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The rate of tumor growth depends on the balance between proliferation and death of tumor cells. It is known that Bax, caspase-3, and p53 proteins are death-promoting factors, whereas Bcl-2 protein is a death antagonist. We immunohistochemically examined the expression of Bax and apoptosis-related proteins such as caspase-3, p53, and Bcl-2 in 76 patients with human esophageal squamous cell carcinoma (SCC) including dysplasia to determine the relationship of expression of each protein to tumor behavior and patients' prognosis. No significant relationships in immunopositivity were found among these proteins in SCCs. Cytoplasmic Bax expression was exhibited in 63 cases of SCCs (82.9%). The apoptotic index of caspase-3-positive lesions was significantly higher than that of caspase-3-negative lesions in both dysplasia and SCC (P =.016, P =.012). On the other hand, the apoptotic index (1.18%) was significantly correlated with Bax overexpression in dysplasia (P =.006), but not in SCC lesions (P =.129). The patients with Bax-positive SCCs were found to have a poor prognosis by the Kaplan-Meier method (P =.043). These findings suggested that Bax expressed in dysplasia may play a role as an apoptotic factor, but that it may be functionally inactive in some cancerous lesions and thus not contribute to suppression of the tumor progression in some cases of human esophageal SCCs.

Background: It is generally accepted that cigarette smoking is closely associated with esophageal squamous cell carcinoma. This study investigated the molecular targets of cigarette smoke in carcinogenesis of the esophagus., Methods: Seventy-four patients with esophageal squamous cell carcinoma (SCC) were grouped according to daily cigarette consumption: heavy smoking group (group H) (n = 26), moderate smoking group (group M) (n = 39) and non-smoking group (group N) (n = 9). We compared p53 and retinoblastoma (RB) expression among the three groups by immunohistochemistry. In addition, fresh tumor tissues from 30 smokers with esophageal SCC were tested for p53 mutations in exons 5-8 by direct sequencing., Results: Staining for the p53 product was positive in 65.4% of group H, 38.5% of group M and 44.4% of group N. The frequency of positive staining in the group H was significantly higher than in group M (p = 0.033) and in group M + group N (p = 0.034). The difference with respect to the frequency of overexpression of RB was not significant. The patterns of p53 base-pair mutations in direct sequencing study were of five types, most commonly G:C to T:A transversion (35.3%)., Conclusions: Our study suggests that one of the molecular targets of cigarette smoke is the p53 gene. The pattern of p53 point mutations involved a wide range of base-pair changes.

Functional defects in the CIP/KIP family of cyclin-dependent kinase inhibitors (CDKIs) have been shown to be associated with human malignancies. We immunohistochemically examined p57KIP2 (p57) expression in 92 patients with human esophageal squamous cell carcinoma (SCC) to determine the relationship between this expression and those of cyclin D1 and E. The p57 labeling index (LI) (defined as the percentage of p57-positive cells) in esophageal SCC was 43.3 +/- 3.2% (mean +/- standard error of the mean). In non-neoplastic esophageal epithelium, p57 staining was more frequently observed in the basal and parabasal cells than in surface layer cells. Immunostaining for cyclin D1 and E was observed in 28.2% (28/92) and 32.6% (30/92) of tumors, respectively. The median p57 LI in cyclin D1-positive cases was 66.2, and significantly higher than that in negative cases (31.9%) (p = 0.0009). There was no significant relationship between p57 LI and cyclin E expression (p = 0.147). As determined using Kaplan-Meier's method, loss of p57 immunoreactivity was not a prognostic factor for esophageal SCC (p = 0.548). Our in vivo findings suggested that p57 protein expression was positively correlated with cyclin D1 expression and that loss of p57 protein expression alone does not affect progression of esophageal SCC.

The expressions of cyclin D1, cyclin E, cyclin-dependent kinase 4 (CDK4), and CDK2 were immunohistochemically examined in 90 patients with human oesophageal squamous cell carcinoma (SCC) to determine their relationship to the tumour behaviour and patient prognosis. Nuclear immunostaining of cyclin D1 and cyclin E was observed in 28 (31.1%) and 27 tumours (30.0%) respectively. Thirty-nine tumours (43.3%) and 31 tumours (34.4%) exhibited both cytoplasmic and nuclear positivity for CDK4 and CDK2 respectively. Of 28 cyclin D1-positive and 27 cyclin E-positive tumours, CDK4 was overexpressed in 12 (42.8%) tumours and CDK2 in seven (25.9%) tumours respectively. There was no significant relationship in immunopositivity between cyclin D1 and CDK4 or between cyclin E and CDK2. Simultaneous immunoreactivity for both cyclin D1 and CDK4 was significantly associated with venous invasion (P < 0.05). In a univariate analysis, the prognosis of patients with tumours that were both cyclin D1- and CDK4-positive was significantly poorer than that of patients with cyclin D1-negative tumours (P < 0.05). In a multivariate analysis, both cyclin D1 and CDK4 immunoreactivities (P < 0.01) and tumour stage (P < 0.001) were recognized as independent risk factors. In this analysis, the hazard ratio for cyclin D1-positive and CDK4-negative cases compared with cyclin D1-negative cases was significant (hazard ratio = 3.128, 95% confidence interval = 1.418-6.899, P = 0.0047). No significant prognostic relevance was detected in both cyclin E and CDK2 immunoreactivity. Our in vivo findings suggest that in human oesophageal SCC, cyclin D1 and cyclin E and their functional partners, CDK4 and CDK2, often exhibit dysregulated overexpression in many cases, and that tumours with simultaneous expression of cyclin D1 and CDK4 are frequently associated with venous invasion and have a worse prognosis, statistically. Moreover, overexpression of cyclin D1 alone may also contribute to tumour progression independent of CDK4 overexpression.

Recent studies have identified a family of proteins referred to as cyclins, which control the cell cycle. Cyclin B1 activates cdc2, which regulates cell progression through the G2 and M phases. The main aim of this study was to examine the relationships between the cyclin B1 expression in human esophageal squamous cell carcinoma (SCC) and clinicopathological factors and prognosis of the patients. Eighty-seven cases of primary human SCC consecutively obtained at esophagectomy were immunohistochemically studied using an anti-human cyclin B1 protein antibody (2H1-H6). The relationship between cyclin B1 expression and clinicopathological factors, including prognosis, were also statistically assessed. Positive immunostaining of cancer cells, mainly in the cytoplasm, was detected in 72.4% (63/87): heterogeneous pattern in 37.9% (33/87) and homogeneous pattern in 34.5% (30/87). The prevalence of cyclin B1 expression was significantly higher in cases with invasion deeper than the muscularis propria (P<0.005) and with venous invasion (P<0.01) than in other cases. Patients whose SCCs expressed high levels of cyclin B1 protein had a significantly poorer prognosis than did the other patients (P<0.05). Multivariate analysis demonstrated that cyclin B1 status was an important factor affecting survival (P<0.05). These findings demonstrated that overexpression of cyclin B1 protein is associated with tumor behavior and prognosis for patients with human esophageal SCC.

Esophageal cancer is frequently found when it is already in the advanced stage and curative surgery for such cases is consequently difficult to perform. The new multi-disciplinary treatment for esophageal cancer presented here was, therefore, conceived to improve both the survival rate and quality of life of these patients. This combined treatment modality consists of limited surgery, external irradiation, intracavitary irradiation with remote-controlled after-loading system (RALS) and peri-operative chemotherapy. In the present series, 45 patients with esophageal cancer received esophagectomy and on another 11 patients bypass operation was performed. All patients were treated with this multi-disciplinary treatment after operation. A 3 cm-wide thin gastric tube was made from the greater curvature of the stomach of the patient using an autosuture apparatus (PLC55 or GIA). In the bypass operation, the jejunum was anastomosed to the original esophagus in the Roux-en Y fashion and jejunostomy was performed on the oral side of the Roux loop. A silastic tube of 9 mm inner diameter was inserted from the jejunostomy and placed into the original esophagus for the purpose of postoperative intracavitary irradiation with RALS. For the patients receiving esophagectomy, a similar silastic tube was also placed in the posterior mediastinum for intracavitary irradiation with RALS. The indication of the bypass operation was i) a tumor length longer than 9 cm on the X-ray film and/or ii) direct invasion to the aortic wall evident by CT or MRI examination. Two weeks after the operation, external irradiation to the mediastinum with Linac 10 MV X-ray, and to the bilateral cervical regions with Linac 15 MeV electron beam, was started. The irradiation doses were 30 Gy (2 Gy/day, 5 times/ week) and 48 Gy (4 Gy/day, 3 times/week), respectively. The intracavitary irradiation with RALS was started shortly before the end of the external irradiation period and was delivered from a 60Co source. The total dose was 24 Gy (6 Gy/day, once a week) for the esophagectomized cases, and 18 Gy for the bypassed cases. Two or three weeks after the termination of the radiotherapy, chemotherapy with cisplatinum and 5-fluorouracil was performed and repeated every 6 months for 2 years. All patients could eat normally and were discharged after finishing the first chemotherapy session. The overall 5-year survival rate was 49% for the esophagectomized cases and 11% for the bypassed cases. The longest survival time in the bypassed cases was 5 years and 4 months. Neither operative death nor severe complications were experienced during the treatment period. The results indicate that this newly developed multi-disciplinary treatment with RALS can improve the prognosis and the quality of life not only in the esophagectomized patients but also in the bypassed patients with advanced esophageal cancer.

p27Kip1, one of the cyclin-dependent kinase (CDK) inhibitors (CDKIs), blocks progression from G1 to S phase by binding cyclin D1-CDK4 and/or cyclin E-CDK2 and inhibiting their activities. Reflecting the function of p27 as a CDKI in vitro, a reduced expression of protein p27 has recently been reported to be associated with tumor aggressiveness in some types of human cancers. In the present study, we examined the relationships between immunohistochemically detected expression of p27, cyclin D1, cyclin E proteins and clinicopathological findings in 77 patients with esophageal squamous cell carcinoma (SCC). Using specific monoclonal antibodies to p27, cyclin DI and cyclin E proteins, positive immunostaining in the nuclei was observed in 32.5% (25/77), 27.3% (21177) and 29.6% (21/71) of patients, respectively. There were no statistically significant relationships among the expressions of these 3 proteins. Using the Kaplan-Meier's method, p27 and cyclin D1 expressions were found to be independently associated with poor prognosis. When all parameters were combined into a multivariate regression analysis using the Cox model, the expressions of p27 and cyclin D1 retained a predictive value for survival. In contrast to former reports supporting a tumor-suppressive function of p27, our results suggest that altered expression of p27 and cyclin D1 may be associated with the progression of human esophageal SCC, in which cyclin E may well not play any central role.

The present study was undertaken in order to investigate the possible involvement of high-risk human papillomavirus (HPV; types 16 and 18) or the overexpression of p53 protein in 123 Japanese patients with esophageal squamous cell carcinoma (SCC) from five different institutions. We detected HPV DNA in 30.1% (37/123) by in situ hybridization (ISH). Of these 123 cases, HPV type 16 was detected in 22 cases and HPV type 18 in 23 cases. In addition, HPV types 16 and 18 were detected simultaneously in eight cases. We found an almost similar incidence of HPV infection at five different places in Japan. Then, among these patients, 24 fresh tumor samples were also examined for screening the presence of HPV DNA using dot blot hybridization (DBH) and polymerase chain reaction (PCR). We detected HPV DNA in 20.8% (5/24) by DBH and in 12.5% (3/24) by PCR. P53 protein overexpression was found in 34.1% (43/123) by immunohistochemistry (IHC). Furthermore, in 43 cases from Kochi Medical School, the group positive for p53 antibody statistically showed worse survival rate than the group negative for both HPV DNA and p53 antibody. Judging from these results obtained in the present study, HPV infection and p53 overexpression are frequently detected and involved in the carcinogenesis of esophageal SCC in Japan. We also found that no significant geographical difference of both HPV infection and p53 overexpression of esophageal SCC was seemingly present in Japan.

The relationship between aberrant expression of cyclin D1 and retinoblastoma (RB) protein and clinicopathological factors was investigated in 80 patients with oesophageal SCC using immunohistochemical analyses. Heterogeneous staining of cancer cell nuclei with antibody to cyclin D1 was found in 31.3% of patients (25 out of 80 patients). Nuclear staining of cancer cells with anti-RB antibody was homogeneous in 10.0% (8 out of 80 patients) and heterogeneous in 58.8% (47 out of 80 patients). Among cases with homogeneous staining for RB protein, 75% (six out of eight patients) exhibited simultaneous positivity for cyclin D1 (P < 0.05). No significant relationship was found between cyclin D1 or RB protein expression and various clinicopathological parameters. The prognosis of patients with cyclin D1-positive tumours was significantly poorer than that of the other patients (P < 0.01). In addition, when patients with cyclin D1-positive and -negative tumours were stratified according to presence or absence of lymph node metastasis and RB status, the cumulative survival rates in the cyclin D1-positive groups were significantly lower for patients without lymph node metastasis (P < 0.01) and for patients whose tumours were positive for RB (P< 0.0001). These findings suggest the possibility that cyclin D1 positivity is a useful prognostic marker related to lymph node metastasis and RB protein expression in human oesophageal SCC, in addition to clinicopathological factors.

Background: Since carcinoma of the esophagus is usually diagnosed at an advanced stage, many cases of esophageal cancer are beyond possible radical resection and only palliative treatment can be performed in such cases. Therefore, a great deal of discussion has taken place concerning indications for treatment modality, and various procedures have been performed to palliate such patients. The prognosis for such patients is still poor, even though many kinds of palliation have already been developed and applied. To improve the prognosis of such patients we developed a multidisciplinary treatment which includes high dose rate intraluminal brachytherapy (HDR-ILBRT) and evaluated its effectiveness, especially the HDR-ILBRT component. PATIENTS AND TREATMENT METHODS: Sixty-six patients with unresectable esophageal cancer enrolled in this study. Twenty-seven patients underwent bypass operations. Seven of the 27 patients received external irradiation only (group BE), 11 received external irradiation and HDR-ILBRT (group BEH), while the remaining 9 did not receive radiotherapy (group B). Another 39 patients without bypass operations were all treated with radiotherapy, 22 with external irradiation only (group E) and 17 were treated with external irradiation and HDR-ILBRT (group EH). After completion of radiotherapy, all patients received chemotherapy with 5-FU (2,500 mg/body) and CDDP (100 mg/body)., Results: Mean survival time and the 5-year survival rate of group BEH (13.3 +/- 1.5 months and 20%) were significantly improved compared with group BE + B (p < 0.05). In the patients without bypass operations, there were significant differences in the mean survival time and the 3-year survival rate in groups EH and E (group EH 16.5 +/- 2.5 months, 21.8%, group E 9.0 +/- 1.3 months, 0%, p < 0.05). The bypass operation itself and chemotherapy did not significantly affect the prognosis of patients with unresectable esophageal cancer., Conclusion: These results strongly suggest the use of HDR-ILBRT, as a component of multidisciplinary treatment for unresectable esophageal cancer, was significantly effective and HDR-ILBRT contributed to improve outcomes in patients with advanced esophageal cancer. HDR-ILBRT should be established as a component of treatments for unresectable esophageal cancer.

The expression of cyclin A protein was retrospectively investigated in 124 patients with human esophageal squamous cell carcinoma with immunohistochemical techniques using antibody to cyclin A protein (BF683). Of 124 tumors, 49 (39.5%) exhibited positive staining in cancer cells with this antibody. Staining was observed predominantly but not exclusively in the nucleus. A significantly higher degree of association of immunoreactivity with this antibody was detected for advanced types of tumors (stages I, II, III, and IV) than for early tumors (stage 0; P < 0.05). Patients with cyclin A immunopositivity had a significantly poorer survival rate than did other patients (P < 0.01). These findings provide the first evidence for frequent and unscheduled overexpression of cyclin A protein in human esophageal cancer and suggest the possibility that alteration of cyclin A overexpression is associated with tumor progression and patient prognosis for esophageal cancers.

A recent study indicates that infection of human papillomavirus (HPV) may cause esophageal cancer and that it takes several decades from infection of HPV to developing cancer. Even in early stage esophageal cancer, lymph node metastasis is usually observed in the mediastinum, abdomen and neck regions. As patients with esophageal cancer are potentially in a malnutritional state because of disturbance of intake before treatment, active nutritional support should be performed before and after surgery. An objective nutritional assessment is necessary for nutritional support. The nutritional assessment index (NAI) can be applied to elderly patients because there is no difference in nutritional parameters between elderly and young patients. From the point of view that surgical treatment has limitations concerning complete curability, we employed a remote after loading system (RALS) into a multidisciplinary treatment called the Kochi system, which consists of a combination of surgery, radiation therapy and chemotherapy. This multidisciplinary method of treatment greatly contributes to survival and quality of life of esophageal cancer patients especially in elderly cases.

The presence and distribution of human papillomavirus (HPV) DNA or of increased expression of the p53 protein were determined in 71 patients with esophageal squamous-cell carcinoma (SCC) by in situ hybridization with biotinylated DNA probes for HPV-16, -18, -31 and -33, and immunohistochemical techniques using antibody to p53 protein. Of 71 patients from Kochi prefecture, 24 (Group I) were positive for HPV DNA, including 10 for HPV type-16 and 14 for HPV type-18; in contrast, none were positive for HPV-31 or -33. Of the remaining 47 patients, 24 (Group II) showed positive nuclear staining in cancer cells with p53 antibody. The group of 23 patients with neither HPV nor p53 expression (Group III) had a significantly better survival rate than Group I or II. These results suggest that HPV-16 and -18 may play a role in the pathogenesis of esophageal SCC, particularly with regard to its striking geographical distribution; that esophageal cancers do occur in the absence of HPV infection when over-expression of p53 is present; and that the presence of HPV infection and over-expression of p53 may each be a factor indicating a relatively poor prognosis.

The distribution of HLA-DR-positive dendritic cells (DR+DCs) and tumor infiltrating lymphocytes (TILs) was investigated in 67 patients with squamous cell carcinoma of the esophagus. DR+DCs were chiefly present among the cancer cell nests, and few were seen in the stroma. As clusters, UCHL-1-positive T-cells (UCHL1-Ts) were detected in the tissue around the cancer cell nests and as individual cells within tumor nests. Some UCHL1-Ts in the tumor nests showed direct contact with DR+DCs. In addition, a few OPD4-positive helper/inducer T cells were found among cancer cells, and were densely present in the stroma. A correlation was demonstrated between the number of DR+DCs and UCHL1-Ts in each patient (R = 0.4547, p < 0.01). In addition, dense tumor infiltration by both DR+DCs and UCHL1-Ts within the cancer cell nests was related to a better prognosis (p < 0.01).

The distribution of immunohistochemically labeled HLA-DR antigen- and S-100 protein-positive dendritic cell (DR+DC and S100+DC) was investigated in 59 human esophageal squamous cell carcinomas (SCCs). A dense infiltration of both DR+DC and S100+DC was detected in 11, only DR+DC in two, and only S100+DC in one. In the remaining 45 tumors infiltrating DC were sparse. By means of double immunostaining or the mirror section method, three different types of DC, namely S-100-negative and HLA-DR-positive DC(S100-DR+DC), S-100-positive and HLA-DR-negative DC(S100+DR-DC) and double-positive DC(S100+DR+DC) were clearly identified. With regard to postoperative survival, these patients with tumors in which there was a dense infiltration of DR+DCs and/or S100+DCs showed a significantly better survival rate than those in which DC were sparse (DR+DCs - P less than 0.001; S100+DCs - P less than 0.01). These results indicate that DC infiltration may be a prognostic factor in esophageal SCCs.

An artificial esophagus consisting of a collagen-silicone copolymer prepared by special chemical and physical procedures proved to be advantageous by both physical and histological examination. The adherence to the host tissue was satisfactory when collagen was used. The esophageal prosthesis was anastomosed to the jejunum in 5 dogs and no leakage occurred during 10-30 days. In subsequent experiments, use of the artificial prosthesis in the thoracic esophagus was followed by leakage in 5 of 14 cases (35 per cent). Five of these dogs survived for over 30 days, and the longest survival was 212 days. The incidence of leakage was acceptable but stricture was a common long-term complication. Further studies are underway to refine the prosthesis, the method of anastomosis, and post-operative management.

We developed a new surgical technique to manually resect the thoracic esophagus without thoracotomy and employed it in 31 patients with cancer of the esophagus who were considered poor risks for resection by traditional thoracotomy. Postoperative complications consisted of left hemothorax in seven patients, hoarseness in five, sternal osteomyelitis in three, a perforated trachea in one, and cardiorenal failure in three patients. No severe pulmonary complications occurred, and there were no hospital deaths. The long-term results of this surgical treatment have not been assessed.