ST3GAL1 promotes malignant phenotypes in intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA) has poor prognosis and elucidation of the molecular mechanisms underlying iCCA malignancy is of great significance. Glycosylation, an important post-translational modification, is closely associated with tumor progression. Altered glycosylation, including aberrant sialylation resulting from abnormal expression of sialyltransferases (STs) and neuraminidases (NEUs), is a significant feature of cancer cells. However, there is limited information on the roles of STs and NEUs in iCCA malignancy. Here, utilizing our proteogenomic resources from a cohort of 262 iCCA patients, we identified ST3GAL1 as a prognostically relevant molecule in iCCA. Moreover, overexpression of ST3GAL1 promoted proliferation, migration and invasion and inhibited apoptosis of iCCA cells in vitro. Through proteomic analyses, we identified the downstream pathway potentially regulated by ST3GAL1, which was the NF-κB signaling pathway and further demonstrated that this pathway was positively correlated with malignancy in iCCA cells. Notably, glycoproteomics showed that O-glycosylation was changed in iCCA cells with high ST3GAL1 expression. Importantly, the altered O-glycopeptides underscored the potential utility of O-glycosylation profiling as a discriminatory marker for iCCA cells with ST3GAL1 overexpression. Additionally, miR-320b was identified as a post-transcriptional regulator of ST3GAL1, capable of suppressing ST3GAL1 expression and then reducing the proliferation, migration and invasion abilities of iCCA cell lines. Taken together, these results suggest ST3GAL1 could serve as a promising therapeutic target for iCCA.

Epigenome-wide development and validation of a prognostic methylation score in intrahepatic cholangiocarcinoma based on machine learning strategies.

Background: Clinical parameter-based nomograms and staging systems provide limited information for the prediction of survival in intrahepatic cholangiocarcinoma (ICC) patients. In this study, we developed a methylation signature that precisely predicts overall survival (OS) after surgery. Methods: An epigenome-wide study of DNA methylation based on whole-genome bisulfite sequencing (WGBS) was conducted for two independent cohorts (discovery cohort, n=164; validation cohort, n=170) from three hepatobiliary centers in China. By referring to differentially methylated regions (DMRs), we proposed the concept of prognostically methylated regions (PMRs), which were composed of consecutive prognostically methylated CpGs (PMCs). Using machine learning strategies (Random Forest and the least absolute shrinkage and selector regression), a prognostic methylation score (PMS) was constructed based on 14 PMRs in the discovery cohort and confirmed in the validation cohort. Results: The C-indices of the PMS for predicting OS in the discovery and validation cohorts were 0.79 and 0.74, respectively. In the whole cohort, the PMS was an independent predictor of OS [hazard ratio (HR) =8.12; 95% confidence interval (CI): 5.48-12.04; P<0.001], and the C-index (0.78) of the PMS was significantly higher than that of the Johns Hopkins University School of Medicine (JHUSM) nomogram (0.69, P<0.001), the Eastern Hepatobiliary Surgery Hospital (EHBSH) nomogram (0.67, P<0.001), American Joint Committee on Cancer (AJCC) tumor-node-metastasis (TNM) staging system (0.61, P<0.001), and MEGNA prognostic score (0.60, P<0.001). The patients in quartile 4 of PMS could benefit from adjuvant therapy (AT) (HR =0.54; 95% CI: 0.32-0.91; log-rank P=0.043), whereas those in the quartiles 1-3 could not. However, other nomograms and staging system failed to do so. Further analyses of potential mechanisms showed that the PMS was associated with tumor biological behaviors, pathway activation, and immune microenvironment. Conclusions: The PMS could improve the prognostic accuracy and identify patients who would benefit from AT for ICC patients, and might facilitate decisions in treatment of ICC patients.

Proteomics of adjacent-to-tumor samples uncovers clinically relevant biological events in hepatocellular carcinoma.

Normal adjacent tissues (NATs) of hepatocellular carcinoma (HCC) differ from healthy liver tissues and their heterogeneity may contain biological information associated with disease occurrence and clinical outcome that has yet to be fully evaluated at the proteomic level. This study provides a detailed description of the heterogeneity of NATs and the differences between NATs and healthy livers and revealed that molecular features of tumor subgroups in HCC were partially reflected in their respective NATs. Proteomic data classified HCC NATs into two subtypes (Subtypes 1 and 2), and Subtype 2 was associated with poor prognosis and high-risk recurrence. The pathway and immune features of these two subtypes were characterized. Proteomic differences between the two NAT subtypes and healthy liver tissues were further investigated using data-independent acquisition mass spectrometry, revealing the early molecular alterations associated with the progression from healthy livers to NATs. This study provides a high-quality resource for HCC researchers and clinicians and may significantly expand the knowledge of tumor NATs to eventually benefit clinical practice.

An Inflammatory Checkpoint Generated by IL1RN Splicing Offers Therapeutic Opportunity for KRAS-Mutant Intrahepatic Cholangiocarcinoma.

KRAS mutations are causally linked to protumor inflammation and are identified as driving factors in tumorigenesis. Here, using multiomics data gathered from a large set of patients, we showed that KRAS mutation was associated with a specific landscape of alternative mRNA splicing that connected to myeloid inflammation in intrahepatic cholangiocarcinoma (iCCA). Then, we identified a negative feedback mechanism in which the upregulation of interleukin 1 receptor antagonist (IL1RN)-201/203 due to alternative splicing confers vital anti-inflammatory effects in KRAS-mutant iCCA. In KRAS-mutant iCCA mice, both IL1RN-201/203 upregulation and anakinra treatment ignited a significant antitumor immune response by altering neutrophil recruitment and phenotypes. Furthermore, anakinra treatment synergistically enhanced anti-PD-1 therapy to activate intratumoral GZMB+ CD8+ T cells in KRAS-mutant iCCA mice. Clinically, we found that high IL1RN-201/203 levels in patients with KRAS-mutant iCCA were significantly associated with superior response to anti-PD-1 immunotherapy. SIGNIFICANCE: This work describes a novel inflammatory checkpoint mediated by IL1RN alternative splicing variants that may serve as a promising basis to develop therapeutic options for KRAS-mutant iCCA and other cancers. This article is featured in Selected Articles from This Issue, p. 2109.

Spatial immunophenotypes predict clinical outcome in intrahepatic cholangiocarcinoma.

Background & Aims: Intrahepatic cholangiocarcinoma (iCCA) is a severe malignant tumour that shows only modest responses to immunotherapy. We aimed to identify the spatial immunophenotypes of iCCA and delineate potential immune escape mechanisms. Method: Multiplex immunohistochemistry (mIHC) was performed to quantitatively evaluate the distribution of 16 immune cell subsets in intratumour, invasive margin and peritumour areas in a cohort of 192 treatment-naïve patients with iCCA. Multiregion unsupervised clustering was used to determine three spatial immunophenotypes, and multiomics analyses were carried out to explore functional differences.Results: iCCA displayed a region-specific distribution of immune cell subsets with abundant CD15+ neutrophil infiltration in intratumour areas. Three spatial immunophenotypes encompassing inflamed (35%), excluded (35%) and ignored (30%) phenotypes were identified. The inflamed phenotype showed characteristics of abundant immune cell infiltration in intratumour areas, increased PD-L1 expression and relatively favourable overall survival. The excluded phenotype with a moderate prognosis was characterized by immune cell infiltration restricted to the invasive margin or peritumour areas and upregulation of activated hepatic stellate cells, extracellular matrix and Notch signalling pathways. The ignored phenotype, with scarce immune cell infiltration across all subregions, was associated with MAPK signalling pathway elevation and a poor prognosis. The excluded and ignored phenotypes, constituting non-inflamed phenotypes, shared features of an increased angiogenesis score, TGF-ß and Wnt-ß catenin pathway upregulation and were enriched for BAP1 mutations and FGFR2 fusions. Conclusion: We identified three spatial immunophenotypes with different overall prognoses in iCCA. Tailored therapies based on the distinct immune evasion mechanisms of the spatial immunophenotypes are needed. Impact and implications: The contribution of immune cell infiltration in the invasive margin and peritumour areas has been proved. We explored the multiregional immune contexture of 192 patients to identify three spatial immunophenotypes in intrahepatic cholangiocarcinoma (iCCA). By integrating genomic and transcriptomic data, phenotype-specific biological behaviours and potential immune escape mechanisms were analysed. Our findings provide a rationale to develop personalized therapies for iCCA.

Genome-Scale CRISPR screen identifies LAPTM5 driving lenvatinib resistance in hepatocellular carcinoma.

ABBREVIATIONS: cld-CASP3: cleaved caspase 3; cld-PARP: cleaved PARP; DTP: drug tolerant persister; GO: Gene Ontology; GTEx: The Genotype-Tissue Expression; HCC: hepatocellular carcinoma; HCQ: hydroxychloroquine; IC50: half maximal inhibitory concentration value; KEGG: Kyoto Encyclopedia of Genes and Genomes; LAPTM5: lysosomal protein transmembrane 5; NT: non-targeting; PDC: patient-derived primary cell lines; PDO: patient-derived primary organoid; TCGA: The Cancer Genome Atlas.

FUT8 is regulated by miR-122-5p and promotes malignancies in intrahepatic cholangiocarcinoma via PI3K/AKT signaling.

BACKGROUND: Intrahepatic cholangiocarcinoma (iCCA) is the second-most lethal primary liver cancer and its prognosis remains dismal. N-glycosylation, which is biosynthesized by a number of glycosyltransferases, plays an important role in a variety of biological processes and is associated with cancer development and progression. METHODS: Based on our previous proteogenomic resources from an iCCA cohort of 262 patients, fucosyltransferases 8 (FUT8) showed significant prognosis relevance in iCCA. Tumor tissues from iCCA patients were used to evaluate the correlation between its expression and clinical information. Gain/loss-of-function experiments in iCCA cell lines were performed to elucidate the biological function of FUT8. In addition, its downstream pathways and post-transcriptional regulators were inferred and verified. RESULTS: Elevated FUT8 expression was clinically associated with worse overall survival in iCCA patients. Its overexpression promoted migration, invasion and proliferation ability of iCCA cells. In addition, miR-122-5p was found to act as a post-transcriptional regulator of FUT8 and proved to inhibit FUT8 expression and then suppress the proliferation and migration ability of iCCA cell lines. Furthermore, FUT8 was observed to promote iCCA development through PI3K/AKT signaling pathway. CONCLUSIONS: These findings demonstrated that FUT8, regulated by miR-122-5p, could be a tumor promoter of iCCA through PI3K/AKT signaling pathway.

The influence of 18F-fluorodeoxyglucose positron emission tomography/computed tomography on the N- and M-staging and subsequent clinical management of intrahepatic cholangiocarcinoma.

Background: Intrahepatic cholangiocarcinoma (ICC) is a highly metastatic cancer. 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) enables sensitive tumor and metastasis detection. Our aim is to evaluate the influence of pre-treatment PET/CT on the N- and M-staging and subsequent clinical management in ICC patients. Methods: Between August 2010 and August 2018, 660 consecutive ICC patients, without prior anti-tumor treatments nor other malignancies, were enrolled. The diagnostic performance of PET/CT on the N- and M-staging was compared with conventional imaging, and the preoperative staging accuracy and treatment re-allocation by PET/CT were retrospectively calculated. Survival difference was compared between patients receiving PET/CT or not after propensity score matching. Results: Patients were divided into group A (n=291) and group B (n=369) according to whether PET/CT was performed. Among 291 patients with both PET/CT and conventional imaging for staging in group A, PET/CT showed significantly higher sensitivity (83.0% vs. 70.5%, P=0.001), specificity (88.3% vs. 74.9%, P<0.001) and accuracy (86.3% vs. 73.2%, P<0.001) than conventional imaging in diagnosing regional lymph node metastasis, as well as higher sensitivity (87.8% vs. 67.6%, P<0.001) and accuracy (93.5% vs. 89.3%, P=0.023) in diagnosing distant metastasis. Overall, PET/CT improved the accuracy of preoperative staging from 60.1% to 71.8% (P<0.001), and modified clinical treatment strategy in 5.8% (17/291) of ICC patients, with unique roles in different tumor-node-metastasis (TNM) stages. High tumor-to-non-tumor ratio (TNR) predicted poor overall survival [hazard ratio (HR) = 2.17; 95% confidence interval (CI): 1.49-3.15; P<0.001]. Furthermore, patients performing PET/CT had longer overall survival compared with those without PET/CT (HR =0.74; 95% CI: 0.58-0.93; P=0.011) after propensity score matching. Conclusions: PET/CT was valuable for diagnosing regional lymph node metastasis and distant metastasis in ICC patients, and facilitated accurate tumor staging and optimal treatment allocation.

Multi-omics characterization reveals the pathogenesis of liver focal nodular hyperplasia.

The molecular landscape and pathogenesis of focal nodular hyperplasia (FNH) have yet to be elucidated. We performed multi-omics approaches on FNH and paired normal liver tissues from 22 patients, followed by multi-level bioinformatic analyses and experimental validations. Generally, FNH had low mutation burden with low variant allele frequencies, and the mutation frequency significantly correlated with proliferation rate. Although no recurrently deleterious genomic events were found, some putative tumor suppressors or oncogenes were involved. Mutational signatures indicated potential impaired mismatch function and possible poison contact. Integrated analyses unveiled a group of FNH specific endothelial cells that uniquely expressed SOST and probably had strong interaction with fibroblasts through PDGFB/PDGFRB pathway to promote fibrosis. Notably, in one atypical FNH (patient No.11) with pronounced copy number variations, we observed a unique immune module. Most FNH are benign, but molecularly atypical FNH still exist; endothelial cell derived PDGFB probably promotes the fibrogenic process in FNH.

Multimodule characterization of immune subgroups in intrahepatic cholangiocarcinoma reveals distinct therapeutic vulnerabilities.

BACKGROUND: Immune microenvironment is well recognized as a critical regulator across cancer types, despite its complex roles in different disease conditions. Intrahepatic cholangiocarcinoma (iCCA) is characterized by a tumor-reactive milieu, emphasizing a deep insight into its immunogenomic profile to provide prognostic and therapeutic implications. METHODS: We performed genomic, transcriptomic, and proteomic characterization of 255 paired iCCA and adjacent liver tissues. We validated our findings through H&E staining (n=177), multiplex immunostaining (n=188), single-cell RNA sequencing (scRNA-seq) (n=10), in vitro functional studies, and in vivo transposon-based mouse models. RESULTS: Integrated multimodule data identified three immune subgroups with distinct clinical, genetic, and molecular features, designated as IG1 (immune-suppressive, 25.1%), IG2 (immune-exclusion, 42.7%), and IG3 (immune-activated, 32.2%). IG1 was characterized by excessive infiltration of neutrophils and immature dendritic cells (DCs). The hallmark of IG2 was the relatively higher tumor-proliferative activity and tumor purity. IG3 exhibited an enrichment of adaptive immune cells, natural killer cells, and activated DCs. These immune subgroups were significantly associated with prognosis and validated in two independent cohorts. Tumors with KRAS mutations were enriched in IG1 and associated with myeloid inflammation-dominated immunosuppression. Although tumor mutation burden was relatively higher in IG2, loss of heterozygosity in human leucocyte antigen and defects in antigen presentation undermined the recognition of neoantigens, contributing to immune-exclusion behavior. Pathological analysis confirmed that tumor-infiltrating lymphocytes and tertiary lymphoid structures were both predominant in IG3. Hepatitis B virus (HBV)-related samples tended to be under-represented in IG1, and scRNA-seq analyses implied that HBV infection indeed alleviated myeloid inflammation and reinvigorated antitumor immunity. CONCLUSIONS: Our study elucidates that the immunogenomic traits of iCCA are intrinsically heterogeneous among patients, posing great challenge and opportunity for the application of personalized immunotherapy.

Geospatial Immune Heterogeneity Reflects the Diverse Tumor-Immune Interactions in Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA) exhibits extensive intratumoral heterogeneity and an extremely high mortality rate. Here, we performed whole-exome sequencing, RNA sequencing, T-cell receptor (TCR) sequencing, and multiplexed immunofluorescence on 207 tumor regions from 45 patients with iCCA. Over half of iCCA displayed intratumoral heterogeneity of immune infiltration, and iCCA were classified into sparsely, heterogeneously, and highly infiltrated subgroups with distinct immunogenomic characteristics. Sparsely infiltrated tumors displayed active copy-number loss of clonal neoantigens, and heterogeneous immune infiltration played an important role in the subclonal evolution across tumor subregions. Highly infiltrated tumors were characterized by extensive immune activation and a similar TCR repertoire across tumor subregions, but counteracted with T-cell exhaustion and pervasive antigen presentation defects. Notably, FGFR2 mutations and fusions correlated with low mutation burden and reduced immune infiltration. Our work delineated the dynamic tumor-immune interactions and developed a robust classification system to divide patients with iCCA into high and low immune evasion groups with different prognoses. SIGNIFICANCE: This study elucidates the impact of spatial immune heterogeneity upon tumor evolution of iCCA and reveals distinct immune evasion mechanisms developed in different immune microenvironments, which can be exploited for the development of personalized immunotherapy strategies. This article is highlighted in the In This Issue feature, p. 2221.

Proteogenomic characterization identifies clinically relevant subgroups of intrahepatic cholangiocarcinoma.

We performed proteogenomic characterization of intrahepatic cholangiocarcinoma (iCCA) using paired tumor and adjacent liver tissues from 262 patients. Integrated proteogenomic analyses prioritized genetic aberrations and revealed hallmarks of iCCA pathogenesis. Aflatoxin signature was associated with tumor initiation, proliferation, and immune suppression. Mutation-associated signaling profiles revealed that TP53 and KRAS co-mutations may contribute to iCCA metastasis via the integrin-FAK-SRC pathway. FGFR2 fusions activated the Rho GTPase pathway and could be a potential source of neoantigens. Proteomic profiling identified four patient subgroups (S1-S4) with subgroup-specific biomarkers. These proteomic subgroups had distinct features in prognosis, genetic alterations, microenvironment dysregulation, tumor microbiota composition, and potential therapeutics. SLC16A3 and HKDC1 were further identified as potential prognostic biomarkers associated with metabolic reprogramming of iCCA cells. This study provides a valuable resource for researchers and clinicians to further identify molecular pathogenesis and therapeutic opportunities in iCCA.

A combined Cox and logistic model provides accurate predictive performance in estimation of time-dependent probabilities for recurrence of intrahepatic cholangiocarcinoma after resection.

BACKGROUND: Intrahepatic cholangiocarcinoma has heterogeneous outcomes after resection. There remains a need for broadly applicable recurrence-specific tool offering precise evaluation on curativeness of resection. METHODS: A four hospital-based clinical cohort involving 1,655 patients with intrahepatic cholangiocarcinoma who received surgical resection were studied. Cox and logistic models were networked into one system containing risk categories with distinctive probabilities of recurrence. Prediction of time-to-recurrence was performed by formulizing time-dependent risk probabilities. The model was validated in three clinical cohorts (n=332). RESULTS: From the training cohort, 10 and 11 covariates, including diabetes, cholelithiasis, albumin, platelet count, alpha fetoprotein, carbohydrate antigen 19-9, carcinoembryonic antigen, hepatitis B virus infection, tumor size and number, resection type, and lymph node metastasis, from Cox and logistic models were identified significant for recurrence-free survival (RFS). The combined Cox & logistic ranking system (CCLRS)-adjusted time-dependent probabilities were categorized into seven ranks (5-yr RFS for lowest and highest ranks were 75% vs. 0%; hazard ratio 18.5, 95% CI: 14.7-24.9, P<0.0001). The CCLRS was validated with a minimum area under curve value of 0.8086. Prediction of time-to-recurrence was validated to be excellent (Pearson r, 0.8204; P<0.0001). CONCLUSIONS: The CCLRS allows precise estimation on risk of recurrence for intrahepatic cholangiocarcinoma after resection. It could be applicative when estimating time-dependent disease status and stratifying individuals who sole resection of the tumor would not be curative.

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.

The distribution of immune cells within combined hepatocellular carcinoma and cholangiocarcinoma predicts clinical outcome.

BACKGROUND: This study aimed to investigate the clinical relevance of the immune microenvironment in patients with combined hepatocellular carcinoma and cholangiocarcinoma (cHCC-ICC). PATIENTS AND METHODS: The density of tumor-infiltrating CD3+ , CD8+ , CD163+ , and Foxp3+ immune cells, as well as Programmed cell death 1, Programmed cell death-ligand 1, and Tumor necrosis factor receptor superfamily member 4, was measured in the peritumor liver, tumor invasive margin, and intratumor subregions of 56 cHCC-ICC by immunohistochemistry. The immune index was established to stratify patients. Prognostic significance of immune cell subsets and immune indices was evaluated. RESULTS: The distribution of immune cells was highly heterogeneous among different subregions of cHCC-ICC. As compared with the hepatocellular carcinoma (HCC) component, the lower density of CD8+ T cells and higher intensity of Foxp3+ Tregs and immune checkpoints in the intrahepatic cholangiocarcinoma (ICC) component may indicate a stronger immune evasive ability of ICC. Based on clustering classification or a combination of random forest and lasso-cox, two models of immune indices were established and both were identified as independent prognostic factors for cHCC-ICC patients. The selected immune variables in the immune prognostic models derived from both HCC and ICC subregions, indicating that the prognosis of cHCC-ICC patients was a complex interaction of both components. CONCLUSIONS: The immune contexture was heterogeneous among different subregions of cHCC-ICC patients and contributed differently to patient prognosis. Immune score based on the densities of immune cells might serve as a promising prognostic predictor for cHCC-ICC patients.

Heterogeneous immunogenomic features and distinct escape mechanisms in multifocal hepatocellular carcinoma.

BACKGROUND & AIMS: The presence of multifocal tumors, developed either from intrahepatic metastasis (IM) or multicentric occurrence (MO), is a distinct feature of hepatocellular carcinoma (HCC). Immunogenomic characterization of multifocal HCC is important for understanding immune escape in different lesions and developing immunotherapy. METHODS: We combined whole-exome/transcriptome sequencing, multiplex immunostaining, immunopeptidomes, T cell receptor (TCR) sequencing and bioinformatic analyses of 47 tumors from 15 patients with HCC and multifocal lesions. RESULTS: IM and MO demonstrated distinct clonal architecture, mutational spectrum and genetic susceptibility. The immune microenvironment also displayed spatiotemporal heterogeneity, such as less T cell and more M2 macrophage infiltration in IM and higher expression of inhibitory immune checkpoints in MO. Similar to mutational profiles, shared neoantigens and TCR repertoires among tumors from the same patients were abundant in IM but scarce in MO. Combining neoantigen prediction and immunopeptidomes identified T cell-specific neoepitopes and achieved a high verification rate in vitro. Immunoediting mainly occurred in MO but not IM, due to the relatively low immune infiltration. Loss of heterozygosity of human leukocyte antigen (HLA) alleles, identified in 17% of multifocal HCC, hampered the ability of major histocompatibility complex to present neoantigens, especially in IM. An integrated analysis of Immunoscore, immunoediting, TCR clonality and HLA loss of heterozygosity in each tumor could stratify patients into 2 groups based on whether they have a high or low risk of recurrence (p = 0.038). CONCLUSION: Our study comprehensively characterized the genetic structure, neoepitope landscape, T cell profile and immunoediting status that collectively shape tumor evolution and could be used to optimize personalized immunotherapies for multifocal HCC. LAY SUMMARY: Immunogenomic features of multifocal hepatocellular carcinoma (HCC) are important for understanding immune-escape mechanisms and developing more effective immunotherapy. Herein, comprehensive immunogenomic characterization showed that diverse genomic structures within multifocal HCC would leave footprints on the immune landscape. Only a few tumors were under the control of immunosurveillance, while others evaded the immune system through multiple mechanisms that led to poor prognosis. Our study revealed heterogeneous immunogenomic landscapes and immune-constrained tumor evolution, the understanding of which could be used to optimize personalized immunotherapies for multifocal HCC.

Splicing Regulator p54nrb /Non-POU Domain-Containing Octamer-Binding Protein Enhances Carcinogenesis Through Oncogenic Isoform Switch of MYC Box-Dependent Interacting Protein 1 in Hepatocellular Carcinoma.

BACKGROUND AND AIMS: Alternative splicing (AS) is a key step that increases the diversity and complexity of the cancer transcriptome. Recent evidence has highlighted that AS has an increasingly crucial role in cancer. Nonetheless, the mechanisms underlying AS and its dysregulation in hepatocellular carcinoma (HCC) remain elusive. Here, we report that the expression of RNA-binding protein p54nrb /non-POU domain-containing octamer-binding protein (NONO) is frequently increased in patients with HCC and is associated with poor outcomes. APPROACH AND RESULTS: Knockdown of NONO significantly abolished liver cancer cell proliferation, migration, and tumor formation. RNA-sequencing revealed that NONO regulates MYC box-dependent interacting protein 1 (or bridging integrator 1 [BIN1]; also known as amphiphysin 2 3P9) exon 12a splicing. In the normal liver, BIN1 generates a short isoform (BIN1-S) that acts as a tumor suppressor by inhibiting the binding of c-Myc to target gene promoters. In HCC, NONO is highly up-regulated and produces a long isoform (BIN1-L, which contains exon 12a) instead of BIN1-S. High levels of BIN1-L promote carcinogenesis by binding with the protein polo-like kinase 1 to enhance its stability through the prevention of ubiquitin/proteasome-dependent cullin 3 degradation. Further analysis revealed that NONO promotes BIN1 exon 12a inclusion through interaction with DExH-box helicase 9 (DHX9) and splicing factor proline and glutamine-rich (SFPQ). Notably, frequent coexpression of DHX9-NONO-SFPQ is observed in patients with HCC. CONCLUSIONS: Taken together, our findings identify the DHX9-NONO-SFPQ complex as a key regulator manipulating the oncogenic splicing switch of BIN1 and as a candidate therapeutic target in liver cancer.

Integrated Proteogenomic Characterization of HBV-Related Hepatocellular Carcinoma.

We performed the first proteogenomic characterization of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) using paired tumor and adjacent liver tissues from 159 patients. Integrated proteogenomic analyses revealed consistency and discordance among multi-omics, activation status of key signaling pathways, and liver-specific metabolic reprogramming in HBV-related HCC. Proteomic profiling identified three subgroups associated with clinical and molecular attributes including patient survival, tumor thrombus, genetic profile, and the liver-specific proteome. These proteomic subgroups have distinct features in metabolic reprogramming, microenvironment dysregulation, cell proliferation, and potential therapeutics. Two prognostic biomarkers, PYCR2 and ADH1A, related to proteomic subgrouping and involved in HCC metabolic reprogramming, were identified. CTNNB1 and TP53 mutation-associated signaling and metabolic profiles were revealed, among which mutated CTNNB1-associated ALDOA phosphorylation was validated to promote glycolysis and cell proliferation. Our study provides a valuable resource that significantly expands the knowledge of HBV-related HCC and may eventually benefit clinical practice.