3540 Background: Retrospective experiences have investigated the potential influence of EGFR intron 1 CA repeats on the efficacy of cetuximab-containing treatments. Different series, adopting different criteria to define short (S) and long (L) variants, have provided contrasting results. Methods: We designed a prospective confirmatory study, to detect a HR for PFS of 1.75 for L- compared to SS genotypes in a population of KRAS and BRAF wild-type irinotecan-resistant mCRC pts treated with cetuximab and irinotecan. Estimating a prevalence of 60% of the SS variant and setting a two-sided alfa=0.05 with a power of 80%, 104 events were required. We defined S and L allelic variants those presenting < and ≥20 CA repeats, respectively. EGFR (CA)_n repeat polymorphism was assessed following a 5’-end [γ-33P] ATP-labeled PCR protocol. Results: One-hundred-fifteen pts were included. At a median follow up of 21.9 months, PFS and OS were 5.2 and 13.4 months, respectively. Thirty-three (29%) out of 114 evaluable pts achieved response. EGFR (CA)_n repeat genotype was L- and SS in 45 (40%) and 68 (60%) out of 113 evaluable cases. No differences in PFS or OS were observed between L- and SS genotypes (median PFS: 4.4 vs. 5.3 months, HR: 1.00 [95%CI: 0.67-1.51], p=0.991; median OS: 11.3 vs. 14.2 months, HR: 1.30 [95%CI: 0.80-2.22], p=0.261). Ten (22%) out of 45 L- pts achieved response compared to 22 (33%) out of 67 SS pts (Fisher’s Exact test: p=0.617). Other exploratory analyses adopting different cut-off values reported in literature led to similar results. Conclusions: This prospective study, including a clinically homogenous and molecularly selected population, does not confirm any predictive or prognostic effect for EGFR (CA)_n repeat allelic variants with respect to the efficacy of cetuximab and irinotecan in advanced lines of treatment. The present experience strengthens the need of prospectively challenging retrospective findings, as an essential step on biomarkers’ way toward clinical practice.