Kouichi Miwa

Expression of p53 protein in colorectal cancer and its relationship to short‐term prognosis

Background and Methods. The expression of p53 protein in 100 large bowel cancers was studied immuno‐histochemically by use of a monoclonal antibody (PAb1801).

Lymphangiogenesis and the vascular endothelial growth factor receptor (VEGFR)-3 in gastric cancer

Vascular endothelial growth factor C (VEGF-C) is the only factor known to cause lymphangiogenesis. We studied the correlation between VEGF-C and vascular endothelial growth factor receptor-3 (VEGFR-3) expression of 85 primary gastric cancers by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry, and the results were correlated with the number of lymphatic vessels, stained with anti-VEGFR-3 antibody. RT-PCR and immunohistology demonstrated that VEGF-C was mainly produced from cancer cells, but not from stromal elements. Morphologically, VEGFR-3 expression was detected in the endothelial cells of the stromal lymphatic vessels. There was a statistically positive correlation between the incidence of VEGF-C and VEGFR-3 mRNA expression in the primary tumours (P=0.0002). The number of VEGFR-3-positive lymphatic vessels in VEGF-C mRNA positive tumours was significantly larger than that in VEGF-C-negative tumours. The number of VEGFR-3-positive vessels in the tumour stroma was closely related to the grade of lymphatic invasion of gastric cancer. These results strongly indicate that VEGF-C may induce the proliferation of lymphatic vessels in the stroma of primary gastric cancer via activation of VEGFR-3, expressed on the endothelial cells of lymphatic vessels. In these circumstances, cancer cells can easily invade the lymphatic vessel, because of the increase of the contact points of cancer cells with the lymphatic vessels.

Expression of C-erbB-2 oncoprotein in gastric carcinoma. Immunoreactivity for C-erbB-2 protein is an independent indicator of poor short-term prognosis in patients with gastric carcinoma

Correlations of c‐erbB‐2 protein expression with clinical outcomes of gastric carcinomas were studied in 189 gastric carcinomas. There were 23 (12.2%) carcinomas with evidence of c‐erbB‐2 protein in which the reaction was localized to the cell membrane. There was no significant association between c‐erbB‐2 staining and the macroscopic or histologic type of the carcinomas. c‐erbB‐2‐stained tumors were more likely to be associated with serosal invasion, nodal involvement, and peritoneal metastasis, than c‐erbB‐2‐unstained ones. In addition, c‐erbB‐2 was stained in none of early gastric carcinomas. The 5‐year survival rates of the c‐erbB‐2 protein‐positive and the protein‐negative group were 11% and 50%, respectively. When the c‐erbB‐2 tissue status and seven clinicopathologic variables as conventional prognostic factors were entered simultaneously into the Cox regression model, serosal invasion, hepatic metastasis, peritoneal metastasis, nodal status, and c‐erbB‐2 tissue status emerged as independent prognostic variables. The results suggested that c‐erbB‐2 protein expression might be enhanced in advanced stages during the progression of gastric carcinoma. In this particular group of patients, immunoreactivity for c‐erbB‐2 protein is an indicator of poor short‐term prognosis.

Amplification of the c-met, c-erbB-2 and Epidermal Growth Factor Receptor Gene in Human Gastric Cancers: Correlation to Clinical Features

We examined amplification of the c-met, c-erbB-2, and epidermal growth factor receptor (EGFR) gene in the patients with primary gastric cancer, and compared the data with clinical features in order to clarify the relationship between oncogenic abnormality and clinical features. Oncogene amplifications were examined by slot blot hybridization using DNAs extracted from formalin-fixed and paraffin-embedded tissues of primary gastric cancers. Seven of the seventy cancers (10.0%) had c-met gene amplification, nine (12.9%) had c-erbB-2 gene amplification, and six (8.6%) had EGFR gene amplification, respectively. Eighteen cases (25.7%) exhibited one or multiple oncogene amplification, and two cases (2.9%) exhibited simultaneous amplification of the three genes. The cases with c-met gene amplification tend to show invasive character and were related to peritoneal dissemination. The cases with c-erbB-2 gene amplification were related to lymph node metastasis. The cases with EGFR gene amplification had large tumors and were in highly advanced stage. The survival rate in patients with oncogene amplification was significantly lower than that in patients without amplification. Our data indicated that these genes were related to growth and metastasis of gastric cancer. Furthermore, this study about the three genes suggested that the type of activated gene might decide on the type of metastasis and clinical features.

Induction of oesophageal and forestomach carcinomas in rats by reflux of duodenal contents.

A study was designed to determine whether oesophageal carcinomas can be induced through reflux of duodenal contents. Male Wistar rats weighing 230-250 g were divided into three groups according to the surgical procedure performed: (1) the duodenal contents were directed into the forestomach through a stoma (duodeno-forestomach reflux); (2) the duodenal contents were regurgitated into the forestomach through the glandular stomach (duodeno-glandular-forestomach reflux); and (3) a sham operation was performed as a control. Animals were fed standard CRF-1 solid food and tap water that was not exposed to carcinogens and were sacrificed 50 weeks post-operatively. While no neoplasia was observed in any of the 32 control rats, 4/11 (36%) with duodeno-forestomach reflux and 3/18 (17%) animals with duodeno-glandular-forestomach reflux developed carcinomas in the lower oesophagus and forestomach. The incidence in each group was significantly higher than in the controls (P < 0.01 and P < 0.05 respectively). Six of the seven lesions consisted of squamous cell carcinomas, and one was a mucinous adenocarcinoma. Oesophageal columnar epithelial metaplasia was observed in two (18%) of the animals with duodeno-forestomach reflux. Carcinomas were always surrounded by chronic inflammatory changes, including regenerative thickening, basal cell hyperplasia and dysplasia. Additional well-differentiated adenocarcinomas were observed in the prepyloric antrum of 6/18 (33%) animals with duodeno-glandular-forestomach reflux. These findings indicate that chronic reflux of duodenal contents may cause oesophageal carcinoma.

Intraperitoneal hyperthermic treatment for peritoneal dissemination of colorectal cancers

Continuous hyperthermic peritoneal perfusion (CHPP) combined with administration of anticancer drugs was performed in eight colorectal cancer patients with peritoneal dissemination. An overall response rate of 50 percent was achieved in the eight patients. Two of three complete responders are long, recurrence-free survivors for 15 and 30 months. The two-year survival has been achieved in 18.8 percent of the patients receiving CHPP, and this rate is significantly higher than the rates in P2 and P3 patients who did not receive CHPP. The complications of CHPP with administration of anticancer drugs were mild bone marrow suppression in two (25 percent) of the eight patients and also a mild grade of renal dysfunction in one (12.5 percent), though not lethal. The results suggest that the combination of CHPP with the administration of anticancer drugs is a safe and effective therapy for peritoneal dissemination of colorectal cancers.

Rationale for extensive lymphadenectomy in early gastric carcinoma.

The incidence of nodal metastasis in early gastric carcinoma (EGC) is 10-20%. However, the optimal nodal dissection for early gastric carcinoma has not been established. A retrospective study was conducted in 392 consecutive patients who underwent potentially curative distal gastrectomy for EGC between 1962 and 1990. Of these 295 patients treated after September 1972 were prospectively entered into an extensive lymphadenectomy protocol. These patients were compared with 97 patients with simple gastrectomy in respect of the causes of death after surgery and the 10 year disease-specific survival rate. The incidence of nodal metastasis in early gastric carcinoma patients was 13.0%. Operative mortality from extensive lymphadenectomy was almost the same as from simple gastrectomy (2.0% and 2.1% respectively). Extensive lymphadenectomy provided a significantly higher 10 year survival rate than limited lymph node dissection (97.9% vs 88.1% respectively; P < 0.005). Among patients with nodal metastasis, the survival rate following extensive lymphadenectomy was significantly higher than that after simple gastrectomy (87.5% vs 55.6%; P = 0.018). Among patients without nodal metastasis, there was no difference between the two groups in the survival rate (99.4% and 96.7% respectively; P = 0.12). Multivariate analysis using the Cox proportional hazards model disclosed two significant independent prognostic factors on disease-specific survival, the nodal involvement (risk ratio: 8.4; P < 0.0001) and the extent of lymph node dissection (risk ratio: 5.8; P < 0.005). Extensive nodel dissection appears to prevent recurrence and to improve the cancer-specific survival in EGC patients with nodal metastasis.

Role of MMP-7 in the formation of peritoneal dissemination in gastric cancer.

Background. Matrix metalloproteinase-7 (MMP-7) is an important matrix-degrading enzyme that has a large role in the invasion and metastasis of cancer. To discover the mechanism of the formation of peritoneal dissemination in gastric cancer, we studied the mRNA and protein expression of MMP-7 in primary gastric cancers and peritoneal dissemination.Methods. MMP-7 expression in primary gastric cancers (136 patients) was studied by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR), and the results were compared with chinicopathological parameters.Results. MMP-7 mRNA was expressed in 28 (53%) of 53 primary gastric cancers, but not in normal gastric mucosa, fibroblasts, or mesothelial cells. An immunohistochemical method demonstrated that MMP-7 immunoreactivity was found on the cell membrane and cytoplasm of cancer cells. Among 136 primary tumors, 70 (53%) tumors overexpressed MMP-7, and MMP-7 tissue status had significant positive correlation with serosal involvement, lymph node metastasis, poor differentiation of cancer, and peritoneal dissemination. Patients with MMP-7-positive tumor had significantly poorer survival and more frequently died of peritoneal recurrence than did those with MMP-7-negative tumors. All 6 examined peritoneal disseminations expressed MMP-7 mRNA, and 13 of 14 peritoneal disseminations showed immunoreactivity to anti-human MMP-7 monoclonal antibody. Logistic regression analysis showed that MMP-7 immunohistological status was an independent risk factor for peritoneal dissemination, and patients with MMP-7 mRNA-positive tumors had a 9.9-fold higher relative risk for peritoneal metastasis.Conclusion. These results strongly suggest that MMP-7 may have a large role in the formation of peritoneal dissemination in gastric cancer, and that clonal selection of cancer cells with MMP-7 overexpression may occur during the invasion of intraperitoneal free cancer cells from the peritoneal surface into the subperitoneal tissue. MMP-7 tissue status in the primary tumor may be a good indicator of peritoneal dissemination.

Quantitative evaluation of metastases in axillary lymph nodes of breast cancer.

We have established a highly sensitive and quantitative reverse transcriptase–polymerase chain reaction (RT–PCR) method to detect axillary lymph node metastases of breast cancer. Amplifying cytokeratin 19 (CK19) mRNA transcripts using real-time TaqMan PCR made it possible to quantify axillary metastatic burden. Metastases in 358 axillary lymph nodes obtained from 23 breast cancers of 22 patients were investigated by conventional haematoxylin and eosin (H&E) staining, immunohistochemical staining and quantitative RT–PCR assay. The detection rates of axillary lymph node metastasis using H&E staining, immunohistochemistry and RT–PCR assay were 4.5, 5.9 and 13.1%, respectively. RT–PCR assay was the most sensitive of these three methods for detecting lymph node metastases. Cytokeratin 19 mRNA expression values of both histologically and immunohistochemically positive lymph nodes were significantly higher than the values for lymph nodes judged to be negative by both histological and immunohistochemical methods (P<0.0001), and those of histologically negative, but immunohistochemically positive lymph nodes were significantly higher than the values for lymph nodes judged to be negative by both histological and immunohistochemical methods (P<0.0001). Furthermore, metastatic rates of sentinel nodes were higher than the rates of nonsentinel lymph nodes as measured by all three methods. These results indicate that quantitative RT–PCR assay is a sensitive and reliable method for detecting lymph node metastasis. Furthermore, quantification of metastases in sentinel lymph nodes by quantitative RT–PCR assay may be useful to assess the entire axillary burden of breast cancer patients.

Expression of c-myc Gene Product in Gastric Carcinoma

The expression of c-myc oncogene product was studied in 213 cases with gastric carcinoma by an immunoperoxidase method using a monoclonal antibody (MYC-1). Fifty (23.5%) of 213 tumors showed immunoreactivity to MYC-1. The distribution of c-myc-product-positive cells was observed mainly at the marginal area of the tumor. Excess reactivity to c-myc product occurred more frequently in invasive cancers than in localized cancers, and c-myc production expression in cancer tissue correlated well with peritoneal dissemination. Patients with c-myc-protein-positive tumor had significantly poorer prognosis than those with c-myc-protein-negative tumor in invasive gastric carcinomas, and the c-myc product status correlated well with the recurrence of cancer by peritoneal dissemination. These results suggest that the expression of c-myc gene product might be related to the proliferative activity of gastric carcinoma and serve as a new biologically relevant tumor marker for determining the prognosis.