-catenin results in oncogenic events. Mutations in β-catenin exon 3 do not appear to be responsible for nuclear translocation of β-catenin in ICCs.

Time trends of surgical treatment and the prognosis for Japanese patients with gastric cancer.

The incidence of gastric cancer is much higher in Japan than in other countries even though diagnostics and treatments of such patients have improved. The objective of this study was to present an overview of the past, present and future of surgical treatment for our patients with gastric cancer. We analysed data on 2152 Japanese men and women with gastric cancer who underwent surgical resection from 1965 to 1995 at Kyushu University in Fukuoka, Japan, based on a univariate and the multivariate analysis. We focused on time trends of surgical treatment and the postoperative outcome. Over the years, there have been favourable changes in the numbers of patients with early gastric cancer. In all cases of gastric cancer, the rate of 18% in the first six year period (group 1) was 57% in the last 5 year period (group 6). Size of the tumour was smaller, well-differentiated tumour tissue was more common, and lymphatic involvement was less frequent. Lymph node metastasis, liver metastasis and peritoneal dissemination all decreased. Extensive lymph node dissection was more frequently done and the rate of curative resection (curability A and B) increased. With increases in identifying the early stage of cancer and better perioperative care, mortality rates 30 days after the surgery greatly decreased. Multivariate analysis revealed that the 10 factors of depth of invasion, lymph node metastasis, lymph node dissection, tumour size, liver metastasis, peritoneal dissemination, lymphatic invasion, vascular invasion, lesion in the whole stomach and lesion in the middle stomach were independent factors for determining the prognosis. Detection of the tumour in an early stage, standardized surgical treatment, including routine lymph node dissection, close follow-up schedules and better perioperative management are expected to increase survival time for patients with this malignancy.

The role of overexpression and gene amplification of cyclin D1 in intrahepatic cholangiocarcinoma

BACKGROUND/AIMSnIntrahepatic cholangiocarcinoma (ICC) is a primary liver malignant tumor with an extremely poor prognosis, but less attention has been directed to factors related to molecular carcinogenesis, including cell cycle proteins. We examined the expression and gene amplification of cyclin D1, the cell cycle regulating protein. Our objective was to evaluate correlations with clinicopathological factors in ICC.nnnMETHODSnCyclin D1 overexpression and cellular proliferative activity (Ki-67 labeling index) were investigated immunohistochemically, and 20 cases were further investigated for cyclin D1 gene amplification, using differential PCR. We examined the correlation between the expression and gene amplification of cyclin D1 and clinicopathological factors, including overall survival in patients with ICC.nnnRESULTSnImmunohistochemical analysis revealed an overexpression of cyclin D1 protein in 28 of 66 subjects with ICCs (42%). The cyclin D1 overexpression was associated with poor histological differentiation (P = 0.04), high cellular proliferative activity (P < 0.01), and a poor prognosis (P = 0.02) by univariate analysis, although it is not an independent prognostic factor by multivariate analysis. Cyclin D1 gene amplification was confirmed in five of the 20 patients. Of those five cases of ICC, all had poor histological differentiation, and four of the five ICCs (80%) showed evidence of cyclin D1 immunoreactivity.nnnCONCLUSIONSnOverexpression and gene amplification of cyclin D1 are frequent and contribute to dedifferentiation and cellular proliferative activity of ICCs, and overexpression also indicates a poor prognosis for patients with ICC.

Diminished expression of ING1 mRNA and the correlation with p53 expression in breast cancers

p33(ING1) is a novel candidate tumor suppressor and its overexpression induces growth arrest or apoptosis in different cell lines. These functions of p33(ING1) depend largely on the activity of p53, and p53-dependent activation of the transcription from the p21/WAF1 promoter also requires p33(ING1). We examined the expression of ING1 mRNA in breast cancer cell lines and clinical breast cancer tissues, using quantitative RT-PCR and real time TaqMan technology. In breast cancer cell lines, ING1 mRNA was expressed at almost the same level. However, in a comparison between the cancer and matched normal tissues, a significant decrease in ING1 mRNA expression was found in 17 of 24 (70.8%) breast cancer tissues. We also examined the correlation between ING1 mRNA expression and p53 expression. There was a significant decrease of ING1 mRNA in nine of 15 tumors negative for p53 immunostaining, most of which were considered to have wild type p53. In these tumors, p53 may not function in case of a decreased expression of p33(ING1), and the lack of cell cycle regulation may correlate with the carcinogenesis and tumor progression.

The instability within: problems in current analyses of microsatellite instability

Microsatellite instability is regarded as one of the phenotypes of defective DNA mismatch repair and, consequently, as a marker of high risk for cancer. Despite numerous studies, the reported rates for positive microsatellite instability differ widely in each human malignancy. These discrepancies may relate to problems in the methods used. To establish a methodology for an accurate microsatellite instability analysis, technical requirements for a precise assay and biological conditions required for positive microsatellite instability were discussed. First, to describe microsatellite changes in detail, a sensitive detection system with linear detection characteristics and electrophoresis with standardised migration and minimised migration errors are considered to be necessary. Therefore, systems using fluorescent labelling and laser scanning are recommended. For reproducible polymerase chain reactions, it is essential to control the terminal deoxynucleotidyl transferase activity in Taq polymerase. Second, as a biological condition for positive microsatellite instability, feasible selection and combination of microsatellite markers, mutations in specific DNA mismatch repair genes and existence of monoclonal populations enriched sufficiently in a sample are essential. Finally, one possible diagnostic criterion for positive microsatellite instability is proposed, that is the existence of one of the patterns shown in the panel (see Fig. 6) at one or more loci in a set of more than five microsatellite markers.

Prognostic factors in patients with submucosal carcinoma of the oesophagus

To clarify the prognostic factors in patients with submucosal carcinoma of the oesophagus, we examined the results of surgical treatment for 78 cases over the last decade. The clinicopathological factors including age, sex, location of the tumour, length of the tumour, histological differentiation, subclassification of depth, lymphatic or blood vessel invasion, intramural metastasis and lymph node metastasis were all analysed. Then the correlation between these factors and prognosis was investigated. As a result, significant differences were observed in the survival rates between the groups regarding lymphatic vessel invasion (P = 0.0003), intramural metastasis (P = 0.0051) and lymph node metastasis (P = 0.0026). According to a multivariate analysis, intramural metastasis (P = 0.0038, relative risk 9.17), vessel invasion (P = 0.0033, relative risk 6.25) and lymph node metastasis (P = 0.0187, relative risk 3.62) were found to be independent prognostic factors. The prognosis of the patients with at least one of these factors was significantly poorer than that without. The five-year survival rate of the patients without these factors was as good as that with mucosal carcinoma of the oesophagus. Based on our findings, vessel invasion, intramural metastasis and lymph node metastasis are thus considered to be significant prognostic factors in patients with submucosal carcinoma of the oesophagus.

Differential growth inhibition by 5-fluorouracil in human colorectal carcinoma cell lines

The effects of 5-fluorouracil (5-FU) on cell growth were investigated using a primary culture of human fibroblasts, MRC-5, and three established human colon cancer cell lines, DLD-1, LoVo and SW620. Detailed flow cytometric analyses revealed differential growth inhibition among these cell lines including three modes of cell growth modulation: (a) loss or accumulation of S phase cells; (b) G2/M block; and (c) G1-S arrest. From analyses on the amount of 5-FU incorporated into cellular RNA and the activity of thymidylate synthase (TS), suppression of TS and depletion of dTTP, a possible consequence of the former, was considered to be the major action of 5-FU in these cells. Differences in the cellular responses to the nucleotide pool imbalance appeared to make the cell growth modulation diverse. Loss of S phase cells and G1-S phase arrest were evident in p53 wild-type cells, MRC-5 and LoVo. Cells proficient in DNA mismatch repair, SW620 and MRC-5, showed marked modulations in S-G2/M progression. These findings suggest that multiple factors, including p53 and DNA mismatch repair, participate in diverse cell growth modulations in cells treated with 5-FU. Cellular resistance to 5-FU correlated well with a loss of modulations in S-G2/M progression, rather than with a defect of G1-S arrest, which suggests the significance of DNA mismatch repair as a factor affecting the sensitivity of cells to 5-FU.

Features of microsatellite instability in colorectal cancer: comparison between colon and rectum

In one third of colorectal cancer patients, tumours occur in the rectum. Unique aetiologies may underlie the increased carcinogenesis in this region of the colorectum. Microsatellite instability (MSI) was analysed in specimens obtained from 121 colorectal carcinoma patients, using five dinucleotide markers and a new fluorescent system. The incidence of microsatellite alterations in the proximal colon, the distal colon and the rectum was 44.4% (16/36), 37.2% (16/43) and 23.8% (10/42), respectively. Patterns of microsatellite alterations could be classified into two subtypes, one showing relatively small changes within 6 bases (type A) and the other exhibiting drastic changes over 8 bases (type B). All the changes observed in tumours in the rectum were type A, and no type B mutation was noted. There was a close correlation between type B mutations and high-frequency MSI (≧2 markers), MSI-H, and between type A mutations and low-frequency MSI (1 marker), MSI-L. The type B/MSI-H phenotype significantly correlated with the proximal localisation of tumours. In the rectum, there was no tumour with the type B/MSI-H phenotype. These findings suggest that cancers occurring in the colon and the rectum have a differential molecular background for carcinogenesis.

Alcohol consumption and cigarette smoking in relation to high frequency of p53 protein accumulation in oesophageal squamous cell carcinoma in the Japanese.

We investigated levels of p53 protein expression in Japanese patients with oesophageal squamous cell carcinoma. A significantly larger proportion of heavy alcohol drinkers and cigarette smokers was evident in the p53-positive group. The combination of drinking and smoking was associated with a high frequency of p53 protein accumulation.