Study Of Expression And Correlation Of RASSF1A, Cyclin D1, ER And PR In Endometrial Carcinoma

Objective:This study was designed to evaluate the expression of RASSF1A and cyclin D1 in normal endometria,endometrial atypical hyperplasias and endometrial carcinoma and to investigate the relationships between their expressions and the clinicopathological parameters and prognosis respectively,and we also analysed the correlation of RASSF1A expressions with cyclin D1,ER,PR in endometrial carcinoma,which may reveal the roles of RASSF1A and cyclin D1 in the pathogenesis and progression of endometrial caricinoma.Thus we will find promising targets for diagnosis and therapy and potentially useful factors for detection the prognosis in endometrial carcinoma.Methods:Hysterectomy specimens(126)containing endometrial carcinoma were obtained from the pathological archives that were operated at Qilu Hospital of Shandong University from 2000 to 2004.Patient age ranged from 25 to 74 years(mean,53.5 years).The clinical follow-up period ranged from 12 to 95 months(mean,62.3 months).According to the International Federation of Gynecology and Obstetrics (FIGO)classification(2000),69 of the 126 patients were stageⅠ,10 were stageⅡ,45 were stageⅢ,and 2 were stageⅣ.Histologically,99 were endometrioid adenocarcinoma,14 were serous papillary adenocarcinoma,11 were clear cell carcinoma,and 2 were squamous cell carcinoma.With regard to histological grade, 58 were grade 1,41 were grade 2,and 27 were grade 3.Of the 126 cases,83 showed no myometrial invasion or invasion with depth of 1/2 or less in the myometrium,and the remaining 43 showed invasion of more than 1/2 of the depth.Positive peritoneal cytology was present in 31 cases.Lymph node metastasis was present in 23 cases. Paraffin-embedded tissue blocks were sectioned and immunostained with the antibodies against the RASSF1A,cyclin D1,ER and PR.We also examined the expression of RASSF1A and cyclin D1 in 20 normal endometria(10 proliferative endometria and secretory endometria),38 endometrial atypical hyperplasias by immunohistochemistry.Positive versus negative immunostaining of RASSF1A in all samples was compared using the x~2 test and the exact Fisher's test where applicable.Cyclin D1 expression levels in all samples were compared using the Kruskal-Wallis test.The expression of RASSF1A and cyclin D1 in ER-positive endometrial carcinomas versus ER-negative carcinomas were compared using the x~2 test and the exact Fisher's test where applicable.The expression of RASSF1A and cyclin D1 in PR-positive carcinomas versus PR-negative carcinomas were compared using the x~2 test and the exact Fisher's test where applicable.Correlations of RASSF1A,cyclin D1,ER and PR expression levels were assessed using the Spearman rank correlation test. Kaplan-Meier survival curves were constructed and compared by calculation of log-rank statistic,and multivariate analysis was performed by Cox regression modeling to estimate the influences of several clinicopathological and immunohistochemical covariates on patient survival.Significance of differences was assumed at P＜0.05 at the two-sided test.Results:1.RASSF1A immunoreactivity was observed in the cytoplasm of cells.Positive RASSF1A immunostaining were observed in 52(41.3%)of 126 endometrial carcinoma samples,19(95.0%)of 20 normal endometria and 27(71.1%)of 38 endometrial atypical hyperplasias.The expression of RASSF1A in endo(?)etrial carcinomas was significantly less than that in normal endometria and endometrial atypical hyperplasias(P＜0.001).RASSF1A expression in endometrial carcinoma samples demonstrated a significant association with surgical stage(P=0.017), positive peritoneal cytology(P=0.044),lymph node metastasis(P=0.035)and histological type(P=0.007).There was no significant association between RASSF1A expression and histological grades,depth of myometrial invasion and age(P＞0.05). Univariate survival analysis revealed that patients with RASSF1A expression had a significant better overall survival compared with the patients with no expression(P= 0.037).2.Immunohistochemistry showed that the nuclei of the cells were positive for cyclin D1.Both proliferative and secretory endometria,and endometrial atypical hyperplasia regardless of type were negligible for cyclin D1 expression.The expression of cyclin D1 in normal endometria and endometrial atypical hyperplasias was significantly less than that in endometrial carcinomas(P＜0.001).Cyclin D1 expression was significantly associated with clinicopathological parameters,including surgical stage(P=0.006),histological grade(P=0.010),lymph node metastasis(P= 0.008)and histological type(P=0.020),but not with age,depth of myometrial invasion and positive peritoneal cytology(P＞0.05).The patients with cyclin D1 expression had a significant poorer overall survival compared with the patients with no expression(P=0.017).3.Positive ER immunostaining were observed in 77(61.1%)of 126 endometrial carcinoma samples.Positive PR immunostaining were observed in 79(62.7%)of 126 endometrial carcinoma samples.The positive rate of RASSF1A expression were 59.2%(29/49)in ER-negative carcinomas,29.9%(23/77)in ER-positive carcinomas. The expression of RASSF1A in ER-positive carcinomas was significantly less than that in ER-negative carcinomas(P=0.009).The positive rate of RASSF1A expression were 68.1%(32/47)in PR-negative carcinomas,25.3%(20/79)in PR-positive carcinomas.The expression of RASSF1A in PR-positive carcinomas was significantly less than that in PR-negative carcinomas(P＜0.001).The positive rate of cyclin D1 expression were 57.1%(28/49)in ER-negative carcinomas,63.6%(49/77)in ER-positive carcinomas.There was no significant difference of cyclin D1 expression between ER-positive carcinomas and ER-negative carcinomas(P＞0.05).The positive rate of cyclin D1 expression were 63.8%(30/47)in PR-negative carcinomas, 59.5%(47/79)in PR-positive carcinomas.There was no significant difference of cyclin D1 expression between PR-positive carcinomas and PR-negative carcinomas(P＞0.05).The expression of RASSF1A was inversely correlated with cyclin D1 expression(r_s=-0.290,P＜0.01).We also detected a significantly inverse correlation between RASSF1A and ER(r_s=-0.233,P＜0.01),RASSF1A and PR (r_s=-0.331,P＜0.001).The expression of ER was significantly correlated with PR expression(r_s=0.529,P＜0.001).Cyclin D1 expression was not correlated with ER and PR(P＞0.05).4.Survival analysis revealed that patients with RASSF1A+/ER+ expression had a significant better overall survival compared with the patients with RASSF1A-/ER+ expression(P=0.049),and patients with RASSF1A+/ER+ expression had a highly significant better overall survival compared with the patients with RASSF1A-/ER-expression (P=0.027).Additionally,patients with RASSF1A+/PR+ expression had a highly significant better overall survival compared with the patients with RASSF 1A-/PR- expression(P=0.021).Survival analysis also revealed patients with cyclin D1-/ER+ expression had a significant better overall survival compared with the patients with cyclin D1+/ER- expression(P=0.042),and patients with cyclin D1-/PR+ expression had a highly significant better overall survival compared with the patients with cyclin D1+/PR+ expression(P=0.026).5.Prognostic factors were also analysed by the multivariate Cox proportional-hazard model.For the Cox regression models,we included the following variables:age, surgical stage,histological grade,histological type,depth of myometrial invasion, lymph node status,positive peritoneal cytology,obesity,high blood pressure,diabetes, radiotherapy,chemotherapy,endocrine therapy,RASSF1A,cyclin D1,ER and PR status.Forward stepwise elimination according to Cox regression results led to a model containing five independent terms that were predictive of overall survival: FIGO surgical stage(P=0.021),histological grade(P=0.009),lymph node involvement (P=0.005),histological type(P=0.001)and PR status(P=0.015).Conclusions:1.The expression of RASSF1A in endometrial carcinomas was significantly less than that in normal endometria and endometrial atypical hyperplasias.RASSF1A expression in endometrial carcinoma samples demonstrated a significant association with surgical stage,positive peritoneal cytology,lymph node metastasis and histological type.Patients with RASSF1A expression had a significant better overall survival compared with the patients with no expression.Our results suggest that absence expression of RASSF1A is a frequent event in endometrial carcinogenesis, which may play an important role in the malignant progression and prognosis of endometrial carcinoma.2.The expression of cyclin D1 in normal endometria and endometrial atypical hyperplasias was significantly less than that in endometrial carcinomas.Cyclin D1 expression was significantly associated with clinicopathological parameters, including surgical stage,histological grade,lymph node metastasis and histological type.cyclin D1 expression had a significant poorer overall survival compared with the patients with no expression.These results imply that cyclin D1 expression may be involved in the development and/or proliferation and differentiation of human endometrial carcinoma.3.We detected a significantly inverse correlation between RASSF1A and ER, RASSF1A and PR.Patients with RASSF1A+/ER+ and RASSF1A+/PR+ expression had a significant better overall survival.This study suggests that RASSF1A gene maybe play an important role in the pathogenesis and progression of endometrial caricinoma through regulating ER and PR expression.4.Cyclin D1 expression was not correlated with ER and PR.Patients with cyclin D1-/ER+ and cyclin D1-/PR+ expression had a significant better overall survival.This study suggests that expression of cyclin D1-/ER+ and cyclin D1-/PR+ might be useful indicators of favorable prognosis in endometrial cancer patients.5.In the present study,we demonstrated RASSF1A expression was inversely correlated with cyclin D1,ER and PR expression.We also detected a significant correlation between ER and PR.The combined analysis of these biopathological markers could provide useful information for the selection of patients with endometrial carcinoma to be enrolled in innovative therapeutic strategies.6.We detected that independent prognostic factors for overall survival were FIGO surgical stage,histological grade,lymph node involvement,histological type and PR status.