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.

Functional characterization of 5p15.33 risk locus in uveal melanoma reveals rs452384 as a functional variant and NKX2.4 as an allele-specific interactor.

The TERT/CLPTM1L risk locus on chromosome 5p15.33 is a pleiotropic cancer risk locus in which multiple independent risk alleles have been identified, across well over ten cancer types. We previously conducted a genome-wide association study in uveal melanoma (UM), which uncovered a role for the TERT/CLPTM1L risk locus in this intraocular tumor and identified multiple highly correlated risk alleles. Aiming to unravel the biological mechanisms in UM of this locus, which contains a domain enriched in active chromatin marks and enhancer elements, we demonstrated the allele-specific enhancer activity of this risk region using reporter assays. In UM, we identified the functional variant rs452384, of which the C risk allele is associated with higher gene expression, increased CLPTM1L expression in UM tumors, and a longer telomere length in peripheral blood mononuclear cells. Electrophoretic mobility shift assays and quantitative mass spectrometry identified NKX2.4 as an rs452384-T-specific binding protein, whereas GATA4 preferentially interacted with rs452384-C. Knockdown of NKX2.4 but not GATA4 resulted in increased TERT and CLPTM1L expression. In summary, the UM risk conferred by the 5p locus is at least partly due to rs452384, for which NKX2.4 presents strong differential binding activity and regulates CLPTM1L and TERT expression. Altogether, our work unraveled some of the complex regulatory mechanisms at the 5p15.33 susceptibility region in UM, and this might also shed light on shared mechanisms with other tumor types affected by this susceptibility region.

Disparity landscapes of viral-induced structural variations in hepatocellular carcinoma: Mechanistic characterization and functional implications.

Oncoviruses can integrate into the host genome and cause tumorigenesis. In particular, hepatitis B virus (HBV) infection accounts for more than 50% of hepatocellular carcinoma (HCC) worldwide. We revealed the global geographical disparity of HBV integration that the landscape of HBV integration between HCC tumor and non-tumorous liver varied in regional cohorts, suggesting the different degrees of clonal enrichment. Most HBV integrations were positionally enriched at telomeres and centromeres (T&C) and they highlighted the novel co-involvement of HBV integration, which likely introduces genomic instability in HCC development. This was confirmed by phospho-H2AX staining. We constructed a large meta-cohort of multiple ethnicities to refine the landscape of HBV integration. This enables the gene set/family level exploration. As TERT is the most frequently integrated gene, we further investigated the underlying mechanistic modulation of TERT transcription activation and revealed the concurrent influence by the orientation and relative distance of HBV integration. Additionally, clonal disparity of HBV integration was observed among patients and the higher level of clonal disparity score can indicate poor patients' prognostication. Taken together, our study uncovered the different levels of clonal enrichment of HBV integration, mechanistic insights, and prognostic biomarker signature, to strengthen our understanding in HBV-associated hepatocarcinogenesis.

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.

Circulating tumour DNA in patients with hepatocellular carcinoma across tumour stages and treatments.

OBJECTIVE: Circulating tumour DNA (ctDNA) is a promising non-invasive biomarker in cancer. We aim to assess the dynamic of ctDNA in patients with hepatocellular carcinoma (HCC). DESIGN: We analysed 772 plasmas from 173 patients with HCC collected at the time of diagnosis or treatment (n=502), 24 hours after locoregional treatment (n=154) and during follow-up (n=116). For controls, 56 plasmas from patients with chronic liver disease without HCC were analysed. All samples were analysed for cell free DNA (cfDNA) concentration, and for mutations in TERT promoter, CTNNB1, TP53, PIK3CA and NFE2L2 by sequencing and droplet-based digital PCR. Results were compared with 232 corresponding tumour samples. RESULTS: In patients with active HCC, 40.2% of the ctDNA was mutated vs 14.6% in patients with inactive HCC and 1.8% in controls (p<0.001). In active HCC, we identified 27.5% of mutations in TERT promoter, 21.3% in TP53, 13.1% in CTNNB1, 0.4% in PIK3CA and 0.2% in NFE2L2, most of the times similar to those identified in the corresponding tumour. CtDNA mutation rate increased with advanced tumour stages (p<0.001). In 103 patients treated by percutaneous ablation, the presence and number of mutations in the ctDNA before treatment were associated with higher risk of death (p=0.001) and recurrence (p<0.001). Interestingly, cfDNA concentration and detectable mutations increased 24 hours after a locoregional treatment. Among 356 plasmas collected in 53 patients treated by systemic treatments, we detected mutations at baseline in 60.4% of the cases. In patients treated by atezolizumab-bevacizumab, persistence of mutation in ctDNA was associated with radiological progression (63.6% vs 36.4% for disappearance, p=0.019). In two patients progressing under systemic treatments, we detected the occurrence of mutations in CTNNB1 in the plasma that was subclonal in the tumour for one patient and not detectable in the tumour for the other one. CONCLUSION: ctDNA offers dynamic information reflecting tumour biology. It represents a non-invasive tool useful to guide HCC clinical management.

Rare primary liver cancers: An EASL position paper.

In recent years, owing to advances in our understanding of hepatocarcinogenesis, rare primary liver cancers (PLCs), including combined hepatocellular-cholangiocarcinoma, fibrolamellar carcinoma, and hepatic epithelioid hemangioendothelioma have garnered increased attention. In this position paper, an international panel of experts representing oncology, hepatology, pathology, radiology, surgery, and molecular biology has summarised the available information and evidence on the pathogenesis, diagnosis, and treatment of rare PLCs. While clinical trials of systemic treatments are underway for some rare PLCs, it is evident that more research, involving national and international collaboration, is required.

Socioeconomic Deprivation Weighs Heavily on Liver Fibrosis and Mortality After Hepatitis C Cure (ANRS CO22 Hepather).

Although Hepatitis C virus (HCV) infection can be cured with direct-acting antivirals (DAA), some cured patients face a serious risk of advanced liver damage and early mortality. In order to avoid these two negative health outcomes, it is important to identify and assess related risk factors. Little is currently known about socioeconomic and behavioural factors in this context. Using data from the ANRS CO22 Hepather cohort, we tested for associations between socioeconomic and behavioural factors and (i) advanced liver fibrosis (defined as an FIB-4 > 3.25) assessed longitudinally using a mixed-effects logistic regression model (both the whole population and stratified on advanced liver fibrosis status at the time of HCV cure) and (ii) all-cause mortality (Cox proportional hazards model), during post-HCV cure follow-up. Among 5833 participants cured of HCV, living in poverty was associated with postcure advanced liver fibrosis in participants without this diagnosis at the time of HCV cure (population attributable fraction-PAF-of 8.6%) and with mortality in the whole study population (PAF of 10.6%). The detrimental effects of unhealthy alcohol use and heavy tobacco smoking, as well as the beneficial effect of living with a stable partner were also highlighted. We highlighted the major role of poverty and behavioural factors in advanced liver fibrosis and all-cause mortality in patients cured of HCV. Encouraging linkage to social support services and healthy behaviours after successful DAA treatment could limit morbidity and increase survival in this population. Clinical Trial Registration: ClinicalTrials.gov: NCT01953458.

A genetic basis of mitochondrial DNAJA3 in nonalcoholic steatohepatitis-related hepatocellular carcinoma.

BACKGROUND AND AIMS: NAFLD is the most common form of liver disease worldwide, but only a subset of individuals with NAFLD may progress to NASH. While NASH is an important etiology of HCC, the underlying mechanisms responsible for the conversion of NAFLD to NASH and then to HCC are poorly understood. We aimed to identify genetic risk genes that drive NASH and NASH-related HCC. APPROACH AND RESULTS: We searched genetic alleles among the 24 most significant alleles associated with body fat distribution from a genome-wide association study of 344,369 individuals and validated the top allele in 3 independent cohorts of American and European patients (N=1380) with NAFLD/NASH/HCC. We identified an rs3747579-TT variant significantly associated with NASH-related HCC and demonstrated that rs3747579 is expression quantitative trait loci of a mitochondrial DnaJ Heat Shock Protein Family (Hsp40) Member A3 ( DNAJA3 ). We also found that rs3747579-TT and a previously identified PNPLA3 as a functional variant of NAFLD to have significant additional interactions with NASH/HCC risk. Patients with HCC with rs3747579-TT had a reduced expression of DNAJA3 and had an unfavorable prognosis. Furthermore, mice with hepatocyte-specific Dnaja3 depletion developed NASH-dependent HCC either spontaneously under a normal diet or enhanced by diethylnitrosamine. Dnaja3 -deficient mice developed NASH/HCC characterized by significant mitochondrial dysfunction, which was accompanied by excessive lipid accumulation and inflammatory responses. The molecular features of NASH/HCC in the Dnaja3 -deficient mice were closely associated with human NASH/HCC. CONCLUSIONS: We uncovered a genetic basis of DNAJA3 as a key player of NASH-related HCC.

Body weight changes and duration of estrogen exposure modulate the evolution of hepatocellular adenomas after contraception discontinuation.

BACKGROUND AND AIMS: The natural history of hepatocellular adenomas (HCAs) remains to be better described, especially in nonresected patients. We aim to identify the predictive factors of HCA evolution after estrogen-based contraception discontinuation. APPROACH AND RESULTS: We retrospectively included patients with a histological diagnosis of HCA from three centers. Clinical, radiological, and pathological data were collected to identify predictive factors of radiological evolution per Response Evaluation Criteria in Solid Tumors, version 1.1, and occurrence of complications (bleeding, malignant transformation). We built a score using variables that modulate estrogen levels: body mass index and duration of estrogen-based contraception. An external cohort was used to validate this score. 183 patients were included in the cohort, including 161 women (89%) using estrogen-based contraception for a median of 12 years. Thirty percent of patients had at least one HNF1A -inactivated HCA, 45.5% at least one inflammatory HCA, and 11% at least one HCA with activation of ß-catenin (bHCA). Twenty-one symptomatic bleedings (11%) and eleven malignant transformations (6%) occurred. Ages < 37 years old ( p = 0.004) and HCA > 5 cm at imaging were independently associated with symptomatic bleeding ( p = 0.003), whereas a bHCA was associated with malignant transformation ( p < 0.001). After a median follow-up of 5 years, radiological regression was observed in 31%, stabilization in 47%, and progression in 22% of patients. Weight loss was associated with regression ( p < 0.0001) and weight gain with progression ( p = 0.02). The estrogen exposure score predicted radiological regression (odds ratio, 2.33; confidence interval 95%, 1.29-4.19; p = 0.005) with a linear relationship between the rate of estrogen exposure and the probability of regression. This result was confirmed in an external cohort of 72 female patients ( p = 0.003). CONCLUSION: Weight variation is strongly associated with radiological evolution after oral contraception discontinuation. A score of estrogen exposure, easily assessable in clinical practice at diagnosis, predicts regression of HCA.

Baseline neutrophil-to-eosinophil-ratio and outcome in metastatic clear-cell renal cell carcinoma treated with nivolumab or ipilimumab/nivolumab.

BACKGROUND AND PURPOSE: This study aims to evaluate neutrophil-to-eosinophil ratio (NER) as a prognostic and/or predictive biomarker in metastatic clear cell renal cell carcinoma (m-ccRCC) treated with nivolumab or ipilimumab/nivolumab. PATIENTS/MATERIALS AND METHODS: We performed a retrospective study on m-ccRCC patients treated with nivolumab or ipilimumab/nivolumab (2012-2022). Baseline NER was calculated and correlated with clinical outcomes: response rate (RR), progression free survival (PFS) and overall survival (OS). Corresponding transcriptomic data were analysed. RESULTS: We included 201 m-ccRCC patients, 76 treated with ipilimumab/nivolumab and 125 with nivolumab. Baseline NER was statistically significantly associated with International Metastatic RCC Database Consortium (IMDC) risk groups. Increased NER was associated with shorter PFS and OS in the total patient series and nivolumab-treated patients. In patients treated with ipilimumab/nivolumab, increased NER was only statistically significantly associated with shorter OS. The impact of baseline NER on PFS and OS was independent of IMDC risk stratification. No clear correlation was found between baseline NER and RECIST response or maximal tumour shrinkage. In two additional databases, NER was also associated with PFS and OS in first-line vascular-endothelial-growth-factor-receptor tyrosine-kinase-inhibitors (VEGFR-TKIs), but not to disease-free survival in the post-nephrectomy setting. Lower NER was associated with intratumoural molecular features possibly associated with better outcome on immune checkpoint inhibitors. INTERPRETATION: Lower baseline NER is associated with better PFS and OS, independent of IMDC risk score, in m-ccRCC patients treated with ipilimumab/nivolumab or nivolumab. It correlates with intratumoural molecular features possibly associated with better outcome on immune checkpoint inhibitors. The predictive power of this biomarker is probably limited and insufficient for patient selection.