Fluorescence In Situ Hybridization (FISH) For Detection Of HER-2/neu Amplification In Breast Cancer: A Methodology Study

BackgroundHer-2/neu (C-erbB-2) gene amplification, identified in 20% to 30% of breast cancers, is a prognostic marker of poor clinical outcome in node-negative and node-positive breast cancer and a predictor of lack of responsiveness to tamoxifen antiestrogen therapy and responsiveness to adjuvant doxorubicin chemotherapy. Thus, HER-2/neu is considered to be a clinically important molecule. It is important for therapy to assay HER-2/neu abnormalities.As indicated above, Her-2/neu has become of particular interest in recent years with development of the Her-2/neu targeting drug Herceptin. Herceptin, a monoclonal humanized murine antibody can specifically bind the extracellullar portion of Her-2/neu(P^185Her-2/neu), leading to cell kill. Now Herceptin has become an important therapeutic option for patients with Her-2/neu overexpressing breast cancer, so testing for HER-2/neu abnormalities is important to patients who want to use Herceptin.Fluorescence in situ hybridization (FISH) is widely used to study the amplification, mutation and translocation in both metaphase and interphase cells. The technology established about FISH performed on formalin-fixed, paraffin-embedded sections is a good platform on the detection of Her-2/neu. ObjectiveTo investigate the optimum conditions of FISH performed on formalin-fixed, paraffin-embedded sections. Secondly, Her-2/neu gene amplification was detected on the breast cancer with node-positive with FISH. Thirdly, method of Her-2/neu detected with FISH was studied on tissue microarray. Finally, Her-2/neu ampliation detected on fine-needle puncture cytologic material was also discussed. MethodsVariations in thickness of section, incubation temperature with sodium thiocyanate, duration of enzymatic pretreatment and denature temperature are important factors in FISH. We also apply the Spherical Symmetric Design toinvestigate the effects of the various parameters on formalin-fixed, paraffin-embedded material and to optimize the technique variable.A total of 40 primary breast carcinomas with positive lymph nodes were analyzed for amplification of the HER-2/neu oncogene by fluorescence in situ hybridization. Results were compared with individual clinicopathologic and follow-up data. Clinic status, tumor size, lymph node status, patient's age, joint ER/PR status, menopausal status, were evaluated, using the [chi]2 test for univariate analysis and logistic regression for multivariate analysis. And examine the concordance between FISH and IHC. Result:The regression equation is Y=11.64-1.5X₂-1.333X3+X/X3-0.5B3Xi, specimen thickness, incubation temperature with sodium thiocyanate, duration of enzymatic pretreatment is independent variable to decision the quality of FISH, The thickness has crossing-interaction with pretreatment duration.Clinic status, tumor size, lymph node status, patient's age, joint ER/PR status, menopausal status do not associated with HER-2/neu status. Her-2/neu gene amplification independently predicts 5 years survival (PO.05) .Amplification of the HER-2/neu gene by FISH was observed in 11 of the 40(27.5%) cases. Protein overexpression and gene amplification were found to correlate in 100% (5 of 5) of the IHC 3+ positive tumors and 5 out of 7 (71.4%) of the IHC 2+ positive tumors and 1 out of 28 (3.57%) of the 1+/0 positive tumors. Neither amplification nor overexpressing was found in 32 cases benign breast tumors. In sum, FISH is concordance with IHC (?=0.00003). Conclusion:1) specimen thickness, incubation temperature with sodium thiocyanate, duration of enzymatic pretreatment is independent variable to decision the quality of FISH, The thickness has crossing-interaction with enzymatic pretreatment duration. The optimized condition are specimen thickness is 3 u m, 70 "C sodium thiocyanate incubated for 20 minus, 0.25 mg/ml proteins K pretreatment for 10 minutes.