| ObjectiveIn order to explore new molecular markers as prognostic factors for gastric carcinoma and to provide more information for clinical practice, guiding treatment and reducing recrudescence and metastasis, the following biological markers, such as CD133, cyclinB1, DNA-PKcs, EGFR, FHIT, Id2, Ki-67, LRP16and NF-κB, were studied by immunohistochemistry. Furthermore, their relationships to tumor biological characteristics and overall survival time of gastric cancer were analyzed.Methods(1) Follow-up study was taken among 336 patients with gastric carcinoma from archival files of the PLA General Hospital during 1998-2001. And all 336 cases of gastric carcinoma tissues were made into 11 tissue microarrays by hand. Expressions of nine biologic markers, CD133, cyclinB1, DNA-PKcs, EGFR, FHIT, Id2, Ki-67, LRP16 and NF-κB/P65, were detected in the tissue microarrays by PV-6000 immunohistochemical method. Their relationships to clinical pathology and prognosis were also analyzed, afterward.(2) To detect gene amplification of HER-2 of 72 gastric cancinoma based on fluorescence in situ hybridization (FISH) with HER-2 probes and a centromeric probe of chromosome 17. And then analyze the correlation between HER-2 gene amplification and HER-2 protein expression.(3) According to the technique of cell culture, the percentage of SP cells were detected in gastric carcinoma cell line SGC-7901 and MGC-803 respectively by using Hoechst33342 staining, and the expressions of ABCG2, CD133, CK20, cyclinB1 and DNA-PKcs in SP cells were compared with those in non-SP cells with fluorescence immunocytochemical method. Results(1) The expressions of CD133, cyclinB1, EGFR, Id2, Ki-67, LRP16 and NF-κB/P65 were increased gradually with the depth of invasion (P< 0.05), but the expressions of FHIT and DNA-PKcs were on the contrary (P<0.05). There were higher positive expression of CD133, cyclinB1, EGFR, Id2, Ki-67 and NF-κB in cancers with lymph node involvement (P<0.05), whereas DNA-PKcs was on the contrary(P=0.031). The positive expressions of CD133, cyclinB1, Id2, Ki-67 and NF-κB in tumors with abdominal and distant metastasis were markedly higher than in those without (P<0.05), but that of DNA-PKcs was on the contrary (P=0.008). The expression of CD133, cyclinB1, EGFR, Id2, Ki-67 and NF-κB was increased gradually, corresponding to TNM stage from I to IV (P<0.05), while that of FHIT and DNA-PKcs presented a inverse correlation with the stage(P<0.05). The expressions of CD133, cyclinB1, EGFR, Id2, Ki-67 and NF-κB/P65 showed an positive correlation with the histological grade (P<0.05), but the expression of FHIT was inversely related to the histological grade (P=0.006). The expression of DNA-PKcs and LRP16 was the highest in the moderately differentiated tissues (P<0.05).(2) The expression of CD133, cyclinB1, EGFR, Id2, Ki-67, LRP16 and NF-κB in carcinoma was significantly higher than that in adjacent normal mucosa (P<0.05), the expression of FHIT was statistically lower than that in normal mucosa (P=0.005), and the expression of DNA-PKcs was not proved distinguished between cancer and normal tissues (P=0.723).(3) By univariant survival analysis, the expressions of CD133, cyclinB1, DNA-PKcs, EGFR, THIT, Id2, Ki-67 and NF-κB, the age of patients, size of tumor, degree of differentiation, histological type, infiltrative deepth, lymphatic invasion, abdominal and distant metastases, TNM stage, and chemoradiotherapy were significantly correlated to prognosis(P<0.05).(4) By Cox multivariant survival analysis, the negative expressions of CD133, EGFR, Id2 and Ki-67, the positive expression of DNA-PKcs, Younger ages. smaller tumors, tubular adenocarcinoma, nonmetastases and chemoradiotherapy were correlated with a longer survival time and better prognosis (P<0.05).(5) HER-2 amplification was detected in 2.78% (2/72) gastric carcinoma. And the ratio of HER-2 amplification is 36.84% (7/19) in gastric cancer with HER-2 protein expression. In this study HER-2 gene amplification was not closely corresponding to HER-2 protein expression (P >0.05).(6) The percentage of SP cells in gastric carcinoma cell line SGC-7901 was 3.8%. The positive expressions of ABCG2 and CK20 in SP cells were significantly lower than those in non-SP cells (P<0.05). There was no marked differences in the expressions of CD133, cyclinB1 and DNA-PKcs between SP cells and non-SP cells (P >0.05).(7) The percentage of SP cells in gastric carcinoma cell line MGC-803 was 2.9%. The expression of cyclinB1 in SP cells was significantly higher than that in non-SP cells (P<0.05), whereas the expressions of ABCG2, CD133, CK20 and DNA-PKcs were on the contrary (P<0.05).Conclusions:(1) The expressions of CD133, cyclinB1, DNA-PKcs, EGFR, THIT, Id2, Ki-67 and NF-κB are significantly correlated to prognosis.(2) CD133, DNA-PKcs, EGFR, Id2, Ki-67, age of patients, tumor size, histological type, abdominal and distant metastases, lymphatic invasion and chemoradiotherapy are independent prognostic factors for survival of gastric cancer.(3) The expressions of CD133, cyclinB1, EGFR, Id2, Ki-67, LRP16 and NF-κB/P65 are higher than those in adjacent normal mucosa, but that of FHIT is on the contrary. It is suggested that the expressions of biological markers above-mentioned may play an important role in the development and progression of the gastric carcinoma.(4) The ratios of HER-2 protein expression and HER-2 amplification are 9.72% and 2.78% in gastric carcinoma, respectively. And the ratio of HER-2 amplification is 36.84% in gastric cancer with HER-2 protein expression. There is no significant correlation between HER-2 gene amplification and HER-2 protein expression in gastric cancer.(5) The percentages of SP cells in gastric carcinoma cell line SGC-7901 and MGC-803 are 3.8% and 2.9%, respectively. Combined with cyclinB1 or CD133 as a positive marker, CK20 may be a specific negative molecular marker to recognize cancer stem cells in gastric carcinoma.
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