Research On Application Of RASSF1A In Diagnosis Of Endometrial Adenocarcinoma

Objective:Endometrial cancer (Endometrial, carcinoma, EC) is the most common gynecologic malignant tumor. The incidence and mortality of endometrial cancer are rising in the worldwide, and the age of onset is younger. Due to lack of effective method in screening of early endometrial cancer, endometrial cancer is still a serious threat to the health of the women of the world. So it is necessary to explore the causes and the pathogenesis of endometrial carcinoma through various research methods and technical means. To help us find and diagnose endometrial cancer in the early stages of the disease and can take timely intervention to achieve the purpose of treatment. Ras-association domain family1A (RASSFIA) gene is a tumor suppressor gene, the reduced or absent expression of gene caused by abnormal methylation of the promoter, suggesting that the RASSFIA gene had a close relationship with tumor. In addition, mutation and loss of expression of epidermal growth factor receptor (EGFR) are closely related to the incidence of malignant tumors, and EGFR is an important molecular targets of tumor personalized treatment.Therefore, research on RASSFIA gene promoter region methylation and expression of RASSFIA protein and EGFR mutation in the occurrence and development of endometrial cancer; it is important to study the personalized diagnosis and treatment of endometrial adenocarcinoma.Methods:During the period of2002January to2009December, we collected63endometrial cancer cases diagnosed and treated in Zhongnan Hospital of Wuhan University.Patients aged35-71years old. All patients were treated by operation of first treatment; they were not carried out preoperative radiotherapy and/or chemotherapy; primary endometrial carcinoma was diagnosed in the postoperative pathology, and metastatic carcinoma was excluded. Endometrial adenocarcinoma pathology staging operation according to the standard made by FIGO in2009by stages,28cases were stage Ⅰ, stage Ⅱ23cases, III in8cases,4cases in stage Ⅳ; According to the1998FIGO formulation of endometrioid carcinoma histological grading and classification method, G1grade21cases, G2grade29cases, G3grade13cases.21cases of uterine muscle in patients with endometrial carcinoma infiltration≤1/2,42cases of muscle in patients with endometrial adenocarcinoma invasive>1/2;11cases with lymph metastasis,52cases without lymph node metastasis. At the same time,20cases of endometrial curettage patients with normal proliferative phase endometrium were used as negative control group. The patients had no abnormal uterine bleeding; hormone therapy is not used within6months. According to the1988International Association of gynecologic pathology (ISGP) the new classification of endometrial carcinoma tumor histology, collect other types of endometrial tissue paraffin embedded samples including24cases of simple hyperplasia,25cases of complex hyperplasia,36cases of complex hyperplasia with atypical hyperplasia (precancerous lesions).We extract DNA samples from the paraffin tissues, using bisulfite to modify DNA sequences, and using methylation specific PCR method to detect the methylation status of the promoter region of RASSFIA gene in different types of endometrial tissue. In addition, we also use quantum dots labeled streptavidin biotin system to detect RASSFIA gene expression of different endometrial tissue.In order to detect the methlation status of the promoter region of RASSF1A in the peripheral blood of the endometrial adenocarcinoma patients, We have also established a specific detection method of mutation fluorescence system to help us determine mutational status of endometrial cancer. EGFR19exon deletion was chosed to be the taget gene.Results:The results show the normal proliferative endometrium RASSF1A methylation positive detection rate was20%, the positive rate of simple hyperplasia of endometrium was12.5%, the positive rate of complex hyperplasia endometrium was24%, complex hyperplasia with atypical hyperplasia (precancerous) positive rate was77.8%, and the positive rate of methylation in endometrioid adenocarcinoma RASSF1A was76.2%. Among them, RASSF1A methylation positive rate in endometrioid adenocarcinoma was higher than that of normal proliferative endometrium, there were statistical significance in the difference; At the same time, RASSF1A methylation positive rate of complex hyperplasia with atypical hyperplasia (precancerous) was higher than the complex hyperplasia endometrium, the difference has statistical significance. While in normal proliferative endometrium, there was no significant difference of the positive rate of RASSF1A methylation in simple endometrial hyperplasia and complex hyperplasia endometrium, the P values were0.498and0.299; The positive rate of RASSF1A methylation in complex hyperplasia with atypical hyperplasia (precancerous) differences and endometrioid adenocarcinoma tissues are also has no statistical significance, P value is0.486.We compare the different pathological staging and histological grading results, shows that the positive rate of RASSF1A methylation in endometrioid adenocarcinoma I was64.3%, Ⅱ was82.6%, Ⅲ+Ⅳ was91.7%; Among them, RASSF1A methylation positive rate showed no significant difference in endometrioid adenocarcinoma Ⅰ phase and Ⅱ phase, no significant difference in endometrioid adenocarcinoma of stage Ⅱ and stage III+IV, however in endometrioid adenocarcinoma of stage Ⅰ and stage Ⅲ+IV, the difference has statistical significance. The positive rate of RASSF1A methylation at the level of G1in endometrial carcinoma was61.9%, G2class82.7%, G3class84.6%; comparisons between the three groups, the difference was not statistically significant. The positive rate of RASSF1A methylation in endometrioid adenocarcinoma of uterine myometrial invasion≤1/2patients was57.1%, at the same time,>1/2infiltration of uterine muscular layer in endometrioid adenocarcinoma patients was85.7%, the difference was statistically significant, P value is0.012; The positive rate of RASSF1A methylation in endometrioid adenocarcinoma with lymph node metastases was81.8%, without the occurrence of lymph node metastases in75%patients, there was no statistically significant difference between the two, the P value is0.813.Expression of RASSF1A gene was detected in different endometrial tissue, results show that the positive rate of RASSF1A expression in normal proliferative endometrium was80%, the positive rate of simple hyperplasia of endometrium was16.7%, the positive rate of complex hyperplasia endometrium was12%, The positive rate in complex hyperplasia with atypical hyperplasia (precancerous) was8.3%, in endometrioid adenocarcinoma was11.1%. The positive rate of RASSF1A in endometrial adenocarcinoma tissue was lower than that of normal proliferative endometrium, there were statistical significance in the difference; At the same time, the positive rate of RASSF1A in simple hyperplasia endometrium was lower than the normal proliferative endometrium, there were statistical significance in the difference; But the positive rate of RASSF1A in a simple endometrial hyperplasia and complex hyperplasia endometrium, in complex endometrial hyperplasia and complex hyperplasia with atypical hyperplasia (precancerous), in complex hyperplasia with dysplasia (precancerous) there was no significant difference and endometrial carcinoma, P=0.641,0.636and0.659.Comparing the results of different pathological staging and grading organizations showed that the positive rate of RASSF1A in endometrial adenocarcinoma Ⅰ+Ⅱ period was11.7%, in stage Ⅲ+Ⅳ was8.3%; the difference was statistically significant, P value is0.734; The positive rate of RASSF1A in G1grade23.8%, G2class6.9%, G3Class0(not found), of which, RASSF1A expression in G1level and G3level, the difference was statistically significant. The positive rate of RASSF1A expression in endometrioid adenocarcinoma of uterine myometrial invasion≤1/2patients was23.8%, at the same time,>1/2infiltration in uterine muscular layer of endometrioid adenocarcinoma patients was4.76%, the difference was statistically significant, P value is0.023; The positive rate of RASSFIA expression in endometrioid adenocarcinoma with lymph node metastases was9.1%, without the occurrence of lymph node metastases in11.5%patients, there was no statistically significant difference between the two, the P value is0.668.In addition, we design the EGFR exon19mutation specific primers and Taqman probe, under different simulation conditions, the detection ability of the system has been tested. Results show that, in the absence of wild signal and other substances interference conditions, the fluorescence detection system can detect1x lO1copies/μ l exon19deletion mutations; in a certain degree of the wild type plasmid as a background, this detection system also reflects the sensitivite ability of detection. Result shows that, the fluorescence detection system can detect the mutation rate were0.1%; we also simulate the plasma samples of different concentration of mutation, the fluorescence detection system can detect a deletion of5x101copies/μ L in human plasma simulated sample. The system can detect EGFR19exon deletion mutation, but it need to be further verified in the sample of patients with endometrial carcinoma.Conclusions:1. RASSFIA hypermethylation rate in normal proliferative endometrium, simple endometrial hyperplasia and complex hyperplasia endometrium in low proportion, In complex hyperplasia with atypical hyperplasia (precancerous) and endometrial adenocarcinoma tissue was significantly increased, RASSFIA hypermethylation may be involved in the occurrence and development of endometrial adenocarcinoma, and RASSFIA hypermethylation may be a heavy influence factor to the malignant progression of endometrioid adenocarcinoma; The positive rate of pathological RASSFIA methylation and endometrioid adenocarcinoma of the learning stage and myometrial invasion degree have significant correlation, when endometrial adenocarcinoma progression to stage III and stage IV, RASSFIA methylation positive rate is on the rise; Methylation positive rate was higher in>1/2myometrial invasion of endometrioid adenocarcinoma patients; results suggest, the positive rate of RASSFIA methylation has certain significance for monitoring and prognosis of endometrioid adenocarcinoma; RASSFIA hypermethylation may be the molecular markers for early diagnosis of endometrioid adenocarcinoma.2. The expression of RASSFIA protein in endometrial tissue can be detected by quantum dot probe.The positive rate was the highest in normal proliferative endometrium express RASSF1A, and simple hyperplasia of endometrium, endometrial complex hyperplasia, and complex hyperplasia with atypical hyperplasia (precancerous), endometrioid adenocarcinoma tissue RASSF1A expression positive rate decreased. Among them, complex hyperplasia with atypical hyperplasia (precancerous), endometrioid adenocarcinoma tissue RASSF1A expression positive rate is low, which is consistent with the results of two groups of corresponding RASSF1A methylation positive rate, was higher. Methylation of promoter region that may be one of the mechanisms for the reduction of RASSF1A expression, the reduced or absent expression product is closely related to gene methylation.3. The positive rate of RASSF1A expression in endometrial carcinoma and histological grade and myometrial invasion has correlation in a certain degree; The decreased or loss of expression of RASSF1A gene has played an important role in occurrence and development of endometrial cancer.4. Deletion of EGFR19exon mutation detection system can be used to detect EGFR19mutations in exons; in the wild type plasmid as interference, the detection sensitivity of EGFR19exon deletion mutation detection system can reach0.1%; in the simulated human plasma samples, the system can detect the5x101copies/μ L deletion mutation.