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Title: MicroRNA-31-3p expression and benefit from anti-EGFR inhibitors in metastatic colorectal cancer patients enrolled in the prospective phase II PROSPECT-C trial. Author’s contribution: DC, IC, SR, NS, DW, KHK, CB and NV recruited patients in the trial. GA, AL, GV, SH, MDD, HL, JCH performed experiments. MF and MR reviewed pathology and scored microRNA expression. KK, RK CP performed statistical analyses. NT, NF and NK performed radiological procedures. JT, RB, IR, AB coordinated the trial and the tissue collection. DC and NV supervised the study. All the authors reviewed the manuscript. work acknowledge and ABSTRACT Purpose: Anti-Epidermal Growth Factor Receptor (EGFR) monoclonal antibodies (mABs) are effective in the treatment of metastatic colorectal cancer (mCRC) patients. RAS status and tumour location (sidedness) are predictive markers of patients’ response to anti-EGFR mABs. Recently, low microRNA-31-3p expression levels have been correlated with clinical benefit from the anti-EGFR mAb cetuximab. Here we aimed to validate the predictive power of microRNA-31-3p in a prospective cohort of chemo-refractory mCRC patients treated with single agent anti-EGFR mABs. Experimental Design: microRNA-31-3p was tested by in-situ hybridization in ninety-one pre-treatment core biopsies from metastatic deposits of forty-five mCRC patients. Sequential tissue biopsies obtained before treatment, at the time of partial response, and at disease progression were tested to monitor changes in microRNA-31-3p expression over treatment. MicroRNA-31-3p expression, sidedness, and RAS status in pre-treatment cell-free DNA were combined in multivariable regression models to assess the predictive value of each variable alone or in combination. Results: Patients with low microRNA-31-3p expression in pre-treatment biopsies showed better overall response rate, as well as better progression free and overall survival, compared to those with high microRNA-31-3p expression. The prognostic effect of microRNA-31-3p was independent from age, gender and sidedness. No significant changes in the expression of microRNA-31-3p were observed when sequential tissue biopsies were tested in long-term or poor responders to anti-EGFR mABs. MicroRNA-31-3p scores were similar when pre-treatment biopsies were compared with treatment-naïve archival tissues (often primary CRC). Conclusions: Our study validates the role of microRNA-31-3p as potential predictive biomarker of selection for anti-EGFR mABs. negative predictive anti-EGFR (mCRC) microRNA-31-3p potential biomarker for the selection of candidates to first line treatment with a combination of chemotherapy and anti-EGFR treatment. we confirm the predictive value of microRNA-31-3p in a prospective cohort of chemo-refractory mCRC patients treated with single agent cetuximab in a phase II trial. We show that: microRNA-31-3p can be scored using in situ hybridization on pre-treatment biopsies; microRNA-31-3p expression is comparable between primary CRC and metastases; microRNA-31-3p expression does not change during cetuximab treatment; and that patients with low microRNA-31-3p expression had better disease control, progression free survival, and overall survival compared to patients with high microRNA-31-3p expression. We suggest that microRNA-31-3p analysis might be incorporated in the work-up of mCRC along with tumour sidedness and RAS testing, in order to further refine the selection of potential responders to anti-EGFR treatments. baseline and, 20 months later at end of treatment due to RECIST 1.1 progression in non-target disease in patient 1005. The images show maintained benefit in the target and a mixed response in the non-target abdominal wall disease with stable appearances of and At baseline the biopsy was obtained from at progression the was of the tract can be seen in the tissue No changes in observed in