Single-cell RNA sequencing-derived signatures define response patterns to atezolizumab + bevacizumab in advanced hepatocellular carcinoma

Background & Aims: The combination of atezolizumab and bevacizumab (atezo+bev) is the current standard of care for advanced hepatocellular carcinoma (HCC), providing a median overall survival (OS) of 19.2 months. Here, we aim to uncover the underlying cellular processes driving clinical benefit vs. resistance to atezo+bev. Methods: We harnessed the power of single-cell RNA sequencing in advanced HCC to derive gene expression signatures recapitulating 21 cell phenotypes. These signatures were applied to 422 RNA-sequencing samples of patients with advanced HCC treated with atezo+bev (n = 317) vs. atezolizumab (n = 47) or sorafenib (n = 58) as comparators. Results: We unveiled two distinct patterns of response to atezo+bev. First, an immune-mediated response characterised by the combined presence of CD8+ T effector cells and pro-inflammatory CXCL10+ macrophages, representing an immune-rich microenvironment. Second, a non-immune, angiogenesis-related response distinguishable by a reduced expression of the VEGF co-receptor neuropilin-1 (NRP1), a biomarker that specifically predicts improved OS upon atezo+bev vs. sorafenib (p = 0.039). Primary resistance was associated with an enrichment of immunosuppressive myeloid populations, namely CD14+ monocytes and TREM2+ macrophages, and Notch pathway activation. Based on these mechanistic insights we define “Immune-competent” and “Angiogenesis-driven” molecular subgroups, each associated with a significantly longer OS with atezo+bev vs. sorafenib (p of interaction = 0.027), and a “Resistant” subset. Conclusion: Our study unveils two distinct molecular subsets of clinical benefit to atezolizumab plus bevacizumab in advanced HCC (“Immune-competent” and “Angiogenesis-driven”) as well as the main traits of primary resistance to this therapy, thus providing a molecular framework to stratify patients based on clinical outcome and guiding potential strategies to overcome resistance. Impact and implications: Atezolizumab + bevacizumab (atezo+bev) is standard of care in advanced hepatocellular carcinoma (HCC), yet molecular determinants of clinical benefit to the combination remain unclear. This study harnesses the power of single-cell RNA sequencing, deriving gene expression signatures representing 21 cell subtypes in the advanced HCC microenvironment. By applying these signatures to RNA-sequencing samples, we reveal two distinct response patterns to atezo+bev and define molecular subgroups of patients (“Immune-competent” and “Angiogenesis-driven” vs. “Resistant”) with differential clinical outcomes upon treatment with atezo+bev, pointing towards the role of immunosuppressive myeloid cell types and Notch pathway activation in primary resistance to atezo+bev. These results may help refine treatment strategies and improve outcomes for patients with advanced HCC, while also guiding future research aimed at overcoming resistance mechanisms.