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.

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.

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.

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.

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.

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.

Recurrent chromosomal rearrangements of ROS1, FRK and IL6 activating JAK/STAT pathway in inflammatory hepatocellular adenomas.

BACKGROUND: Inflammatory hepatocellular adenomas (IHCAs) are benign liver tumours characterised by an activation of the janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway caused by oncogenic activating mutations. However, a subset of IHCA lacks of identified mutation explaining the inflammatory phenotype. METHODS: 657 hepatocellular adenomas developed in 504 patients were analysed for gene expression of 17 genes and for mutations in seven genes by sequencing. 22 non-mutated IHCAs were analysed by whole-exome and/or RNA sequencing. RESULTS: We identified 296 IHCA (45%), 81% of them were mutated in either IL6ST (61%), FRK (8%), STAT3 (5%), GNAS (3%) or JAK1 (2%). Among non-mutated IHCA, RNA sequencing identified recurrent chromosome rearrangement involving ROS1, FRK or IL6 genes. ROS1 fusions were identified in 8 IHCA, involving C-terminal part of genes highly expressed in the liver (PLG, RBP4, APOB) fused with exon 33-35 to 43 of ROS1 including the tyrosine kinase domain. In two cases a truncated ROS1 transcript from exon 36 to 43 was identified. ROS1 rearrangements were validated by fluorescence in situ hybridisation (FISH) and led to ROS1 overexpression. Among the 5 IHCA with FRK rearrangements, 5 different partners were identified (MIA3, MIA2, LMO7, PLEKHA5, SEC16B) fused to a common region in FRK that included exon 3-8. No overexpression of FRK transcript was detected but the predicted chimeric proteins lacked the auto-inhibitory SH2-SH3 domains. In two IHCA, we identified truncated 3'UTR of IL6 associated with overexpression of the transcript. CONCLUSION: Recurrent chromosomal alterations involving ROS1, FRK or IL6 genes lead to activation of the JAK/STAT pathway in IHCAs.

PTEN expression and mutations in TSC1, TSC2 and MTOR are associated with response to rapalogs in patients with renal cell carcinoma.

The mammalian target of rapamycin (mTOR) pathway inhibitors are key drugs for the treatment of many tumor types, however, there are no predictive biomarkers in clinical use. Here, we performed a molecular and immunohistochemical characterization of key mTOR pathway components in a series of 105 renal cell carcinoma patients treated with rapalogs, aimed at identifying markers of treatment response. Mutational analysis in MTOR, TSC1 and TSC2 was performed through targeted next-generation sequencing (NGS), and immunohistochemistry (IHC) was performed for PTEN, pAKT, pS6K1, pS6 and p21. Among patients with NGS data, 11 of 87 (13%) had mTOR pathway mutations (8 in MTOR, 1 in TSC1 and 2 in TSC2). When comparing the molecular data to the response of the patients, we found that partial response was more frequent in cases with mTOR pathway mutations than in those without mutations (odds ratio [OR] = 0.08, 95% confidence interval [CI] = 0.008-0.79, p = 0.030 univariate; p = 0.038 multivariable). Regarding IHC, negative PTEN staining was detected in 58% of the tumors, and it was more frequent in rapalog responder patients (OR = 0.24, 95% CI = 0.065-0.86, p = 0.029 univariate; p = 0.029 multivariable). Mutations and PTEN IHC were not mutually exclusive events and its combination improved response prediction (OR = 0.16, 95% CI = 0.04-0.62, p = 0.008 univariate; p = 0.013 multivariable). The staining of other proteins did not show and association with response and no association with PFS was observed in unselected patients. In conclusion, our findings suggest that mTOR pathway mutations, negative PTEN IHC and their combination are potential markers of rapalog response.

BAP1 mutations define a homogeneous subgroup of hepatocellular carcinoma with fibrolamellar-like features and activated PKA.

BACKGROUND & AIMS: DNAJB1-PRKACA fusion is a specific driver event in fibrolamellar carcinoma (FLC), a rare subtype of hepatocellular carcinoma (HCC) that occurs in adolescents and young adults. In older patients, molecular determinants of HCC with mixed histological features of HCC and FLC (mixed-FLC/HCC) remain to be discovered. METHODS: A series of 151 liver tumors including 126 HCC, 15 FLC, and 10 mixed-FLC/HCC were analyzed by RNAseq and whole-genome- or whole-exome sequencing. Western blots were performed to validate genomic discoveries. Results were validated using the TCGA database. RESULTS: Most of the mixed-FLC/HCC RNAseq clustered in a robust subgroup of 17 tumors, which all had mutations or translocations inactivating BAP1, the gene encoding BRCA1-associated protein-1. Like FLC, BAP1-HCC were significantly enriched in females, patients with a lack of chronic liver disease, and fibrotic tumors compared to non-BAP1 HCC. However, patients were older and had a poorer prognosis than those with FLC. BAP1 tumors were immune hot, showed progenitor features and did not show DNAJB1-PRKACA fusion, while almost none of these tumors had mutations in CTNNB1, TP53 and TERT promoter. In contrast, 80% of the BAP1 tumors showed a chromosome gain of PRKACA at 19p13, combined with a loss of PRKAR2A (coding for the inhibitory regulatory subunit of PKA) at 3p21, leading to a high PRKACA/PRKAR2A ratio at the mRNA and protein levels. CONCLUSION: We have characterized a subgroup of BAP1-driven HCC with fibrolamellar-like features and a dysregulation of the PKA pathway, which could be at the root of the clinical and histological similarities between BAP1 tumors and DNAJB1-PRKACA FLCs. LAY SUMMARY: Herein, we have defined a homogeneous subgroup of hepatocellular carcinomas in which the BAP1 gene is inactivated. This leads to the development of cancers with features similar to those of fibrolamellar carcinoma. These tumors more frequently develop in females without chronic liver disease or cirrhosis. The presence of PKA activation and T cell infiltrates suggest that these tumors could be treated with PKA inhibitors or immunomodulators.

Analysis of Liver Cancer Cell Lines Identifies Agents With Likely Efficacy Against Hepatocellular Carcinoma and Markers of Response.

BACKGROUND AND AIMS: Hepatocellular carcinomas (HCCs) are heterogeneous aggressive tumors with low rates of response to treatment at advanced stages. We screened a large panel of liver cancer cell lines (LCCLs) to identify agents that might be effective against HCC and markers of therapeutic response. METHODS: We performed whole-exome RNA and microRNA sequencing and quantification of 126 proteins in 34 LCCLs. We screened 31 anticancer agents for their ability to decrease cell viability. We compared genetic, RNA, and protein profiles of LCCLs with those of primary HCC samples and searched for markers of response. RESULTS: The protein, RNA and mutational signatures of the LCCLs were similar to those of the proliferation class of HCC, which is the most aggressive tumor type. Cell lines with alterations in genes encoding members of the Ras-MAPK signaling pathway and that required fibroblast growth factor (FGF)19 signaling via FGF receptor 4 for survival were more sensitive to trametinib than to FGF receptor 4 inhibitors. Amplification of FGF19 resulted in increased activity of FGF19 only in tumor cells that kept a gene expression pattern of hepatocyte differentiation. We identified single agents and combinations of agents that reduced viability of cells with features of the progenitor subclass of HCC. LCCLs with inactivating mutations in TSC1 and TSC2 were sensitive to the mammalian target of rapamycin inhibitor rapamycin, and cells with inactivating mutations in TP53 were sensitive to the Aurora kinase A inhibitor alisertib. Amplification of MET was associated with hypersensitivity to cabozantinib and the combination of sorafenib and inhibitors of MAP kinase 1 and MAP kinase2 had a synergistic antiproliferative effect. CONCLUSION: LCCLs can be screened for drugs and agents that might be effective for treatment of HCC. We identified genetic alterations and gene expression patterns associated with response to these agents. This information might be used to select patients for clinical trials.

Lect2 Controls Inflammatory Monocytes to Constrain the Growth and Progression of Hepatocellular Carcinoma.

Leukocyte cell-derived chemotaxin-2 (LECT2) was originally identified as a hepatocyte-secreted chemokine-like factor and a positive target of ß-catenin signaling. Here, we dissected out the mechanisms by which LECT2 modulates hepatocellular carcinoma (HCC) development using both HCC mouse models and human HCC samples. We have demonstrated that LECT2 exhibits dual abilities as it has profound repercussions on the tumor phenotype itself and the immune microenvironment. Its absence confers Ctnnb-1-mutated tumor hepatocytes a stronger ability to undergo epithelial to mesenchymal transition and fosters the accumulation of pejorative inflammatory monocytes harboring immunosuppressive properties and strong tumor-promoting potential. Consistent with our HCC mouse model, a low level of LECT2 in human HCC is strongly associated with high tumor grade and the presence of inflammatory infiltrates, emphasizing the clinical value of LECT2 in human liver tumorigenesis. Conclusion: Our findings have demonstrated that LECT2 is a key player in liver tumorigenesis because its absence reshapes the tumor microenvironment and the tumor phenotype, revealing LECT2 as a promising immunotherapeutic option for HCC.

Dynamics and predicted drug response of a gene network linking dedifferentiation with beta-catenin dysfunction in hepatocellular carcinoma.

BACKGROUND & AIMS: Alterations of individual genes variably affect the development of hepatocellular carcinoma (HCC). Thus, we aimed to characterize the function of tumor-promoting genes in the context of gene regulatory networks (GRNs). METHODS: Using data from The Cancer Genome Atlas, from the LIRI-JP (Liver Cancer - RIKEN, JP project), and from our transcriptomic, transfection and mouse transgenic experiments, we identify a GRN which functionally links LIN28B-dependent dedifferentiation with dysfunction of ß-catenin (CTNNB1). We further generated and validated a quantitative mathematical model of the GRN using human cell lines and in vivo expression data. RESULTS: We found that LIN28B and CTNNB1 form a GRN with SMARCA4, Let-7b (MIRLET7B), SOX9, TP53 and MYC. GRN functionality is detected in HCC and gastrointestinal cancers, but not in other cancer types. GRN status negatively correlates with HCC prognosis, and positively correlates with hyperproliferation, dedifferentiation and HGF/MET pathway activation, suggesting that it contributes to a transcriptomic profile typical of the proliferative class of HCC. The mathematical model predicts how the expression of GRN components changes when the expression of another GRN member varies or is inhibited by a pharmacological drug. The dynamics of GRN component expression reveal distinct cell states that can switch reversibly in normal conditions, and irreversibly in HCC. The mathematical model is available via a web-based tool which can evaluate the GRN status of HCC samples and predict the impact of therapeutic agents on the GRN. CONCLUSIONS: We conclude that identification and modelling of the GRN provide insights into the prognosis of HCC and the mechanisms by which tumor-promoting genes impact on HCC development. LAY SUMMARY: Hepatocellular carcinoma (HCC) is a heterogeneous disease driven by the concomitant deregulation of several genes functionally organized as networks. Here, we identified a gene regulatory network involved in a subset of HCCs. This subset is characterized by increased proliferation and poor prognosis. We developed a mathematical model which uncovers the dynamics of the network and allows us to predict the impact of a therapeutic agent, not only on its specific target but on all the genes belonging to the network.

Argininosuccinate synthase 1 and periportal gene expression in sonic hedgehog hepatocellular adenomas.

Genetic alterations define different molecular subclasses of hepatocellular adenoma (HCA) linked with risk factors, histology and clinical behavior. Recently, Argininosuccinate Synthase 1 (ASS1), a major periportal protein, was proposed as a marker of HCA with a high risk of hemorrhage. We aimed to assess the significance of ASS1 expression through the scope of the HCA molecular classification. ASS1 expression was evaluated using RNAseq, quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and Immunohistochemistry. ASS1 and glioma-associated oncogene 1 (GLI1) expression were analyzed in vitro after modulation of GLI1 expression. Using RNAseq in 27 HCA and five nontumor liver samples, ASS1 expression was highly correlated with GLI1 expression (P<0.0001, R=0.75). In the overall series of 408 HCA, ASS1 overexpression was significantly associated with sonic hedgehog HCA (shHCA) compared to other molecular subgroups (P<0.0001), suggesting that sonic hedgehog signaling controls ASS1 expression. GLI1 expression silencing by siRNA induced a downregulation of ASS1 in PLC/PFR5 and SNU878 cell lines. In 390 HCA, we showed that ASS1 expression belonged to the periportal expression program that was maintained in shHCA but down-regulated in all the other HCA subtypes. In contrast, HCA with ß-catenin activation showed an activation of a perivenous program. Despite the significant association between GLI1 and ASS1 expression, ASS1 mRNA expression was not associated with specific clinical features. At the protein level using immunohistochemistry, prostaglandin D synthase (PTGDS) was strongly and specifically overexpressed in shHCA. CONCLUSION: ASS1 is associated with sonic hedgehog activation as part of a periportal program expressed in shHCA, a molecular subgroup defined by INHBE-GLI1 gene fusion. (Hepatology 2018).

Molecular Classification of Hepatocellular Adenoma Associates With Risk Factors, Bleeding, and Malignant Transformation.

BACKGROUND & AIMS: Hepatocellular adenomas (HCAs) are benign liver tumors that can be assigned to molecular subtypes based on inactivating mutations in hepatocyte nuclear factor 1A, activating mutations in ß-catenin, or activation of inflammatory signaling pathways. We aimed to update the classification system for HCA and associate the subtypes with disease risk factors and complications. METHODS: We analyzed expression levels of 20 genes and sequenced exon regions of 8 genes (HNF1A, IL6ST, CTNNB1, FRK, STAT3, GNAS, JAK1, and TERT) in 607 samples of 533 HCAs from 411 patients, collected from 28 centers mainly in France from 2000 and 2014. We performed gene expression profile, RNA sequence, whole-exome and genome sequence, and immunohistochemical analyses of select samples. Molecular data were associated with risk factors, histopathology, bleeding, and malignant transformation. RESULTS: Symptomatic bleeding occurred in 14% of the patients (85% of cases were female, median age, 38 years); 7% of the nodules were borderline between HCA and hepatocellular carcinoma, and 3% of patients developed hepatocellular carcinoma from HCA. Based on molecular features, we classified HCA into 8 subgroups. One new subgroup, composed of previously unclassified HCA, represented 4% of HCAs overall and was associated with obesity and bleeding. These tumors were characterized by activation of sonic hedgehog signaling, due to focal deletions that fuse the promoter of INHBE with GLI1. Analysis of genetic heterogeneity among multiple HCAs, from different patients, revealed a molecular subtype field effect; multiple tumors had different mutations that deregulated similar pathways. Specific molecular subtypes of HCA associated with various HCA risk factors, including imbalances in estrogen or androgen hormones. Specific molecular subgroup of HCA with ß-catenin and sonic hedgehog activation associated with malignant transformation and bleeding, respectively. CONCLUSIONS: Using sequencing and gene expression analyses, we identified a subgroup of HCA characterized by fusion of the INHBE and GLI1 genes and activation of sonic hedgehog pathway. Molecular subtypes of HCAs associated with different patients' risk factors for HCA, disease progression, and pathology features of tumors. This classification system might be used to select treatment strategies for patients with HCA.

Pro-angiogenic gene expression is associated with better outcome on sunitinib in metastatic clear-cell renal cell carcinoma.

OBJECTIVES: Clear-cell renal cell carcinomas (ccRCC) are characterized by hyper-vascularization and can respond to vascular endothelial growth factor receptor (VEGFR) inhibitors such as sunitinib. We aimed to study the predictive value of the expression of genes in the hypoxia induced factor (HIF) - vascular endothelial growth factor (VEGF) - VEGFR-pro-angiogenic pathway in metastatic ccRCC (m-ccRCC) patients treated with sunitinib and the correlation between the expression of these genes and the molecular ccrcc-classification, the expression of genes involved in the immune-suppressive microenvironment and Von Hippel-Lindau (VHL) - and Polybromo-1 (PBRM1) - mutational status. MATERIAL AND METHODS: m-ccRCC patients treated with sunitinib as first-line targeted therapy were included. Gene expression was studied in the primary nephrectomy sample by qRT-PCR, VHL- and PBRM1-mutational status by sequencing. Response rate by RECIST, progression-free survival (PFS) and overall survival (OS) were study endpoints. RESULTS: One hundred and four patients were included. On multivariate-analysis, HIF2A-, platelet derived growth factor receptor beta (PDGFRB)-, VEGFC-, VEGFR1- and VEGFR2-expression were correlated with PFS and HIF1A-, HIF2A-, VEGFR1- and VEGFR2-expression with OS. VEGFR2-expression showed the strongest association with outcome, being significantly correlated with all outcome parameters. HIF2A, VEGFA, VEGFR1, VEGFR2 and VEGFR3 were highly expressed in the transcriptomic ccrcc2-subtype of tumors, known to be highly sensitive to sunitinib. In the total tumor series, there was no correlation nor inverse correlation between the expression of genes involved in angiogenesis and in the immune-suppressive microenvironment. In tumors with a bi-allelic PBRM1-inactivation, HIF2A-, VEGFA-, VEGFR1- and VEGFR2-expression were higher, compared to tumors with one or two functional PBRM1-alleles. CONCLUSIONS: Intratumoral expression of genes involved in the HIF-VEGF-VEGFR-pro-angiogenic pathway, especially VEGFR2, is associated with favorable outcome on sunitinib in m-ccRCCs. Several genes involved in this pathway are upregulated in the molecular ccrcc2-subgroup, which usually responds well to sunitinib.

Histological subtypes of hepatocellular carcinoma are related to gene mutations and molecular tumour classification.

BACKGROUND & AIMS: Our increasing understanding of hepatocellular carcinoma (HCC) biology holds promise for personalized care, however its translation into clinical practice requires a precise knowledge of its relationship to tumour phenotype. METHODS: We aimed at investigating molecular-phenotypic correlations in a large series of HCC. To this purpose, 343 surgically resected HCC samples were investigated by pathological review, immunohistochemistry, gene expression profiling and sequencing. RESULTS: CTNNB1 (40%) and TP53 (21%) mutations were mutually exclusive and defined two major groups of HCC characterized by distinct phenotypes. CTNNB1 mutated tumours were large (p=0.002), well-differentiated (p<0.001), cholestatic (p<0.001), with microtrabecular (p<0.001) and pseudoglandular (p<0.001) patterns and without inflammatory infiltrates (p<0.001). TP53 mutated tumours were poorly differentiated (p<0.001) with a compact pattern (p=0.02), multinucleated (p=0.01) and pleomorphic (p=0.02) cells and frequent vascular invasion (p=0.02). World Health Organization (WHO) classification of histological subtypes were also strongly related to molecular features. The scirrhous subtype was associated with TSC1/TSC2 mutations (p=0.005), epithelial-to-mesenchymal transition and a progenitor expression profile. The steatohepatitic subtype showed frequent IL-6/JAK/STAT activation without CTNNB1, TERT and TP53 pathway alterations (p=0.01). Pathological review identified a novel subtype, designated as "macrotrabecular-massive" associated with poor survival (p<0.001), high alpha-fetoprotein serum level (p=0.02), vascular invasion (p<0.001), TP53 mutations (p<0.001) and FGF19 amplifications (p=0.02), features also validated in The Cancer Genome Atlas (TCGA) data. Finally, integration of HCC pathological characteristics with its transcriptomic classification showed phenotypically distinct tumour subclasses closely related to G1-G6 subgroups. CONCLUSION: HCC phenotypes are tightly associated with gene mutations and transcriptomic classification. These findings may help in translating our knowledge of HCC biology into clinical practice. Lay summary: HCC is a very heterogenous tumour, both at the pathological and molecular levels. We show here that HCC phenotype is tightly associated to its molecular alterations and underlying oncogenic pathways.

Germline and somatic DICER1 mutations in familial and sporadic liver tumors.

BACKGROUND & AIMS: Growing evidence suggests that genetic predisposition significantly increases the risk of hepatocellular carcinoma (HCC), independently from the presence of other risk factors. Here, we report a novel germline DICER1 mutation associated with familial recurrent liver tumors. We then aimed to investigate the contribution of constitutional and somatic DICER1 mutations on HCC occurrence. METHODS: We investigated two individuals of a single family that developed recurrent well-differentiated hepatocellular tumors over the years. Histological slides from surgically resected tumors were reviewed. Exome sequencing was performed on constitutional DNA from circulating lymphocytes in both patients. The presence of somatic DICER1 mutations was analyzed in 243 liver tumors. MicroRNA (miRNA) sequencing was performed in 50 liver tumors to identify groups of tumors with similar profiles and differentially expressed miRNAs (DEMs). RESULTS: A pathological study identified hepatocellular adenomas and well-differentiated carcinomas in both patients. Tumors exhibited Wnt/ß-catenin pathway activation, with strong and diffuse glutamine synthetase expression. Interestingly, non-tumor liver tissues showed abnormal liver zonation as previously reported in Dicer1 knockout mouse livers. Screening for DICER1 mutations in 243 sporadic liver tumors identified six tumors with somatic DICER1 mutations. In HCCs, DICER1 mutations were significantly associated with CTNNB1 mutations (p=0.03). miRNA profiling identified a specific expression profile in DICER1-mutated tumors with a decreased expression of mature miRNAs compared to the other samples. Among the DEMs, downregulation of let-7a and miR-365b was closely related to DICER1 mutations. CONCLUSIONS: Our results highlight the role of DICER1 mutations in liver carcinogenesis in a specific subtype of familial and sporadic hepatocellular carcinomas associated with ß-catenin activation. LAY SUMMARY: DICER1 germline mutations are known to predispose individuals to the development of malignant tumors, mainly pleuropulmonary blastoma and ovarian Sertoli-Leydig cell tumor. Here, we described familial HCC associated with a novel DICER1 germline mutation and altered liver zonation. Familial and sporadic HCCs carrying DICER1 mutations are associated with CTNNB1 mutation and characterized by a reduced expression of specific mature miRNAs.

The liver-specific microRNA-122*, the complementary strand of microRNA-122, acts as a tumor suppressor by modulating the p53/mouse double minute 2 homolog circuitry.

The tumor suppressor p53 is a central regulator of signaling pathways that controls the cell cycle and maintains the integrity of the human genome. p53 level is regulated by mouse double minute 2 homolog (Mdm2), which marks p53 for proteasomal degradation. The p53-Mdm2 circuitry is subjected to complex regulation by a variety of mechanisms, including microRNAs (miRNAs). We found a novel effector of this regulatory circuit, namely, miR-122*, the passenger strand of the abundantly expressed liver-specific miR-122. Here, we demonstrate that miR-122* levels are reduced in human hepatocellular carcinoma (HCC). We found that miR-122* targets Mdm2, thus participating as an important player in the p53-Mdm2 circuitry. Moreover, we observed significant negative correlation between levels of miR-122* and Mdm2 in a large set of human HCC samples. In vivo tumorigenicity assays demonstrate that miR-122* is capable of inhibiting tumor growth, emphasizing the tumor-suppressor characteristics of this miRNA. Furthermore, we show that blocking miR-122 in murine livers with an antagomiR-122 (miRNA inhibitor) results in miR-122* accumulation, leading to Mdm2 repression followed by elevated p53 protein levels. CONCLUSION: miR-122*, the passenger strand of miR-122, regulates the activity of p53 by targeting Mdm2. Importantly, similarly to miR-122, miR-122* is significantly down-regulated in human HCC. We therefore propose that miR-122* is an important contributor to the tumor suppression activity previously attributed solely to miR-122. (Hepatology 2016;64:1623-1636).

Modeling a human hepatocellular carcinoma subset in mice through coexpression of met and point-mutant ß-catenin.

Hepatocellular cancer (HCC) remains a significant therapeutic challenge due to its poorly understood molecular basis. In the current study, we investigated two independent cohorts of 249 and 194 HCC cases for any combinatorial molecular aberrations. Specifically we assessed for simultaneous HMET expression or hMet activation and catenin ß1 gene (CTNNB1) mutations to address any concomitant Met and Wnt signaling. To investigate cooperation in tumorigenesis, we coexpressed hMet and ß-catenin point mutants (S33Y or S45Y) in hepatocytes using sleeping beauty transposon/transposase and hydrodynamic tail vein injection and characterized tumors for growth, signaling, gene signatures, and similarity to human HCC. Missense mutations in exon 3 of CTNNB1 were identified in subsets of HCC patients. Irrespective of amino acid affected, all exon 3 mutations induced similar changes in gene expression. Concomitant HMET overexpression or hMet activation and CTNNB1 mutations were evident in 9%-12.5% of HCCs. Coexpression of hMet and mutant-ß-catenin led to notable HCC in mice. Tumors showed active Wnt and hMet signaling with evidence of glutamine synthetase and cyclin D1 positivity and mitogen-activated protein kinase/extracellular signal-regulated kinase, AKT/Ras/mammalian target of rapamycin activation. Introduction of dominant-negative T-cell factor 4 prevented tumorigenesis. The gene expression of mouse tumors in hMet-mutant ß-catenin showed high correlation, with subsets of human HCC displaying concomitant hMet activation signature and CTNNB1 mutations. CONCLUSION: We have identified cooperation of hMet and ß-catenin activation in a subset of HCC patients and modeled this human disease in mice with a significant transcriptomic intersection; this model will provide novel insight into the biology of this tumor and allow us to evaluate novel therapies as a step toward precision medicine. (Hepatology 2016;64:1587-1605).