Background: REVERCE demonstrated longer overall survival (OS) with the therapeutic sequence of regorafenib (R) followed by cetuximab (C) ± irinotecan (R-C arm) compared with that of C ± irinotecan followed by R (C-R arm) for patients (pts) with pretreated metastatic colorectal cancer (mCRC; median OS 17.4 vs. 11.6 months, HR 0.61; Shitara K, et al. GI Symposium 2018). Biomarker analyses assessed the association of multiple candidate biomarkers with clinical outcomes. Methods: Pts with KRAS exon 2 wild-type mCRC after failure of fluoropyrimidine, oxaliplatin, and irinotecan treatment were randomized. The primary endpoint was OS. Key secondary endpoints included progression-free survival (PFS) with initial treatment (PFS1), PFS with second treatment (PFS2), safety, and QOL. Sequential biomarker analysis including oncogenic alterations from ctDNA or multiple serum proteins related to the EGF or VEGF pathway was an exploratory endpoint. Results: Among the randomized 101 pts, plasma and serum samples were collected from 98 pts. OS HR in the biomarker cohort (n = 98, HR 0.61) and all RAS/RAF wild types (n = 86, HR 0.60) at baseline were consistent with that of the overall cohort. R-C arm consistently showed longer OS in all subgroups according to serum proteins biomarkers (median value as cut off). Significant interactions between PlGF or VEGFR-3 level and PFS1 were observed, favoring R over C in higher PlGF and VEGFR-3 level. Among RAS/RAF wild types at baseline, emerging RAS, BRAF, EGFR S492R mutations, and HER2 and MET amplification were observed in 1,0,0,2,2 patients after R and 11,1,2,4,4 patients (overlapping) after C. Conclusions: This trial consistently showed longer OS with R followed by C versus the reverse sequence for pretreated mCRC pts regardless of biomarker subgroups. Emerging oncogenic alterations are more frequently observed after C treatment than R. Clinical trial information: UMIN000011294.