Comparision Of HER2/neu Oncogene Detected By Chromogenic In Situ Hybridization And Immunohistochemistry And Enzyme-lined Immunosorbent Assay In Breast Cancer

Background and ObjectivesBreast cancer is a common malignancy in our country, and severely threatenswomen's lives.Its clinical outcome will be affected by many factors. Along with the thorough study of the molecular mechanism of the tumor occurrence, HER2 is conceidered to be an important prognostic factor. HER2 /neu gene overexpressing is prognostic marker of poor clinical outcome in breast cancer and a predictor of responsiveness to tamoxifen antistrogen therapy and resistance to adjuvant doxorubicin chemotherapy. The extracellular domain (ECD) of the HER2/neu oncoprotein is shed from cancer cells into the circulation and is called serum HER2. Serum HER2 oncoprotein concentrations are currently being examined as biomarkers, and might be useful in breast cancer for detecting early recurrence or metastasis and for predicting response to therapy So, it is important for therapy to assay HER2/neu abnormalities.Many researches about HER2/neu expression in breast cancer have been present, but the result had much difference in the expression level, even reverse.This difference was caused by different kit and testing methods and samples of the researches, further research is necessary to establish accurate HER2/neu expression level. To solve the problems mentioned above, we select HER2/neu gene as study target to explore how to dedect HER2/neu expression level in patients with breast cancer accurately by chromogenic in situ hybridization (CISH) and immunohistochemistry (IHC) and enzyme-lined immunosorbent assay (ELISA). To detct serum HER2 oncoprotein levels in patients with operable and netastatic breast cancer, and to study the correlations between serum HER2 level and tumor size as well as other clinical parameters. Through comparision of HER2/neu oneogene detected by CISH and IHC in breast cancer to explore the effect of CISH on detecting gene amplification of HER2/neu.Materials and MethodsA total 83 women were studied consisting of 10 healthy volunteers, 19 benign breast disease, 50 operable breast cancer, and 13 patients with metastatic breast cancer. Fasting blood was drawn and centrifuged immediately, the sera was stored at -70℃. The paraffin-embedded sections of breast cancer tissues from 40 operable breast cancer patients were collected. All patients with breast cancer and benign breast diseases were initially presented to the Henan Tumor Hospital during the period 2005 to 2006.The levels of serum HER2 were assayed by ELISA to determine the expression level of serum HER2. IHC was used to analyze the staining in the malignant cancer tissue segment of operable breast cancer. CISH for HER2 gene expression status was performed using SPOT-Light HER2 CISH^TM on the paraffin-embedded sections of breast cancer tissues from 40 operable breast cancer patients with IHC staining scores of (+++),(++),(+) and (-). Statistical analysis was performed with SPSS10.0 software, using T test and x² test.Statistical significant level was considered as "a =0.05".Results1. We observed the positive rate and level of HER2 in sera of healthy subjects and benign breast disease patients was no difference to those of operal breast cancer patients (P> 0.05), but significant difference to those of metastatic breast cancer patients (P< 0.05).2. Serum HER2 levels, tumor size, lymph nodes and tissue HER2/neu expression of operable breast cancer: There was a significant correlation between tumor size and serum HER2 positivity. But this research failed to demonstrate a relationship between lymph nodes and tissue HER2/neu overexpression and serum HER2.3. Seven of the 8 patients with score (+++) by IHC were positive for HER2/neu by CISH, one other case were negative. Five of the 13 patients with score (++) by IHC showed HER2/neu gene amplification, the remaining 8 cases were CISH-negative. Two of nine patients with score (+) by IHC were positive for HER2/neu by CISH, the remaining 8 cases were CISH-negative. All the patients with scores 0 (number=9) by IHC failed to show amplification. Two kinds of methods have concordance to the examination of HER2/neu status (kappa=0.458 p=0.003).Conclusion1. There is a correlation between serum HER2 and tumor loads of breast cancer. It iscomplementary to HER2/neu tissue results and could be helpful in monitoring recurrent or metastatic breast cancer and predicting patients' response to Herceptin therapy.2. CISH is a novel technique for detecting HER2/neu gene amplification. The results of HER2/neu gene amplification detected by CISH have concordance with the results detectd by IHC. IHC is useful as an initial screening tool for HER2/neu expression status. Because of the certain discrepancies between protein expression and gene amplification, patients with score (+++⁺) by IHC should undergo CISH testing.