Background: Rechallenge therapy with epidermal growth factor receptor (EGFR) inhibitors in chemo-refractory metastatic colorectal cancer (mCRC) is an emerging therapeutic approach. Trifluridine-tipiracil is approved for treatment of chemo-refractory mCRC patients. Methods: Chemo-refractory RAS WT mCRC patients, that had a major response (partial or complete response) to first-line chemotherapy plus an anti-EGFR monoclonal antibody and had an-anti-EGFR drug-free interval during second-line therapy of four or more months were randomized in a phase II trial to assess the addition of the anti-EGFR monoclonal antibody panitumumab to trifluridine-tipiracil as third-line rechallenge therapy. The primary endpoint was progression free survival (PFS). Baseline plasma was analyzed for circulating free tumor (ct) DNA by using Biocartis Idylla platform to detect mutations in KRAS, NRAS, BRAF (V600E) and EGFRextracellular domain (S492R). In 24 patients with baseline RAS/BRAF wild type (WT) ctDNA, Foundation One liquid CDx (324 gene profiling) was also performed before treatment and at disease progression. Results: 62 patients were treated with trifluridine-tipiracil (31 patients, arm A) or with trifluridine-tipiracil plus panitumumab (31 patients, arm B). As of September 16, 2022, 1 patient in arm A and 2 patients in arm B were on treatment. The primary endpoint was met. Median PFS (mPFS) was 4 months in arm B versus 2.5 months in arm A [hazard ratio (HR): 0.48; 95% CI 0.28-0.82; P = 0.007]. Baseline plasma RAS/BRAF WT ctDNA was found in 23/31 patients in Arm A and in 26/31 patients in Arm B. In this group, mPFS was 4.5 months in Arm B versus 2.6 months in Arm A (HR: 0.48; 95% CI 0.26-0.89; P = 0.019). Disease control (major responses plus stable disease) was higher for patients in Arm B compared to Arm A (81% versus 48%), whereas disease progression was the best response in 19% versus 52% patients, respectively. PFS rates at 6 and 12 months were 38.5% and 15.4% in arm B versus 13% and 0% in arm A. At disease progression, Foundation One liquid CDx detected several mutations within the EGFR pathway, which could correlate with cancer cell resistance to panitumumab. Conclusions: This is the first prospective randomized trial which evaluated anti-EGFR monoclonal antibody (panitumumab) in addition to standard of care (trifluridine-tipiracil) as third-line rechallenge therapy in chemo-refractory RASWT mCRC patients. Baseline liquid biopsy allows selection of RAS/BRAF WT ctDNA patients who could have a relevant clinical benefit. Clinical trial information: NCT05468892.