The Expression Of CD44v6 And Nm23-H1 In Endometrial Carcinoma

PrefaceUnder the influence of increasing proportion of aged population, infertility, less accouchement, and the wide application of exogenous estrogen etc, endom-etrial carcinoma has now become the most frequent gynecologic cancer in the world. The judgment of the malignant level and the metastasis potential which directly affect the biological behavior of carcinoma is essential for evaluating the prognosis of the patients. In this study, we adopted RT — PCR and immunohisto-chemical technique to detect the expression of nm23 - HI and CD44v6 in 60 cases of endometrial cancer tissue samples. And try to elucidate the correlation between the expression level of nm23H1,CD44v6 and the relative clinicopathologic features. Thereafter to judge the feasibility of taking the expression of nm23H1, CD44v6 as a factor to evaluate the prognosis of patients suffering from endometrial carcinoma.Materials and Methods1. Case selectionA total of 60 paraffin - embedded tissue blocks of endometrial carcinoma were retrieved from the surgical pathology department at Chinese Medical University during 1998 to 2004. The selected clinical cases were confirmed by pathological diagnosis . All of the patients were preoperatively untreated and with no combination of other neoplasm.2. MaterialsBoth anti - CD44v6 mouse monoclonal antibody and anti - nm23H1 mousemonoclonal antibody were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd. Trizol Reagent purchased from Shanghhai Sangon Biological Engineering Technology and Service Co. Ltd. The RT - PCR kit and primer was provided by TaKaRa Biotechnology (Dalian) Co. , Ltd.3. Methods(1) RT-PCREight — micrometer - thick sections were cut from tissue blocks, deparaffinized in xylene, and dehydration in absolute alcohol. The total tissue mRNA was extracted by Trizol total RNA extract kit. The purity of RNA was measured by absorption at 260/280 nm with a spectrophotometer. The two - step RT -PCR was performed to reverse transcript mRNA, and amplify the cDNA product. Then the cloned fragments was confirmed of the identity by agarose gel e-lectrophoresis, and following EB stain, the pictures of gel image were recorded under the ultraviolet lamp.(2) ImmunohistochemistryTissue samples embedded in paraffin were cut into 4m — thick sections. These sections were deparaffinized with xylene, and rehydrated with a series of graded alcohols. The slides were immersed in methanol containing 3% H2O2 to block endogenous peroxidase activity. After heat antigen retrieve processing, the sections were treated with normal serum to block nonspecific staining. Sections were then incubated with anti - CD44v6 and anti - nm23 primary antibodies respectively at 4 °C overnight in a moisture chamber. This was followed by incubation with a biotinylated secondary antibody at room temperature for 35 minutes and thereafter incubation with horseradish peroxidase conjugated avidin - biotin complex at room temperature for 1 hour. Immunostaining was visualized using 3 , 3V - diaminobenzidine tetrahydrochloride substrate. Slides were counter -stained with hematoxylin. Then the sections were dehydrated and cleared and mounted with resin. Tissue ample of breast cancer and pancreas cancer with known antigen of CD44v6 and nm23 - HI were used as positive control of CD44v6 and nm23 - HI immunoassaying respectively . PBS replacing the primary antibodies was used as negatives controls.4. Results JudgmentThe introcytoplasmic brown granule was considered as nm23 — HI immu-nopositive and clear membrane stain is considered as CD44v6 positive. The amplified cDNA was evaluated by electrophoresis, and the brand appeared at the site of target molecular weight was considered as positive.5. Statistical AnalysisAll the data was processed by chi - square test or Fisher exact probability test using SPSS 13.0 software package.Results1. the Expression of CD44v6Both of immunohistochemical and RT - PCR results show that the expression level of CD44v6 is greater in adeno - squamous than glandular carcinoma. The expression of CD44v6 is positively correlated with clinicopathologic stage and lymph node metastasis, with observed low expression in patients who have no uterine adnexa invasion. The extent of CD44v6 immunostaining was correlated with the depth of myometrium invasion, and the same incline is also noticed in RT - PCR, but have no significance statistically. The result of RT - PCR in well - differenciated endometrial cancer showed that the expression of CD44v6 is low, but immunohistochemical results didnt show the tendency. Both of the two evaluation methods demonstrated no correlation with the extent of cervical invasion.2. the Expression of nm23 — HIBoth of immunohistochemical and RT - PCR results show that the expression level of nm23 - HI is low in poorly differentiated carcinoma. There are very significant difference between the expression level of nm23 - HI and differentiated level of endometrium cancer. The results of nm23 - HI RT - PCR demonstrated negative correlation with clinicopathologic stage, uterine adnexa invasion and lymph node metastasis, while immunohistochemical results showed the same tendency which have no statistical difference. There are no obvious correlation between the expression level of nm23 -HI and myometrium invasion, cervical invasion and histological type.3. the Correlation between Immunohistochemistry and RT - PCR Compareing the immunohistochemical and RT - PCR results of nm23 - HIand CD44v6 respectively, there are significant correlation between the two testing methods(P <0.001).4. The Correlation between the Expression of nm23 - HI and CD44v6 Statistical outcome show there are no significant correlation between the expression level of the two genes.Conclusion1. The expression of CD44v6 may associated with the development, invasion and metastasis of endometrial carcinoma, thus CD44v6 may be a useful tool for the diagnosis and the prognosis of endometrial carcinoma.2. The expression of nm23 - HI may associated with the differentiative level of endometrial carcinoma.3. Its valid to extract short clip of target mRNA from paraffin - embedded tissue for RT - PCR, thus it may be useful in studying the rare pathological tissue embedded by paraffin.4. There are highly positive correlation between the data obtain by immunohistochemistry and RT - PCR, which imply both of the two methods are valid for detecting the expression level of the two genes, and may suggestthat the two gene have the similar expression in gene and protein level.5. There are no correlation between the expression level of nm23 - HI and CD44v6 either in gene or protein level. Detection the expression level of CD44v6 and nm23 - HI may be useful in making the prognosis of patients with EC.