Opposing roles of hepatic stellate cell subpopulations in hepatocarcinogenesis.

Hepatocellular carcinoma (HCC), the fourth leading cause of cancer mortality worldwide, develops almost exclusively in patients with chronic liver disease and advanced fibrosis1,2. Here we interrogated functions of hepatic stellate cells (HSCs), the main source of liver fibroblasts3, during hepatocarcinogenesis. Genetic depletion, activation or inhibition of HSCs in mouse models of HCC revealed their overall tumour-promoting role. HSCs were enriched in the preneoplastic environment, where they closely interacted with hepatocytes and modulated hepatocarcinogenesis by regulating hepatocyte proliferation and death. Analyses of mouse and human HSC subpopulations by single-cell RNA sequencing together with genetic ablation of subpopulation-enriched mediators revealed dual functions of HSCs in hepatocarcinogenesis. Hepatocyte growth factor, enriched in quiescent and cytokine-producing HSCs, protected against hepatocyte death and HCC development. By contrast, type I collagen, enriched in activated myofibroblastic HSCs, promoted proliferation and tumour development through increased stiffness and TAZ activation in pretumoural hepatocytes and through activation of discoidin domain receptor 1 in established tumours. An increased HSC imbalance between cytokine-producing HSCs and myofibroblastic HSCs during liver disease progression was associated with increased HCC risk in patients. In summary, the dynamic shift in HSC subpopulations and their mediators during chronic liver disease is associated with a switch from HCC protection to HCC promotion.

DLK1/DIO3 locus upregulation by a ß-catenin-dependent enhancer drives cell proliferation and liver tumorigenesis.

The CTNNB1 gene, encoding ß-catenin, is frequently mutated in hepatocellular carcinoma (HCC, ∼30%) and in hepatoblastoma (HB, >80%), in which DLK1/DIO3 locus induction is correlated with CTNNB1 mutations. Here, we aim to decipher how sustained ß-catenin activation regulates DLK1/DIO3 locus expression and the role this locus plays in HB and HCC development in mouse models deleted for Apc (ApcΔhep) or Ctnnb1-exon 3 (ß-cateninΔExon3) and in human CTNNB1-mutated hepatic cancer cells. We identified an enhancer site bound by TCF-4/ß-catenin complexes in an open conformation upon sustained ß-catenin activation (DLK1-Wnt responsive element [WRE]) and increasing DLK1/DIO3 locus transcription in ß-catenin-mutated human HB and mouse models. DLK1-WRE editing by CRISPR-Cas9 approach impaired DLK1/DIO3 locus expression and slowed tumor growth in subcutaneous CTNNB1-mutated tumor cell grafts, ApcΔhep HB and ß-cateninΔExon3 HCC. Tumor growth inhibition resulted either from increased FADD expression and subsequent caspase-3 cleavage in the first case or from decreased expression of cell cycle actors regulated by FoxM1 in the others. Therefore, the DLK1/DIO3 locus is an essential determinant of FoxM1-dependent cell proliferation during ß-catenin-driven liver tumorigenesis. Targeting the DLK1-WRE enhancer to silence the DLK1/DIO3 locus might thus represent an interesting therapeutic strategy to restrict tumor growth in primary liver cancers with CTNNB1 mutations.

Artificial intelligence-based pathology as a biomarker of sensitivity to atezolizumab-bevacizumab in patients with hepatocellular carcinoma: a multicentre retrospective study.

BACKGROUND: Clinical benefits of atezolizumab plus bevacizumab (atezolizumab-bevacizumab) are observed only in a subset of patients with hepatocellular carcinoma and the development of biomarkers is needed to improve therapeutic strategies. The atezolizumab-bevacizumab response signature (ABRS), assessed by molecular biology profiling techniques, has been shown to be associated with progression-free survival after treatment initiation. The primary objective of our study was to develop an artificial intelligence (AI) model able to estimate ABRS expression directly from histological slides, and to evaluate if model predictions were associated with progression-free survival. METHODS: In this multicentre retrospective study, we developed a model (ABRS-prediction; ABRS-P), which was derived from the previously published clustering-constrained attention multiple instance learning (or CLAM) pipeline. We trained the model fit for regression analysis using a multicentre dataset from The Cancer Genome Atlas (patients treated by surgical resection, n=336). The ABRS-P model was externally validated on two independent series of samples from patients with hepatocellular carcinoma (a surgical resection series, n=225; and a biopsy series, n=157). The predictive value of the model was further tested in a series of biopsy samples from a multicentre cohort of patients with hepatocellular carcinoma treated with atezolizumab-bevacizumab (n=122). All samples in the study were from adults (aged ≥18 years). The validation sets were sampled between Jan 1, 2008, to Jan 1, 2023. For the multicentre validation set, the primary objective was to assess the association of high versus low ABRS-P values, defined relative to cross-validation median split thresholds in the first biopsy series, with progression-free survival after treatment initiation. Finally, we performed spatial transcriptomics and matched prediction heatmaps with in situ expression profiles. FINDINGS: Of the 840 patients sampled, 641 (76%) were male and 199 (24%) were female. Across the development and validation datasets, hepatocellular carcinoma risk factors included alcohol intake, hepatitis B and C virus infections, and non-alcoholic steatohepatitis. Using cross-validation in the development series, the mean Pearson's correlation between ABRS-P values and ABRS score (mean expression of ABRS genes) was r=0·62 (SD 0·09; mean p<0·0001, SD<0·0001). The ABRS-P generalised well on the external validation series (surgical resection series, r=0·60 [95% CI 0·51-0·68], p<0·0001; biopsy series, r=0·53 [0·40-0·63], p<0·0001). In the 122 patients treated with atezolizumab-bevacizumab, those with ABRS-P-high tumours (n=74) showed significantly longer median progression-free survival than those with ABRS-P-low tumours (n=48) after treatment initiation (12 months [95% CI 7-not reached] vs 7 months [4-9]; p=0·014). Spatial transcriptomics showed significantly higher ABRS score, along with upregulation of various other immune effectors, in tumour areas with high ABRS-P values versus areas with low ABRS-P values. INTERPRETATION: Our study indicates that AI applied on hepatocellular carcinoma digital slides is able to serve as a biomarker for progression-free survival in patients treated with atezolizumab-bevacizumab. This approach could be used in the development of inexpensive and fast biomarkers for targeted therapies. The combination of AI heatmaps with spatial transcriptomics provides insight on the molecular features associated with predictions. This methodology could be applied to other cancers or diseases and improve understanding of the biological mechanisms that drive responses to treatments. FUNDING: Institut National du Cancer, Fondation ARC, China Scholarship Council, Ligue Contre le Cancer du Val de Marne, Fondation de l'Avenir, Ipsen, and Fondation Bristol Myers Squibb Pour la Recherche en Immuno-Oncologie.

RSK2 inactivation cooperates with AXIN1 inactivation or ß-catenin activation to promote hepatocarcinogenesis.

BACKGROUND & AIMS: Recurrent somatic mutations of the RPS6KA3 gene encoding for the serine/threonine kinase RSK2 were identified in hepatocellular carcinomas (HCCs), suggesting its tumour-suppressive function. Our goal was to demonstrate the tumour suppressor role of RSK2 in the liver and investigate the functional consequences of its inactivation. METHODS: We analysed a series of 1,151 human HCCs for RSK2 mutations and 20 other driver genetic alterations. We then modelled RSK2 inactivation in mice in various mutational contexts recapitulating or not those naturally found in human HCC, using transgenic mice and liver-specific carcinogens. These models were monitored for liver tumour appearance and subjected to phenotypic and transcriptomic analyses. Functional consequences of RSK2 rescue were also investigated in a human RSK2-deficient HCC cell line. RESULTS: RSK2-inactivating mutations are specific to human HCC and frequently co-occur with AXIN1-inactivating or ß-catenin-activating mutations. Modelling of these co-occurrences in mice showed a cooperative effect in promoting liver tumours with transcriptomic profiles recapitulating those of human HCCs. By contrast, there was no cooperation in liver tumour induction between RSK2 loss and BRAF-activating mutations chemically induced by diethylnitrosamine. In human liver cancer cells, we also showed that RSK2 inactivation confers some dependency to the activation of RAS/MAPK signalling that can be targeted by MEK inhibitors. CONCLUSIONS: Our study demonstrates the tumour suppressor role of RSK2 and its specific synergistic effect in hepatocarcinogenesis when its loss of function is specifically combined with AXIN1 inactivation or ß-catenin activation. Furthermore, we identified the RAS/MAPK pathway as a potential therapeutic target for RSK2-inactivated liver tumours. IMPACT AND IMPLICATIONS: This study demonstrated the tumour suppressor role of RSK2 in the liver and showed that its inactivation specifically synergises with AXIN1 inactivation or ß-catenin activation to promote the development of HCC with similar transcriptomic profiles as found in humans. Furthermore, this study highlights that activation of the RAS/MAPK pathway is one of the key signalling pathways mediating the oncogenic effect of RSK2 inactivation that can be targeted with already available anti-MEK therapies.

Borderline Hepatocellular Adenomas: A Practical Diagnostic Approach Based on Pathologic and Molecular Features.

Borderline hepatocellular adenomas (BL-HCA) are characterized by focal architectural/cytologic atypia and reticulin loss, features that are insufficient for a definitive diagnosis of hepatocellular carcinoma (HCC). The diagnosis and management of BL-HCA are challenging as their biological behavior, especially in terms of malignant potential, is still debated. We aimed to compare the clinicopathologic and molecular features of BL-HCA with those of typical HCA (T-HCA), HCA with malignant transformation (HCC on HCA), and HCC to assess the risk of malignancy. One hundred six liver resection specimens were retrospectively selected from 2 reference centers, including 39 BL-HCA, 42 T-HCA, 12 HCC on HCA, and 13 HCC specimens. Somatic mutations, including TERT promoter mutations associated with HCA malignant transformation and the gene expression levels of 96 genes, were investigated in 93 frozen samples. Additionally, TERT promoter mutations were investigated in 44 formalin-fixed, paraffin-embedded samples. The clinical features of patients with BL-HCA were similar to those of patients with T-HCA, patients being mainly women (69%) with a median age of 37 years. The median tumor size was 7.5 cm, 64% of patients had a single nodule, and no recurrence was observed. Compared with T-HCA, BL-HCA was significantly enriched in ß-catenin-mutated HCA in exon 3 (41% vs 6%; P < .001). Unsupervised statistical analysis based on gene expression showed that BL-HCA overlapped with T-HCA and HCC on HCA, favoring a molecular continuum of the tumors. TERT promoter mutations were observed only in HCC on HCA (42%) and in HCC (38%). In conclusion, these results suggest that despite their worrisome morphologic features, the clinicopathologic and molecular features of BL-HCA are much closer to those of T-HCA than those of HCC on HCA or HCC. This strongly supports the usefulness of combining morphologic and molecular analyses in a practical diagnostic approach for guiding the management of BL-HCA.

Omicron induced distinct immune respiratory transcriptomics signatures compared to pre-existing variants in critically ill COVID-19 patients.

Severe coronavirus disease 2019 (COVID-19) is related to dysregulated immune responses. We aimed to explore the effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants on the immune response by nasopharyngeal transcriptomic in critically-ill patients. This prospective monocentric study included COVID-19 patients requiring intensive care unit (ICU) admission between March 2020 and 2022. Patients were classified according to VOC (ancestral, Alpha, Delta, and Omicron). Eighty-eight patients with severe COVID-19 were included after matching (on prespecified clinical criteria). Profiling of gene expression markers of innate and adaptive immune responses were investigated by respiratory transcriptomics at ICU admission. Eighty-eight patients were included in the study after matching (ancestral [n = 24], Alpha [n = 24], Delta [n = 22], and Omicron [n = 18] variants). Respiratory transcriptomic analysis revealed distinct innate and adaptive immune profiling between variants. In comparison with the ancestral variant, there was a reduced expression of neutrophil degranulation, T cell activation, cytokines signalling pathways in patients infected with Alpha and Delta variants. In contrast, there was a higher expression of neutrophil degranulation, T and B cells activation, and inflammatory interleukins pathways in patients infected with Omicron. To conclude, Omicron induced distinct immune respiratory transcriptomics signatures compared to pre-existing variants in patients with severe COVID-19, pointing to an evolving pathophysiology of severe COVID-19 in the Omicron era.

Hepatitis B virus integrations promote local and distant oncogenic driver alterations in hepatocellular carcinoma.

OBJECTIVE: Infection by HBV is the main risk factor for hepatocellular carcinoma (HCC) worldwide. HBV directly drives carcinogenesis through integrations in the human genome. This study aimed to precisely characterise HBV integrations, in relation with viral and host genomics and clinical features. DESIGN: A novel pipeline was set up to perform viral capture on tumours and non-tumour liver tissues from a French cohort of 177 patients mainly of European and African origins. Clonality of each integration event was determined with the localisation, orientation and content of the integrated sequence. In three selected tumours, complex integrations were reconstructed using long-read sequencing or Bionano whole genome mapping. RESULTS: Replicating HBV DNA was more frequently detected in non-tumour tissues and associated with a higher number of non-clonal integrations. In HCC, clonal selection of HBV integrations was related to two different mechanisms involved in carcinogenesis. First, integration of viral enhancer nearby a cancer-driver gene may lead to a strong overexpression of oncogenes. Second, we identified frequent chromosome rearrangements at HBV integration sites leading to cancer-driver genes (TERT, TP53, MYC) alterations at distance. Moreover, HBV integrations have direct clinical implications as HCC with a high number of insertions develop in young patients and have a poor prognosis. CONCLUSION: Deep characterisation of HBV integrations in liver tissues highlights new HBV-associated driver mechanisms involved in hepatocarcinogenesis. HBV integrations have multiple direct oncogenic consequences that remain an important challenge for the follow-up of HBV-infected patients.

Common genetic variation in alcohol-related hepatocellular carcinoma: a case-control genome-wide association study.

BACKGROUND: Hepatocellular carcinoma is a frequent consequence of alcohol-related liver disease, with variable incidence among heavy drinkers. We did a genome-wide association study (GWAS) to identify common genetic variants for alcohol-related hepatocellular carcinoma. METHODS: We conducted a two-stage case-control GWAS in a discovery cohort of 2107 unrelated European patients with alcohol-related liver disease aged 20-92 years recruited between Oct 22, 1993, and March 12, 2017. Cases were patients with alcohol-related hepatocellular carcinoma diagnosed by imaging or histology. Controls were patients with alcohol-related liver disease without hepatocellular carcinoma. We used an additive logistic regression model adjusted for the first ten principal components to assess genetic variants associated with alcohol-related hepatocellular carcinoma. We did another analysis with adjustment for age, sex, and liver fibrosis. New candidate associations (p<1 × 10-6) and variants previously associated with alcohol-related hepatocellular carcinoma were evaluated in a validation cohort of 1933 patients with alcohol-related liver disease aged 29-92 years recruited between July 21, 1995, and May 2, 2019. We did a meta-analysis of the two case-control cohorts. FINDINGS: The discovery cohort included 775 cases and 1332 controls. Of 7 962 325 variants assessed, we identified WNT3A-WNT9A (rs708113; p=1·11 × 10-8) and found support for previously reported regions associated with alcohol-related hepatocellular carcinoma risk at TM6SF2 (rs58542926; p=6·02 × 10-10), PNPLA3 (rs738409; p=9·29 × 10-7), and HSD17B13 (rs72613567; p=2·49 × 10-4). The validation cohort included 874 cases and 1059 controls and three variants were replicated: WNT3A-WNT9A (rs708113; p=1·17 × 10-3), TM6SF2 (rs58542926; p=4·06 × 10-5), and PNPLA3 (rs738409; p=1·17 × 10-4). All three variants reached GWAS significance in the meta-analysis: WNT3A-WNT9A (odds ratio 0·73, 95% CI 0·66-0·81; p=3·93 × 10-10), TM6SF2 (1·77, 1·52-2·07; p=3·84×10-13), PNPLA3 (1·34, 1·22-1·47; p=7·30 × 10-10). Adjustment for clinical covariates yielded similar results. We observed an additive effect of at-risk alleles on alcohol-related hepatocellular carcinoma. WNT3A-WNT9A rs708113 was not associated with liver fibrosis. INTERPRETATION: WNT3A-WNT9A is a susceptibility locus for alcohol-related hepatocellular carcinoma, suggesting an early role of the Wnt-ß-catenin pathway in alcohol-related hepatocellular carcinoma carcinogenesis. FUNDING: Ligue Nationale contre le Cancer, Bpifrance, INSERM, AFEF, CARPEM, Labex OncoImmunology, and Agence Nationale de la Recherche.

Nivolumab, nivolumab-ipilimumab, and VEGFR-tyrosine kinase inhibitors as first-line treatment for metastatic clear-cell renal cell carcinoma (BIONIKK): a biomarker-driven, open-label, non-comparative, randomised, phase 2 trial.

BACKGROUND: We previously reported a 35-gene expression classifier identifying four clear-cell renal cell carcinoma groups (ccrcc1 to ccrcc4) with different tumour microenvironments and sensitivities to sunitinib in metastatic clear-cell renal cell carcinoma. Efficacy profiles might differ with nivolumab and nivolumab-ipilimumab. We therefore aimed to evaluate treatment efficacy and tolerability of nivolumab, nivolumab-ipilimumab, and VEGFR-tyrosine kinase inhibitors (VEGFR-TKIs) in patients according to tumour molecular groups. METHODS: This biomarker-driven, open-label, non-comparative, randomised, phase 2 trial included patients from 15 university hospitals or expert cancer centres in France. Eligible patients were aged 18 years or older, had an Eastern Cooperative Oncology Group performance status of 0-2, and had previously untreated metastatic clear-cell renal cell carcinoma. Patients were randomly assigned (1:1) using permuted blocks of varying sizes to receive either nivolumab or nivolumab-ipilimumab (ccrcc1 and ccrcc4 groups), or either a VEGFR-TKI or nivolumab-ipilimumab (ccrcc2 and ccrcc3 groups). Patients assigned to nivolumab-ipilimumab received intravenous nivolumab 3 mg/kg plus ipilimumab 1 mg/kg every 3 weeks for four doses followed by intravenous nivolumab 240 mg every 2 weeks. Patients assigned to nivolumab received intravenous nivolumab 240 mg every 2 weeks. Patients assigned to VEGFR-TKIs received oral sunitinib (50 mg/day for 4 weeks every 6 weeks) or oral pazopanib (800 mg daily continuously). The primary endpoint was the objective response rate by investigator assessment per Response Evaluation Criteria in Solid Tumors version 1.1. The primary endpoint and safety were assessed in the population who received at least one dose of study drug. This trial is registered with ClinicalTrials.gov, NCT02960906, and with the EU Clinical Trials Register, EudraCT 2016-003099-28, and is closed to enrolment. FINDINGS: Between June 28, 2017, and July 18, 2019, 303 patients were screened for eligibility, 202 of whom were randomly assigned to treatment (61 to nivolumab, 101 to nivolumab-ipilimumab, 40 to a VEGFR-TKI). In the nivolumab group, two patients were excluded due to a serious adverse event before the first study dose and one patient was excluded from analyses due to incorrect diagnosis. Median follow-up was 18·0 months (IQR 17·6-18·4). In the ccrcc1 group, objective responses were seen in 12 (29%; 95% CI 16-45) of 42 patients with nivolumab and 16 (39%; 24-55) of 41 patients with nivolumab-ipilimumab (odds ratio [OR] 0·63 [95% CI 0·25-1·56]). In the ccrcc4 group, objective responses were seen in seven (44%; 95% CI 20-70) of 16 patients with nivolumab and nine (50% 26-74) of 18 patients with nivolumab-ipilimumab (OR 0·78 [95% CI 0·20-3·01]). In the ccrcc2 group, objective responses were seen in 18 (50%; 95% CI 33-67) of 36 patients with a VEGFR-TKI and 19 (51%; 34-68) of 37 patients with nivolumab-ipilimumab (OR 0·95 [95% CI 0·38-2·37]). In the ccrcc3 group, no objective responses were seen in the four patients who received a VEGFR-TKI, and in one (20%; 95% CI 1-72) of five patients who received nivolumab-ipilimumab. The most common treatment-related grade 3-4 adverse events were hepatic failure and lipase increase (two [3%] of 58 for both) with nivolumab, lipase increase and hepatobiliary disorders (six [6%] of 101 for both) with nivolumab-ipilimumab, and hypertension (six [15%] of 40) with a VEGFR-TKI. Serious treatment-related adverse events occurred in two (3%) patients in the nivolumab group, 38 (38%) in the nivolumab-ipilimumab group, and ten (25%) patients in the VEGFR-TKI group. Three deaths were treatment-related: one due to fulminant hepatitis with nivolumab-ipilimumab, one death from heart failure with sunitinib, and one due to thrombotic microangiopathy with sunitinib. INTERPRETATION: We demonstrate the feasibility and positive effect of a prospective patient selection based on tumour molecular phenotype to choose the most efficacious treatment between nivolumab with or without ipilimumab and a VEGFR-TKI in the first-line treatment of metastatic clear-cell renal cell carcinoma. FUNDING: Bristol Myers Squibb, ARTIC.

Gene expression signature as a surrogate marker of microvascular invasion on routine hepatocellular carcinoma biopsies.

BACKGROUND & AIMS: Microvascular invasion (MVI), a major risk factor for tumor recurrence after surgery in hepatocellular carcinoma (HCC), is only detectable by microscopic examination of the surgical specimen. We aimed to define a transcriptomic signature associated with MVI in HCC than can be applied to formalin-fixed paraffin-embedded (FFPE) biopsies for use in clinical practice. METHODS: To identify a gene expression signature related to MVI by using NanoString technology, we selected a set of 200 genes according to the literature and RNA-sequencing data obtained from a cohort of 150 frozen HCC samples previously published. We used 178 FFPE-archived HCC samples, including 109 surgical samples for the training set and 69 paired pre-operative biopsies for the validation set. In 14 cases of the training set, a paired biopsy was available and was also analyzed. RESULTS: We identified a 6-gene signature (ROS1, UGT2B7, FAS, ANGPTL7, GMNN, MKI67) strongly associated with MVI in the training set of FFPE surgical HCC samples, with 82% accuracy (sensitivity 82%, specificity 81%, AUC 0.82). The NanoString gene expression was highly correlated in 14 paired surgical/biopsy HCC samples (mean R: 0.97). In the validation set of 69 FFPE HCC biopsies, the 6-gene NanoString signature predicted MVI with 74% accuracy (sensitivity 73%, specificity 76%, AUC 0.74). Moreover, on multivariate analysis, the MVI signature was associated with overall survival in both sets (hazard ratio 2.29; 95% CI 1.03-5.07; p = 0.041). CONCLUSION: We defined a 6-gene signature that can accurately predict MVI in FFPE HCC biopsy samples, which is also associated with overall survival, although its survival impact must be confirmed by extensive study with further clinical data. LAY SUMMARY: Microvascular invasion, a major risk factor for tumor recurrence after surgery in hepatocellular carcinoma, is only detectable by microscopic examination of a surgical specimen. In this study, we defined a relevant surrogate signature of microvascular invasion in hepatocellular carcinoma that may be applied in clinical practice with routine tumor biopsy and integrated into the therapeutic strategy.

Deleting the ß-catenin degradation domain in mouse hepatocytes drives hepatocellular carcinoma or hepatoblastoma-like tumor growth.

BACKGROUND & AIMS: One-third of hepatocellular carcinomas (HCCs) harbor mutations activating the ß-catenin pathway, predominantly via mutations in the CTNNB1 gene itself. Mouse models of Apc loss-of-function are widely used to mimic ß-catenin-dependent tumorigenesis. Given the low prevalence of APC mutations in human HCCs, we aimed to generate liver tumors through CTNNB1 exon 3 deletion (ßcatΔex3). We then compared ßcatΔex3 liver tumors with liver tumors generated via frameshift in exon 15 of Apc (Apcfs-ex15). METHODS: We used hepatocyte-specific and inducible mouse models generated through either a Cre-Lox or a CRISPR/Cas9 approach using adeno-associated virus vectors. Tumors generated by the Cre-Lox models were phenotypically analyzed using immunohistochemistry and were selected for transcriptomic analysis by RNA-sequencing (RNAseq). Mouse RNAseq data were compared to human RNAseq data (8 normal tissues, 48 HCCs, 9 hepatoblastomas) in an integrative analysis. Tumors generated via CRISPR were analyzed using DNA sequencing and immuno-histochemistry. RESULTS: Mice with CTNNB1 exon 3 deletion in hepatocytes developed liver tumors indistinguishable from Apcfs-ex15 liver tumors. Both Apcfs-ex15 and ßcatΔex3 mouse models induced growth of phenotypically distinct tumors (differentiated or undifferentiated). Integrative analysis of human and mouse tumors showed that differentiated mouse tumors cluster with well-differentiated human CTNNB1-mutated tumors. Conversely, undifferentiated mouse tumors cluster with human mesenchymal hepatoblastomas and harbor activated YAP signaling. CONCLUSION: Apcfs-ex15 and ßcatΔex3 mouse models both induce growth of tumors that are transcriptionally similar to either well-differentiated and ß-catenin-activated human HCCs or mesenchymal hepatoblastomas. LAY SUMMARY: New and easy-to-use transgenic mouse models of primary liver cancers have been generated, with mutations in the gene encoding beta-catenin, which are frequent in both adult and pediatric primary liver cancers. The mice develop both types of cancer, constituting a strong preclinical model.

Artificial intelligence predicts immune and inflammatory gene signatures directly from hepatocellular carcinoma histology.

BACKGROUND & AIMS: Patients with hepatocellular carcinoma (HCC) displaying overexpression of immune gene signatures are likely to be more sensitive to immunotherapy, however, the use of such signatures in clinical settings remains challenging. We thus aimed, using artificial intelligence (AI) on whole-slide digital histological images, to develop models able to predict the activation of 6 immune gene signatures. METHODS: AI models were trained and validated in 2 different series of patients with HCC treated by surgical resection. Gene expression was investigated using RNA sequencing or NanoString technology. Three deep learning approaches were investigated: patch-based, classic MIL and CLAM. Pathological reviewing of the most predictive tissue areas was performed for all gene signatures. RESULTS: The CLAM model showed the best overall performance in the discovery series. Its best-fold areas under the receiver operating characteristic curves (AUCs) for the prediction of tumors with upregulation of the immune gene signatures ranged from 0.78 to 0.91. The different models generalized well in the validation dataset with AUCs ranging from 0.81 to 0.92. Pathological analysis of highly predictive tissue areas showed enrichment in lymphocytes, plasma cells, and neutrophils. CONCLUSION: We have developed and validated AI-based pathology models able to predict the activation of several immune and inflammatory gene signatures. Our approach also provides insights into the morphological features that impact the model predictions. This proof-of-concept study shows that AI-based pathology could represent a novel type of biomarker that will ease the translation of our biological knowledge of HCC into clinical practice. LAY SUMMARY: Immune and inflammatory gene signatures may be associated with increased sensitivity to immunotherapy in patients with advanced hepatocellular carcinoma. In the present study, the use of artificial intelligence-based pathology enabled us to predict the activation of these signatures directly from histology.

DNA Methylation Signatures Reveal the Diversity of Processes Remodeling Hepatocellular Carcinoma Methylomes.

BACKGROUND AND AIMS: DNA methylation patterns are highly rearranged in HCCs. However, diverse sources of variation are intermingled in cancer methylomes, precluding the precise characterization of underlying molecular mechanisms. We developed a computational framework (methylation signature analysis with independent component analysis [MethICA]) leveraging independent component analysis to disentangle the diverse processes contributing to DNA methylation changes in tumors. APPROACH AND RESULTS: Applied to a collection of 738 HCCs, MethICA unraveled 13 stable methylation components preferentially active in specific chromatin states, sequence contexts, and replication timings. These included signatures of general processes associated with sex and age but also signatures related to specific driver events and molecular subgroups. Catenin beta 1 mutations were major modulators of methylation patterns in HCC, characterized by a targeted hypomethylation of transcription factor 7-bound enhancers in the vicinity of Wnt target genes as well as a widespread hypomethylation of late-replicated partially methylated domains. By contrast, demethylation of early replicated highly methylated domains was a signature of replication stress, leading to an extensive hypomethylator phenotype in cyclin-activated HCC. Inactivating mutations of the chromatin remodeler AT-rich interactive domain-containing protein 1A were associated with epigenetic silencing of differentiation-promoting transcriptional networks, also detectable in cirrhotic liver. Finally, a hypermethylation signature targeting polycomb-repressed chromatin domains was identified in the G1 molecular subgroup with progenitor features. CONCLUSIONS: This study elucidates the diversity of processes remodeling HCC methylomes and reveals the epigenetic and transcriptional impact of driver alterations.

Expression of NKG2D ligands is downregulated by ß-catenin signalling and associates with HCC aggressiveness.

BACKGROUND & AIMS: The NKG2D system is a potent immunosurveillance mechanism in cancer, wherein the activating NK cell receptor (NKG2D) on immune cells recognises its cognate ligands on tumour cells. Herein, we evaluated the expression of NKG2D ligands in hepatocellular carcinoma (HCC), in both humans and mice, taking the genomic features of HCC tumours into account. METHODS: The expression of NKG2D ligands (MICA, MICB, ULBP1 and ULBP2) was analysed in large human HCC datasets by Fluidigm TaqMan and RNA-seq methods, and in 2 mouse models (mRNA and protein levels) reproducing the features of both major groups of human tumours. RESULTS: We provide compelling evidence that expression of the MICA and MICB ligands in human HCC is associated with tumour aggressiveness and poor patient outcome. We also found that the expression of ULBP1 and ULBP2 was associated with poor patient outcome, and was downregulated in CTNNB1-mutated HCCs displaying low levels of inflammation and associated with a better prognosis. We also found an inverse correlation between ULBP1/2 expression levels and the expression of ß-catenin target genes in patients with HCC, suggesting a role for ß-catenin signalling in inhibiting expression. We showed in HCC mouse models that ß-catenin signalling downregulated the expression of Rae-1 NKG2D ligands, orthologs of ULBPs, through TCF4 binding. CONCLUSIONS: We demonstrate that the expression of NKG2D ligands is associated with aggressive liver tumorigenesis and that the downregulation of these ligands by ß-catenin signalling may account for the less aggressive phenotype of CTNNB1-mutated HCC tumours. LAY SUMMARY: The NKG2D system is a potent immunosurveillance mechanism in cancer. However, its role in hepatocellular carcinoma development has not been widely investigated. Herein, we should that the expression of NKG2D ligands by tumour cells is associated with a more aggressive tumour subtype.

Telomere length is key to hepatocellular carcinoma diversity and telomerase addiction is an actionable therapeutic target.

BACKGROUND & AIMS: Telomerase activation is the earliest event in hepatocellular carcinoma (HCC) development. Thus, we aimed to elucidate the role of telomere length maintenance during liver carcinogenesis. METHODS: Telomere length was measured in the tumor and non-tumor liver tissues of 1,502 patients (978 with HCC) and integrated with TERT alterations and expression, as well as clinical and molecular (analyzed by genome, exome, targeted and/or RNA-sequencing) features of HCC. The preclinical efficacy of anti-TERT antisense oligonucleotides (ASO) was assessed in vitro in 26 cell lines and in vivo in a xenograft mouse model. RESULTS: Aging, liver fibrosis, male sex and excessive alcohol consumption were independent determinants of liver telomere attrition. HCC that developed in livers with long telomeres frequently had wild-type TERT with progenitor features and BAP1 mutations. In contrast, HCC that developed on livers with short telomeres were enriched in the non-proliferative HCC class and frequently had somatic TERT promoter mutations. In HCCs, telomere length is stabilized in a narrow biological range around 5.7 kb, similar to non-tumor livers, by various mechanisms that activate TERT expression. Long telomeres are characteristic of very aggressive HCCs, associated with the G3 transcriptomic subclass, TP53 alterations and poor prognosis. In HCC cell lines, TERT silencing with ASO was efficient in highly proliferative and poorly differentiated cells. Treatment for 3 to 16 weeks induced cell proliferation arrest in 12 cell lines through telomere shortening, DNA damage and activation of apoptosis. The therapeutic effect was also obtained in a xenograft mouse model. CONCLUSIONS: Telomere maintenance in HCC carcinogenesis is diverse, and is associated with tumor progression and aggressiveness. The efficacy of anti-TERT ASO treatment in cell lines revealed the oncogenic addiction to TERT in HCC, providing a preclinical rationale for anti-TERT ASO treatment in HCC clinical trials. LAY SUMMARY: Telomeres are repeated DNA sequences that protect chromosomes and naturally shorten in most adult cells because of the inactivation of the TERT gene, coding for the telomerase enzyme. Here we show that telomere attrition in the liver, modulated by aging, sex, fibrosis and alcohol, associates with specific clinical and molecular features of hepatocellular carcinoma, the most frequent primary liver cancer. We also show that liver cancer is dependent on TERT reactivation and telomere maintenance, which could be targeted through a novel therapeutic approach called antisense oligonucleotides.

AICAR and compound C negatively modulate HCC-induced primary human hepatic stellate cell activation in vitro.

Tumor stroma and microenvironment have been shown to affect hepatocellular carcinoma (HCC) growth, with activated hepatic stellate cells (HSC) as a major contributor in this process. Recent evidence suggests that the energy sensor adenosine monophosphate-activated kinase (AMPK) may mediate a series of essential processes during carcinogenesis and HCC progression. Here, we investigated the effect of different HCC cell lines with known TP53 or CTNBB1 mutations on primary human HSC activation, proliferation, and AMPK activation. We show that conditioned media obtained from multiple HCC cell lines differently modulate human hepatic stellate cell (hHSC) proliferation and hHSC AMPK activity in a paracrine manner. Pharmacological treatment of hHSC with AICAR and Compound C inhibited the HCC-induced proliferation/activation of hHSC through AMPK-dependent and AMPK-independent mechanisms, which was further confirmed using mouse embryonic fibroblasts (MEFs) deficient of both catalytic AMPKα isoforms (AMPKα1/α2-/-) and wild type (wt) MEF. Both compounds induced S-phase cell-cycle arrest and, in addition, AICAR inhibited the mTORC1 pathway by inhibiting phosphorylation of 4E-BP1 and S6 in hHSC and wt MEF. Data mining of the Cancer Genome Atlas (TCGA) and the Liver Cancer (LICA-FR) showed that AMPKα1 (PRKAA1) and AMPKα2 (PRKAA2) expression differed depending on the mutation (TP53 or CTNNB1), tumor grading, and G1-G6 classification, reflecting the heterogeneity in human HCC. Overall, we provide evidence that AMPK modulating pharmacological agents negatively modulate HCC-induced hHSC activation and may therefore provide a novel approach to target the mutual, tumor-promoting interactions between hHSC and HCC.NEW & NOTEWORTHY HCC is marked by genetic heterogeneity and activated hepatic stellate cells (HSC) are considered key players during HCC development. The paracrine effect of different HCC cell lines on the activation of primary hHSC was accompanied by differential AMPK activation depending on the HCC line used. Pharmacological treatment inhibited the HCC-induced hHSC activation through AMPK-dependent and AMPK-independent mechanisms. This heterogenic effect on HCC-induced AMPK activation was confirmed by data mining TCGA and LICA-FR databases.

Clinical Impact of Genomic Diversity From Early to Advanced Hepatocellular Carcinoma.

To date, genomic analyses of hepatocellular carcinoma (HCC) have been limited to early stages obtained from liver resection. We aim to describe the genomic profiling of HCC from early to advanced stages. We analyzed 801 HCC from 720 patients (410 resections, 137 transplantations, 122 percutaneous ablations, and 52 noncurative) for 190 gene expressions and for 31 gene mutations. Forty-one advanced HCC and 156 whole exome of Barcelona Clinic Liver Cancer (BCLC) 0/A were analyzed by whole-exome sequencing. Genomic profiling was correlated with tumor stages, clinical features, and survival. Our cohort included patients classified in BCLC stage 0 (9.4%), A (59.5%), B (16.2%), and C (14.9%). Among the overall 801 HCC, the most frequently mutated genes were telomerase reverse transcriptase (TERT) (58.1%), catenin beta 1 (CTNNB1) (30.7%), tumor protein 53 (TP53; 18.7%), AT-rich interaction domain 1A (ARID1A) (13%), albumin (11.4%), apolipoprotein B (APOB) (9.4%), and AXIN1 (9.2%). Advanced-stage HCC (BCLC B/C) showed higher frequencies of splicing factor 3b subunit 1 (SF3B1) (P = 0.0003), TP53 (P = 0.0006), and RB Transcriptional Corepressor 1 mutations (P = 0.03). G1-G6 transcriptomic classification and the molecular prognostic 5-gene score showed different distributions according to the stage of the disease and the type of treatment with an enrichment of G3 (P < 0.0001), poor prognostic score (P < 0.0001), and increased proliferation and dedifferentiation at the transcriptomic level in advanced HCC. The 5-gene score predicted survival in patients treated by resection (P < 0.0001) and ablation (P = 0.01) and in advanced HCC (P = 0.04). Twenty-two percent of advanced HCC harbored potentially druggable genetic alterations, and MET amplification was associated with complete tumor response in patients with advanced HCC treated by a specific MET inhibitor. Conclusion: Genomic analysis across the different stages of HCC revealed the mechanisms of tumor progression and helped to identify biomarkers of response to targeted therapies.

Molecular underpinnings of glandular tropism in metastatic clear cell renal cell carcinoma: therapeutic implications.

BACKGROUND: Glandular metastases (GM) have been associated with improved survival in metastatic clear cell renal cell carcinoma (m-ccRCC). We aimed to molecularly characterize m-ccRCC with GM. MATERIAL AND METHODS: We performed a retrospective cohort study on all m-ccRCC patients with available tissue at our institution, diagnosed with metastatic disease from 2000 to 2019. We determined previously described angiogenesis- and immune-related gene expression signatures (GES) and ccrcc molecular subtypes through whole transcriptome RNA sequencing of primary tumors and metastases. We tested differences in GES and molecular subtypes across groups and studied overall (OS) and progression-free survival (PFS) using Kaplan-Meier survival analysis and Cox regression models. RESULTS: Primary tumors of patients who developed GM (n = 55) had higher IMmotion Angio (p < 0.001) and JAVELIN Angio (p = 0.003) GES as well as a higher proportion of angiogenic ccrcc2 molecular subtypes (p = 0.008) than primary tumors of patients with non-GM (n = 128). Metastatic lesions in glandular organs (n = 32) also had higher IMmotion Angio (p = 0.008) and JAVELIN Angio (p = 0.02) GES and were more frequently of the ccrcc2 molecular subtype (p = 0.03), compared to metastatic lesions in non-glandular organs in patients who did not develop any GM (n = 231), but not compared to metastatic lesions in non-glandular organs in patients who also developed GM (n = 18). Patients with GM had better OS (HR 0.49, p < 0.001) and PFS on first-line vascular endothelial growth factor receptor tyrosine kinase inhibitors (VEGFR-TKIs) (HR 0.64, p = 0.045) than patients with non-GM. PFS on first- or any-line immuno-oncology (IO) was not different. IMmotion Angio, JAVELIN Angio GES, and ccrcc2 molecular subtype were associated with better OS and PFS on first-line VEGFR-TKIs, but not PFS on first or any-line IO. CONCLUSIONS: Patients with m-ccRCC who develop GM are molecularly characterized by heightened angiogenesis, translating into better prognosis and better outcomes on VEGFR-TKIs, but not IO. Based on these findings, VEGFR-TKIs should be included in the first-line treatment of m-ccRCC patients with GM.

Adeno-associated virus in the liver: natural history and consequences in tumour development.

OBJECTIVE: Adeno-associated virus (AAV) is a defective mono-stranded DNA virus, endemic in human population (35%-80%). Recurrent clonal AAV2 insertions are associated with the pathogenesis of rare human hepatocellular carcinoma (HCC) developed on normal liver. This study aimed to characterise the natural history of AAV infection in the liver and its consequence in tumour development. DESIGN: Viral DNA was quantified in tumour and non-tumour liver tissues of 1461 patients. Presence of episomal form and viral mRNA expression were analysed using a DNAse/TaqMan-based assay and quantitative RT-PCR. In silico analyses using viral capture data explored viral variants and new clonal insertions. RESULTS: AAV DNA was detected in 21% of the patients, including 8% of the tumour tissues, equally distributed in two major viral subtypes: one similar to AAV2, the other hybrid between AAV2 and AAV13 sequences. Episomal viral forms were found in 4% of the non-tumour tissues, frequently associated with viral RNA expression and human herpesvirus type 6, the candidate natural AAV helper virus. In 30 HCC, clonal AAV insertions were recurrently identified in CCNA2, CCNE1, TERT, TNFSF10, KMT2B and GLI1/INHBE. AAV insertion triggered oncogenic overexpression through multiple mechanisms that differ according to the localisation of the integration site. CONCLUSION: We provided an integrated analysis of the wild-type AAV infection in the liver with the identification of viral genotypes, molecular forms, helper virus relationship and viral integrations. Clonal AAV insertions were positive selected during HCC development on non-cirrhotic liver challenging the notion of AAV as a non-pathogenic virus.

Polyploidy spectrum: a new marker in HCC classification.

OBJECTIVES: Polyploidy is a fascinating characteristic of liver parenchyma. Hepatocyte polyploidy depends on the DNA content of each nucleus (nuclear ploidy) and the number of nuclei per cell (cellular ploidy). Which role can be assigned to polyploidy during human hepatocellular carcinoma (HCC) development is still an open question. Here, we investigated whether a specific ploidy spectrum is associated with clinical and molecular features of HCC. DESIGN: Ploidy spectra were determined on surgically resected tissues from patients with HCC as well as healthy control tissues. To define ploidy profiles, a quantitative and qualitative in situ imaging approach was used on paraffin tissue liver sections. RESULTS: We first demonstrated that polyploid hepatocytes are the major components of human liver parenchyma, polyploidy being mainly cellular (binuclear hepatocytes). Across liver lobules, polyploid hepatocytes do not exhibit a specific zonation pattern. During liver tumorigenesis, cellular ploidy is drastically reduced; binuclear polyploid hepatocytes are barely present in HCC tumours. Remarkably, nuclear ploidy is specifically amplified in HCC tumours. In fact, nuclear ploidy is amplified in HCCs harbouring a low degree of differentiation and TP53 mutations. Finally, our results demonstrated that highly polyploid tumours are associated with a poor prognosis. CONCLUSIONS: Our results underline the importance of quantification of cellular and nuclear ploidy spectra during HCC tumorigenesis.