Biology Contribution Short-Course Treatment With Gefitinib Enhances Curative Potential of Radiation Therapy in a Mouse Model of Human Non-Small Cell Lung Cancer Sivan M. Bokobza, PhD, Yanyan Jiang, PhD, Anika M. Weber, MSc, Aoife M. Devery, PhD, and Anderson J. Ryan, PhD Department of Oncology, Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, United Kingdom Received Oct 14, 2013, and in revised form Dec 19, 2013. Accepted for publication Dec 21, 2013. Summary We show that an epidermal growth factor receptor (EGFR) tyrosine kinase in- hibitor (gefitinib) increases clonogenic cell killing by radiation in non-small cell lung cancer (NSCLC) cell lines that are highly sensitive to gefitinib alone. Further- more, in a mouse xenograft model of human EGFR mu- tation- positive NSCLC, a short course of gefitinib treatment markedly enhanced the antitumor ef- fects of a single dose of ra- diation, producing effective cures in a model of NSCLC, suggesting that this combi- nation may be of clinical in- terest for stereotactic radiation. Purpose: To evaluate the combination of radiation and an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) in preclinical models of human non-small cell lung can- cer. Methods and Materials: Sensitivity to an EGFR TKI (gefitinib) or radiation was assessed using proliferation assays and clonogenic survival assays. Effects on receptor signal transduction path- ways (pEGFR, pAKT, pMAPK) and apoptosis (percentage of cleaved PARP Poly (ADP-ribose) polymerase (PARP)) were assessed by Western blotting. Radiation-induced DNA damage was assessed by gH2AX immunofluorescence. Established (100 mm 3 ) EGFR-mutated (HCC287) or EGFR wild-type (A549) subcutaneous xenografts were treated with radiation (10 Gy, day 1) or gefitinib (50 mg/kg, orally, on days 1-3) or both. Results: In non-small cell lung cancer (NSCLC) cell lines with activating EGFR mutations (PC9 or HCC827), gefitinib treatment markedly reduced pEGFR, pAKT, and pMAPK levels and was associated with an increase in cleaved PARP but not in gH2AX foci. Radiation treat- ment increased the mean number of gH2AX foci per cell but did not significantly affect EGFR signaling. In contrast, NSCLC cell lines with EGFR T790M (H1975) or wild-type EGFR (A549) were insensitive to gefitinib treatment. The combination of gefitinib and radiation treatment in cell culture produced additive cell killing with no evidence of synergy. In xenograft models, a short course of gefitinib (3 days) did not significantly increase the activity of radiation treatment in wild-type EGFR (A549) tumors (PZ.27), whereas this combination markedly increased the activity of radiation (P< .001) or gefitinib alone (PZ.002) in EGFR-mutated HCC827 tumors, producing sustained tumor regressions. Conclusions: Gefitinib treatment increases clonogenic cell killing by radiation but only in cell lines sensitive to gefitinib alone. Our data suggest additive rather than synergistic interactions between gefitinib and radiation and that a combination of short-course gefitinib and high- dose/-fraction radiation may have the greatest potential against the subsets of lung cancers harboring activating mutations in the EGFR gene. Ó 2014 Elsevier Inc. Reprint requests to: Anderson J. Ryan, PhD, Department of Oncology, Gray Institute for Radiation Oncology and Biology, University of Oxford, Roosevelt Dr, Headington, Oxford, OX3 7DQ, United Kingdom. Tel: (44) 1865 617409; E-mail: anderson.ryan@oncology.ox.ac.uk Conflict of interest: none. Supplementary material for this article can be found at www.redjournal.org. Int J Radiation Oncol Biol Phys, Vol. 88, No. 4, pp. 947e954, 2014 0360-3016/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ijrobp.2013.12.038 Radiation Oncology International Journal of biology physics www.redjournal.org