Background: RNA expression of epiregulin (EREG) ligands EREG and/or amphiregulin (AREG) has shown correlation with the efficacy of EGFR-targeted therapy in advanced colorectal cancer (aCRC). This finding requires validation, and interaction with MEK-AKT pathway mutations clarified. We examined both ligands and mutations in patients (pts) in a large randomised trial of panitumumab. The a priori hypothesis was that high expression of either AREG or EREG, in RAS-wild type (wt) pts, would predict panitumumab benefit. Methods: AREG and EREG expression and RAS (KRAS and NRAS) and BRAF mutations were assessed in archival tumor from 323 pts randomised to irinotecan (Ir) or irinotecan/panitumumab (IrPan) as second-line treatment of aCRC (PICCOLO trial, Lancet Oncol 14: 749-759). A predefined dichotomous model classified ligand expression as “high” (either EREG or AREG in top tertile for mRNA level) or “low” (neither EREG nor AREG in top tertile). Prognostic effect was assessed, then predictive effect by testing interaction with drug impact. The primary population was RAS-wt (n=220); primary endpoint was progression-free survival (PFS). Results: High ligand expression was not a significant prognostic marker for PFS (HR 0.93, 95% CI 0.68-1.27, p=0.64) or overall survival (OS) (HR 0.79, 95% CI, 0.58-1.09, p=0.15). However, it was significantly predictive. For RAS-wt pts with high ligand expression, median PFS was 8.3 months (IrPan) vs 4.4 months (Ir) (HR=0.62, 95%CI 0.49-0.78, p<0.001). Conversely, RAS-wt pts with low expression gained no benefit: 3.2 months (IrPan) vs. 4 months (Ir) (HR=0.97, 95%CI 0.8-1.17, p=0.73). The ligand-treatment interaction was p=0.01. Less effect was seen on the secondary endpoints response rates (interaction p=0.09) and overall survival (interaction p=0.11). Ligand expression was markedly lower in BRAF-mutated than all-wt tumours (p<0.001), however ligand expression remained predictive of panitumumab effect following adjustment for BRAF. Conclusions: The hypothesis was confirmed: high ligand expression predicts panitumumab benefit on PFS in RAS-wt pts; there was no evidence of benefit with low ligand expression. Optimisation of the ligand model for clinical use is needed, however this work confirms that EREG and AREG are potentially useful biomarkers for anti-EGFR therapy.