Toshikazu Kanai

Anti-tumor and anti-metastatic effects of human-vascular-endothelial-growth-factor-neutralizing antibody on human colon and gastric carcinoma xenotransplanted orthotopically into nude mice

In order to determine whether the inhibition of vascular‐endothelial‐growth‐factor (VEGF) activity by administration of an immunoneutralizing antibody could suppress tumor growth and metastasis in spontaneous metastatic models of human colon and gastric carcinoma, 4 human carcinoma xenografts, 2 human colon carcinomas (TK4 and TK13) and 2 gastric carcinomas (MT2 and MT5) were transplanted orthotopically into nude mice. The anti‐VEGF antibody (MV833, 100 μg/mouse) or the same volume of saline was administered i.p. on alternative days from day 10 after transplantation. With each of the 4 xenografts, administration of MV833 significantly inhibited not only primary tumor growth but also macroscopic liver metastasis, although the growth rate varied. The inhibitory effect of MV833 on primary tumor growth appeared to have no correlation with the level of VEGF in tumor. Body‐weight gain in each treated group was comparable with that in the control group. No toxicity of the antibody was observed. These results suggest that an anti‐VEGF antibody can be effective against a wide variety of cancers, and that VEGF may be a possible target for cancer therapy. Int. J. Cancer 77:933–936, 1998.© 1998 Wiley‐Liss, Inc.

Efficacy of an angiogenesis inhibitor, TNP-470, in xenotransplanted human colorectal cancer with high metastatic potential

The summation of gene mutations increases the metastatic potential of colorectal cancer. The genetic characterization and hepatic metastatic potential of five xenotransplanted human colon carcinoma strains were investigated. Furthermore, the therapeutic effect of the angiogenesis inhibitor, TNP‐470, was evaluated.

Effect of angiogenesis inhibitor TNP-470 on the progression of human gastric cancer xenotransplanted into nude mice.

The effect of an angiogenesis inhibitor, TNP‐470, on primary tumor growth, liver metastasis and peritoneal dissemination of gastric cancer was investigated by means of an orthotopic xenotransplanted model of 2 human gastric cancers, MT‐2 and MT‐5. TNP‐470 showed a significant inhibitory effect on the growth of primary tumors after orthotopic transplantation of both xenografts when given at a dose of 30 mg/kg on alternate days from day 7 after transplantation (early treatment). However, growth of the MT‐2 primary tumor was not inhibited by administration from day 14 after transplantation (late treatment). Liver metastasis was prevented significantly by early treatment of TNP‐470. In particular, early treatment of MT‐2 completely inhibited the development of macroscopic foci in the liver and was significantly more effective than late treatment. Peritoneal dissemination also was inhibited. Thus, TNP‐470 was revealed to have strong inhibitory activity not only on primary tumors and liver metastases but also against peritoneal dissemination. These results suggest that this agent may provide a new approach to the treatment of gastric cancer. Int. J. Cancer 71: 838‐841, 1997.

Antitumor Effect of a Neutralizing Antibody to Vascular Endothelial Growth Factor on Liver Metastasis of Endocrine Neoplasm

Distant metastasis of gastrointestinal endocrine neoplasm is resistant to currently available treatments. Because hematogenic metastasis is dominant, anti‐angiogenic drugs are expected to be a novel therapy for this neoplasm. In the present study, the therapeutic effect of vascular endothelial growth factor neutralizing antibody (VEGFAb) on liver metastasis of an endocrine neoplasm was investigated experimentally. Cecal transplantation into nude mice of small pieces of EN‐1, a xenotransplanted human intestinal endocrine neoplasm, resulted in liver metastasis. A treated group (n=19) received 100 μg/mouse of VEGFAb intraperitoneally on alternate days from day 10 after tumor transplantation, and the control group (n=19) received saline. Five of the 19 control mice died of tumor progression, of which 2 could not be evaluated. The cecal tumor weighed 6316±2333 mg (n=17) in the control group and 1209±837 mg (n=19) in the treated group (P<0.01) 6 weeks after transplantation. Liver metastasis developed in 16 of 17 control mice and in 2 of 19 treated mice (P<0.01). The VEGF level of the whole cecal tumor in the control group was significantly higher than that in the treated group (305.1±174.1 vs. 54.7±41.2 mg; P<0.001). VEGFAb did not cause any body weight loss (28.52±1.63 in the control vs. 28.44±1.71 g in the treated group). These results indicate that VEGFAb may be a novel therapeutic agent for endocrine neoplasm with distant metastasis.

Combination therapy with vascular endothelial growth factor neutralizing antibody and mitomycin C on human gastric cancer xenograft

Antiangiogenic therapy has been proposed as a new strategy for the treatment of solid tumors. To enhance the therapeutic effect of antiangiogenic agents, combination with conventional anticancer therapy should be investigated. In the present study, we investigated the therapeutic effect of the combination of vascular endothelial growth factor neutralizing antibody (VEGF Ab) and mitomycin C (MMC) on MT‐2, a human gastric cancer xenograft. When small pieces of MT‐2 were transplanted orthotopically into 62 nude mice, liver metastasis developed 6 weeks after transplantation. The VEGF Ab (100 μg/mouse) was administered i.p. in the VEGF Ab group (n=14) and the combination group (n=16) twice a week from day 10 after transplantation. MMC (2 mg/kg) was administered in the MMC group (n=16) and the combination group (n=16) on days 10, 17 and 24 after transplantation. Compared with the control group, in which saline solution was administered i.p., all three treatments inhibited tumor growth significantly and the effects of MMC and combination therapy were potent. Liver metastases were also inhibited significantly by the administration of VEGF Ab alone, MMC alone or combination therapy. Liver metastasis developed in 9 mice of the control group, 3 of the VEGF Ab group, and 4 of the MMC group, but no mice had liver metastasis in the combination therapy group. However a significant body weight loss and a decrease in spleen weight were observed in the MMC and combination groups, with no significant difference between the two groups. These results suggest that combination therapy with VEGF Ab and MMC may be a potent therapy for human gastric cancer.

p53 Overexpression and Proliferative Activity Do Not Correlate with Lymph Node Metastasis in Early Gastric Cancer

We investigated p53 overexpression and the proliferative activity of the primary lesion as well as the clinicopathological features of 75 patients with gastric cancer invading the submucosa (sm cancer), of whom 14 (18.7%) had lymph node metastasis. Among the clinicopathologic features studied, only lymphatic invasion by the primary tumor was related to lymph node metastasis. There was no relationship between immunohistochemical staining for p53 protein or Ki-67 and lymph node metastasis. The p53-positive rate was 35.7 and 57.1% in patients with and without metastasis, respectively, while the mean Ki-67 labeling index was 38.9 and 38.1%, respectively. Our results suggest that p53 mutation or the proliferative activity of sm cancer do not influence lymph node metastasis, even though p53 mutation may enhance the proliferative activity and metastatic potential of advanced gastric cancer.

Effect of loss of heterozygosity of the c-kit gene on prognosis after hepatectomy for metastatic liver gastrointestinal stromal tumors

The authors have previously reported that loss of heterozygosity (LOH) of the c‐kit gene could be responsible for the gain in high proliferative activity in some gastrointestinal stromal tumors (GIST), resulting in enhanced metastatic potential. In the present study, an attempt was made to identify the factors that might predict the postoperative prognosis of patients with metastatic liver GIST. The clinicopathologic or genetic features of resected liver GIST in 14 patients who had undergone a hepatectomy for metachronous liver metastases and who had not received adjuvant imatinib treatment were examined. LOH of the c‐kit gene was observed in seven of 12 metastatic liver GIST (58.3%), of which DNA suitable for testing could be extracted. Ten patients had recurrence after hepatectomy and four had none. The median post‐recurrent disease‐free survival (PRDFS) after hepatectomy was 27.5 months (range 8–104). The tumor‐specific PRDFS was examined using clinicopathologic features, c‐kit mutation and LOH of the c‐kit gene. No single clinicopathologic or genetic finding was significantly associated with PRDFS. However, patients with ‘Ki67 labeling index <5% and LOH(–)’ had a significantly longer PRDFS than those with ‘Ki67 ≥5% or LOH(+)’ (P = 0.032), and there was no correlation between the presence of LOH of the c‐kit gene and the Ki67 labeling index. LOH of the c‐kit gene in metastatic liver seems to be a common event, and LOH of the c‐kit gene in resected liver GIST may be a helpful factor in the prediction of the post‐recurrent prognosis of patients with liver metastasis. (Cancer Sci 2007; 98: 1734–1739)

Relationship of p53 and Vascular Endothelial Growth Factor Expression to Clinicopathological Factors in Human Scirrhous Gastric Cancer

Although scirrhous cancer has the highest malignant potential among various types of gastric cancer, its pathogenesis is still unclear. The relationship between expression of p53 or vascular endothelial growth factor (VEGF) and clinicopathological variables was investigated by immunohistochemical analysis of archival specimens from 40 patients with scirrhous gastric cancer. Staining for p53 and VEGF was observed in the nuclei and cytoplasm of the tumor cells, respectively. There was no significant association between expression of p53 or VEGF and sex, age, depth of invasion, lymph node metastasis or histological stage. Peritoneal dissemination was the most frequent mode of recurrence, and the depth of tumor invasion was a crucial factor. The recurrence rate was 83.9% (26/32) in patients with serosal invasion, whereas it was 22.2% (2/9) in patients without serosal invasion. Only 7 out of 40 patients (17.5%) survived without recurrence. Among them, the VEGF-positive rate was 14.3% (1/7), whereas it was 52.6% (10/19) in the patients with recurrence. There was no correlation between p53 and VEGF staining. These findings suggest that the progression of scirrhous gastric cancer may be promoted by VEGF overexpression, which is not upregulated by p53 mutation.

Prevention of Hepatic and Peritoneal Metastases by the Angiogenesis Inhibitor FR‐118487 after Removal of Growing Tumor in Mice

We established a mouse “primary tumor resection model” in which a transplanted tumor was resected after an orthotopic transplantation of colorectal cancer tissue to estimate the therapeutic effect of an angiogenesis inhibitor on metastasis. The angiogenesis inhibitor FR‐118487 is a member of the fumagUlin family. Here, 1 mg/kg/day of FR‐118487 was subcutaneously administered to nude mice for 1 week, 2 weeks, or 4 weeks through an osmotic pump. Liver metastasis developed in 7 of 9 control mice, 2 of 6 mice that underwent the tumor resection 2 weeks after transplantation (early resection), and in all 7 of the mice that underwent the tumor resection 4 weeks after transplantation (late resection). In the short treatment trial, the FR‐118487 administration immediately after the early resection completely inhibited both hepatic and peritoneal metastases, whereas its administration after the late resection had no effect on liver metastasis. In the prolonged treatment trial, inhibitory effects of prolonged treatment with FR‐118487 on both hepatic and peritoneal metastases after the late resection were clearly demonstrated. The mice of the resection‐alone group all died within 106 days after tumor inoculation, due to metastases of colon carcinoma. In contrast, half of the mice that underwent resection and then received antiangiogenic therapy were alive at the end of the observation period (160 days after transplantation). In conclusion, the combination of surgery and subsequent antiangiogenic therapy may be useful to prevent the distant metastasis of colorectal cancer and to improve the prognosis of patients with colorectal cancer.

Therapeutic Effect of 1 M Tegafur-0.4 M 5-Chloro-2,4-dihydroxypyridine-1 M Potassium Oxonate (S-1) on Liver Metastasis of Xenotransplanted Human Colon Carcinoma

S‐1 [1 M tegafur (FT)‐0.4 M 5‐chloro‐2,4‐dihydroxypyridine (CDHP)‐1 M potassium oxonate (Oxo)], was developed as a new oral antineoplastic agent based on biochemical modulation of fluorouracil (5‐FU) by CDHP and Oxo. The therapeutic effect of S‐1 on human colon cancer xenografts (TK‐13) with high metastatic potential to the liver was evaluated. Small pieces of TK‐13 were sutured into the cecal wall of 52 nude mice, and the animals were randomly divided into 3 groups [control (n=17), UFT (combination of 1 M FT and 4 M uracil) (n=18) and S‐1 (n=17)]. S‐1 or UFT was administered orally at an equitoxic dose (S‐1, 7.5 mg/kg; UFT, 17.5 mg/kg as FT) for 37 consecutive days beginning 10 days after the transplantation. S‐1 showed higher tumor growth inhibition than UFT (P < 0.05) and also showed a significant anti‐metastatic effect on liver metastasis, while UFT did not. Liver metastasis developed in only 2 of the 17 mice (12%) in the S‐1 group, whereas it developed in 9 of the 17 (53%) and 7 of the 18 (39%) in the control and UFT group, respectively. Analysis of AUC (area under the curve) revealed that S‐1 yielded higher 5‐FU levels in both tumor tissue (1.6 times) and plasma (2.5 times) than UFT. These results suggest that S‐1 will show a higher clinical therapeutic effect against human colorectal cancer than UFT.