Response of non-small cell lung cancer harboring different epidermal growth factor receptor mutations to ablative radiotherapy

Abstract

Background: Stereotactic ablative radiation therapy (SABR) provides an alternative treatment for patients with inoperable early-stage lung cancer (ES-LC). The epidermal growth factor receptor (EGFR) plays an important role in tumor progression and treatment resistance in non-small cell lung cancer (NSCLC). EGFR-targeted therapies in combination with radiotherapy (RT) have not been successful at enhancing RT's response or improving tumor control. The response of NSCLCs carrying EGFR mutations to SABR has not been well investigated, although worse overall survival is seen among patients with L858R-EGFR mutations. We aim to evaluate the effect of different EGFR-mutant lung cancers to SABR in vitro and in vivo and provide a deeper understanding of the mechanisms of response and resistance to SABR.

Methods: A549 cells were stably transfected with either wild-type-EGFR (WT), deleted-EGFR (DEL), or L858R-EGFR (L858R) constructs to generate isogenic cell lines. In vitro assessment included colony formation, cell viability, and proliferation assays. Tumor formation was assessed by subcutaneous injection of pre-irradiated cells in yellow fluorescent protein (YFP)/severe combined immunodeficiency (SCID) mice. All mice were sourced from the Animal Resource Division at the McGill University Healthcare Centre. Response to SABR was evaluated in mice injected subcutaneously with isogenic cells and followed with sham or 34 Gy treatment. Tumors collected from both groups were evaluated for SABR effect histologically.

Results: EGFR-mutant cell lines displayed a similar in vitro response to SABR: reduced colony formation, cell viability, and cell cycle arrest in G2. Pre-irradiated WT-EGFR and L858R-EGFR NSCLC cell lines maintained their ability to initiate tumor growth in vivo, whilst pre-irradiated DEL-EGFR cells were unable to form tumors upon injection. Subcutaneous DEL-EGFR xenograft tumors had a significant decrease in tumor volume post-SABR treatment compared to WT and L858R-EGFR xenografts. Histological assessment demonstrated less necrosis and a decrease (P=0.049) of apoptotic cells in DEL-EGFR-treated tumors compared to L858R-EGFR.

Conclusions: Novel demonstration of DEL-EGFR mutation imparting better response to SABR compared to WT-EGFR or L858R-EGFR mutations, consistent with findings from The Cancer Genome Atlas (TCGA), suggesting L858R-EGFR mutations are associated with worse overall survival. Radiation dose fractionation should be investigated further to establish an optimal SABR regimen in the context of LCs and possible overall survival with EGFR mutations.

Keywords: Non-small cell lung cancer (NSCLC); epidermal growth factor receptor (EGFR); stereotactic ablative radiation therapy (SABR).

Figure 1

Patients with L858R-EGFR mutation have a lower overall survival when compared to patients carrying WT (A) and DEL (B) subpopulations. DEL, deletion; EGFR, epidermal growth factor receptor; WT, wild type.

Figure 2

Response to ablative radiation in isogenic EGFR-mutant cell lines. (A) Clonogenic assay of isogenic EGFR-mutant cells. (B) Total number of viable cells in isogenic EGFR mutant cells following SABR. (C) Colony formation assay with colonies staining positive for methylene blue. (D) Cell cycle analysis. (E-G) Proliferation rate analysis. *, P<0.05; **, P<0.01; ***, P<0.001. DEL, deletion; EGFR, epidermal growth factor receptor; PE-A, 2-dimensional phycoerythrin area; SABR, stereotactic ablative radiation therapy; WT, wild type.

Figure 3

Tumor development of irradiated EGFR mutant cell lines. BLI of animals injected with EGFR mutant cells that were pre-irradiated at 34 Gy. Day 0 is when animals received treatment of 34 Gy (treated group). The response and tumor volume were measured in all groups 9 days after treatment. BLI, bioluminescence imaging; DEL, deletion; EGFR, epidermal growth factor receptor; IR, irradiation; WT, wild type.

Figure 4

Response to SABR treatment in animals injected with cells harboring different EGFR mutations. Animals were injected subcutaneously with either WT, DEL or L858R cell lines and divided into two groups: control and treated group with a single dose of 34 Gy. (A) BLI measurements at day 9 after treatment in Ctrl and treated groups. (B) Tumor volume following nine days of treatment. **, P<0.01. BLI, bioluminescence imaging; Ctrl, control; DEL, deletion; EGFR, epidermal growth factor receptor; SABR, stereotactic ablative radiation therapy; WT, wild type.

Figure 5

Histological assessment of collected tumors from control and treated groups of isogenic EGFR mutant cell lines. (A-C) Necrosis analysis: (A) 20× magnification of collected tumor showing 60% of necrosis; (B) 20× magnification of collected tumor showing absence of necrosis; (C) percentage of necrotic area in all control and irradiated cell lines. (D-F) Solid tumor analysis: (D) 40× magnification of collected tumor showing 20% solid tumor; (E) 40× magnification of collected tumor showing 40% solid tumor; (F) solid tumor area % in control and irradiated cell lines. (G-I) Apoptotic cells analysis: (G,H) 40× magnification of collected tumor with black arrows identifying apoptotic cell and 100× magnification (box) of apoptotic cell; (I) number of apoptotic cells in all three cell lines, present in collected tumors. Sides were stained by hematoxylin and eosin. *, P<0.05. DEL, deletion; EGFR, epidermal growth factor receptor; IR, irradiation; WT, wild type.

Figure 6

Proteins expression assessment of collected tumor tissues. (A) Tissues were collected from WT-EGFR (control n=3, treated n=6), DEL-EGFR (control n=2, treated n=6), and L858R-EGFR (control n=3, treated n=6). (B) Densitometry analysis of p-ERK/total-ERK ratio normalized to beta-actin. M#, mouse number. DEL, deletion; EGFR, epidermal growth factor receptor; IR, irradiation; WT, wild type.