Expression Of BCRP, MRP And P-gp And Their Relationship With Esophageal Carcinoma Prognosis In The Patients From The High-incidence Area For Esophageal Cancer In Linzhou, Henan

Background and objectivesEsophageal carcinoma (EC) is one of the six most common malignant cancers worldwide. Linzhou city (formerly Linxian county) and the nearby Huixian county in Henan province, have been well recognized as the one of highest incidence and mortality areas for EC in the world. Scientists have done systemic research in the high-incidence area for EC for many years and confirmed the occurrence of EC was a multistep progressive process. Now surgery and radiation therapy are the effective and primary methods. The five-year survival rate for early EC patients is more than 90% after surgical treatment. However, the five-year survival rate for middle and advanced stages patients is less than 10%. Chemotherapy was used in adjuvant therapy after surgical treatment, and the effect of single drug is very poor. Effect of combining chemotherapy has been improved than single drug. Whereas, status in quo of chemotherapy has not be satisfied. Resistance to anti-cancer drugs is responsible for chemotherapy failure of tumors. Multidrug resistance is a major type of resistance. Many studies have confirmed that ATP-binding cassette transporter proteins (mayincluding BCRP, MRP and P-gp) are involved in multidrug resistance by transport anticancer drugs through cell membrane in many tumors, and overexprcssion of them maybe lead to multidrug resistance. EC is a malignant cancer, which obviously correlated with multidrug resistance. Studies had showed that P-gp and MRP may play very important roles in multidrug resistance of EC. But, the relationship between BCRP and the prognosis as well as multidrug resistance of EC have not been reported by domestic and foreign scholars. This study used histopathological and immunohisto-chemical methods to determine expression of BCRP, MRP and P-gp in EC specimens. Combining with clinical information and follow-up results, the significance of prognosis and mechanism of multidrug resistance in EC were detected, and it can be provided the theoretical basis and new therapy method for clinical chemotherapy. So far, differentiation degree and TNM classificantion are still used to evaluate the prognosis of EC patients by clinician. However, it has been recognized that there were discorded between differentiation degree and /or TNM classificantion and the actual prognosis, indicating the limitation of these markers in evaluating EC prognosis. Therefore, combining several factors and biomarkers together may provide more accurate information in predicting the prognosis of EC patients.Materials and Methods76 patients with 40 males and 36 females, who had undergone esophagectomy at Yaocun Esophageal Carcinoma Hospital of Linzhou city between 1993 and 1996, were enrolled in this study. The range of ages were 40-72 years. The average ages were 55.1+8.3 years in males and 55.2±7.6 years in females, respectively. All patients had not received radiation therapy and/or chemotherapy before surgical treatment. Primary esophageal squamous cell carcinoma (ESCC) had been confirmed by histopathological diagnosis in all tissue samples. The patients were local residents of Linzhou city, and were followed up at least 5 years after surgery until October 2001. 47 patients who survived less than 5 years were all died of recurrence or metastasis, without otherserious diseases, accident and obvious causation, and 17 cases of normal oesophagus mucosa were served as normal control from the high-incidence area. All tumor samples were fixed with 10% formalin and embedded with paraffin. Each block was sectioned serially at 5μm, one was stained with hematoxylin and eosin for histopathological analysis by two pathologists and the others were used for immunostaining. Immunohistochemistry method (ABC) was applied to determine expression of BCRP, MRP and P-gp in each sample, which had been resected by surgery. The datum were analysed by statistical software SPSS 10.0. Chi-squared test and Spearman's rank correlation were performed to compare difference between groups and relativity analysis respectively. Survival analysis was calculated by the Kaplan-Meier method and Survival rate was compared by the Log-rank test. And prognosis factors of EC patients were confirmed by Cox proportional hazard regression model. The significant difference was considered when the p value was less than 0.05.Results(1) All of the 76 esophageal carcinoma patients were followed up, and 29 cases have survived more than 5 years (38.2%), 47 cases had died within 5 years after surgical treatment (61.8%).(2) The positive and negative rates of BCRP were 36.8% (28/76) and 63.2% (48/76) respectively in EC and the positive rate in normal oesophagus mucosa is only 11.8% (2/17), and the difference was statistic significant (p=0.042). The mean survival periods (month) and 95% confidence interval of those with positive expression of BCRP and with negative expression were 26 (18, 34) and 64 (62, 67), respectively. Patients with positive expression survived less than those with negative expression obviously and the difference was statistic significant (P=0.000). The positive expression of BCRP showed obvious correlation with local lymph node and distant metastasis, TNM classificantion and infiltration degree by relativity analysis.(3) The positive and negative rates of MRP were 59.2% (45/76) and 40.8% (31/76) respectively in EC and the positive rate in normal esophagus mucosa is only 23,5% (4/17), and the difference was statistic significant (P=0.008). The mean survival periods (month) and 95% confidence interval of those with positive expression of MRP and with negative expression were 41 (33, 49) and 66 (64, 68) respectively. The difference was no statistic significant (p=0.490). The positive expression of MRP showed irrespective with sex, local lymph node and distant metastasis, differentiation degree, TNM classification and infiltration degree by relativity analysis.(4) The positive and negative rates of P-gp were 56.6% (43/76) and 43.4% (33/76) respectively in EC and the positive rate in normal oesophagus mucosa is 23.5% (4/17), and the difference was statistic significant (P=0.013). The mean survival periods (month) and 95% confidence interval of those with positive expression of P-gp and with negative expression were 35 (28, 43) and 66 (64, 69) respectively. Patients with positive expression survived less than those with negative expression obviously and the difference was statistic significant (p=0.010). The positive expression of P-gp showed irrespective with sex, local lymph node and distant metastasis, differentiation degree, TNM classification and infiltration degree by relativity analysis.(5) Expression of BCRP in EC cells was irrespective with MRP and P-gp. But, the correlation of MRP and P-gp were significant.(6) Sex, age, local lymph node metastasis, distant metastasis, TNM classifieantion, differentiated grade, infiltration degree, the positive expression of BCRP, MRP and P-gp, which may affect EC prognosis were served as independent variable. Survival time was served as dependent variable, and Cox's regression model was created. The results revealed that local lymph node metastasis and the positive expression of BCRP were the risk factors, which affected survival time of EC patients. Regression coefficients were 1.565 and 0.725 respectively.Conclusions(1) The positive expression of BCRP showed obvious correlation with local lymph node and distant metastasis, TNM classificantion and infiltration degree by relativity analysis and irrespective with the expression of MRP, P-gp. These suggested that the positive expression of BCRP may be a new multidrug resistance mechanism and an independent prognostic indicator for poor prognosis of EC from the high-incidence area. The positive expression of BCRP was able to predict the prognosis of EC as an independent prognosis marker.(2) EC patients with positive expression of MRP were seemed to live shorter survival time than those with negative expression, but the difference was not significant, and its value to evaluate EC prognosis may need further laboratorial and clinical studies.(3) EC patients with positive expression of P-gp were seemed to live shorter survival time than those with negative expression. It suggested that the positive expression of P-gp may be a marker for poor prognosis of EC from the high-incidence area.(4) Expression of MRP and P-gp showed obvious correlation in primary esophageal squamous cell carcinoma. It suggested that the cooperative expression of them has the same modulatory mechanism and plays a cooperation role in multidrug resistance.(5) Combining several molecular markers together may provide more accurate information in predicting the prognosis and might be important in clinical application to evaluate EC prognosis and might be helpful in individualizing chemotherapy.