Programmed cell death protein 1 (PD-1) inhibitors have modest efficacy as a monotherapy in hepatocellular carcinoma (HCC). A personalized therapeutic cancer vaccine (PTCV) may enhance responses to PD-1 inhibitors through the induction of tumor-specific immunity. We present results from a single-arm, open-label, phase 1/2 study of a DNA plasmid PTCV (GNOS-PV02) encoding up to 40 neoantigens coadministered with plasmid-encoded interleukin-12 plus pembrolizumab in patients with advanced HCC previously treated with a multityrosine kinase inhibitor. Safety and immunogenicity were assessed as primary endpoints, and treatment efficacy and feasibility were evaluated as secondary endpoints. The most common treatment-related adverse events were injection-site reactions, observed in 15 of 36 (41.6%) patients. No dose-limiting toxicities or treatment-related grade ≥3 events were observed. The objective response rate (modified intention-to-treat) per Response Evaluation Criteria in Solid Tumors 1.1 was 30.6% (11 of 36 patients), with 8.3% (3 of 36) of patients achieving a complete response. Clinical responses were associated with the number of neoantigens encoded in the vaccine. Neoantigen-specific T cell responses were confirmed in 19 of 22 (86.4%) evaluable patients by enzyme-linked immunosorbent spot assays. Multiparametric cellular profiling revealed active, proliferative and cytolytic vaccine-specific CD4⁺ and CD8⁺ effector T cells. T cell receptor β-chain (TCRβ) bulk sequencing results demonstrated vaccination-enriched T cell clone expansion and tumor infiltration. Single-cell analysis revealed posttreatment T cell clonal expansion of cytotoxic T cell phenotypes. TCR complementarity-determining region cloning of expanded T cell clones in the tumors following vaccination confirmed reactivity against vaccine-encoded neoantigens. Our results support the PTCV’s mechanism of action based on the induction of antitumor T cells and show that a PTCV plus pembrolizumab has clinical activity in advanced HCC. ClinicalTrials.gov identifier: NCT04251117.

Background: Novel immunotherapy combination therapies have improved outcomes for patients with hepatocellular carcinoma (HCC), but responses are limited to a subset of patients. Little is known about the inter- and intra-tumor heterogeneity in cellular signaling networks within the HCC tumor microenvironment (TME) that underlie responses to modern systemic therapy. Methods: We applied spatial transcriptomics (ST) profiling to characterize the tumor microenvironment in HCC resection specimens from a prospective clinical trial of neoadjuvant cabozantinib, a multi-tyrosine kinase inhibitor that primarily blocks VEGF, and nivolumab, a PD-1 inhibitor in which 5 out of 15 patients were found to have a pathologic response at the time of resection. Results: ST profiling demonstrated that the TME of responding tumors was enriched for immune cells and cancer-associated fibroblasts (CAF) with pro-inflammatory signaling relative to the non-responders. The enriched cancer-immune interactions in responding tumors are characterized by activation of the PAX5 module, a known regulator of B cell maturation, which colocalized with spots with increased B cell marker expression suggesting strong activity of these cells. HCC-CAF interactions were also enriched in the responding tumors and were associated with extracellular matrix (ECM) remodeling as there was high activation of FOS and JUN in CAFs adjacent to the tumor. The ECM remodeling is consistent with proliferative fibrosis in association with immune-mediated tumor regression. Among the patients with major pathologic responses, a single patient experienced early HCC recurrence. ST analysis of this clinical outlier demonstrated marked tumor heterogeneity, with a distinctive immune-poor tumor region that resembles the non-responding TME across patients and was characterized by HCC-CAF interactions and expression of cancer stem cell markers, potentially mediating early tumor immune escape and recurrence in this patient. Conclusions: These data show that responses to modern systemic therapy in HCC are associated with distinctive molecular and cellular landscapes and provide new targets to enhance and prolong responses to systemic therapy in HCC.

Purpose: Pancreatic ductal adenocarcinoma (PDAC) patients with tumors enriched for the basal-like molecular subtype exhibit enhanced resistance to standard-of-care treatments and have significantly worse overall survival compared with patients with classic subtype–enriched tumors. It is important to develop genomic resources, enabling identification of novel putative targets in a statistically rigorous manner. Experimental Design: We compiled a single-cell RNA sequencing (scRNA-seq) atlas of the human pancreas with 229 patient samples aggregated from publicly available raw data. We mapped cell type–specific scRNA-seq gene signatures in bulk RNA-seq (n = 744) and spatial transcriptomics (ST; n = 22) and performed validation using multiplex immunostaining. Results: Analysis of tumor cells from our scRNA-seq atlas revealed nine distinct populations, two of which aligned with the basal subtype, correlating with worse overall survival in bulk RNA-seq. Deconvolution identified one of the basal populations to be the predominant tumor subtype in nondissociated ST tissues and in vitro tumor cell and patient-derived organoid lines. We discovered a novel enrichment and spatial association of CXCL10+ cancer-associated fibroblasts with basal tumor cells. We identified that besides immune cells, ductal cells also express CXCR3, the receptor for CXCL10, suggesting a relationship between these cell types in the PDAC tumor microenvironment. Conclusions: We show that our scRNA-seq atlas (700,000 cells), integrated with ST data, has increased statistical power and is a powerful resource, allowing for expansion of current subtyping paradigms in PDAC. We uncovered a novel signaling niche marked by CXCL10+ cancer-associated fibroblasts and basal tumor cells that could be explored for future targeted therapies.