Abstract
e13577 Background: Inhibitors of Topo1 abrogate DNA replication and induce apoptosis in tumor cells. Topo1 inhibitors are effective cancer therapeutics however improved pharmacokinetic profiles have been sought. NKTR-102 is a next generation topoisomerase I-inhibitor with a unique pharmacokinetic profile that provides continuous exposure of the active metabolite, SN38. NKTR-102 is in phase III clinical development for patients with metastatic breast cancer. Monitoring DNA damage response markers in CTCs may be an informative, minimally invasive approach to detect drug activity in patients. Objectives of this study are to: 1) develop a multiplex immunofluorescent assay for measuring DNA damage effects of SN38 in CTCs; 2) obtain data to determine whether measuring DNA damage biomarkers in CTCs isolated from blood of cancer patients treated with SN38 ex vivo could be applied clinically to identify the best responders to NKTR-102. Methods: Solid tumor cell lines were treated with SN38 for 24 hr. Expression levels of gH2AX, survivin, BRCA1, BRCA2, RAD51, ATM and TUNEL were measured by quantitative laser scanning cytometry (LSC).Cytotoxcity was confirmed by MTT assay. Blood samples from 3 cancer patients were treated ex vivo with SN38 and CTCs were recovered and analyzed for expression of DNA damage biomarkers by LSC. Results: Treatment of H460 and HCT116 cells with SN38 led to a dose-dependent increase in the level of protein expression and/or frequency of positive cells for all DNA damage markers. Ex vivo treatment of breast cancer patients' blood with SN38 for 24 hr induced a dose-dependent increase in the expression of ATM (7.8-fold) and RAD51(188-fold) in one patients’ blood sample. An increase in the percent TUNEL-positive cells (0-20% in untreated to 44-54% in drug-treated samples) was observed in 2 patients’ samples without affecting the total number of isolated CTCs. Conclusions: Quantitative multiplex immunofluorescent assays for monitoring changes in gH2AX, ATM, RAD51 protein expression and percent TUNEL positive CTCs have been developed using LSC. These assays have been incorporated into an ongoing phase III clinical study of NKTR-102.
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