The overall aim of this application is to identify the clinically relevant mechanisms of resistance which develop to irinotecan- based chemotherapy in patients with locally advanced non small cell lung cancer (NSCLC). Irinotecan is a camptothecin derivative with significant activity in NSCLC. Current management of locally advanced NSCLC incorporates surgical staging at diagnosis, treatment with chemotherapy, and then surgical resection of the tumor, thus providing a unique opportunity to obtain sufficient pathologic material before and after pre-operative chemotherapy in order to conduct the laboratory studies proposed. Camptothecins stabilize the topoisomerase (topo) I: DNA covalent complex in a single strand protein linked DNA break (PLDB). PLDBs can subsequently be converted to dsDNA breaks which are potentially lethal. Cellular resistance to camptothecin may be related to steps occurring before the formation of the PLDB, such as reduction in topo I, or result from steps occurring after formation of the PLDB, such as an enhanced capacity to repair DNA damage. In order to characterize how resistance to camptothecin develops in vivo, NSCLC tumor tissue obtained before and after three cycles of pre- operative chemotherapy consisting of irinotecan and cisplatin will be analyzed. In single cell suspensions prepared from tumor and incubated in camptothecin in vitro, dsDNA damage and PLDB formation will be analyzed. Comparison of drug sensitivity will determine whether limited exposure to non-curative chemotherapy induces resistance and whether resistance develops before or after formation of the PLDB. The concentration, activity, intracellular distribution, and topoisomerase I affinity for irinotecan will be characterized to determine which of these specific biochemical alterations contribute to resistance to irinotecanin vivo. Additional studies will be directed according to whether analysis of DNA lesions suggest that resistance acquired during treatment occurs before of after formation of the PLDB. Following completion of this first clinical trial, a second study will be performed utilizing the combination of irinotecan plus paclitaxel. Biochemical studies to analyze clinically relevant mechanisms of resistance to camptothecins will be repeated. Comparison of the phenotype changes observed in the tumor will provide insight into the mechanisms of resistance which are invoked toirinotecan when it is combined with different classes of chemotherapeutic agents. In addition, since multidrug resistance (MDR) may be relevant to the activity of paclitaxel, we will evaluate these tumors for the expression of MDR before and after exposure to pre-operative chemotherapy. The overexpression of these drug efflux associated proteins will be measured by immunocytochemistry and functionally by in-vivo scanning with 99Tc-sestamidi. If the expression of MDR is increased in NSCLC following pre-operative chemotherapy containing paclitaxel, then selection of clones within the tumor which express MDR may be relevant to ultimate treatment failure. This would support additional trials in which modulators are included in the initial treatment to eradicate these clones. These biochemical approaches should result in a better understanding of the mechanisms of acquired resistance to chemotherapy in NSCLC and lead to new strategies to treat this disease.
