HBV-infected Hepatocellular Carcinoma Patients Research Articles

Sponging of five tumour suppressor miRNAs by lncRNA-KCNQ1OT1 activates BMPR1A/BMPR1B-ACVR2A/ACVR2B signalling and promotes chemoresistance in hepatocellular carcinoma

Diverse mechanisms have been established to understand the chemoresistance of hepatocellular carcinoma (HCC), but the contribution of non-coding RNAs is not surveyed well. Here, we aimed to explore the lncRNA-miRNA axis in Hepatitis C and B virus (HCV and HBV) infected HCC to investigate the molecular mechanism of chemoresistance and to identify a potential therapeutic target for HCC. The small RNA transcriptome analysis followed by qRT-PCR validation with the liver tissues of both HCV and HBV infected HCC patients revealed that miR-424-5p, miR-136-3p, miR-139-5p, miR-223-3p, and miR-375-3p were the most downregulated miRNAs in HCC compared to normal (log2 fold change ≤−1.5, Padj ≤ 0.05). In silico pathway analysis with the validated targets of each miRNA revealed that the signalling pathway regulating pluripotency of stem cells is commonly targeted by these five miRNAs. Subsequent validation by 3′UTR-luciferase assay and western blot analysis unveiled that these five miRNAs impeded either same or diverse genes, but all linked to BMP signalling pathway such as BMPR1A/BMPR1B by miR-139-5p, miR-136-3p, and miR-375-3p, and ACVR2A/ACVR2B by miR-424-5p and miR-223-3p. Furthermore, restoration of each miRNA in Huh7/SNU449 cells inhibited phosphorylation of downstream SMAD1/5 and ERK1/2, and attenuated Epithelial-mesenchymal transition, stemness, spheroid formation, chemoresistance, invasion and migration of cells. To investigate the mechanism of suppression of these miRNAs, “DIANA” tool was employed and lncRNA-KCNQ1OT1 was retrieved as interacting partner of all the five miRNAs. In vitro RNA pull-down assay revealed that lncRNA-KCNQ1OT1 physically interacted and sequestered these five miRNAs in the cytoplasm. Hence, KCNQ1OT1 was suppressed in Huh7/SNU449 cells using CRISPR technology and observed regression of oncogenic properties with enhanced chemosensitivity and reduced metastasis in cancer cells. Shrinkage of tumour size and volume in NOD-SCID mice injected with KCNQ1OT1-sgRNA cells further strengthened our observations. Thus, lncRNA-KCNQ1OT1 is the main regulator, which reduces the level of beneficiary miRNAs in the tumour milieu and modulates BMP signalling pathway to promote chemoresistance in HCC, suggesting lncRNA-KCNQ1OT1 might have robust potential to be a therapeutic target in HCC.

HBV-infected hepatocellular carcinoma can be robustly classified into three clinically relevant subgroups by a novel analytical protocol.

Liver cancer is the third leading cause of cancer-related death worldwide, and hepatocellular carcinoma (HCC) accounts for a relatively large proportion of all primary liver malignancies. Among the several known risk factors, hepatitis B virus (HBV) infection is one of the important causes of HCC. In this study, we demonstrated that the HBV-infected HCC patients could be robustly classified into three clinically relevant subgroups, i.e. Cluster1, Cluster2 and Cluster3, based on consistent differentially expressed mRNAs and proteins, which showed better generalization. The proposed three subgroups showed different molecular characteristics, immune microenvironment and prognostic survival characteristics. The Cluster1 subgroup had near-normal levels of metabolism-related proteins, low proliferation activity and good immune infiltration, which were associated with its good liver function, smaller tumor size, good prognosis, low alpha-fetoprotein (AFP) levels and lower clinical stage. In contrast, the Cluster3 subgroup had the lowest levels of metabolism-related proteins, which corresponded with its severe liver dysfunction. Also, high proliferation activity and poor immune microenvironment in Cluster3 subgroup were associated with its poor prognosis, larger tumor size, high AFP levels, high incidence of tumor thrombus and higher clinical stage. The characteristics of the Cluster2 subgroup were between the Cluster1 and Cluster3 groups. In addition, MCM2-7, RFC2-5, MSH2, MSH6, SMC2, SMC4, NCPAG and TOP2A proteins were significantly upregulated in the Cluster3 subgroup. Meanwhile, abnormally high phosphorylation levels of these proteins were associated with high levels of DNA repair, telomere maintenance and proliferative features. Therefore, these proteins could be identified as potential diagnostic and prognostic markers. In general, our research provided a novel analytical protocol and insights for the robust classification, treatment and prevention of HBV-infected HCC.

Association of PNPLA3 SNP With the Development of HBV-related Hepatocellular Carcinoma.

Concomitant nonalcoholic fatty liver disease (NAFLD)/hepatic steatosis (HS) is suggested to increase the risk of hepatocellular carcinoma (HCC) in hepatitis virus B (HBV)-infected patients. Patatin-like phospholipase domain-containing 3 (PNPLA3) rs738409 gene single-nucleotide polymorphism (SNP) is well-known to be associated with the development of NAFLD/HS; however, it is still unclear whether this SNP is related to the development of HCC in HBV-infected patients. We investigated a total of 202 HBV-infected patients who received percutaneous liver biopsy, and simultaneously assessed biopsy-proven HS, insulin resistance, and the PNPLA3 SNP status. We further investigated the relationships of these factors with the development of HCC in HBV-infected patients. Most of the enrolled cases (196/202: 97.0%) were non-cirrhotic patients. One hundred seventy-three patients (85.6%) received antiviral therapy. A Kaplan-Meier analysis showed that the incidence of HCC development in patients with HS was higher than that in patients without HS (p<0.01). An increased homeostasis model assessment as an index of insulin resistance (HOMA-IR) value (≥1.6) was associated not only with the presence of HS (p<0.0001) but also with the development of HCC (p<0.01). The PNPLA3 rs738409 SNP was also associated with the presence of HS (p<0.01) and the development of HCC (p<0.05) in HBV-infected patients. In addition to HS and IR, PNPLA3 rs738409 SNP was suggested to be associated with the development of HCC in Japanese patients with HBV infection.

MiR-135a inhibits the proliferation of HBV-infected hepatocellular carcinoma cells by targeting HOXA10.

The incidence of hepatocellular carcinoma (HCC) in patients with hepatitis B virus (HBV) is extremely high. MicroRNAs (miRNAs) are a type of endogenous non-coding small RNA with novel molecular therapeutic mechanisms that plays an important role in the occurrence and development of cancers. This study aimed to explore the regulation mechanism of miR-135a and HOXA10 in the proliferation, invasion, and apoptosis of HCC cells. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis was used to detect the expression level of miR-135a. Overexpression of miR-135a was used to measure the roles of miR-135a in the proliferation, invasion, and apoptosis of HCC cells. A dual luciferase experiment was performed to assess the relationship between HOXA10 and miR-135a. Western blot was applied to observe the protein levels of p-p38, p-ERK, and p-JNK. The expression levels of miR-135a were significantly decreased in HCC tissues and cells. Overexpression of miR-135a inhibited the proliferation and invasion but promoted the apoptosis of HCC cells. Importantly, our results confirmed that HOXA10 was a direct target of miR-135a. Under HBV infection, silencing of HOXA10 significantly blocked the proliferation and invasion and promoted the apoptosis of HCC cells. In addition, miR-135a/HOXA10 regulated the expressions of p-p38, p-ERK, and p-JNK through the miR-135a/HOXA10 axis, thereby inhibiting the activation of the MAPK pathway. HBV promoted the proliferation and invasion, and inhibited the apoptosis of HCC cells by regulating the miR-135a/HOXA10 pathway. These findings provide a theoretical basis for improving the treatment of HBV-infected HCC patients.

Post-operative recurrence of liver cancer according to antiviral therapy for detectable hepatitis B viremia: A nationwide study

BackgroundHigh postoperative recurrence of hepatitis B virus (HBV)-infected hepatocellular carcinoma (HCC) remains a significant challenge. Here, we aimed to compare the postoperative HCC recurrence between patients with AVT for detectable serum HBV-DNA vs. those without. MethodsData of patients undergoing curative resection of HBV-infected HCC as an initial therapy from 2015 to 2017 were obtained from the National Health Insurance Service database in South Korea. AVT was initiated when serum HBV-DNA was detectable. The primary outcome was HCC recurrence. The cumulative risk of HCC recurrence between AVT users and non-users was estimated using the Kaplan–Meier method. ResultsDuring follow-up (median 2.7 years) with 3034 patients, 25.7% and 23.6% of AVT users and non-users experienced HCC recurrence, respectively. The 1-, 2-, and 3-year cumulative recurrence rates were similar (p = 0.57): 15.6%, 23.3%, and 26.4% in AVT users versus 15.3%, 22.0%, and 24.9% in non-users, respectively. After adjusting for covariates, multivariable Cox regression analysis showed comparable outcomes between the two groups with adjusted hazard ratios (aHR 1.08, 95% confidence interval [CI] 0.89–1.31; p = 0.439). Similar outcomes between the two groups were reproduced after stratification of liver cirrhosis. Inverse probability treatment weighting analysis also showed comparable outcomes between the two groups in the subgroups with liver cirrhosis (aHR 1.01, 95% CI 0.80–1.29; p = 0.92) and non-cirrhosis (aHR 1.08, 95% CI 0.87–1.34; p = 0.472). ConclusionsInitiating AVT based on detectable serum HBV-DNA provided the similar risk of postoperative HCC recurrence in HBV-infected HCC patients with and without detectable serum HBV-DNA.

Characteristics of Hepatitis B Virus Genotype and Sub-Genotype in Hepatocellular Cancer Patients in Vietnam.

Untreated chronic hepatitis B virus (HBV) infection can lead to chronic liver disease and may progress to cirrhosis or hepatocellular carcinoma (HCC). HBV infection has been prevalent in Vietnam, but there is little information available on the genotypes, sub-genotypes, and mutations of HBV in patients with HBV-related HCC confirmed by histopathological diagnosis. We studied the molecular characteristics of HBV and its genetic variants in Vietnamese HCC patients after liver tumor resection. We conducted a descriptive cross-sectional study on 107 HBV-related HCC hospitalized patients from October 2018 to April 2019. The specimens collected included EDTA anticoagulant blood and liver tissues. Extracted HBV DNA was subjected to whole genome sequencing by the Sanger method. We discovered 62 individuals (57.9%) with genotype B and 45 patients (42.1%) with genotype C, with only sub-genotypes B4 and C1. Among the mutations, the double mutation, A1762T-G1764A, had the most significant frequency (73/107 samples; 68.2%) and was higher in genotype C than in genotype B (p < 0.001). The most common genotypes found in HCC patients in this investigation were B and C, with sub-genotypes B4 and C1 for each. The prevalence of genotype B4 was greater in HBV-infected Vietnamese HCC patients.

Exosomal miR-142-3p secreted by hepatitis B virus (HBV)-hepatocellular carcinoma (HCC) cells promotes ferroptosis of M1-type macrophages through SLC3A2 and the mechanism of HCC progression.

Most patients with hepatitis B virus (HBV) infection will develop hepatocellular carcinoma (HCC). This study aimed to explore the potential mechanism of miR-142-3p in HCC caused by HBV infection. HepG2 cells and M1 macrophages were cocultured and then infected with HBV to establish an in vitro model. MicroRNA (miRNA) and messenger RNA (mRNA) expression was analyzed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot. The protein expressions of COX2, ACSL4, PTGS2, GPX4, and NOX1 were analyzed by Western blot. Flow cytometry and TUNEL assays were used to assess cell reactive oxygen species (ROS) and ferroptosis, respectively. Cell invasion and migration were measured by Transwell assay. To evaluate the ferroptosis of M1-type macrophages, glutathione (GSH), malondialdehyde (MDA), and Fe2+ content was detected by corresponding kits. Dual luciferase reporter gene detection verified the targeting relationship between miR-142-3p and SLC3A2. MiR-142-3p was highly expressed in HBV-infected HCC patients and HBV-infected M1-type macrophages. Inhibition of miR-142-3p or overexpression of SLC3A2 reversed ferroptosis and inhibited the proliferation, migration, and invasion of HCC cells. Our findings indicated that miR-142-3p promoted HBV-infected M1-type macrophage ferroptosis through SLC3A2, affecting the production of GSH, MDA, and Fe2+ and accelerating the development of HCC. The regulation of miR-142-3p and its target genes will help to clarify the pathogenesis of HCC induced by HBV infection and provide new theoretical foundations and therapeutic targets.

Blood-Based Biomarkers in Hepatitis B Virus-Related Hepatocellular Carcinoma, Including the Viral Genome and Glycosylated Proteins

Hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC) development and is a global public health issue. High performance biomarkers can aid the early detection of HCC development in HBV-infected individuals. In addition, advances in the understanding of the pathogenesis of HBV infection and in clinical laboratory techniques have enabled the establishment of disease-specific tests, prediction of the progression of liver diseases, including HCC, and auxiliary diagnosis of HCC, using blood-based methods instead of biopsies of liver or HCC tissues. Viral factors such as the HBV genotype, HBV genetic mutations, HBV DNA, and HBV-related antigens, as well as host factors, such as tumor-associated proteins and post-translational modifications, especially glycosylated proteins, can be blood-based, disease-specific biomarkers for HCC development in HBV-infected patients. In this review, we describe the clinical applications of viral biomarkers, including the HBV genome and glycosylated proteins, for patients at a risk of HBV-related HCC, based on their molecular mechanisms. In addition, we introduce promising biomarker candidates for practical use, including colony stimulating factor 1 receptor (CSF1R), extracellular vesicles, and cell-free, circulating tumor DNA. The clinical use of such surrogate markers may lead to a better understanding of the risk of disease progression and early detection of HCC in HBV-infected patients, thereby improving their prognosis.

Comprehensive genomic profiling analysis of HBV-infected hepatocellular carcinoma patients.

e16180 Background: Hepatocellular carcinoma (HCC) is one of the leading causes of tumor-related death worldwide which is dominantly caused by chronic infection of hepatitis B virus (HBV) and hepatitis C virus (HCV). Recent studies have shown that immune checkpoint inhibitors may enter the first-line treatment of HCC. Comprehensive investigation of the genetic alterations and their correlation with microsatellite instability (MSI) and PD-L1 expression in HBV-infected HCC is helpful to determine the therapy strategy for patients. Methods: The genomic information of HCC patients was obtained from HapLab database. HaploX 605- gene panel was performed to analyze the genomic data of patients. PD-L1 expression was detected by immunochemistry. Results: A total of 349 mutations were found by analyzing the genomic profiles of 115 HBV-infected HCC patients. The frequently mutated genes included TP53 (63%), TERT (27%), CTNNB1 (16%), CSMD3 (15%), LRP1B (14%), AXIN1 (13%), CDKN2A (13%), KIT (13%), MDM4 (12%) and CCND1 (12%). Besides, 279 mutations were identified by analyzing the genomic profiles of 82 HCC patients without HBV-infection. Mutation frequencies for several genes were relatively higher in the HBV-positive patients than that in HBV-negative HCC patients, including TP53 (63% versus 39%), CCND1 (12% versus 5%), RB1(12% versus 4%) and BRCA2 (11% versus 4%). The proportion of MSI-H/L in HBV-infection patients were higher than that in HBV-negative ones (7.83% versus 4.88%). Notably, the proportion of high PD-L1 expression in patients with HBV-infection was 40.57% (43/106), while it was 28.95% (22/76) in patients without HBV-infection. Conclusions: These results highlighted a potential impact of viral infection on the mutational signatures in hepatocarcinogenesis. The PD-L1 expression status and MSI were closely related to HBV-infected HCC. These results may contribute to understanding the mechanism of HBV-infected HCC and the selection of therapy for relative patients.

The genetic polymorphism down-regulating HLA-DRB1 enhancer activity facilitates HBV persistence, evolution and hepatocarcinogenesis in the Chinese Han population.

Genetic predisposition of human leucocyte antigen (HLA)-DR has been linked to nonresponse to hepatitis B virus (HBV) vaccination. We sought to reveal their effects on chronic infection and evolution of HBV and development of hepatocellular carcinoma (HCC). Genetic polymorphisms at HLA-DR enhancer regions were genotyped in 4588 participants using quantitative PCR. HBV mutations were determined by sequencing. A dual-luciferase assay was applied to detect the enhancer activity. Associations between HLA-DR polymorphisms and postoperative prognosis were investigated in another cohort of 397 HBV-infected HCC patients. Variant alleles (rs3135395-T, rs3135338-C and rs477515-T) were significantly associated with a decreased risk of HBV persistence in Chinese patients. rs3135395-T, rs3135338-C, rs477515-T and rs2395178-G also significantly decreased HCC risk. rs3135395-T, rs477515-T and rs2395178-G were inversely associated with the generation of A1762T/G1764A, T1753V and C1653T, the HCC-risk HBV mutations. Multiplicative interactions of the variant genotypes with the HCC-risk HBV mutations were significantly associated with a decreased risk of HCC. In multivariate Cox analysis, rs477515-T independently predicted a favourable prognosis, with a hazard ratio of 0.48 (P=.002). The activity of the HLA-DRB1 enhancer with rs477515-T was significantly higher than that with rs477515-C. The activity of the HLA-DRB1 enhancer with rs477515-T and that with rs477515-C was significantly up-regulated by interferon-γ and interleukin-4, respectively. Interleukin-6 significantly inhibited the HLA-DRB1 enhancer activity, and this effect was more evident in those carrying rs477515-T. Polymorphisms predisposing to down-regulation of HLA-DR facilitate the Th1-to-Th2 transition and promote HCC development, possibly via selecting the HCC-risk HBV mutations. This can be transformed into specific prophylaxis of HCC.

Anti-PD-1/PD-L1 Blockade Immunotherapy Employed in Treating Hepatitis B Virus Infection-Related Advanced Hepatocellular Carcinoma: A Literature Review.

Hepatitis B virus (HBV) infection is regarded as the main etiological risk factor in the process of hepatocellular carcinoma (HCC), as it promotes an immunosuppressive microenvironment that is partially mediated by the programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) signaling pathway. The tumor microenvironment (TME) of HBV–related HCC is indeed more immunosuppressive than microenvironments not associated with viruses. And compared to TME in hepatitis C virus (HCV) infected HCC, TME of HBV-related HCC is less vascularized and presents different immune components resulting in similar immunosuppression. However, few studies are focusing on the specific side effects and efficacy of PD-1/PD-L1 blockade immunotherapy in HBV-related HCC patients, as well as on the underlying mechanism. Herein, we reviewed the basic research focusing on potential TME alteration caused by HBV infection, especially in HCC patients. Moreover, we reviewed PD-1/PD-L1 blockade immunotherapy clinical trials to clarify the safety and efficacy of this newly developed treatment in the particular circumstances of HBV infection. We found that patients with HBV-related HCC displayed an acceptable safety profile similar to those of non-infected HCC patients. However, we could not determine the antiviral activity of PD-1/PD-L1 blockade because standard anti-viral therapies were conducted in all of the current clinical trials, which made it difficult to distinguish the potential influence of PD-1/PD-L1 blockade on HBV infection. Generally, the objective response rates (ORRs) of PD-1/PD-L1 blockade immunotherapy did not differ significantly between virus-positive and virus-negative patients, except that disease control rates (DCRs) were obviously lower in HBV-infected HCC patients.

The mutation profiles of HBV or HCV infected hepatocellular carcinoma patients underlie the varied responses to second-line immunotherapy.

e16629 Background: HBV and HCV infections facilitate the development of hepatocellular carcinoma (HCC) via different mechanisms, given that the DNA sequence of HBV but not HCV can integrate into the host genome. However, it remains unclear that whether the altered genomic landscape of HCC patients caused by HBV or HCV infection may result in different responses to treatments. Methods: The MSK panel sequencing results and treatment data of 127 HCC patients were downloaded from the TCGA database (MSK, Clin Cancer Res 2018). Profiles of HBV positive and HCV positive patients were extracted. Based on the treatment received, those patients were classified to sorafenib treated groups and sorafenib + immunotherapy (IT) groups. The progression-free survival (PFS, months) and overall survival (OS, months) were compared, and the mutational profiles of patients were analyzed. Results: Among patients who received sorafenib treatment alone, the mean PFS of HBV positive patients (n = 12, 4.9 ±1.1) is similar to that of the HCV positive patients (n = 21, 5.1±1.8). However, the mean OS of HBV positive patients (n = 12, 18.3 ±6.6) was longer than the mean OS of HCV positive patients (n = 21, 10.0 ±1.9). Among the patients who received immunotherapy following sorafenib treatment, the mean IT PFS of HBV positive patients was (n = 5, 4.4 ±1.8), which was longer than the mean IT PFS of HCV positive patients (n = 5, 2.5 ±0.5). Consistently, the mean IT OS of HBV positive patients (n = 5, 10.9 ±4.4) was longer than the mean IO PFS of HCV positive patients (n = 5, 4.6 ±1.0). Overall it seems that the HBV positive patients responded better to second-line immunotherapy, despite the fact that these comparisons did not reach statistical difference due to the limited sample size. By comparing the MSK panel sequencing results, mutations exclusive to the HBV or HCV infected groups were identified. Enrichment analysis showed that 7/35 mutations exclusively found in the HBV positive group are responsible for lymphocyte activation (p &lt; 0.00001), which may underlie the better IT treatment outcome. Among the mutations restricted to the HCV infected group, 9/50 were involved in histone modification, 7/50 were involved in Hepatitis B pathways (p &lt; 0.0001), suggesting that HCV infection may interfere with diverse molecular processes. Conclusions: HBV and HCV infected HCC patients may respond differently to second-line immunotherapy. This difference is perhaps related to the altered genetic landscape of the patients resulted from virus infections by HBV or HCV.

Distinct clinical and genetic characteristics of AFP-negative hepatocellular carcinoma patients infected by HBV or HCV.

e16620 Background: The serum level of alpha-fetoprotein (AFP) is a key biomarker in hepatocellular carcinoma (HCC) screening regardless of the high false-negative rates. Studies have characterized AFP negative HCC patients, however, the differences between HBV and HCV infected AFP negative HCC patients have not been discussed. Methods: The clinical profiles and whole-exome sequencing data of 243 patients were downloaded from the TCGA database. Patients were divided into 4 groups based on their virus infection status and serum AFP measurements as indicated in the table. The mutation profiles and clinical features of the patients were analyzed. Results: In the HBV infected groups, the AFP+ patients carried significantly larger tumors (n = 17, 85.88±11.87 mm) compared to the AFP- patients (n = 13, 49.08±8.933 mm, p = 0.026). The HBV+ AFP+ group exhibited the prevalence of vascular invasion (VI, 76%, 13/17) while less than half of the patients in the HBV+ AFP- group developed VI (46%, 6/13). The WES results identified shared mutations in more than 10% of patients in each group. It appeared that 126 shared mutations were restricted to the HBV+ AFP+ group, they are involved in the regulation of ion transmembrane transport (p &lt; 0.001) and interaction between L1 and Ankyrins (p &lt; 0.0001). Only 51 shared mutations were exclusive to the HBV+ AFP- group, they majorly affect developmental growth (p &lt; 0.001) and skeletal system development (p &lt; 0.01). In contrast, the tumor sizes of HCV+ AFP+ and HCV+ AFP- patients are similar (55.18±7.781 vs 48.19±5.564, p = 0.47), while the VI rate is higher in the HCV+ AFP- group (63%, 17/29, Table). In total, 11 shared mutations were restricted to the HCV+ AFP+ group, several of the genes were involved in the generation of precursor metabolites and energy (p &lt; 0.01) and activation of immune response (p &lt; 0.01). 27 shared mutations were exclusive to the HCV+ AFP- group, the enrichment analysis showed that they are related to the regulation of synapse organization (p &lt; 0.0001) and the regulation of chromosome organization (p &lt; 0.01). Conclusions: HBV and HCV infection were associated with distinct clinical features among the AFP positive and AFP negative patients. The differences were underpinned by the mutations exclusive to each group, and they were involved in distinct molecular and cellular functions. [Table: see text]

Genomic characterization of HBV-infected hepatocellular carcinoma patients using circulating tumor DNA.

575 Background: Hepatocellular carcinoma (HCC) is a leading cause of mortality, with Hepatitis B virus (HBV) infection as a dominant etiology. Surgery or ablation may be curative for early-stage HCC. Thus, effective detection strategies are needed. We investigated genomic aberrations in circulating tumor DNA (ctDNA) as a potential diagnostic marker of HCC in HBV-infected patients. Methods: We identified early stage (BCLC 0-A) HCC cases (n = 21) and cancer-free controls (n = 15) from a cohort of Asian patients with HBV, undergoing surveillance at Thomas Jefferson University Hospital between 2013-2017. Blood samples were collected. Circulating cell-free DNA was isolated from plasma and assayed by capture-based next-generation sequencing of a targeted panel of 23 genes implicated in HCC pathogenesis. Sequencing data analysis and somatic mutation identification were conducted using a computational pipeline. Using area under the curve (AUC) in receiver operating characteristic analysis, we evaluated gene alterations and clinical factors (age, gender, cirrhosis) in an exploratory early detection HCC model. Results: Mutant ARID1A, ATM, CDKN2A, CTNNB1, ERBB2, TP53 genes were increased in HCC cases relative to non-cancer patients (85.7% vs 53.3%, P = 0.058; 42.9% vs 6.7%, P = 0.025; 38.1% vs 6.7%, P = 0.051; 42.9% vs 0%, P = 0.005; 52.4% vs 13.3%, P = 0.016; 100% vs 66.7%, P = 0.008, respectively). HCC patients had higher prevalence of cirrhosis than controls (90.5% vs. 60%, P = 0.046). Using the 6 mutant genes alone, the AUC for discriminating HCC from non-cancer patients was 0.827 (95% confidence interval [CI]: 0.701-0.953), which was greater than the AUC for discriminating cirrhosis from non-cirrhosis (0.531). When the 6 mutant genes were combined with clinical factors, the AUC of the exploratory HCC detection model increased to 0.914 ( P= 0.045). Conclusions: We identified 6 genomic aberrations in ctDNA that were more prevalent in HCC patients compared with non-cancer patients. Combining these alterations with clinical factors may identify HCC in HBV-infected patients at an early stage. These findings warrant further validation in future studies.

Genomic analysis of hepatocellular carcinoma (HCC) with active hepatitis B virus (HBV) replication.

e15593 Background: HBV replication contributes to HCC initiation and is associated with worse patient outcomes. Prior tumor genomic studies of HBV-positive and -negative (HBV+/-) HCC have used detection of HBV surface antigen (HBsAg) in serum to annotate HBV status. However, a substantial proportion of HBsAg+ patients lack HBV replication in tumor, suggesting a potentially distinct patient subset. In this study, we determined HBV status by measuring tumor HBV RNA, a proxy for active replication. We then investigated HBV RNA+/- association with somatic mutations, gene sets, homologous recombination deficiency (HRD) and tumor mutation burden (TMB). Methods: RNA-Seq data for 371 HCC tumors were obtained from TCGA. Tumors were classified as HBV RNA+ if they harbored more than 1 HBV RNA read per million human reads, as measured using GATK PathSeq software. Associations between HBV RNA status and somatic mutations, gene sets, HRD and TMB were investigated. HRD score was calculated as the sum of 3 independent HRD measures (large scale state transitions, loss of heterozygosity and telomeric allelic imbalance). Results: HBV RNA+ status was associated with a higher rate of nonsynonymous somatic mutations in multiple genes, including the tumor suppressors TP53, CDKN2A, CHD5 and TET1, as well as AXIN2 and the proto-oncogene BCL11A ( p &lt; 0.05 for all), while HBV RNA- status was associated with a higher rate of nonsynonymous mutations in the chromatin modifier BAP1 ( p = 0.03). In gene set enrichment analysis of normalized RNA-Seq expression data, HBV RNA+ status was associated with increased transcription of DNA repair genes, as well as genes upregulated by mTORC1 and MYC (FDR &lt; 0.03 for all). HBV RNA status was also associated with HRD score (22.19 for HBV RNA+ vs. 15.97 for HBV RNA-, p = 1e-6), but not with TMB. A substantial subset of HBV RNA+ patients (33/100) were not annotated as HBV+ in the TCGA clinical database. Conclusions: HBV status based on tumor HBV RNA detection identifies a genetically distinct subset within all HBV-infected HCC patients that is associated with nonsynonymous somatic mutations in several genes and differential transcription of gene sets, some of which have not been previously reported, as well as with HRD score. These findings suggest potential for differential responsiveness to targeted therapies.

Vitamin D Receptor Fok I Polymorphism and Risk of Hepatocellular Carcinoma in HBV-Infected Patients

Context: We conducted a meta-analysis to evaluate the role of the vitamin D receptor (VDR) polymorphism in the risk of hepatocellular carcinoma (HCC) in patients infected with hepatitis B virus (HBV). Objective: To develop new preventions, mechanisms, and therapies for HCC, it is important to investigate the underlying causes of HCC. Recently, there has been increasing attention to the relationship between VDR polymorphism and the risk of HBV-related HCC, but no specific conclusion has been made. Data Sources: PubMed/Medline, EMBASE, Cochrane Library, Chinese National Knowledge Infrastructure, VIP Database for Chinese Technical Periodicals, Wanfang Data, and SINOMED databases were searched until August 2018. Studies reporting VDR polymorphism and HBV-related HCC risks were included and the quality was assessed by Newcastle-Ottawa scale (NOS). Odds ratio (OR) and 95% confidence interval (CI) were calculated to compare the pooled data between HCC risks and different genotypes, such as FF, Ff, F, f, and ff, with each other. Results: Three case-control studies with totally 728 HBV-related HCC cases and 920 HBV controls were included. When comparing HBV-related HCC cases with HBV controls, our data showed that all genotypes of Fok I polymorphism significantly increased the risk of HCC in the overall population (ff vs. FF: OR = 1.816, 95% CI = 1.161 - 2.841, P = 0.0009; Ff vs. FF: OR = 1.315, 95% CI = 1.037 - 1.667, P = 0.024; ff/Ff vs. FF, OR = 1.504, 95% CI = 1.206 - 1.876, P < 0.001; ff vs. FF/Ff: OR = 1.591, 95% CI = 1.270 - 1.992, P < 0.001; ff vs. Ff: OR = 1.435, 95% CI = 1.127 - 1.827, P = 0.003; f vs. F: OR = 1.375, 95% CI = 1.075 - 1.759). Conclusion: The results of our analysis suggest the “f” allele of Fok I polymorphism might be a risk factor for HCC in HBV-infected patients and it could be a predictive factor to screen HCC in HBV-infected patients. However, more accurate analyses and larger studies are needed.

Hepatitis B virus promotes β-catenin-signalling and disassembly of adherens junctions in a Src kinase dependent fashion.

Hepatitis B virus (HBV) infection is a prominent cause of hepatocellular carcinoma (HCC) but the underlying molecular mechanisms are complex and multiple pathways have been proposed such as the activation of the Wnt−/β-catenin-signalling and dysregulation of E-cadherin/β-catenin adherens junctions. This study aimed to identify mechanisms of how HBV infection and replication as well as HBV X protein (HBx) gene expression in the context of an HBV genome influence Wnt−/β-catenin-signalling and formation of adherens junctions and to which extent HBx contributes to this.Regulation of E-cadherin/β-catenin junctions and β-catenin-signalling as well as the role of HBx were investigated using constructs transiently or stably inducing replication of HBV+/−HBx in hepatoma cell lines. In addition, HCC and adjacent non-tumorous tissue samples from HBV-infected HCC patients and drug interference in HBV-infected cells were studied.Although HBV did not alter overall expression levels of E-cadherin or β-catenin, it diminished their cell surface localization resulting in nuclear translocation of β-catenin and activation of its target genes. In addition, HBV gene expression increased the amount of phosphorylated c-Src kinase. Treatment with Src kinase inhibitor Dasatinib reduced HBV replication, prevented adherens junction disassembly and reduced β-catenin-signalling, while Sorafenib only did so in cells with mutated β-catenin. Interestingly, none of the HBV induced alterations required HBx.Thus, HBV stimulated β-catenin-signalling and induced disassembly of adherens junctions independently of HBx through Src kinase activation. These pathways may contribute to hepatocellular carcinogenesis and seem to be more efficiently inhibited by Dasatinib than by Sorafenib.

Development and Validation of a Nomogram to Detect Hepatocellular Carcinoma in Patients with Chronic Hepatitis B: A Prospective, Multicenter Study

Background More than 50% of hepatocellular carcinoma (HCC) are caused by hepatitis B virus (HBV) infection and almost one third of carriers of hepatitis B virus (HBV) world-wide are in China. Early detection of HCC in HBV-infected patients remains a challenge. Methods Consecutive participants with HBV-related HCC, chronic hepatitis B (CHB), liver cirrhosis with HBV infection (HBV-LC), benign hepatic tumors and healthy controls at 11 hospitals in China from June 2016 to April 2017 were recruited to form a training cohort. Participants from the same hospitals from May 2017 to September 2017 were recruited to form a validation cohort. All HCCs were diagnosed by ultrasound, characteristic radiologic features on CT/MRI, and confirmed by histopathological examination from biopsy or surgically resected specimens. Findings 908 of 2207 (41.1%) participants in the training cohort and 289 of 730 (39.6%) participants in the validation cohort were diagnosed to have HBV-related HCC. The serum levels of PIVKA-II and AFP were significantly higher in patients with HBV-related HCC than in those with CHB, HBV-LC or in controls (P<0.0001). A combination of PIVKA-II and AFP showed improved diagnostic accuracy in differentiating patients with HBV-related HCC from those with CHB and HBV-LC (area under the curve [AUC] 0.922 [95% CI 0.908-0.935], sensitivity 88.3% and specificity 85.1% for the training cohort; the corresponding figures were 0.902 [0.875, 0.929], 87.8% and 81.0%) respectively for the validation cohort. The variables which were included in the nomogram were AFP, PIVKA-II, age and sex. The nomogram performed well in predicting HBV-related HCC in calibration and in discrimination in the training cohort (AUC, 0.941 [0.929, 0.952]); as well as in the validation cohort (0.931 [0.909, 0.953]). The nomogram also performed well in detecting early-stage HBV-related HCC. Interpretation A nomogram and an online tool, which included AFP, PIVKA-II, age and sex in predicting the presence of HBV-related HCC were built and validated with good discrimination and calibration. This nomogram can help clinicians to detect early HCC in patients with HBV infection. Trial Registration Number: The study was registered with ClinicalTrials.gov, number NCT03047603. Funding Abbott Diagnostics (ADD-China-2016), and in part by National Natural Science Foundation of China (No. 81472284 and 81672699). Declaration of Interest: None. Ethical Approval: The study was approved by the Institutional Review Boards at each study center. Informed consent was obtained from each participant.

LAP+CD4+ T cells are elevated among the peripheral blood mononuclear cells and tumor tissue of patients with hepatocellular carcinoma.

The purpose of the present study was to investigate the role of latency-associated peptide (LAP)+CD4+T cells in hepatocellular carcinoma (HCC) immunity. Flow cytometric analysis was performed to detect the proportion of LAP+CD4+ T cells among the peripheral blood mononuclear cells (PBMCs) of 30 HBV-infected HCC patients at the pre-operative and post-operative stages, as well as 30 hepatitis B virus (HBV)-infected volunteers as a control group. Furthermore, tumor tissues and peri-tumor tissues from 28 patients with HCC, as well as hepatic tissues from 28 HBV-infected patients with benign lesions were subjected to immunohistochemical analysis with double staining for LAP and CD4, and the average number of the LAP+CD4+T cells in each visual field was quantified. The results indicated that the proportion of LAP+CD4+ T cells in the PBMCs of patients with HCC was significantly higher than that in the control group (1.84±0.85 vs. 0.73±0.39%, P=0.019), while it was significantly reduced after the operation (1.07±0.35, P=0.021), but still slightly, if not significantly, higher compared with that in the control group (P=0.342). Furthermore, the number of LAP+CD4+ T cells per high-magnification microscopic field (magnification, ×400) in the HCC tissues was 11.25±3.00, which was significantly higher than that in the peri-cancer tissues (5.75±1.00) and that in the HBV-infected hepatic tissues around benign lesions (2.61±0.83). In peri-cancer tissues, LAP+CD4+ T cells were also significantly more abundant than in control tissues. Furthermore, in the HCC tissues, LAP+CD4+ T cells were present as clusters in the tumor stroma and closely associated with CD4+ T lymphocytes. By contrast, in the peri-cancer liver tissues and HBV-infected hepatic tissues around benign lesions, LAP+CD4+ T cells were sparsely distributed. LAP+CD4+ T cells have marked inhibitory effects, and in the peripheral blood and tumor tissues of patients with HCC, they have an important role in the suppression of anti-tumor immunity and in the immune evasion of tumor cells.

Selective serotonin reuptake inhibitors and the risk of hepatocellular carcinoma in hepatitis B virus-infected patients.

BackgroundThis study aimed to investigate the association between the use of selective serotonin reuptake inhibitors (SSRIs) and the risk of hepatocellular carcinoma (HCC) in patients with chronic hepatitis B virus (HBV) infection.MethodsWe conducted a population-based cohort study by using claims data from the Taiwan National Health Insurance Research Database (NHIRD). The study cohort comprised 1380 newly diagnosed HBV-infected patients with SSRI use who were frequency matched by age, sex, liver cirrhosis, and index year with HBV-infected patients without SSRI use in the comparison cohort. Each patient case was followed from 2000 to 2012 to identify incident HCC cases. Cox proportional hazards regression was performed to evaluate the association between SSRI use and HCC risk. The further sensitivity analysis used case-control study design. A total of 9070 HCC subjects retrieved from NHIRD, and equal non-HCC subjects were analyzed after matching for age and sex.ResultsWe identified 9 and 24 HCC cases in the study and comparison cohorts during the follow-up period of 7056 and 6845 person-years, respectively. The incidence rate of HCC was 1.28 and 3.51 per 1000 person-years for SSRI and non-SSRI users, respectively. After adjusting for potential confounders, the adjusted hazard ratio (HR) for SSRI use was 0.28 (95% confidence interval [CI], 0.12–0.64; p = 0.0027). For SSRI users with a cumulative defined daily dose (cDDD) of 28–89, 90–364, and ≥365, the adjusted HRs were 0.51, 0.22, and 0.12, respectively, (95% CI, 0.21–1.25, 0.05–0.94, and 0.02–0.90, respectively) compared with non-SSRI users (<28 cDDD). The sensitivity analysis showed that the SSRI presented with a dose-response protective effect for HCC in the multivariate analysis.ConclusionSSRIs use may possibly reduce the risk of HCC in HBV-infected patients in a dose-responsive manner.