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In human breast cancer, overexpression of HER2 protein (score 3+) is a good indication for herceptin (trastuzumab) administration and can expect a favorable prognosis. In this study, we examined the correlation between the expression of HER2 protein and the status of mRNA amplification in human breast cancer tissues prepared using laser assisted microdissection (LAM) technique. Twenty breast cancers were selected for this study. The surgical specimens were obtained under informed consent. Fresh tumor tissues were cut into no more than 10x5x5 mm sections, and frozen immediately in hexane/dry-ice acetone. Eight-micrometer thick sections were cut and mounted on a membrane film-coated slide glass, fixed in methanol and stained with toluidine blue. Microdissection was carried out using a LAM instrument (LS 337 Laser Scissors(TM), Cell Robotics Inc. USA). RNA was extracted from the microdissected tumor tissues by means of guanidium thiocyanate method, and transcribed to cDNA. HER2 and GAPDH (internal control) mRNAs were analyzed by real time polymerase chain reaction (PCR) based on a fluoro-genic TaqMan method. Amplification of HER2 mRNA was standardized with the copy number of GAPDH mRNA. Immunohistochemistry (IHC) for HER2 protein was carried out using LSAB (labeled streptavidine biotin) method. Regarding the status of HER2 mRNA amplification, we obtained 4 cases of invalid score and 4 cases of distinct and unequivocal amplification. The status of HER2 mRNA amplification of these 8 cases was well correlated with the expression of HER2 protein evaluated as score 0 and 3+. The cases evaluated as score 1+ and 2+ in the immunohistochemistry were quite heterogeneous and showed a relatively wide range of HER2 amplification. A combination of LAM technique and real time PCR is a powerful and reliable method for the quantitative analysis of mRNA. Our results indicated that the groups of HER2 score 1+ and 2+ include candidates for herceptin therapy.

Comparison of HER2 mRNA Amplification with Immunohistochemistry in Human Breast Cancer Using Laser Assisted Microdissection Technique