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EGFR is a therapeutic target in NSCLC for EGFR-mutant patients. Proximity ligation assay (PLA) is a method to detect functional signaling associated protein complexes. Growth factor receptor bound protein 2 (GRB2) is an adaptor protein that binds to the phosphorylated residues of active EGFR. Interaction of EGFR and GRB2 correlates with active EGFR signaling and leads to activation of the MAPK/ERK pathway.A PLA developed to detect EGFR-GRB2 interaction was measured by quantitative immunofluorescence using Automated Quantitative Analysis technology. EGFR pathway activation was assessed in patients with NSCLC with different mutation status along with overall EGFR expression. Additionally, the PLA to detect EGFR-GRB2 interaction was evaluated as a prognostic marker in two cohorts of patients with lung adenocarcinoma.The PLA to detect EGFR-GRB2 interaction was unrelated to overall EGFR expression or mutation in a series of patients with NSCLC with known mutation status. EGFR-mutant (p = 0.04) and EGFR/KRAS wild-type tumors (p = 0.0049) had significantly higher EGFR pathway activation compared with KRAS-mutant cases, with no significant difference shown between mutation sites. In two series of patients with lung adenocarcinoma, the PLA to detect EGFR-GRB2 interaction was independently associated with longer survival (hazard ratio = 0.46, 95% confidence interval: 0.2–0.78, p = 0.0085 and hazard ratio = 0.48, 95% confidence interval: 0.2–0.85, p = 0.017). Total EGFR protein expression alone was not correlated with outcome.EGFR colocalization with GRB2 as assessed by PLA is not correlated with EGFR expression levels or mutation status, defining a patient group that may show EGFR pathway activation, as illustrated by its prognostic value. Future studies may determine whether this group is more likely to respond to EGFR-targeted therapies.