BACKGROUND.Gemcitabine incorporation into DNA enhances cleavage complexes in vitro when combined with topoisomerase I inhibitors and demonstrates synergy in cancer cells when given with irinotecan. Topoisomerase I inhibitors require that topoisomerase I interacts with DNA to exert activity.METHODS.Patients who had received previous anthracycline therapy or were not candidates for anthracycline therapy received gemcitabine at a dose of 1000 mg/m2 intravenously over 30 minutes followed by irinotecan at a dose of 100 mg/m2 over 90 minutes on Days 1 and 8 of a 21‐day cycle. The primary endpoint was improvement in response from that historically observed with gemcitabine (from 25% to 45%) as measured by Response Evaluation Criteria in Solid Tumors. Correlative studies included characterization of cellular levels and nuclear distribution of topoisomerase I and pharmacokinetic analysis of gemcitabine and irinotecan.RESULTS.Forty‐nine patients were assessed for response. The response rate was approximately 25% (all partial responses [PRs], 12 patients; 95% confidence interval [95% CI], 13‐39). Six patients had stable disease (SD) for ≥6 months for a clinical benefit rate (PR + SD) of 39%. The median time to disease progression was 3.7 months (95% CI, 2.5 months‐4.6 months), and median survival was 11.6 months (95% CI, 8.9 months‐15 months). Toxicities included neutropenia, nausea, and vomiting. Seven of 9 tissue biopsies were assessable for topoisomerase I. Tumors with the 2 lowest nuclear to cytoplasmic ratios demonstrated no response to irinotecan.CONCLUSIONS.Gemcitabine and irinotecan are active in metastatic breast cancer, but response did not meet predetermined response parameters, and the null hypothesis was accepted. Topoisomerase I localization can be measured in metastatic breast cancer. Further validation is needed to determine whether this assay can predict response. Cancer 2008. © 2008 American Cancer Society.