Background: Angiogenesis-factors (AFs) have been reported as prognostic biomarkers for metastatic colorectal cancer (mCRC). RAS status in circulating-tumor DNA (ctDNA) is considered to be associated with resistance and potential efficacy of re-challenge treatment of anti-EGFR antibody in mCRC with RAS wild-type tumors. However, there were few evidence to prospectively evaluate the dynamic change of AFs and association of RAS status in ctDNA with clinical outcomes in anti-angiogenic therapies after anti-EGFR therapy for mCRC. Methods: Plasma samples were collected at pre-, day 15 after, and post-treatments in the pre-planned biomarker study of the JACCRO CC-16 trial which evaluated 2nd-line FOLFIRI plus RAM after anti-EGFR therapy in mCRC patients with RAS wild-type tumors (UMIN000034885). In 42 enrolled patients (median age, 68-y; ECOG PS 0/1, 31/11; all pts with left-sided tumors; median PFS, 6.8 months; median OS, 21.4 months; ORR, 10.3 %), some AFs (VEGF-A, VEGF-D, PlGF, HGF, IL-8, and angiopoietin-2) and RAS status in plasma were measured using the multiplex assay with Luminex technology and OncoBEAM RAS CRC Kit, respectively. The associations between clinical outcomes and AFs/RAS status were investigated at timepoint of pre-treatment, using t-test and log-rank test. The median value was used as the cut-off value of AFs for high or low. The associations between AFs and RAS status were investigated using Brunner-Munzel test. Results: AFs and RAS status were evaluable in 38 and 41 patients, respectively. The OS was significantly longer in patients with IL-8 low compared to patients with IL-8 high (32.2 vs. 14.5 months, HR 0.36, p= 0.024). However, there was no difference in PFS between patients with IL-8 high and low. On the contrary, RAS mutations were detected at pre-treatment in 18 (44%) patients: KRAS codon12 83%, codon13 17%, codon61 67%, codon117 6%, codon146 11%, NRAS codon12 39%, codon61 78% in RAS mutations. Median PFS was 6.4 months in patients with RAS mutations in ctDNA, while it was 7.4 months in patients without RAS mutations (p = 0.10). Median OS was significantly longer in patients without RAS mutations compared to patients with RAS mutations (25.4 vs. 14.5 months, HR 0.39, p = 0.024). The ORR was 14.3% and 5.6% in patients without RAS mutations and with RAS mutations, respectively. In an exploratory analysis regarding the relationship between AFs and RAS status, IL-8 level was significantly higher in patients with RAS mutations than those without in ctDNA. Conclusions: This biomarker study demonstrated the associations between clinical outcomes and IL-8/RAS status in blood before treatment in RAS wild-type mCRC treated with 2nd-line FOLFIRI plus RAM after anti-EGFR antibody therapy. Clinical trial information: UMIN000034885.