Tokiharu Miyahara

In oesophageal Squamous cell carcinoma vascular endothelial growth factor is associated with p53 mutation, advanced stage and poor prognosis.

Vascular endothelial growth factor (VEGF) affects malignant tumours by promoting angiogenesis. The tumour-suppressor gene p53 has been thought to regulate VEGF. We investigated the effect of VEGF on oesophageal carcinoma and the connection between VEGF and p53. One hundred and nine resected oesophageal squamous cell carcinomas were examined. VEGF expression was analysed by immunohistochemical staining. Sixty-five tumours (59.6%, 65 out of 109) were classified as VEGF positive. A significant correlation was found between the VEGF expression and both the depth of invasion (P = 0.0001) and lymph node metastasis (P < 0.0001). With regard to p53, we compared the expression of VEGF with the mutation of p53, examined using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and direct sequencing in tumour samples obtained from 36 patients who we have reported previously. The VEGF expression was significantly correlated to p53 mutation (P = 0.0291). To evaluate the angiogenesis, microvascular density (MVD) was counted, and endothelial cells were stained immunohistochemically using anti-CD34 monoclonal antibody against 29 cases with invasion limited to the submucosal layer. The average MVD had a tendency to correlate to VEGF expression (P = 0.1626). The prognoses of patients with VEGF-positive primary tumours were significantly worse than for those with VEGF-negative primary tumours (P = 0.0077). We have assumed that VEGF contributes to aggressive characteristics in oesophageal carcinomas and that VEGF expression might be affected by p53 status.

Genetic alterations in patients with esophageal cancer with short- and long-term survival rates after curative esophagectomy.

OBJECTIVE The objective of this study was to ascertain the exact relation between specific oncogenes and long- and short-term survival rates in patients with esophageal cancer. SUMMARY BACKGROUND DATA Recent developments in molecular biology have shown that several oncogenes and suppressor genes are involved in the development of esophageal cancer. However, the role of these genes still is unknown. METHODS The clinical outcome of 84 cases of R0-resected esophageal carcinomas (from 1986-1993) and the molecular and biologic characteristics of these tumors were studied. The patients studied were divided into three groups, which were designated as follows: shortest term survivors (up to 6 months), short-term survivors (7-12 months), and long-term survivors (>5 years). These groups included 23, 17, and 44 subjects, respectively. For the genomic analysis, CyclinD1, int-2, murine double minute 2 (MDM2), retinoblastoma, p53, adenomatous polyposis coli (APC), deleted in colorectal carcinoma (DCC), and human papillomavirus were studied in these patients. The regrowth capability of primary cultures and the clinicopathologic characteristics of these patients also were analyzed. RESULTS The CyclinD1 and int-2 genes, which are located in the 11q13 chromosome, and the MDM2 gene were related to short survival. However, the p53 mutation and human papillomavirus infection were not related to short-term survival. The average ratio of genomic abnormalities to genes examined was higher in the shortest and short-term survival groups than in the long-term survival group. Regrowth capability in primary cultures also was related to short-term survival. Among the long-term survival patients, 7 (16%) of 44 cases suffered further cancer after esophagectomy. CONCLUSIONS These results suggest that the 11q13 amplicon and MDM2 may play an important role in the progression of esophageal cancer, and an accumulation of genomic abnormalities may result in poor prognosis. Careful follow-up testing for double cancer is needed in long-term survivors of esophageal cancer.

Hemodynamic changes after resection of thoracic duct for en bloc resection of esophageal cancer

An en bloc resection of esophageal cancer is one of the most radical forms of esophagectomy, and includes the resection of the thoracic duct, but a relatively high hospital motality rate has been reported. There is very little knowledge on the pathophysiological changes after resection of the thoracic duct. We examined 24 patients who underwent en bloc resection. Some patients developed severe tachycardia or shock postoperatively which subsided after a massive infusion of plasma. Analysis of the fluid balance revealed that much more fluid was necessary during surgery and the postoperative 24 h than in patients treated by a standard esophagectomy. Postoperative lymphangiography or CT revealed abnormal collateral lymphatics around the kidneys or in the pelvic cavity. This suggests the development of the lymphaticovenous shunts, which differed depending on the anatomy of each patient. One patient with chronic hepatitis developed uncontrollable ascites. These are important findings which can hopefully reduce the high rate of hospital death after this operation.

Hyperthermic Enhancement of Cytotoxicity and Increased Uptake of cis‐Diamminedichloroplatinum(II) in Cultured Human Esophageal Cancer Cells

Thermal enhancement of cytotoxicity of cis‐diamminedichloroplatinum(II) (CDDP) has been well recognized and applied clinically to chemotherapy of various malignancies, but its fundamental mechanism remains to be elucidated. In order to obtain a clue to this mechanism, we analyzed the effect of hyperthermia on the uptake and subsequent distribution of [195mPt]CDDP in two lines of esophageal cancer cells (KYSE‐150 and KYSE‐170) established from clinical patients. First, we observed a significant increase in [195mPt]CDDP uptake by both types of cells at increasingly higher temperatures. The incorporated CDDP was distributed between the nucleus and the cytosol at a ratio of approximately 3:1, and the ratio remained the same at various temperatures. The CDDP was found in all four molecular fractions, i.e., DNA, RNA, protein, and TCA‐soluble, with a slight preference for DNA at higher temperatures. Enhancement of cytotoxicity required simultaneous, and not sequential, treatments with CDDP and hyperthermia; hyperthermia after CDDP treatment increased the efflux of CDDP from the cells, and rather reduced the cytotoxicity of CDDP. These results suggest that thermal enhancement of the cytotoxicity of CDDP is caused mainly by acceleration of the drug entry into the cell, probably due to increased permeability, and a consequent increase in the amount of CDDP binding to DNA. This mechanism gives support for clinical trial of simultaneous treatment with CDDP and hyperthermia.

Unresponsiveness of insulinoma cells to secretin: significance of the secretin test in patients with insulinoma.

Summary It is well known that B cells in the pancreas release insulin when stimulated by secretin, but there have been few reports on the response of insulinoma cells to secretin. In five patients with insulinoma, changes in serum immunoreactive insulin (IRI) concentration were measured after the intravenous injection of secretin into the peripheral vein before and after extirpation of the insulinoma. The extirpated insulinomas were cultured and tested for their response to secretin. The rise in serum IRI in response to secretin in patients with insulinoma was significantly slower and smaller than in normal volunteers. After removal of the insulinoma, the response to secretin became prompt and increased with time. Cultured insulinoma cells did not release insulin when stimulated by secretin. Therefore, it is concluded that the response of insulinoma cells to secretin is quite different from that of normal β cells, and that the function of β cells in the insulinoma-bearing pancreas is suppressed by the autonomous hypersecretion of insulin by the insulinoma. The extent of the decrease in function of the β cells in patients with insulinoma can be estimated by the intravenous secretin test. Thus, the secretin test is sometimes useful in the differentiation of hypoglycemia due to insulinoma from that due to β cell hyperplasia or alimentary hyperinsulinemia.

Binding Characteristics of (–)-(R)-2-Aminomethylpyrrolidine(1,1-cyclobutanedi-carboxylato)-2-platinum(II) to DNA, RNA and Protein Molecules in HeLa Cells and Its Lethal Effect: Comparison with cis- and trans-Diammmedichloroplatinums(II)

HeLa S‐3 cells were treated with 195mPt‐radiolabeled (–)‐(R)‐2‐aminomethylpyrrolidine(1,1‐cyclobutanedicarboxylato)‐2‐platinum(II) (DWA2114R) under various conditions, and the relationship between the lethal effect of the agent and the number of platinum (Pt) atoms binding to DNA, RNA and proteins was examined. The values of mean lethal concentration for the cells treated with DWA2114 at 37°C for 1, 2 and 3 h were 137.3, 75.10 and 51.17 μM, respectively. Cells were treated identically and the numbers of Pt atoms combined with DNA, RNA and protein molecules were determined after fractionation of the cells. In this way, the D0 values (D0, dose that would give an average of one lethal event per member of the population), expressed as the drug concentration, were substituted for the number of Pt atoms combined with each fraction. The target volumes, the efficacy of Pt atom to kill cells expressed as the reciprocals of the D0 values, were then calculated for each fraction. Our findings suggested that DNA was the primary target molecule for cell killing by DWA‐2114R. The target volumes for DNA were 3.36 × 104, 4.00 × 104 and 4.10 × 104 nucleotides for 1‐, 2‐and 3‐h treated cells, respectively. The cell‐killing effects of DWA2114R were lower than those of cis‐dianuninedichloroplatinum(II) (CDDP) by factors of 1.54, 1.42 and 2.51 for 1‐, 2‐ and 3‐h treatments at 37°C, respectively, in terms of the target volume, while those in terms of the mean lethal dose (D0) were 14.8, 11.2 and 16.0, respectively. The efficacy of DWA2114R in killing the cells was 2.6 times greater than that of CDDP in the 3‐h treatment at 0°C.

Usefulness of preoperative low dose cisplatin treatment for advanced esophageal cancer.

In order to decrease the perioperative complications by preoperative cisplatin chemotherapy, the preoperative single administration of cisplatin (30 mg/m2) was performed weekly from one to six times in 36 consecutive patients with esophageal cancer classified as higher than Stage II. The survival curve of 17 patients in Stage III was significantly better (P<0.05) than that of patients who had been treated without preoperative cisplatin treatment. In 3 of the 12 patients who had locally invasive cancer, either the main tumors or the metastatic lymph nodes, which had invaded the trachea or the left main bronchus, sufficiently receded, so that a curative esophagectomy became possible; 2 of them have survived over 33 months while 1 died of pneumonia 33 months after surgery. The number of perioperative complications was minimal, and thus, we consider that the postoperative use of cisplatin and fluorouracil is indicated in patients in whom a histological response is noted in the resected specimens.

Determination of the target volume of HeLa cells treated with platinum-195m radiolabeled trans-diaminedichloroplatinum(II): a comparison with cis-diaminedichloroplatinum(II)

HeLa S-3 cells were treated with 195mPt-radiolabeled trans-diaminedichloroplatinum(II) (TDDP) under various conditions, and the relationship between lethal effect and the number of Pt atoms binding to DNA, RNA and proteins was examined. The mean lethal concentrations for the cells treated with TDDP at 37 degrees C for 1, 2 and 3 h were 163.7, 65.8 and 24.9 microM, respectively. By using identically treated cells, the number of Pt atoms combined with DNA, RNA and protein molecules was determined after the cells were fractionated using the method of Schneider. In this way, the D0 values given as the drug concentration were substituted for the number of Pt atoms combined with each fraction, then the target volumes, expressed as the reciprocals of the D0 values, were calculated for each fraction. The results suggested that DNA and high molecular weight RNAs (except t-RNA), under some limited condition, could be the target molecules for cell killing by TDDP. The target volumes for DNA were 1.31 x 10(3), 3.01 x 10(3) and 6.26 x 10(3) nucleotides for 1, 2 and 3 h treated cells, respectively. Cell killing effects of TDDP were lower than CDDP by a factor of 39.5, 19.0 and 16.5 for 1, 2 and 3 h treatments at 37 degrees C, respectively, when viewed from the stand point of the target volume, while those from the mean lethal dose (D0) were 17.6, 9.8 and 6.7, respectively.

Determination of the target volume of hela cells treated with tetra‐ and hexa‐ chloroplatinates

HeLa S‐3 cells were treated with l95mPt‐radiolabeled tetra‐ and hexa‐chloroplatinums (Pt(II) and Pt(IV)) under various conditions, and the relationship between lethal effect and the number of Pt‐atoms binding to DNA, RNA and proteins was examined. The mean lethal concentrations for the cells treated with Pt(II) at 37°C for 1, 2 and 3 h were 163.5, 126.2 and 67.3 μM, while those for Pt(IV) were 78.6, 53.4 and 35.9, respectively. By using identically treated cells, the number of Pt‐atoms combined with DNA, RNA and protein molecules were determined after the cell were fractionated using the method of Schneider [ref. 1]. In this way, the Do values given as the drug concentration were substituted for the number of Pt‐atoms combined with each fraction, then, the target volumes expressed as the reciprocals of Do values were calculated for each fraction. The results suggested that DNA and high molecular weight RNAs (except t‐RNA), under some limited condition, can be the target molecules for cell killing by both ...

Determination of the target volume of HeLa cells treated with platinum-195 m radiolabeled cis-diammine(1,1-cyclobutane-dicarboxylato)-platinum(II); comparison with cis- and trans-diamminedichloroplatinums(II)

HeLa S-3 cells were treated with 195mPt-radiolabeled cis-diammine(1, 1-cyclobutane-dicarboxylato)platinum(II) (carboplatin) under various conditions, and the relationship between lethal effect and the number of Pt atoms binding to DNA, RNA and proteins was examined. The mean lethal concentrations for the cells treated with carboplatin at 37 degrees C for 1, 2 and 3 h were 553.4, 194.3 and 68.7 microM, respectively. By using identically treated cells, the numbers of Pt-atoms combined with DNA, RNA and protein molecules were determined after fractionation of the cells. In this way the D0 values (D0, dose that would give an average of one lethal event per member of the population), expressed as the drug concentration, were substituted for the number of Pt atoms combined with each fraction. The target volumes, the efficiency of Pt atom to kill cells expressed as the reciprocals of the D0 values, were then calculated with each fraction. The results suggested that DNA was the primary target for cell killing by carboplatin. The target volumes for DNA were 0.891 x 10(4), 2.01 x 10(4) and 3.96 x 10(4) nucleotides for 1, 2 and 3 h treated cells, respectively. The cell killing effects of carboplatin were lower than those of cis-diamminedichloroplatinum(II) (CDDP) by factors of 6.0, 2.8 and 2.6 for 1, 2 and 3 h treatments at 37 degrees C, respectively, in terms of the target volume, while those in terms of the mean lethal dose (D0) were 59.5, 29.0 and 21.5, respectively.