Wei Wei

Intrahepatic cholangiocarcinoma (iCCA) is a highly lethal malignancy with limited therapeutic options. The spatial architecture and functional diversity of tertiary lymphoid structures (TLSs) in iCCA remain unclear. Here, we present a multimodal spatial atlas of TLSs and identified intratumoral TLSs (iTLSs) as independent prognostic markers. Bulk proteomic profiling of 214 discovery and 155 validation cases identified a four-tier TLS-based tumor microenvironment classification system and supported development of a TLS-predictive random forest classifier. Imaging mass cytometry revealed that iTLS ⁺ tumors harbor structured immune architectures, where M1-like tissue-resident macrophages (RTMs), dendritic cells, and CXCL13 ⁺ CD4 ⁺ T cells colocalize to form antigen-presenting neighborhoods (apc-CNs) spatially coupled to TLS core regions (TLScore-CNs). Single-cell spatial transcriptomics further resolved 61 TLSs into 14 spatial niches and defined a pseudotemporal maturation continuum: aggregated, activated, and postactivated. Intraniche communication, primarily mediated by ifnCAFs, iCAFs, and CXCL12 ⁺ macrophages, evolved dynamically with maturation. Single-nucleus RNA sequencing combined with Tangram-based spatial mapping revealed CXCL12 ⁺ macrophages and iCAFs forming a peripheral band in aggregated TLSs, whereas ifnCAFs infiltrated TLS interiors during activation. These findings define TLS heterogeneity and provide insights for stroma-directed immunotherapy.

Supplementary Table 4. Immune-related adverse events.

Purpose:Immune checkpoint inhibitor (ICI)–related trials in hepatocellular carcinoma (HCC) have strict restrictions on hepatitis B (HBV) DNA load because of HBV reactivation. This study aimed to compare the HBV reactivation between patients with HCC with low or high HBV DNA loads receiving ICIs and antiviral therapy.Patients and Methods:This prospective observational study (NCT04680598) recruited hepatitis B surface antigen–positive patients with HCC who received concurrent antiviral therapy with initial ICI treatment. Participants were divided into HBV DNA–low (≤500 IU/mL) and HBV DNA–high (>500 IU/mL) groups. The primary endpoint was the HBV reactivation rate.Results:Between December 25, 2020, and February 23, 2024, 356 and 659 participants were enrolled in the HBV DNA–low and HBV DNA–high groups. The HBV DNA–high group included significantly higher proportions of patients with hepatitis Be antigen positivity (24.1% vs. 7.0%, P < 0.001), albumin–bilirubin grade 2 to 3 (49.9% vs. 33.7%, P < 0.001), and Barcelona Clinic Liver Cancer stage C (83.3% vs. 72.5%, P < 0.001). The HBV reactivation rate (4.5% vs. 6.1%, relative risk, 1.24; 95% confidence interval, 0.81–1.89; P = 0.29), frequencies of HBV reactivation–associated hepatitis (1.7% vs. 2.3%, P = 0.53), and interruptions in ICI treatment (25.8% vs. 30.5%, P = 0.12) were comparable in the HBV DNA–low group and HBV DNA–high group.Conclusions:There was no significant difference in the risk of HBV reactivation between patients with HCC with HBV DNA ≤500 IU/mL and those with HBV DNA >500 IU/mL when treated with ICIs and concurrent antiviral prophylaxis.

Supplementary Table 8: Representativeness of study participants

Supplementary Figure 2. Survival analysis. Kaplan‒Meier curves of overall survival of patients in HBV DNA-low and HBV DNA-high groups, using (A) 1000 IU/mL and (B) 2000 IU/mL as cutoff, respectively.

Supplementary Table 1. Detailed characteristics of participants with HBV reactivation.

Supplementary Table 6. Tumor response in patients received ICIs monotherapy or combination therapy

Supplementary Table 3. Treatment-related adverse events.

Supplementary file 1: Study protocol.

Supplementary Table 7. Univariate and multivariate analysis of HBV DNA reactivation among two cohorts.