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<h4>Background</h4>There is a need for improved selection of patients for adjuvant chemotherapy after resection of non-metastatic colorectal cancer (CRC). Regulator of chromosome condensation 2 (RCC2) is a potential prognostic biomarker. We report on the establishment of a robust protocol for RCC2 expression analysis and prognostic tumour biomarker evaluation in patients who did and did not receive adjuvant chemotherapy.<h4>Materials and methods</h4>RCC2 was analysed in 2916 primary CRCs from the QUASAR2 randomised trial and two single-hospital Norwegian series. A new protocol using fluorescent antibody staining and digital image analysis was optimised. Biomarker value for 5-year relapse-free survival was analysed in relation to tumour stage, adjuvant chemotherapy and the molecular markers microsatellite instability, <i>KRAS</i>/<i>BRAF</i> <sup>V600E</sup>/<i>TP53</i> mutations and <i>CDX2</i> expression.<h4>Results</h4>Low RCC2 expression was scored in 41% of 2696 evaluable samples. Among patients with stage I-III CRC who had not received adjuvant chemotherapy, low RCC2 expression was an independent marker of inferior 5-year relapse-free survival in multivariable Cox models including clinicopathological factors and molecular markers (HR 1.45, 95% CI 1.09 to 1.94, p=0.012, N=521). RCC2 was not prognostic in patients who had received adjuvant chemotherapy, neither in QUASAR2 nor the pooled Norwegian series. The interaction between RCC2 and adjuvant chemotherapy for prediction of patient outcome was significant in stage III, and strongest among patients with microsatellite stable tumours (p<sub>interaction</sub>=0.028).<h4>Conclusions</h4>Low expression of RCC2 is a biomarker for poor prognosis in patients with stage I-III CRC and seems to be a predictive biomarker for effect of adjuvant chemotherapy.

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Department of Molecular Oncology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway.