Abstract 2521: In vitro and in vivo antitumor efficacy of sequentially combined vinorelbine and gefitinib in non-small cell lung cancer
Authors: Alama Angela , Dal Bello Maria Giovanna , Vanni Irene , Truini Anna , Coco Simona , Rijavec Erika , Genova Carlo , Biello Federica , Marini Cecilia , Bottoni Gianluca , Sambuceti Gianmario , Grossi Francesco 
Published: August 2015

Abstract
Introduction: The current standard of care for advanced non-small cell lung cancer (NSCLC) is systemic chemotherapy or treatment with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) for EGFR-mutated tumors. Preclinical studies suggest that the combination of cytotoxic agents and EGFR-TKIs may increase the inhibitory effect albeit influenced by the type of drug and the order of administration. Whether these combinations may exert similar effects in tumors poorly responsive to EGFR-TKIs is still unclear. The antitumor efficacy of different protocols with vinorelbine (VNB) and gefitinib (GEF) were investigated in two NSCLC cell lines, in vitro and in vivo, as a rationale to be potentially exploited in the clinical setting.
Methods: The EGFR-wild-type A549 and the EGFR-mutated (exon 21 L858R/exon 20 T790M) H1975 cell lines were treated for 72 hrs with VNB and GEF according to different schemes and concentrations: GEF followed by VNB, VNB followed by GEF and the two drugs given individually or concurrently. The cytotoxicity was determined by MTT assay (short-term). In addition, the efficacy of repeated weekly VNB treatment along with sequential or continuous GEF administration was investigated by cell count (long-term). The expression levels of activated EGFR and downstream pathway molecules were investigated by western blot. The in vivo effects of single or combined treatments were studied in a CD1-Nude mouse model by subcutaneous injection of the H1975 cells. Cancer growth was determined by measurements of tumor diameters with a Vernier caliper, while lesion glucose consumption was estimated by means of a dynamic micro Positron Emission Tomography scans. To this purpose, a bolus of 3-6 MBq of 18F-fluorodeoxyglycose was injected through a tail vein during a list mode acquisition lasting one hour. After framing rate optimization, pixelwise maps of cancer glucose consumption (MRGlc) were created using dedicated software (PMOD) and applying Patlak graphical approach.
Results: In vitro short and long-term studies demonstrated that the sequence of VNB followed by GEF was significantly more active than GEF followed by VNB or the concurrent administration of the two drugs. Western blot analyses indicated that the increased cytotoxic effect of the VNB and GEF sequence was accompanied by inhibition of p-EGFR, p-AKT and p-ERK1/2 expression levels, mainly in the EGFR-mutated H1975. Moreover, the increased antitumor efficacy of the sequence VNB followed by GEF was also confirmed in vivo by a significant inhibition of the H1975 tumor growth (p&lt;0.0001) that was paralleled by a corresponding decrease in cancer glucose consumption.
Conclusions: These preclinical findings in NSCLC cell lines, poorly responsive to EGFR-TKIs, showed that the sequential treatment with VNB followed by GEF induced a significant antitumor efficacy supporting the application to clinical trials.
Citation Format: Angela Alama, Maria Giovanna Dal Bello, Irene Vanni, Anna Truini, Simona Coco, Erika Rijavec, Carlo Genova, Federica Biello, Cecilia Marini, Gianluca Bottoni, Gianmario Sambuceti, Francesco Grossi. In vitro and in vivo antitumor efficacy of sequentially combined vinorelbine and gefitinib in non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2521. doi:10.1158/1538-7445.AM2015-2521