Analysis of HER2/neu Gene Amplification in Microdissected Breast Cancer Tumour Samples

One in nine women in Canada will develop breast cancer during their lifetime. Generally, breast cancer progresses sequentially from atypical hyperplasia to ductal carcinoma in situ (DCIS) to an invasive stage, which eventually metastasizes to distant sites. Our laboratory has been interested in studying the role of HER2/neu amplification and its correlation with breast cancer progression. The HER2/neu oncogene has been reported to be amplified in>20 % of sporadic breast tumours. However, it is not clearly understood at what stage HER2/neu amplification occurs. In order to investigate HER2/neu status in different stages of tumour progression, we chose to utilize the Laser Capture Microdissection (LCM) system to isolate pure cell populations for gene amplification analysis. Five-micron formalin fixed paraffin embedded tissue sections from an archival collection at the Sunnybrook and Women’s College Health Sciences Centre were stained with hematoxylin and eosin, to visualize cell morphology. The Pixcell IIe Laser Capture Microdissection System was used to capture specific tumour cell populations. Desired tissue areas were identified and captured on a plastic film after pulsing the region with laser light for a duration of six milliseconds, at a power of twenty-one to thirty-three milliwatts. Genomic DNA was isolated by a modified proteinase K- phenol extraction method. β-globin was used as a housekeeping gene, and HER2/neu as the primary oncogene in this study. The control genomic DNA was obtained from the HT29 tumour cell line. We preformed a serial dilution of control genomic DNA to determine the lower limit of HER2/neu gene detection. Tissue samples corresponding to normal, ductual carcinoma in situ (DCIS), an