| Purpose:The HER-2 protein, which is targeted by drug Herceptin, could fuel the growth of breast carcinoma. First of all, the Immunohistochemistry (IHC) is used as the tool in early screening of HER-2 detection. The positive expression (the result showed 2+) of HER-2 in IHC is further detected by fluorescence in situ hybridization (FISH) to verify the presence of DNA amplification. Currently, Herceptin is the anti-HER2 drug that has demonstrated a survival benefit in these cases. In this study, the real-time quantitative PCR (qPCR) was used to detect HER-2 expression level in paraffin-embedded tissues of non-special invasive breast carcinoma, and then compare these with the corresponding results from IHC and FISH, to verify the feasibility ofqPCR using in HER-2 detection. The aim of this work was to find a simpler, better reliable and easier practical method for HER-2 clinical detection in breast carcinoma.Methods:Paraffin-embedded tissues of non-special invasive breast carcinoma from 2010.01 to 2014.06 were collected for HER-2 detection using IHC method according to the standard operating procedure. The specifical wax block was picking out, from which tissue contains high proportions of invasive carcinoma tissue and normal breast tissue. The tissue’s section, of which thickness is 3 μm, was stained by automatic immunohistochemical instrument. After the diagnosis from the experienced pathologists according to the new ASCO/CAP and Chinese guide for HER-2 judgement. The candidate tissues were divided into four different groups:HER-2(0), HER-2(1+), HER-2(2+) and HER-2(3+). And 100 cases were selected from each group for further detection.The paraffin-embedded tissues were sliced into 4μm, in which Her-2 gene was amplified and stained with dual- color FISH in accordance with the manufacturer’s procedures. After operated in a dark room, we observed results with the help of fluorescence microscope, the results were diagnosed by the expert pathologists according to the newest ASCO/CAP and Chinese guide for HER-2 judgement.The tissues of the related patients were serial sectioned into 2-3 pieces (8-10μm), which were trimed to remove normal tissues according to the HE slices. And the residual tumor tissues were used for RNA isolation according to the standard instruction manual. RNA isolations were used as the templates inqPCR for HER-2 detection. Results fromqPCR were compared with those from IHC and FISH.The comparision of results would provide a novel method for the detectiong of HER-2 gene in breast carcinoma.Results:UsingqPCR analysis, the positive expression rate of HER-2 gene in cancer tissue was found to be full compliance willmigerl with FISH’s result in HER-2(0) and HER-2(1+) groups. While in HER-2(2+) group,20 cases were detected to be positive by FISH (positive rate,20%) and 25 cases were positive byqPCR (positive rate,25%). In HER-2(3+) group,92 cases (positive rate,92%) were diagnosed to be overexpression of HER-2 using FISH and 90cases (positive rate,90%) were diagnosed usingqPCR. The Kappa test was used to measure the consistency among results from different methods. In HER-2(0), HER-2(1+), and HER-2(3+) groups by IHC, there was well consistency dignosis in results compared in pairs (IHC, FISH and qPCR methods) (P> 0.05). For the studied population in HER-2(2+) group by IHC, there is no significant difference between FISH andqPCR (P > 0.05).Using IHC analysis andqPCR analysis, The overexpression of Her-2 was significantly comformed to pathology classification of tissues, the expression of ER,PR and Ki67 (P<0.05).Using FISH test, the overexpression of Her-2 was signifcantly correlated with age, clinical stage, pathology classification of tissues,the expression of ER,PR and Ki67 (P＜0.05). From the above results, there is good consistency in the results of IHC andqPCR.Conclusions:For HER-2(0,1) and HER-2 (3+) specimens detected by IHC in non-special invasive breast carcinoma, there is a well correlation between qPCR and FISH analysis. For HER-2(2+) specimens by IHC in non-special invasive breast carcinoma qPCR well correlated with FISH method and combination of the two methods may improve the sisitivity of detection.Compararing with FISH,there is better consistency in qPCR and IHC at evaluation the clinicopathological parameters in breast carcinoma. |
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