Deep whole-genome analysis of 494 hepatocellular carcinomas.

Over half of hepatocellular carcinoma (HCC) cases diagnosed worldwide are in China1-3. However, whole-genome analysis of hepatitis B virus (HBV)-associated HCC in Chinese individuals is limited4-8, with current analyses of HCC mainly from non-HBV-enriched populations9,10. Here we initiated the Chinese Liver Cancer Atlas (CLCA) project and performed deep whole-genome sequencing (average depth, 120×) of 494 HCC tumours. We identified 6 coding and 28 non-coding previously undescribed driver candidates. Five previously undescribed mutational signatures were found, including aristolochic-acid-associated indel and doublet base signatures, and a single-base-substitution signature that we termed SBS_H8. Pentanucleotide context analysis and experimental validation confirmed that SBS_H8 was distinct to the aristolochic-acid-associated SBS22. Notably, HBV integrations could take the form of extrachromosomal circular DNA, resulting in elevated copy numbers and gene expression. Our high-depth data also enabled us to characterize subclonal clustered alterations, including chromothripsis, chromoplexy and kataegis, suggesting that these catastrophic events could also occur in late stages of hepatocarcinogenesis. Pathway analysis of all classes of alterations further linked non-coding mutations to dysregulation of liver metabolism. Finally, we performed in vitro and in vivo assays to show that fibrinogen alpha chain (FGA), determined as both a candidate coding and non-coding driver, regulates HCC progression and metastasis. Our CLCA study depicts a detailed genomic landscape and evolutionary history of HCC in Chinese individuals, providing important clinical implications.

Integrated Multi-Omics Landscape of Liver Metastases.

BACKGROUND & AIMS: Lack of thorough knowledge about the complicated immune microenvironment (IM) within a variety of liver metastases (LMs) leads to inappropriate treatment and unsatisfactory prognosis. We aimed to characterize IM subtypes and investigate potential mechanisms in LMs. METHODS: Mass cytometry was applied to characterize immune landscape of a primary liver cancers and liver metastases cohort. Transcriptomic and whole-exome sequencing were used to explore potential mechanisms across distinct IM subtypes. Single-cell transcriptomic sequencing, multiplex fluorescent immunohistochemistry, cell culture, mouse model, Western blot, quantitative polymerase chain reaction, and immunohistochemistry were used for validation. RESULTS: Five IM subtypes were revealed in 100 LMs and 50 primary liver cancers. Patients featured terminally exhausted (IM1) or rare T-cell-inflamed (IM2 and IM3) immune characteristics showed worse outcome. Increased intratumor heterogeneity, enriched somatic TP53, KRAS, APC, and PIK3CA mutations and hyperactivated hypoxia signaling accounted for the formation of vicious subtypes. SLC2A1 promoted immune suppression and desert via increasing proportion of Spp1+ macrophages and their inhibitory interactions with T cells in liver metastatic lesions. Furthermore, SLC2A1 promoted immune escape and LM through inducing regulatory T cells, including regulatory T cells and LAG3+CD4+ T cells in primary colorectal cancer. CONCLUSIONS: The study provided integrated multi-omics landscape of LM, uncovering potential mechanisms for vicious IM subtypes and confirming the roles of SLC2A1 in regulating tumor microenvironment remodeling in both primary tumor and LM lesions.

Application and effect of tension-reducing suture in surgical treatment of hypertrophic scar.

PURPOSE: To investigate the application and effectiveness of tension-reducing suture in the repair of hypertrophic scars. METHODS: A retrospective analysis of clinical data was conducted on 82 patients with hypertrophic scars treated at the Department of Burns and Plastic Surgery of Nanjing Drum Tower Hospital from September 2021 to December 2022. Patients were operated with combination of heart-shaped tension-reducing suturing technique and looped, broad, and deep buried (LBD) suturing technique or conventional suture method. Outcomes of surgical treatment were assessed before and 6 months after surgery using the Patient and Observer Scar Assessment Scale (POSAS) and the Vancouver Scar Scale (VSS). RESULTS: Improvements were achieved on scar quality compared to that preoperatively, with a reduction in scar width (1.7 ± 0.6 cm vs. 0.7 ± 0.2 cm, P < 0.001). Assessment using the POSAS and VSS scales showed significant improvements in each single parameter and total score compared to preoperative values (P < 0.05). The Combination method group achieved better score in total score of VSS scale, in color, stiffness, thickness and overall opinion of PSAS scale, and in vascularity, thickness, pliability and overall opinion of OSAS scale. CONCLUSION: The amalgamation of the heart-shaped tension-reducing suturing technique and the LBD suturing technique has shown promising outcomes, garnering notably high levels of patient satisfaction in the context of hypertrophic scar repair. Patients have exhibited favorable postoperative recoveries, underscoring the clinical merit and the prospective broader applicability of this approach in the realm of hypertrophic scar management.

Discovery of a Carbamoyl Phosphate Synthetase 1-Deficient HCC Subtype With Therapeutic Potential Through Integrative Genomic and Experimental Analysis.

BACKGROUND AND AIMS: Metabolic reprogramming plays an important role in tumorigenesis. However, the metabolic types of different tumors are diverse and lack in-depth study. Here, through analysis of big databases and clinical samples, we identified a carbamoyl phosphate synthetase 1 (CPS1)-deficient hepatocellular carcinoma (HCC) subtype, explored tumorigenesis mechanism of this HCC subtype, and aimed to investigate metabolic reprogramming as a target for HCC prevention. APPROACH AND RESULTS: A pan-cancer study involving differentially expressed metabolic genes of 7,764 tumor samples in 16 cancer types provided by The Cancer Genome Atlas (TCGA) demonstrated that urea cycle (UC) was liver-specific and was down-regulated in HCC. A large-scale gene expression data analysis including 2,596 HCC cases in 7 HCC cohorts from Database of HCC Expression Atlas and 17,444 HCC cases from in-house hepatectomy cohort identified a specific CPS1-deficent HCC subtype with poor clinical prognosis. In vitro and in vivo validation confirmed the crucial role of CPS1 in HCC. Liquid chromatography-mass spectrometry assay and Seahorse analysis revealed that UC disorder (UCD) led to the deceleration of the tricarboxylic acid cycle, whereas excess ammonia caused by CPS1 deficiency activated fatty acid oxidation (FAO) through phosphorylated adenosine monophosphate-activated protein kinase. Mechanistically, FAO provided sufficient ATP for cell proliferation and enhanced chemoresistance of HCC cells by activating forkhead box protein M1. Subcutaneous xenograft tumor models and patient-derived organoids were employed to identify that blocking FAO by etomoxir may provide therapeutic benefit to HCC patients with CPS1 deficiency. CONCLUSIONS: In conclusion, our results prove a direct link between UCD and cancer stemness in HCC, define a CPS1-deficient HCC subtype through big-data mining, and provide insights for therapeutics for this type of HCC through targeting FAO.

Spatial single-cell protein landscape reveals vimentinhigh macrophages as immune-suppressive in the microenvironment of hepatocellular carcinoma.

Tumor microenvironment heterogeneity in hepatocellular carcinoma (HCC) on a spatial single-cell resolution is unclear. Here, we conducted co-detection by indexing to profile the spatial heterogeneity of 401 HCC samples with 36 biomarkers. By parsing the spatial tumor ecosystem of liver cancer, we identified spatial patterns with distinct prognosis and genomic and molecular features, and unveiled the progressive role of vimentin (VIM)high macrophages. Integration analysis with eight independent cohorts demonstrated that the spatial co-occurrence of VIMhigh macrophages and regulatory T cells promotes tumor progression and favors immunotherapy. Functional studies further demonstrated that VIMhigh macrophages enhance the immune-suppressive activity of regulatory T cells by mechanistically increasing the secretion of interleukin-1ß. Our data provide deep insights into the heterogeneity of tumor microenvironment architecture and unveil the critical role of VIMhigh macrophages during HCC progression, which holds potential for personalized cancer prevention and drug discovery and reinforces the need to resolve spatial-informed features for cancer treatment.

Hyperactivation of ß-catenin signal in hepatocellular carcinoma recruits myeloid-derived suppressor cells through PF4-CXCR3 axis.

The high mutation rate of CTNNB1 (37 %) and Wnt-ß-catenin signal-associated genes (54 %) has been notified in hepatocellular carcinoma (HCC). The activation of Wnt-ß-catenin signal pathway was reported to be associated with an immune "desert" phenotype, but the underlying mechanism remains unclear. Here we mainly employed orthotopic HCC models to explore on it. Mass cytometry depicted the immune contexture of orthotopic HCC syngeneic grafts, unveiling that the exogenous expression of ß-catenin significantly increased the percentage of myeloid-derived suppressor cells (MDSCs) and decreased the percentage of CD8+ T-cells. Flow cytometry and immunohistochemistry further confirmed the findings. The protein microarray analysis, Western blot and PCR identified PF4 as its downstream regulating cytokine. Intratumorally injection of cytokine PF4 enhanced the accumulation of MDSCs. Knockout of PF4 abolished the effect of ß-catenin on recruiting MDSCs. Chromatin immunoprecipitation and luciferase reporter assay demonstrated that ß-catenin increases the mRNA level of PF4 via binding to PF4's promoter region. In vitro chemotaxis assay and in vivo administration of specific inhibitors identified CXCR3 on MDSCs as receptor for recruiting PF4. Lastly, the significant correlations across ß-catenin, PF4 and MDSCs and CD8+ T-cells infiltration were verified in HCC clinical samples. Our results unveiled HCC tumor cell intrinsic hyperactivation of ß-catenin can recruit MDSC through PF4-CXCR3, which contributes to the formation of immune "desert" phenotype. Our study provided new insights into the development of immunotherapeutic strategy of HCC with CTNNB1 mutation. SIGNIFICANCE: This study identifies PF4-CXCR3-MDSCs as a downstream mechanism underlying CTNNB1 mutation associated immune "desert" phenotype.

Integrated characterization of hepatobiliary tumor organoids provides a potential landscape of pharmacogenomic interactions.

Despite considerable efforts to identify human liver cancer genomic alterations that might unveil druggable targets, the systematic translation of multiomics data remains challenging. Here, we report success in long-term culture of 64 patient-derived hepatobiliary tumor organoids (PDHOs) from a Chinese population. A divergent response to 265 metabolism- and epigenetics-related chemicals and 36 anti-cancer drugs is observed. Integration of the whole genome, transcriptome, chromatin accessibility profiles, and drug sensitivity results of 64 clinically relevant drugs defines over 32,000 genome-drug interactions. RUNX1 promoter mutation is associated with an increase in chromatin accessibility and a concomitant gene expression increase, promoting a cluster of drugs preferentially sensitive in hepatobiliary tumors. These results not only provide an annotated PDHO biobank of human liver cancer but also suggest a systematic approach for obtaining a comprehensive understanding of the gene-regulatory network of liver cancer, advancing the applications of potential personalized medicine.

A Noninvasive Approach to Evaluate Tumor Immune Microenvironment and Predict Outcomes in Hepatocellular Carcinoma.

It is widely recognized that tumor immune microenvironment (TIME) plays a crucial role in tumor progression, metastasis, and therapeutic response. Despite several noninvasive strategies have emerged for cancer diagnosis and prognosis, there are still lack of effective radiomic-based model to evaluate TIME status, let alone predict clinical outcome and immune checkpoint inhibitor (ICIs) response for hepatocellular carcinoma (HCC). In this study, we developed a radiomic model to evaluate TIME status within the tumor and predict prognosis and immunotherapy response. A total of 301 patients who underwent magnetic resonance imaging (MRI) examinations were enrolled in our study. The intra-tumoral expression of 17 immune-related molecules were evaluated using co-detection by indexing (CODEX) technology, and we construct Immunoscore (IS) with the least absolute shrinkage and selection operator (LASSO) algorithm and Cox regression method to evaluate TIME. Of 6115 features extracted from MRI, five core features were filtered out, and the Radiomic Immunoscore (RIS) showed high accuracy in predicting TIME status in testing cohort (area under the curve = 0.753). More importantly, RIS model showed the capability of predicting therapeutic response to anti-programmed cell death 1 (PD-1) immunotherapy in an independent cohort with advanced HCC patients (area under the curve = 0.731). In comparison with previously radiomic-based models, our integrated RIS model exhibits not only higher accuracy in predicting prognosis but also the potential guiding significance to HCC immunotherapy. Supplementary Information: The online version contains supplementary material available at 10.1007/s43657-023-00136-8.

Hepatobiliary Tumor Organoids Reveal HLA Class I Neoantigen Landscape and Antitumoral Activity of Neoantigen Peptide Enhanced with Immune Checkpoint Inhibitors.

Neoantigen-directed therapy lacks preclinical models recapitulating neoantigen characteristics of original tumors. It is urgent to develop a platform to assess T cell response for neoantigen screening. Here, immunogenic potential of neoantigen-peptides of tumor tissues and matched organoids (n = 27 pairs) are analyzed by Score tools with whole genome sequencing (WGS)-based human leukocyte antigen (HLA)-class-I algorithms. The comparisons between 9203 predicted neoantigen-peptides from 2449 mutations of tumor tissues and 9991 ones from 2637 mutations of matched organoids demonstrate that organoids preserved majority of genetic features, HLA alleles, and similar neoantigen landscape of original tumors. Higher neoantigen load is observed in tumors with early stage. Multiomics analysis combining WGS, RNA-seq, single-cell RNA-seq, mass spectrometry filters out 93 candidate neoantigen-peptides with strong immunogenic potential for functional validation in five organoids. Immunogenic peptides are defined by inducing increased CD107aCD137IFN-γ expressions and IFN-γ secretion of CD8 cells in flow cytometry and enzyme-linked immunosorbent assay assays. Nine immunogenic peptides shared by at least two individuals are validated, including peptide from TP53R90S . Organoid killing assay confirms the antitumor activity of validated immunogenic peptide-reactive CD8 cells, which is further enhanced in the presence of immune checkpoint inhibitors. The study characterizes HLA-class-I neoantigen landscape in hepatobiliary tumor, providing practical strategy with tumor organoid model for neoantigen-peptide identification in personalized immunotherapy.

M6A Demethylase ALKBH5 Regulates PD-L1 Expression and Tumor Immunoenvironment in Intrahepatic Cholangiocarcinoma.

N6-methyladenosine (m6A) has been reported as an important mechanism of posttranscriptional regulation. Programmed death-ligand 1 (PD-L1) is a primary immune inhibitory molecule expressed on tumor cells that promotes immune evasion. Here we report ALKBH5 as an important m6A demethylase that orchestrates PD-L1 expression in intrahepatic cholangiocarcinoma (ICC). Regulation of PD-L1 expression by ALKBH5 was confirmed in human ICC cell lines. Sequencing of the m6A methylome identified PD-L1 mRNA as a direct target of m6A modification whose levels were regulated by ALKBH5. Furthermore, ALKBH5 and PD-L1 mRNA were shown to interact. ALKBH5 deficiency enriched m6A modification in the 3'UTR region of PD-L1 mRNA, thereby promoting its degradation in a YTHDF2-dependent manner. In vitro and in vivo, tumor-intrinsic ALKBH5 inhibited the expansion and cytotoxicity of T cells by sustaining tumor cell PD-L1 expression. The ALKBH5-PD-L1-regulating axis was further confirmed in human ICC specimens. Single-cell mass cytometry analysis unveiled a complex role of ALKBH5 in the tumor immune microenvironment by promoting the expression of PD-L1 on monocytes/macrophages and decreasing the infiltration of myeloid-derived suppressor-like cells. Analysis of specimens from patients receiving anti-PD1 immunotherapy suggested that tumors with strong nuclear expression patterns of ALKBH5 are more sensitive to anti-PD1 immunotherapy. Collectively, these results describe a new regulatory mechanism of PD-L1 by mRNA epigenetic modification by ALKBH5 and the potential role of ALKBH5 in immunotherapy response, which might provide insights for cancer immunotherapies. SIGNIFICANCE: This study identifies PD-L1 mRNA as a target of ALKBH5 and reveals a role for ALKBH5 in regulating the tumor immune microenvironment and immunotherapy efficacy.

Single-Cell Transcriptome Analysis Uncovers Intratumoral Heterogeneity and Underlying Mechanisms for Drug Resistance in Hepatobiliary Tumor Organoids.

Molecular heterogeneity of hepatobiliary tumor including intertumoral and intratumoral disparity always leads to drug resistance. Here, seven hepatobiliary tumor organoids are generated to explore heterogeneity and evolution via single-cell RNA sequencing. HCC272 with high status of epithelia-mesenchymal transition proves broad-spectrum drug resistance. By examining the expression pattern of cancer stem cells markers (e.g., PROM1, CD44, and EPCAM), it is found that CD44 positive population may render drug resistance in HCC272. UMAP and pseudo-time analysis identify the intratumoral heterogeneity and distinct evolutionary trajectories, of which catenin beta-1 (CTNNB1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and nuclear paraspeckle assembly transcript 1 (NEAT1) advantage expression clusters are commonly shared across hepatobiliary organoids. CellphoneDB analysis further implies that metabolism advantage organoids with enrichment of hypoxia signal upregulate NEAT1 expression in CD44 subgroup and mediate drug resistance that relies on Jak-STAT pathway. Moreover, metabolism advantage clusters shared in several organoids have similar characteristic genes (GAPDH, NDRG1 (N-Myc downstream regulated 1), ALDOA, and CA9). The combination of GAPDH and NDRG1 is an independent risk factor and predictor for patient survival. This study delineates heterogeneity of hepatobiliary tumor organoids and proposes that the collaboration of intratumoral heterogenic subpopulations renders malignant phenotypes and drug resistance.

Comprehensive analysis of spatial architecture in primary liver cancer.

Heterogeneity is the major challenge for cancer prevention and therapy. Here, we first constructed high-resolution spatial transcriptomes of primary liver cancers (PLCs) containing 84,823 spots within 21 tissues from seven patients. The progressive comparison of spatial tumor microenvironment (TME) characteristics from nontumor to leading-edge to tumor regions revealed that the tumor capsule potentially affects intratumor spatial cluster continuity, transcriptome diversity, and immune cell infiltration. Locally, we found that the bidirectional ligand-receptor interactions at the 100-µm-wide cluster-cluster boundary contribute to maintaining intratumor architecture and the PROM1+ and CD47+ cancer stem cell niches are related to TME remodeling and tumor metastasis. Last, we proposed a TLS-50 signature to accurately locate tertiary lymphoid structures (TLSs) spatially and unveiled that the distinct composition of TLSs is shaped by their distance to tumor cells. Our study provides previous unknown insights into the diverse tumor ecosystem of PLCs and has potential benefits for cancer intervention.

The identification and functional analysis of CD8+PD-1+CD161+ T cells in hepatocellular carcinoma.

Immunotherapy is a powerful therapeutic strategy for end-stage hepatocellular carcinoma (HCC). It is well known that T cells, including CD8+PD-1+ T cells, play important roles involving tumor development. However, their underlying phenotypic and functional differences of T cell subsets remain unclear. We constructed single-cell immune contexture involving approximate 20,000,000 immune cells from 15 pairs of HCC tumor and non-tumor adjacent tissues and 10 blood samples (including five of HCCs and five of healthy controls) by mass cytometry. scRNA-seq and functional analysis were applied to explore the function of cells. Multi-color fluorescence staining and tissue micro-arrays were used to identify the pathological distribution of CD8+PD-1+CD161 +/- T cells and their potential clinical implication. The differential distribution of CD8+ T cells subgroups was identified in tumor and non-tumor adjacent tissues. The proportion of CD8+PD1+CD161+ T cells was significantly decreased in tumor tissues, whereas the ratio of CD8+PD1+CD161- T cells was much lower in non-tumor adjacent tissues. Diffusion analysis revealed the distinct evolutionary trajectory of CD8+PD1+CD161+ and CD8+PD1+CD161- T cells. scRNA-seq and functional study further revealed the stronger immune activity of CD8+PD1+CD161+ T cells independent of MHC class II molecules expression. Interestingly, a similar change in the ratio of CD8+CD161+/ CD8+CD161- T cells was also found in peripheral blood samples collected from HCC cases, indicating their potential usage clinically. We here identified different distribution, function, and trajectory of CD8+PD-1+CD161+ and CD8+PD-1+CD161- T cells in tumor lesions, which provided new insights for the heterogeneity of immune environment in HCCs and also shed light on the potential target for immunotherapy.

Autoantibody signature in hepatocellular carcinoma using seromics.

BACKGROUND: Alpha-fetoprotein (AFP) is a widely used biomarker for hepatocellular carcinoma (HCC) early detection. However, low sensitivity and false negativity of AFP raise the requirement of more effective early diagnostic approaches for HCC. METHODS: We employed a three-phase strategy to identify serum autoantibody (AAb) signature for HCC early diagnosis using protein array-based approach. A total of 1253 serum samples from HCC, liver cirrhosis, and healthy controls were prospectively collected from three liver cancer centers in China. The Human Proteome Microarray, comprising 21,154 unique proteins, was first applied to identify AAb candidates in discovery phase (n = 100) and to further fabricate HCC-focused arrays. Then, an artificial neural network (ANN) model was used to discover AAbs for HCC detection in a test phase (n = 576) and a validation phase (n = 577), respectively. RESULTS: Using HCC-focused array, we identified and validated a novel 7-AAb panel containing CIAPIN1, EGFR, MAS1, SLC44A3, ASAH1, UBL7, and ZNF428 for effective HCC detection. The ANN model of this panel showed improvement of sensitivity (61.6-77.7%) compared to AFP (cutoff 400 ng/mL, 28.4-30.7%). Notably, it was able to detect AFP-negative HCC with AUC values of 0.841-0.948. For early-stage HCC (BCLC 0/A) detection, it outperformed AFP (cutoff 400 ng/mL) with approximately 10% increase in AUC. CONCLUSIONS: The 7-AAb panel provides potentially clinical value for non-invasive early detection of HCC, and brings new clues on understanding the immune response against hepatocarcinogenesis.

Trajectory and Functional Analysis of PD-1high CD4+CD8+ T Cells in Hepatocellular Carcinoma by Single-Cell Cytometry and Transcriptome Sequencing.

The spatial heterogeneity of immune microenvironment in hepatocellular carcinoma (HCC) remains elusive. Here, a single-cell study involving 17 432 600 immune cells of 39 matched HCC (T), nontumor (N), and leading-edge (L) specimens by mass cytometry is conducted. The tumor-associated CD4/CD8 double-positive T (DPT) cells are found enriched in L regions with synergetic expression of PD-1/HLA-DR/ICOS/CD45RO and exhibit a higher level of IFN-γ, TNF-α, and PD-1 upon stimulation. The enrichment of DPT and PD-1+DPT in L regions indicates favorable prognosis. These tumor-associated DPT cells with similar phenotype are also verified in other tumors and HCC animal models. Single-cell RNA-seq further characterizes the molecular features of DPT cells and uncovers 11 clusters with different cytotoxicity, exhaustion, and activation scores. TCR-based trajectory analysis reveals that tumor-associated DPT clusters share separated ancestries with local CD4+ or CD8+SPT cells rather than CD3+PBMC cells. TCR clones with frequency above 10 are mainly found coexisting in DPT and CD8+SPT cells. Specifically, PD-1highDPT cluster (TDPT_10) shares the same ancestry with exhausted CD8+SPT cluster (TCD8T_2) and shows higher expression similarity and closer pathological location to PD-1+CD8+ than PD-1+CD4+T cells. Together, this study systematically characterizes the unique distribution of PD-1+DPTs in HCC and puts forward new insights for the function and origin of tumor-associated DPT cells.