Abstract A154: Preclinical investigation of gefitinib combined with radiation in EGFR-mutation positive non-small cell lung cancer.
Authors: Bokobza Sivan , Weber Anika , Devery Aoife , Ryan Anderson 
Published: June 2012

A subset of non-small cell lung cancer (NSCLC) patients with EGFR mutation positive tumors gain marked clinical benefit from treatment with EGFR tyrosine kinase inhibitors (TKIs), although the benefits can be short-lived as drug resistance emerges. This preclinical study investigated the potential of EGFR TKI treatment to augment the activity of radiotherapy in EGFR mutation positive human NSCLC cell lines.

Sensitivity to the EGFR TKI gefitinib (0−3.0 μM) or radiotherapy (0–6 Gy) was assessed using clonogenic survival assays. For combination studies, clonogenic assays were performed on NSCLC cells exposed to a single dose of radiation (3 Gy) and 24 h exposure to gefitinib (0.1 μM). Effects on receptor signal transduction pathways (pEGFR, pAKT, pMAPK) and apoptosis (percentage cleaved/total PARP) were assessed by western blotting. DNA double strand break repair was assessed by counting residual H2AX foci by immunofluorescence 24 h after irradiation (3 Gy).

NSCLC cell lines with activating EGFR mutations (PC-9 and HCC-827) were sensitive to both gefitinib (IC50 ± SEM 0.052 ± 0.007 M and 0.014 ± 0.004μM, respectively, n=6) and radiation (surviving fraction at 3 Gy ± SEM, SF3 = 0.76 ± 0.07 and 0.61 ± 0.03, respectively, n=6). Gefitinib treatment markedly reduced pEGFR (–72.1%), pAKT (−60.1%) and pMAPK (−92.5%) levels 4 h after treatment, and was associated with an increase in cleaved PARP (15.7%) but not in H2AX foci. Radiation treatment increased mean H2AX foci per cell (± SEM) from 9.3 ± 0.09 to 13.7 ± 0.31, (n=3, p=&lt;0.01) and cleaved PARP (20%) but did not significantly affect pEGFR signaling. In contrast, NSCLC cell lines with an EGFR T790M mutation (H1975), or null for EGFR expression (H520), were insensitive to gefitinib (IC50 &gt;3 μM) but were sensitive to radiation (SF3 = 0.43 ± 0.05 and 0.34 ± 0.05, respectively, n=6). Gefitinib treatment had no significant effect on EGFR signaling, cleaved PARP, or H2AX foci in H1975 or H520 cells, whereas radiation treatment increased levels of cleaved PARP (30.7%), compared with non-irradiated controls.

In PC-9 and HCC-827 cells, the addition of gefitinib (0.1 μM, 24 h) to radiation reduced clonogenic survival (± SEM) by −30.8 ± 2.9% (P=0.03) and −46.2 ± 6% (P &lt;0.01), respectively, compared with radiation alone, but had no significant effect on the radiation sensitivity of H1975 or H520. Following combination treatment in PC-9, there was a small increase (5.4%) in the levels of cleaved PARP compared with irradiation alone. However, there was no evidence of increased effects on EGFR signaling (pEGFR, pAKT, pMAPK) or DNA repair (H2AX foci) in the combination treatment in any of the cell lines tested.

In conclusion, our data demonstrate that gefitinib treatment increases clonogenic cell killing by radiation, but only in cell lines sensitive to gefitinib alone. Our data provide no evidence for synergistic or mechanistic interactions between gefitinib and radiation and suggest that the combination of gefitinib and radiation may have the greatest potential against the subsets of lung cancers that are most sensitive to EGFR-TKIs, such as those harboring activating mutations in the EGFR gene.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A154.