Multiplexed P21/MCM-2 detection predicts relapse and may identify tyrosine kinase inhibitor-resistant patients in clear cell renal cell carcinoma.

Current clinical tools for predicting relapse and guiding adjuvant therapy in clear cell renal cell carcinoma (ccRCC) lack precision, especially in intermediate-risk disease. This study evaluated whether a non-proliferative tumour cell phenotype, P21 (CDKN1A inhibitor) positive and MCM2 (DNA replication protein) negative (P21⁺/MCM2⁻), could serve as a robust biomarker to improve prognostic stratification and guide post-nephrectomy treatment decisions. We used multiplex immunofluorescence and AI-based image analysis on nephrectomy specimens from three independent ccRCC cohorts: UK arm of the SORCE trial (n=382), Korean (n=71), and Scottish (n=88). An optimal 2% cut-off for P21⁺/MCM2⁻ cells was determined using X-tile software. Additional analyses assessed endoglin/CD105 co-expression, paired primary-metastatic samples (n=41), and associations with adjuvant sorafenib therapy. In two intermediate-risk ccRCC cohorts (SORCE, n=63; Korean, n=71), patients with >2% P21⁺/MCM2⁻ cells had significantly longer time to relapse (HR=0.17, 95% CI: 0.06-0.54; HR=0.27, 95% CI: 0.10-0.72). Prognostic value was confirmed in high-risk (SORCE, HR=0.43, 95% CI: 0.19-0.99) and all-risk (Scottish, HR=0.37, 95% CI: 0.14-0.98) cohorts. Notably, patients with high P21⁺/MCM2⁻ levels on placebo fared better than those receiving adjuvant TKI therapy (HR=0.29, 95% CI: 0.16-0.50). In 41 paired samples, 85% showed higher P21⁺/MCM2⁻ abundance in metastases than in primary tumours. As a conclusion, P21⁺/MCM2⁻ cell count is a robust biomarker that refines relapse risk stratification in ccRCC and identifies patients who may not benefit from adjuvant tyrosine kinase inhibitor therapy. High levels of these non-proliferative, senescent-like cells suggest tumour dormancy and a more favourable outcome without treatment.