Exploratory biomarker findings from regorafenib plus toripalimab in patients with refractory metastatic colorectal cancer (REGOTORI study).

The REGOTORI study showed that some metastatic colorectal cancer (mCRC) patients benefited from programmed death 1 (PD-1) antibody toripalimab plus anti-angiogenic tyrosine-kinase inhibitor regorafenib. However, biomarkers for this combined therapy in mCRC remain unclear. To address this gap, we performed an integrated multi-omics biomarker analysis in REGOTORI. Next-generation sequencing was performed on formalin-fixed, paraffin-embedded (FFPE) tissue and paired plasma samples. Multiplex immunohistochemistry was performed to analyze the tumor microenvironment (TME) on FFPE samples. Variant allele frequency, somatic alterations, tumor mutational burden (TMB), circulating tumor DNA (ctDNA), and TME markers were jointly assessed from both FFPE and plasma samples to explore their associations with clinical outcomes under regorafenib plus toripalimab. A total of 35 patients were included. Patients with lower ctDNA maximum somatic allele frequency (maxAF), high-allele-frequency blood-based TMB (HAF-bTMB), blood-based intratumor heterogeneity (bITH), or HAF-bITH had longer survival time than their respective counterparts. Somatic alterations in SMAD4 (progression-free survival: 2.4 vs 1.6 months; overall survival: not reached vs 5.1 months) or PIK3CA (overall survival: 15.5 vs 5.7 months) were associated with shorter survival time. ctDNA dynamics appeared related to treatment response. High positive rates of CD3+, CD3+CD8+, CD3+CD8-, and PD-1+CD3+ T cells in the stroma area correlated with prolonged progression-free survival and favorable disease control; however, neither the positive rate of PD-L1 cells nor the density of it in the tumor area was associated with survival and response. In this combination-therapy-focused multi-omics analysis integrating tissue genomics, ctDNA features/dynamics and TME profiling, we identified ctDNA maxAF, HAF-bTMB, bITH, HAF-bITH, mutational status of SMAD4 or PIK3CA and TME markers as predictive or prognostic biomarkers for regorafenib plus toripalimab in refractory mCRC.