The Study Of Real-time PCR And Immunological Methods For The HER2 Determination And It's Application To Evulate The Therapeutic Effect In Patients With Breast Cancer

Background and objectiveIt was shown that the HER2(c-erb-b2) gene amplification and/or protein over- expression were associated with poor prognosis ,shorter overall survival time. HER2 is an important prognostic factor and cellular target for the targeted therapies for breast cancer patients, and it is meaningful to direct therapy and evaluate prognosis, so it is very importment to evaluate the her-2/neu status accurately.Currently , methods for tumor analysis such as immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH) are routinely used to determine the HER-2/neu status of patients with breast cancer. However, both IHC and FISH analyses are invasive methods performed on tumor tissues sample , thus they do not allow the evaluationw when there is no tumor tissues sample or the monitoring of Her2neu status during the follow up of the patient.This subject aims to evaluate the her-2/neu status through the serum and real time PCR ,study the clinical value of serum her-2/neu level of the metastatic breast cancer patients,and to study the relationship between the change of the concentration of serum HER2 level after chemotherapy and response of chemotherapy ; We expect to develop and evaluate a Taqman Real-Time fluorescence RT-PCR diagnostic techniques to evaluate the her-2/neu status,and compare with the ELISA assessments.Methodsserum her-2/neu level detection are as follows:HER2 was measured in serum samples from 30 health controls women the and the serum samples from metastatic breast cancer patients before chemotherapy and after 4 cycles chemotherapy with the method of ELISA. Serum HER2 levels in healthy individuals and tissue HER2 status positive patients were used to design a ROC curve,an optimal serum HER2 level yielded based on the ROC curve obtained. We want toevulate the serum HER2 method, study the relationship between the serum HER2 level and clicial factors, to compare the serum HER2 baseline level is in accordance or not with tissue HER2 status, and to know whether serum HER2 baseline level can influence the response of chemotherapy,and also to study the relationship between the change of the concentration of serum HER2 level and response of chemotherapy.PCR detection method is as follows: RNA from peripheral blood were evaluated in 72 breast cancer patients (42 tissue HER2 positive and 30 tissue HER2 negative) and 30 healthy controls. According to the GenBank database, we searched highly conservative sequence of HER2 with the Blast and DNASIS tools. One pair of Real-Time PCR primers and fluorescent probes of HER2 were designed by Primer Express 2.0 and Primer Express software, The standards were established and used to establish standard curves. For each sample, the level of HER2 mRNA transcript was determined as the ratio of the number of copy of HER2 to the number of copies of 28S rRNA. The gene copy number ratios in healthy controls and in tissue HER2 positive cases was used to design ROC curve to determine the optimal cut off values. The Real-Time PCR method were used to detected in clinical patients.ResultsSerum HER2 determination results:(1)Based on the ROC curve obtained, an optimal serum HER2 level of 18.4 ng/ml yielded a 73.4% sensitivity and a 86.1% specificity .(2)the serum HER2 baseline level is in accordance with tissue HER2 status and the values of the serum HER2 concentration was significantly higher in tissue HER2 positive cases than in tissue HER2 negative cases and in healthy controls. (3)The serum HER2 baseline level was not correlated with the response of chemotherapy.(4)the change of serum HER2 level is associated with response of therapy.Real-Time PCR determination results: (1) RNA extraction from peripheral blood can obtain a good result by real-time PCR and general PCR and analysis. (2)The technological platform for detection of HER2 mRNA was established using Real-Time PCR, The value of the HER2/28SEC copy number ratios was significantly higher in tissue HER2 positive cases than in tissue HER2 negative cases and in healthy controls. Based on the ROC curve an optimized gene copy number ratio of 5.95 yielded a 76.6% sensitivity and a 91.6% specificity.ConclusionAccording to the above results, we got the conclusions as follows:(1) the serum HER2 baseline level is in accordance with tissue HER2 status, can be considered as a complementary method besides tissue methods.(2)There was no relationship between the serum HER2 level and some clicial factors,such as age. (3) The serum HER2 baseline level can not influence the response of chemotherapy, but the change of serum HER2 level is associated with response of therapy and the serum HER2 level could be used as a tumor maker. (4) The Real-Time Reverse transcription quantitative PCR techniques for HER2 mRNA was preliminary established. This method was useful for evulating the HER2 status in clinical metastatic breast cancer patients.(5) QPCR performs a litter better than ELISA in terms of sensitivity and specificity.