Tumor biopsies of 18 patients with HER2 positive breast cancer were collected in our previous neoadjuvant study as published [6 (link)]. They consisted of eight HER2 and ten luminal-HER2 cases that were collected from patients after neoadjuvant treatment with four cycles of epirubicin (E, 90 mg/m2) and cyclophosphamide (C, 600 mg/m2), every 3 weeks prior to surgery. Pretreatment biopsies were also collected and stored as either formalin-fixed or paraffin embedded blocks. The study was approved by the Yokohama City University Medical Center in 2006.
The TOP2A gene status was examined by fluorescence in-situ hybridization (FISH) (TOP2A FISH pharmDx™ Kit; DAKO) in pre-treatment tumor biopsies. TOP2A copy number was then determined in a minimum of 20 interphase, non-overlapping, tumor cell nuclei and compared with that of chromosome 17 centromeres (CEP17) in the same nuclei. The ratio of TOP2A to CEP17 signals was then calculated. In accordance with previous reports, a TOP2A/CEP17 ratio greater than 2.0 was defined as gene amplification [4 (link), 5 (link)].
The TOP2A gene status was examined by fluorescence in-situ hybridization (FISH) (TOP2A FISH pharmDx™ Kit; DAKO) in pre-treatment tumor biopsies. TOP2A copy number was then determined in a minimum of 20 interphase, non-overlapping, tumor cell nuclei and compared with that of chromosome 17 centromeres (CEP17) in the same nuclei. The ratio of TOP2A to CEP17 signals was then calculated. In accordance with previous reports, a TOP2A/CEP17 ratio greater than 2.0 was defined as gene amplification [4 (link), 5 (link)].