Somatic mutations and clonal dynamics in healthy and cirrhotic human liver.
ABSTRACT: The most common causes of chronic liver disease are excess alcohol intake, viral hepatitis and non-alcoholic fatty liver disease, with the clinical spectrum ranging in severity from hepatic inflammation to cirrhosis, liver failure or hepatocellular carcinoma (HCC). The genome of HCC exhibits diverse mutational signatures, resulting in recurrent mutations across more than 30 cancer genes1-7. Stem cells from normal livers have a low mutational burden and limited diversity of signatures8, which suggests that the complexity of HCC arises during the progression to chronic liver disease and subsequent malignant transformation. Here, by sequencing whole genomes of 482 microdissections of 100-500 hepatocytes from 5 normal and 9 cirrhotic livers, we show that cirrhotic liver has a higher mutational burden than normal liver. Although rare in normal hepatocytes, structural variants, including chromothripsis, were prominent in cirrhosis. Driver mutations, such as point mutations and structural variants, affected 1-5% of clones. Clonal expansions of millimetres in diameter occurred in cirrhosis, with clones sequestered by the bands of fibrosis that surround regenerative nodules. Some mutational signatures were universal and equally active in both non-malignant hepatocytes and HCCs; some were substantially more active in HCCs than chronic liver disease; and others-arising from exogenous exposures-were present in a subset of patients. The activity of exogenous signatures between adjacent cirrhotic nodules varied by up to tenfold within each patient, as a result of clone-specific and microenvironmental forces. Synchronous HCCs exhibited the same mutational signatures as background cirrhotic liver, but with higher burden. Somatic mutations chronicle the exposures, toxicity, regeneration and clonal structure of liver tissue as it progresses from health to disease.
Project description:The role of chronic hepatitis C virus (HCV) in the pathogenesis of HCV-associated hepatocellular carcinoma (HCC) remains controversial. To understand the transition from benign to malignant, we studied the gene expression patterns in liver tissues at different stages, including normal, cirrhosis, and different HCC stages. We studied 108 liver tissue samples obtained from 88 distinct patients (41 HCV-cirrhotic tissues, 17 HCV-cirrhotic tissues from patients with HCC, and 47 HCV-HCC tissues). Differentially expressed genes (DEG) were studied by use of high-density oligonucleotide arrays. Among probe sets identified as differentially expressed via the F test, all pairwise comparisons were performed. Cirrhotic tissues with and without concomitant HCC were further evaluated, and a classifier was used to predict whether the tissue type was associated with HCC. Differential expression profiles were analyzed using Interaction Networks and Functional Analysis. Characteristic gene signatures were identified when normal tissue was compared with cirrhosis, cirrhosis with early HCC, and normal with HCC. Pathway analysis classified the cellular and biological functions of the DEG as related to cellular growth and proliferation, cell death and inflammatory disease in cirrhosis; cell death, cell cycle, DNA replication, and immune response in early HCCs; and cell death, cell growth and proliferation, cell cycle, and DNA repair in advanced HCCs. Characteristic gene signatures were identified at different stages of HCV-HCC progression. A set of genes were identified to predict whether the cirrhotic tissue was associated with HCC.
Project description:Macroautophagy and chaperone-mediated autophagy (CMA) represent two major lysosomal degradation processes and often compensate for one another to facilitate cell survival. The aim of this study was to determine whether these autophagy pathways could compensate for one another to promote HCC cell survival in the cirrhotic liver. Analysis of normal liver tissue showed no expression of glypican-3 or p62 proteins, suggesting that macroautophagy is the major contributor to autophagic flux under non-pathological conditions. Of 46 cirrhotic livers with HCC examined, 39 (84%) of HCCs showed increased expression of p62, and 36 (78%) showed increased expression of glypican-3, while adjacent non-tumorous hepatocytes were negative for expression of p62 and glypican-3, similar to normal liver tissue. These results suggest that macroautophagy flux is impaired in HCC. Furthermore, more than 95% of HCCs showed altered expression of LAMP-2A compared to the surrounding non-tumorous cirrhotic liver, consistent with induction of CMA in HCC. Elevated expression of glucose-regulated protein 78 (GRP78) and heat shock cognate protein (Hsc70) were detected in 100% of HCC and adjacent non-tumorous cirrhotic livers, suggesting that unresolved ER-stress is associated with HCC risk in liver cirrhosis. Interestingly, inhibition of lysosomal degradation using hydroxychloroquine (HCQ) induced expression of the tumor suppressor p53, promoted apoptosis, and inhibited HCC growth, whereas activation of autophagy using an mTOR inhibitor (Torin1) promoted HCC growth. Results of this study suggest that induction of CMA compensates for the impairment of macroautophagy to promote HCC survival in the cirrhotic liver.
Project description:Dysplastic nodules are premalignant neoplastic nodules found in explanted livers with cirrhosis. Genetic signatures of premalignant dysplastic nodules (DNs) with concurrent hepatocellular carcinoma (HCC) may provide an insight in the molecular evolution of hepatocellular carcinogenesis. We analyzed four patients with multifocal nodular lesions and cirrhotic background by whole-exome sequencing (WES). The genomic profiles of somatic single nucleotide variations (SNV) and copy number variations (CNV) in DNs were compared to those of HCCs. The number and variant allele frequency of somatic SNVs of DNs and HCCs in each patient was identical along the progression of pathological grade. The somatic SNVs in DNs showed little conservation in HCC. Additionally, CNVs showed no conservation. Phylogenetic analysis based on SNVs and copy number profiles indicated a nonlinear segregation pattern, implying independent development of DNs and HCC in each patient. Thus, somatic mutations in DNs may be developed separately from other malignant nodules in the same liver, suggesting a nonlinear model for hepatocarcinogenesis from DNs to HCC.
Project description:Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide; however, the mutational properties of HCC-associated carcinogens remain largely uncharacterized. We hypothesized that mechanisms underlying chemical-induced HCC can be characterized by evaluating the mutational spectra of these tumors. To test this hypothesis, we performed exome sequencing of B6C3F1/N HCCs that arose either spontaneously in vehicle controls ( n = 3) or due to chronic exposure to gingko biloba extract (GBE; n = 4) or methyleugenol (MEG; n = 3). Most archived tumor samples are available as formalin-fixed paraffin-embedded (FFPE) blocks, rather than fresh-frozen (FF) samples; hence, exome sequencing from paired FF and FFPE samples was compared. FF and FFPE samples showed 63% to 70% mutation concordance. Multiple known (e.g., Ctnnb1T41A, BrafV637E) and novel (e.g., Erbb4C559S, Card10A700V, and Klf11P358L) mutations in cancer-related genes were identified. The overall mutational burden was greater for MEG than for GBE or spontaneous HCC samples. To characterize the mutagenic mechanisms, we analyzed the mutational spectra in the HCCs according to their trinucleotide motifs. The MEG tumors clustered closest to Catalogue of Somatic Mutations in Cancer signatures 4 and 24, which are, respectively, associated with benzo(a)pyrene- and aflatoxin-induced HCCs in humans. These results establish a novel approach for classifying liver carcinogens and understanding the mechanisms of hepatocellular carcinogenesis.
Project description:Hedgehog (Hh) pathway activation promotes many processes that occur during fibrogenic liver repair. Whether the Hh pathway modulates the outcomes of virally mediated liver injury has never been examined. Gene-profiling studies of human hepatocellular carcinomas (HCCs) demonstrate Hh pathway activation in HCCs related to chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV). Because most HCCs develop in cirrhotic livers, we hypothesized that Hh pathway activation occurs during fibrogenic repair of liver damage due to chronic viral hepatitis, and that Hh-responsive cells mediate disease progression and hepatocarciongenesis in chronic viral hepatitis. Immunohistochemistry and qRT-PCR analysis were used to analyze Hh pathway activation and identify Hh-responsive cell types in liver biopsies from 45 patients with chronic HBV or HCV. Hh signaling was then manipulated in cultured liver cells to directly assess the impact of Hh activity in relevant cell types. We found increased hepatic expression of Hh ligands in all patients with chronic viral hepatitis, and demonstrated that infection with HCV stimulated cultured hepatocytes to produce Hh ligands. The major cell populations that expanded during cirrhosis and HCC (ie, liver myofibroblasts, activated endothelial cells, and progenitors expressing markers of tumor stem/initiating cells) were Hh responsive, and higher levels of Hh pathway activity associated with cirrhosis and HCC. Inhibiting pathway activity in Hh-responsive target cells reduced fibrogenesis, angiogenesis, and growth. In conclusion, HBV/HCV infection increases hepatocyte production of Hh ligands and expands the types of Hh-responsive cells that promote liver fibrosis and cancer.
Project description:Hepatocellular carcinoma (HCC) typically develops in cirrhosis, a condition characterized by Hedgehog (Hh) pathway activation and accumulation of Hh-responsive myofibroblasts. Although Hh signaling generally regulates stromal-epithelial interactions that support epithelial viability, the role of Hh-dependent myofibroblasts in hepatocarcinogenesis is unknown. Here, we used human HCC samples, a mouse HCC model, and hepatoma cell/myofibroblast cocultures to examine the hypothesis that Hh signaling modulates myofibroblasts' metabolism to generate fuels for neighboring malignant hepatocytes. The results identify a novel paracrine mechanism whereby malignant hepatocytes produce Hh ligands to stimulate glycolysis in neighboring myofibroblasts, resulting in release of myofibroblast-derived lactate that the malignant hepatocytes use as an energy source. This discovery reveals new diagnostic and therapeutic targets that might be exploited to improve the outcomes of cirrhotic patients with HCCs.
Project description:Liver cirrhotic patients suffer from a seemingly unpredictable risk of hepatocellular carcinoma (HCC). Here, an HCC risk score R (0???R???1) was derived from commonly tested haematological and biochemical parameters. In the score-derivation Taiwanese cohort (144 cirrhosis versus 48 HCC-remission patients), the score had an area-under-the-curve (AUC) of 0.70 (95% confidence interval [CI], 0.61-0.78, P?<?0.001). When validated in a Korean cohort (78 cirrhosis versus 23 HCC-remission patients), the AUC was 0.68 (CI, 0.56-0.80, P?=?0.009). In a multicentre prospective cohort (478 cirrhotic patients prospectively followed for HCC occurrence), the hazard ratio with respect to R was 2.344 (CI?=?1.183-4.646, P?=?0.015). The cumulative incidences of HCC at two years after patient enrolment were 9.6% and 1.7% for the high-risk (R???0.5) and low-risk (R?<?0.5) groups, respectively (P?<?0.001). At the end of the study, the incidences were 10.9% and 5.0%, respectively (P?=?0.012). The majority of HCCs (23/26) in the high-risk group emerged within the first two years of follow-up. In conclusion, an HCC risk score was developed for cirrhotic patients that effectively predicted HCC in a prospective cohort study.
Project description:Recurrent somatic mutations in the promoter region of telomerase reverse transcriptase (TERT) gene and in the exon 3 of CTNNB1 gene have been recognized as common events in hepatocellular carcinoma (HCC) with variable frequencies depending on etiology and geographical region. We have analyzed TERT promoter and CTNNB1 gene mutations in 122 cases of hepatitis B (HBV) and hepatitis C (HCV) related HCCs, in 7 cases of cholangiocarcinoma (CC) and hepatocholangiocarcinoma (HCC-CC) as well as in autologous cirrhotic tissues. Overall, 50.4% and 26% of HCC as well as 14.3% and none of CC and HCC-CC were mutated in TERT promoter and in CTNNB1 exon 3, respectively. TERT and CTNNB1 mutations were found more frequently in HCV related (53.6% and 26.4%, respectively) than HBV related (41.7% and 16.7%, respectively) HCCs and coexisted in 57.6% of CTNNB1 mutated tumors. Mutations in TERT and CTNNB1 were not associated with the functional promoter polymorphism rs2853669. No mutations were detected in the 129 non-HCC cirrhotic tissues. In conclusion, mutations in TERT promoter and in CTNNB1 gene represent specific cancer signatures in the pathogenesis of viral related HCC and could be promising early biomarkers as well as targets for tailored therapies.
Project description:Introduction:Cirrhosis is one of the most important risk factors for development of hepatocellular carcinoma (HCC). Recent studies have shown that removal or well control of the underlying cause could reduce but not eliminate the risk of HCC. Therefore, it is important to elucidate the molecular mechanisms that drive the progression of cirrhosis to HCC. Materials and Methods:Microarray datasets incorporating cirrhosis and HCC subjects were identified from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were determined by GEO2R software. Functional enrichment analysis was performed by the clusterProfiler package in R. Liver carcinogenesis-related networks and modules were established using STRING database and MCODE plug-in, respectively, which were visualized with Cytoscape software. The ability of modular gene signatures to discriminate cirrhosis from HCC was assessed by hierarchical clustering, principal component analysis (PCA), and receiver operating characteristic (ROC) curve. Association of top modular genes and HCC grades or prognosis was analyzed with the UALCAN web-tool. Protein expression and distribution of top modular genes were analyzed using the Human Protein Atlas database. Results:Four microarray datasets were retrieved from GEO database. Compared with cirrhotic livers, 125 upregulated and 252 downregulated genes in HCC tissues were found. These DEGs constituted a liver carcinogenesis-related network with 272 nodes and 2954 edges, with 65 nodes being highly connected and formed a liver carcinogenesis-related module. The modular genes were significantly involved in several KEGG pathways, such as "cell cycle," "DNA replication," "p53 signaling pathway," "mismatch repair," "base excision repair," etc. These identified modular gene signatures could robustly discriminate cirrhosis from HCC in the validation dataset. In contrast, the expression pattern of the modular genes was consistent between cirrhotic and normal livers. The top modular genes TOP2A, CDC20, PRC1, CCNB2, and NUSAP1 were associated with HCC onset, progression, and prognosis, and exhibited higher expression in HCC compared with normal livers in the HPA database. Conclusion:Our study revealed a highly connected module associated with liver carcinogenesis on a cirrhotic background, which may provide deeper understanding of the genetic alterations involved in the transition from cirrhosis to HCC, and offer valuable variables for screening and surveillance of HCC in high-risk patients with cirrhosis.
Project description:Chronic liver injury predisposes to cirrhosis and hepatocellular carcinoma, but how somatic mutations accumulate in liver disease is unexplored. We sequenced whole genomes of 400 microdissections of 100-500 hepatocytes from 5 normal and 6 cirrhotic livers. Compared to normal liver, cirrhotic liver had higher mutation burden, especially structural variants, including chromothripsis. Cirrhotic nodules were oligoclonal; sometimes entirely derived from a single, recent common ancestor. Clonal expansions millimeters in diameter occurred in cirrhosis in the absence of known driver mutations. Endogenous mutational processes predominated, although signatures of polycyclic aromatic hydrocarbon and aristolochic acid exposure occurred in some samples. Up to 10-fold within-patient variation in activity of exogenous signatures existed between adjacent cirrhotic nodules, with both clone-specific and microenvironmental forces shaping this heterogeneity. Synchronous hepatocellular carcinomas drew from the same repertoire of mutational signatures as background cirrhotic liver, but with higher burden. Somatic mutations chronicle the exposures, toxicity, regeneration and clonal structure of liver tissue as it progresses from health to disease.