11588 Background: We performed pharmacodynamic biomarker analysis for response to a panel of three indenoisoquinolines (LMP776, LMP400 and NSC706744, J. Med. Chem. 49:7740, 2006) that have demonstrated anti-tumor activity in dogs. In preclinical xenograft models treated with indenoisoquinolines, we observed that gH2AX was not a useful biomarker for the biological activity of compound 706744, but was a reasonable biomarker of drug activity (Clin. Canc. Res. 16:5447, 2010) for the other two compounds, even though in vitro data indicated that all 3 compounds inhibited TOP1 and killed tumor cells. It has been reported that irinotecan activates autophagy (Pharm. Rev. 65:1162, 2013) which correlated with its metabolism to SN-38; we therefore developed, validated and tested an immunofluorescence microscopy assay for LC3 as a marker of autophagy. Methods: Assays were developed to evaluate apoptosis by co-localization of cleaved caspase 3 and gH2Ax, and autophagy by LC3 immunofluorescence on formalin fixed, paraffin-embedded tissue sections of xenografts models or lymphomas from outbred dogs. Percent positive cells containing LC3 puncta were quantitated using a spot morphology algorithm. Analysis of gH2AX and cleaved caspase 3 cellular co-localization was developed using a blebbing morphology algorithm (Definiens). Results: LC3 reported that indenoisoquinoline 706744 activates autophagy in vitro with the absence of cleaved caspase 3-dependent apoptosis while the -776 and -400 compounds do not activate autophagy, but instead demonstrate apoptosis in response to drug treatment. Results in animal models confirmed that both autophagy and apoptosis were active. Clinical readiness of the assays was confirmed on canine biopsy FFPE slides. Conclusions: 1,Structurally-related TOP1 inhibitors may trigger alternative pathways of cell destruction; 2,Autophagy may report drug anti-tumor activity or tumor drug resistance according to current literature. This assay may be useful for determination of pharmacodynamic pathways associated with anti-tumor activity to elucidate mechanism of action of investigational agents used in clinical trials.