Regorafenib plus nivolumab (RegoNivo) combination has shown promising anti-cancer activity in multiple cancer types. REBNOBATE trial is a single-arm multicenter phase 2 trial of first-line RegoNivo in patients (pts) with uHCC (NCT04310709). Here we report the clinical outcomes of the RENOBATE study and integrative biomarker analysis using circulating tumor DNA (ctDNA) and single cell RNA sequencing (scRNA-seq) analysis. Method: Adult pts with ECOG PS 0 or 1, BCLC stage B or C, and no prior systemic therapy were eligible for the study. Pts received nivolumab 480 mg iv, every 4 wks, and regorafenib 80 mg daily po, 3 wks on/1 wk off, every 4 wks. Tumor response was assessed according to RECIST v1.1 every 8 wks. ctDNA analysis was performed using the Guardant 360 CDx (n=42). scRNA-seq was performed using PBMCs at baseline (e.g., C1D1) and on treatment (eg., C1D15) from early progression (EP) group (14 pts with PD or tumor burden increases towards PD on the 1st evaluation) and long-term response (LR) group (15 pts with PR or SD > 6 months). Results: 42 pts were enrolled. Response rates were 31.0% and median progression-free survival (was 7.4 mo (95% CI, 4.2-13.0) and 1-year OS rate was 80.5% (95% CI, 63.0-90.3%). Aberrations of Wnt/β-Catenin and PI3K/mTOR pathways identified in ctDNA were not associated with efficacy outcomes. scRNA-seq revealed that genes upregulated in immune cells on C1D15 (vs. C1D1) were associated with immune activation and regulation of angiogenesis. Unsupervised clustering identified 13 immune subsets; among them, classical monocyte showed the most prominent changes in their proportion and gene expression profiles. After RegoNivo treatment, CD8 clusters had enhanced expressions of genes related to cytotoxicity, tissue homing (i.e., CXCR6, CX3CR1, and CXCR3) and proliferation (MKI67), and monocyte clusters showed an enrichment of the gene signatures representing polarization towards M1-related features. Importantly, the enrichment of cytotoxic features of the T cell clusters and M1-polarizing features of monocyte clusters were more pronounced in LR group than in EP group. From the analysis of differentially expressed genes monocyte clusters between the EP and LR groups, a surface molecule ‘X’ was identified as a potential biomarker involved in resistance to RegoNivo. RegoNivo showed promising efficacy outcomes in pts with uHCC. Through scRNA-seq of serial PBMCs, we identified enhanced cellular immune responses induced by RegoNivo and a potential predictive marker for immunotherapy in pts with uHCC. Our study highlighted that regorafenib might synergize with anti-PD-1 in pts with uHCC. Association between differential features of T cell and monocyte clusters induced by this combination and distinct clinical outcomes highlights the importance of immune-modulation in HCC pts suggesting a potential to further harness these signals Citation Format: Hyung-Don Kim, Seyoung Jung Jung, Baek-Yeol Ryoo, Min-Hee Ryu, Beodeul Kang, Hong Jae Chon, Jung Yong Hong, Ho Yeong Lim, Jeong Seok Lee, June-Young Koh, Changhoon Yoo. Integrative biomarker analysis of regorafenib plus nivolumab (RegoNivo) in unresectable hepatocellular carcinoma (uHCC): A multicenter phase 2 RENOBATE trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1392.

Recent technical advances have enabled unbiased transcriptomic and epigenetic analysis of each cell, known as "single-cell analysis". Single-cell analysis has a variety of technical approaches to investigate the state of each cell, including mRNA levels (transcriptome), the immune repertoire (immune repertoire analysis), cell surface proteins (surface proteome analysis), chromatin accessibility (epigenome), and accordance with genome variants (eQTLs; expression quantitative trait loci). As an effective tool for investigating robust immune responses in coronavirus disease 2019 (COVID-19), many researchers performed singlecell analysis to capture the diverse, unbiased immune cell activation and differentiation. Despite challenges elucidating the complicated immune microenvironments of chronic inflammatory diseases using existing experimental methods, it is now possible to capture the simultaneous immune features of different cell types across inflamed tissues using various single-cell tools. In this review, we introduce patient-based and experimental mouse model research utilizing single-cell analyses in the field of chronic inflammatory diseases, as well as multi-organ atlas targeting immune cells.

In this phase 2 REBNOBATE trial, we evaluated regorafenib-nivolumab as front-line treatment for unresectable hepatocellular carcinoma (uHCC). Patients (n = 42) received nivolumab 480 mg every 4 weeks, and regorafenib 80 mg daily (3-weeks on/1-week off schedule). Single-cell RNA sequencing was performed using peripheral blood mononuclear cells from early progressors (progressively increased tumor burden) and long-term responders (response/stable disease for ≥ 10 months). The overall response rate in the study was 31.0%, with a median progression-free survival of 7.4 months and a 1-year overall survival rate of 80.5%. Regorafenib-nivolumab was well-tolerated (no new safety signal). Long-term responders exhibited T-cell receptor repertoire diversification; enrichment of genes representing immunotherapy-responsiveness and cytotoxicity in MKI67+ proliferating CD8+ T cells; and interaction between MKI67+ proliferating CD8+ T cells and classical monocytes through IFN-γ pathways with a higher probability of M1-directed polarization of monocytes. Classical monocytes from early progressors exhibited upregulation of TMEM176A/B coupled with an ineffective inflammasome response. Regorafenib-nivolumab is feasible as front-line treatment for uHCC. Our findings may support the development of biomarkers, or novel immunotherapies to overcome resistance in uHCC.