Abstract Topoisomerase I (Top1) is the molecular target for a number of drugs used in cancer chemotherapy. Several third generation Top1 inhibitors are being evaluated, including the indenoisoquinolines (NSC 725776 and NSC743400) which are currently in clinical trials at the National Cancer Institute. We previously reported the development and validation of a quantitative ELISA to measure Top1 levels in cells and solid tissues [PloS ONE (2012) 7(12)]. We observed a large variation in baseline Top1 levels in xenografts and human patient biopsies using the published method. Baseline Top1 levels in needle biopsies for some patients were too low to allow for the reporting of a drug effect. We have modified our tissue processing procedures to improve assay sensitivity. We have also demonstrated the fitness of the assay in a clinical trial of the indenoisoquinolines for canine lymphoma as a stepping stone to human clinical trials. Baseline Top1 levels in canine lymphoma patients varied greatly, consistent with our previous findings in xenografts and a limited number of human tumor biopsies. These data suggest that it may not be possible to measure drug effect based on post-dose Top1 levels alone, but will require paired pre-dose and post-dose specimens from the same patient. Decreased Top1 levels following therapy (compared to pre-dose) were detected in tumors from a number of dogs, each treated with one of three indenoisoquinolines (NSC706744, NSC 725776 and NSC743400), demonstrating evidence of target engagement as predicted by in vitro and xenograft models. Measurement of baseline Top1 levels in this trial will be correlated with the degree of drug-related target reduction and evidence of drug efficacy in the dogs enrolled on this study. Funded by NCI Contract No. HHSN261200800001E. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C278. Citation Format: Thomas D. Pfister, Katerine V. Ferry-Galow, Tariq Mohabbat, Robert J. Kinders, Chand Khanna, Christina Mazcko, Ralph E. Parchment, Yves Pommier, Joseph E. Tomaszewski, James H. Doroshow. Topoisomerase 1 immunoassay provides proof of target engagement by the indenoisoquinoline class of topoisomerase 1 inhibitors in canine lymphomas. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C278.