| Objective 1 To test the expression of thymidylate synthase(TS),thymidine phosphorylase(TP)and dihydropyrimidine dehydrogenase(DPD)in gastric cancer tissues and normal gastric mucosal tissues and the activity of DPD in gastric cancer patients'blood,and analysis the relationship between their expression and the clinical and pathological features,chemotherapy effect,toxicity and prognosis,to assess their feasibility as sensitivity and toxicity indicator of 5-FU chemotherapy ,and provide a reference of the development of individualized and reasonable chemotherapy in clinical therapy,and to explore whether they are the molecular target of determining the prognosis of gastric cancer.2 To study the relationship of the expression of TS,TP and DPD in gastric cancer tissues and tumor angiogenesis.3 Compare the results of the expression of TS,TP and DPD in gastric cancer tissues measured by immunohistochemistry and western blot.Methods 1 Tissues of 107 cases of gastric cancer patients ,including 77 cases of regular radical gastrectomy and 30 cases of advanced gastric cancer were detected their expression of TS, TP and DPD by Immunohistochemistry (IHC), taking 25 cases of adjacent normal tissue(from cancer tissue≥5cm)as control. CD34 marks the capillaries. 2 Choosed the tissues of 40 cases of gastric cancer and 20 cases of adjacent normal tissue(from cancer tissue≥5cm) and detected the expression of TS, TP and DPD by western blot.The patients have had gastric cancer surgery and haven't had radiotherapy , chemotherapy and any anti-tumor therapy before.After surgery they all received 5-FU chemotherapy. 3 Tissues of 66 gastric cancer patients who were receiving the FOLFOX4 chemotherapy after radical gastrectomy for the first time.Before the chemotherapy we detected the dihydrouracil(UH2)and uracil(U) and calculated the ratio by High Performance Liquid Chromatography(HPLC),the ratio stands for the activity of DPD indirectly.Results 1 Immunohistochemistry experiment part:(1) The positive expression rate of TS,TP,DPD in 77cases of gastric cancer tissues were 70.13%,59.74%,85.71%,and 24.0%,12.0%,48.0% in 25 cases of adjacent normal tissues.The expressions of TS,TP,DPD in gastric cancer were significantly higher than that in adjacent normal tissues,the differences are all significant( x 2are 16.580,17.232and14.919,p <0.01).(2) The expression of TS in gastric cancer has positive relationship with lymph node metastasis( r =0.743, p <0.05),but the expression of TS in gastric cancer was not significantly differert between other clinical and pathological feature(s p >0.05);The expression of TP in gastric cancer was significantly different in differentiation,depth of invasion,lymph node metastasis and staging of gastric cancer( x 2are 6.196,16.235,9.701 and 37.774,p <0.05),correlation analysis showed that the expression of TP has a positive relationship with depth of invasion,lymph node metastasis and staging of gastric cancer( r are 0.426,0.355 and 0.619, p <0.01) ; There was no significant difference between DPD expression in gastric cancer tissues and the clinical and pathological features( p >0.05).(3) Logistic regression analysis showed that,the expression of TP has relationship with the staging( p =0.001),the regression equation classification test ability is 76.6% , x 2 is 43.130, p =0.000.(4) 30 cases were evaluable for the efficacy of chemotherapy,TS negative expression and positive expression of chemotherapy were 77.78% and 28.57%,the difference was statistically significant ( x 2=4.370 , p <0.05 ) ;TP negative expression of chemotherapy effective rate was 25.00%,positive expression rate was 66.67% ,there was significant differerce between them( x 2=5.000,p <0.05);DPD positive expression rate of chemotherapy was 21.43%,while DPD negative expression was 62.5%,there was significant differerce( x 2=5.129, p <0.05).(5) The expression of TS and TP in gastric cancer has no significant difference with the toxicity of 5-FU-based chemotherapy ( p >0.05 ) ,there is statistically significant differences between the expression of DPD in gastric cancer and the side effects caused by 5-FU-based chemotherapy( p <0.01) ,Spearman rank correlation analysis showed that the expression of DPD in gastric cancer has negative correlation with 5-FU-based chemotherapy toxicity( p <0.01). (6) In the 75 follow-up cases of gastric cancer patients with radical surgery,the occurrence and(or)transfer are 42cases,accounting for 56%,all of the occurrence and transfer patients are died during the follow-up,the average survival time is 39 months.The positive rates of TS,TP and DPD were 78.57%(33/42),78.57%(33/42)and 85.71%(36/42),the positive rate ofTS and TP between the two groups was statistically significant( p are 0.007,0.001),there was no statistically significant with DPD expression rates( p >0.05). Kaplan Meier analysis showed,the average survival time of 75 patients was 48 months,95%CI for 42.685～53.082,the median OS was 52 moths,95%CI for 47.046～56.954.1-year survival rate is 94.6%,3-year survival rate is 67.4%,5-year survival rate is 26.2%. Single factor survival analysis showed that the average survival times of TS negative and positive expression were 62 months and 42 months,the median survival time were 59 months and 39 months,the difference was statistically significant( x 2=7.800, p =0.005), the average survival times of TP negative and positive expression were 65 months and 37 months,the median survival time were 52 months and 35 months,the differerce was significant( x 2=27.080, p =0.000),DPD negative and positive expression's average survival times were 58 months and 45 months ,the median survival time were 59 months and 47 months,the differerce was significant( x 2=4.305,p =0.038).Adopting COX regression model to risk ratio analysis the overall survival,we found stage of gastric cancer,lymph node metastasis,TS expression were the independent prognostic factor.The later stage,with lymph node metastasis ,the higher TS expression has the pooer prognosis.(7) Using Spearman rank correlation anylasis showed that TS and TP,TS and DPD,TP and DPD had no correlation( r are 0.073,0.023,0.043, p >0.05).(8) The expression of TS and DPD in gastric cancer had no correlation with MVD( r are 0.064,0.010,p >0.05),MVD of TP positive expression is higher than that of TP negative expression, t is 10.031 , p <0.01,the difference was statistically significance,Spearman rank correlation analysis showed that the expression of TP has positive correlation with MVD( p <0.01).2 Western blot experiment part:(1) The expression of TS,TP and DPD in gastric cancer tissues were significantly higher than that in normal gastric mucosa,the differerce was statistically significant( t are -4.360,-4.224,-2.997, p are 0.000,0.000,0.004).(2) The expression of TS in gastric cancer patients has positive relationship with lymph node metastasis( p <0.01).TP expression in gastric cancer has a positive correlation with tumor type, invasion depth,lymph node metasta(s p <0.05).There was no significant differert between the expression of DPD and clincopathologic features( p >0.05).(3) There was no significant difference between the expression of TS and TP and the toxicity with 5-FU-based chemotherapy( p >0.05),DPD expression in gastric cancer had correlationship with 5-FU based chemotherapy toxicity( p <0.01),there were negative correlated between the expression of DPD and the toxicity such as nausea and vomiting,bone marrow ,diarrhea,oral mucosa( p <0.01).(4) Spearman rank correlation analysis showed that TS and TP,TS and DPD,TP and DPD were positive related, r are 0.683,0.527,0.577, p are all 0.000.(5) Dctected the expression of TS,TP and DPD in gastric cancer by IHC and western blot ,the relative coefficient of the positive and negative expression has statistically significant( t are 5.920,6.879,2.708,p are 0.000,0.000,0.014).Divided the relative coefficient of TS,TP and DPD by the mean relative coefficient into low-expression group and high-expression group,Kappa analysis showed that the Kappa values of the three enzymes are 0.802,0.794,0.794,are bigger than 0.75,the results are consistent.3 High Performance Liquid Chromatography experiment part:(1) UH2/U of 66 gastric cancer patients showed a normal distribution,the average was 2.3109,the midian was 2.2750 and in differert patients there was large variation,the standard deviation was 0.86733,the range was from 0.47 to 3.96.(2) The differences between UH2/U of gastric cancer patients as well as DPD activity and the toxicity of 5-FU-based chemotherapy( p <0.05), UH2/U of peripheral blood has negative correlated with nausea and vomiting,bone marrow suppression,diarrhea and oral mucositis( p <0.01).Conclusion 1 TS,TP,DPD expressed in gastric cancer tissue and adjacent normal tissue,while the expression in gastric cancer was higher than that in normal tissue,TS localized in the cytoplasm and nucleus of gastric cancer cells,TP mainly expressed in cytoplasm,and DPD was also localized in the cytoplasm,which suggested that the expression of three enzymes related to the occurrence of gastric cancer. 2 TS can be used as predictor of lymph node metastasis;the expression of TS in gastric cancer tissue has negatively correlation with 5-FU-based chemotherapy and survival.3 The expression of TP is closely related with MVD, it can be one of the reliable prognostic indicators.4 The expression of DPD can be used as evaluation of 5-FU-based chemotherapy efficacy , reduce toxicity.5 We detected the expression of TS,TP and DPD by IHC and western blot both on qualitation and quantitation,the results are consistent.6 UH2/U of patients can predict the toxicities of 5-FU-based chemotherapy and clinical dose adjustment. |
PDF Full Text Request