Abstract 1715: High ALK gene copy number as a predictor of response to crizotinib in non-small cell lung cancer cell lines

Abstract

Abstract Introduction: The anaplastic lymphoma kinase (ALK) gene translocation is a rare event, described in approximately 4-6% of lung adenocarcinoma. ALK translocation is a robust predictive factor for ALK inhibitors sensitivity. However, increased ALK gene copy number seems to be a frequent event, described in 13-17% of Non-small cell lung cancer (NSCLC). The goal of this study was to explore the predictive value of ALK High copy number for 2 ALK inhibitors, crizotinib and TAE684 in NSCLC cell lines. Methods: 27 NSCLC cell lines were screened for ALK copy number by FISH after cytospin. ALK high copy number (HCN) was defined as the presence of ≤ 5 copies of ALK. FISH with CEP2 was performed to determine the ploïdy status in cell lines with HCN. As crizotinib is a dual c-MET and ALK inhibitor, FISH c-MET was performed in selected cell lines for in vitro studies. C-MET scoring was based on Cappuzzo criteria. In vitro sensitivity was evaluated through WST1 assays and clonogenic tests. Kelly, an ALK mutated neuroblastic cell line, was added as a positive control. Cells were seeded in triplicates in 96-well plates 18h before the treatment with TAE 684 or crizotinib with concentrations varying from 50 to 4000nM and 100 to 10000nM respectively. The half maximal inhibitory concentration of the two drugs (IC50) was estimated after 72h of treatment with 4 and 5 parameters logistic regression models. In clonogenic tests, crizotinib was tested with three schedules of concentrations: low dose (50nM), intermediate (200nM) and high (1000nM). Results: Among NSCLC cell lines, 6 (22%) displayed more than 5 copies of ALK, 19 (70%) presented a gain of 3 or 4 ALK copy number, only one cell line exhibited normal ALK copies and one harbored EML4-ALK translocation. FISH with CEP2 revealed a polysomy of chromosome 2 in cases with ALK HCN. C-MET amplification was observed in only three cell lines. Based on ALK FISH status, 9 NSCLC cell lines were selected to undergo in vitro assays, 6 exhibiting ALK HCN (H2030, H661, A427, H3255, BEN, H1299) and 3 exhibiting low ALK copy number (LCN) (H1975, H1651, H1650). Kelly and A549 cell lines were added as sensitive and resistant controls respectively. In ALK HCN cell lines, two (H661 and A427) were as sensitive as Kelly for crizotinib through clonogenic and WST1 assays. In addition, ALK HCN cell lines exhibited lower IC50 for anti-ALK drugs than cell lines with ALK LCN (median IC50 with crizotinib values: 1750nM [300-2800nM] in ALK HCN cell lines versus 4500nM [800-8000nM] in ALK LCN cell lines, p=0.35). The trend of crizotinib activity was not due to c-MET inhibition. Conclusion: The in vitro assays suggest that ALK HCN may be a predictive marker for sensitivity to crizotinib. The predictive value of ALK HCN would be investigated in in vivo models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1715. doi:1538-7445.AM2012-1715