Background: Higher levels of vascular endothelial growth factor-D (VEGF-D) is a potential predictive biomarker for ramucirumab (Ram) efficacy on overall survival and progression-free survival (PFS) in Ram+FOLFIRI vs Placebo+FOLFIRI for bevacizumab (Bev) refractory metastatic colorectal cancer (mCRC). However, it is not clear whether VEGF-D acts as a predictive biomarker in pts who pretreated without Bev. RAINCLOUD showed the clinical benefit of FOLFIRI+ plus Ram for pts with recurrent CRC refractory to adjuvant chemotherapy (median PFS was 6.2 months (M); ESMO-GI 2022). RAINCLOUD-TR was designed as a prospective biomarker study estimating the association of biomarkers with Ram efficacy. Methods: Plasma sample collections were done at time points of pre-/ post-treatments in pts receiving FOLFIRI+Ram. Measurements of 17 factors (including VEGF-A, VEGF-D and PlGF) were performed by multiplex assay with Luminex technology. The high/ low groups were defined bases on the median plasma level at the baseline. Results: From September 2017 to September 2021, 81 plasma samples were collected from 48 pts and 32 pts in both pre/ and post treatment. Level of VEGF-A, VEGF-D, PlGF and TSP-2 were all significantly higher in post vs pre-treatment [Table]. The ORR analyses were as follows; VEGF-A, VEGF-D and PlGF in low vs high were 50.0%/33.3% (p=0.35, HR 1.97), 55.0% /33.3% (p=0.22, HR 2.39), and 60.0%/28.6% (p=0.35, HR 1.95). The PFS analyses were as follows; VEGF-A, VEGF-D, and PlGF in low vs high were 6.1M/6.0M (p=0.815, HR 0.92), 7.6M /5.6M (p=0.095, HR 0.56), and 8.0M/4.2M (p=0.014, HR 0.40). In other factors, low TSP-2 were better ORR (low vs high: 66.7%/20.0% (p=0.004, HR 7.54) and PFS 7.5M/4.3M (p=0.022, HR 0.45). Conclusions: In this exploratory study, PFS was numerically greater for pts with low vs high VEGF-D for Ram in pts with mCRC pretreated without Bev. VEGF-D may not be predictive biomarker, but TSP-2 may be a potential prognostic biomarker, further studies in larger cohorts are needed to confirm our results. Clinical trial information: UMIN000028678.