Tumor biomarkers increasingly provide information for predicting outcomes with chemotherapeutic regimens (
personalized medicine). Topo2A is
a DNA helicase targeted by
anthracyclines, cytotoxic
therapeutics used in both adjuvant and
palliative treatments of
breast cancer. TOP2A gene amplification/deletion is implicated in response to
anthracycline-based
chemotherapy. We describe an approach for analyzing
formalin-fixed,
paraffin-embedded
breast tumors on tissue microarrays with TOP2A fluorescence in situ hybridization coupled with
cytokeratin immunofluorescence to target
tumor cells. Stained tissue from patient specimens was imaged and analyzed using Metafer/Metacyte (Metasystems, Waltham, MA, USA), including customized image classifiers. TOP2A/CEN17 ratios of 2.0 or greater (amplified) and 0.8 or less (deleted) were observed for 10.0% and 6.1% of the patients, respectively. Patient outcomes for
adjuvant chemotherapy (
cyclophosphamide-
epirubicin-
fluorouracil,
cyclophosphamide-
methotrexate-
fluorouracil, no
chemotherapy) were evaluated. No statistical significance was achieved for clinical end points regarding TOP2A status in
anthracycline-treated patients. However, patients with TOP2A aberrations receiving
methotrexate-based
therapy exhibited a significant decrease in 5-year distant disease-free survival and
breast cancer-specific overall survival, especially for patients with TOP2A deletions (disease-free survival: hazard ratio, 5.31 [P = .001], and
breast cancer-specific overall survival: hazard ratio, 6.45 [P ≤ .001]). No significant differences were seen in patients included in the no-
chemotherapy group. Topo2A
protein levels were assessed by immunohistochemistry with no correlative statistical relevance to immunofluorescence/fluorescence in situ hybridization-based prognosis for
cyclophosphamide-
epirubicin-
fluorouracil or
cyclophosphamide-
methotrexate-
fluorouracil groups. Interestingly, aberrant (under)expressing patients in the no-
chemotherapy group exhibited better 5-year distant disease-free survival (hazard ratio, 0.39; P = .004), trending toward more favorable
breast cancer-specific overall survival (hazard ratio, 0.61;
P = .11). Our results indicate a strategy by which fluorescence in situ hybridization scoring targeted to
cytokeratin-positive
tumor cells may provide a tool for added precision and efficiency in TOP2A evaluation from
tumor tissue.