Intratumoral microbial heterogeneity affected tumor immune microenvironment and determined clinical outcome of HBV-related HCC.

BACKGROUND AND AIMS: The intratumoral microbiome has been reported to regulate the development and progression of cancers. We aimed to characterize intratumoral microbial heterogeneity (IMH) and establish microbiome-based molecular subtyping of HBV-related HCC to elucidate the correlation between IMH and HCC tumorigenesis. APPROACH AND RESULTS: A case-control study was designed to investigate microbial landscape and characteristic microbial signatures of HBV-related HCC tissues adopting metagenomics next-generation sequencing. Microbiome-based molecular subtyping of HCC tissues was established by nonmetric multidimensional scaling. The tumor immune microenvironment of 2 molecular subtypes was characterized by EPIC and CIBERSORT based on RNA-seq and verified by immunohistochemistry. The gene set variation analysis was adopted to explore the crosstalk between the immune and metabolism microenvironment. A prognosis-related gene risk signature between 2 subtypes was constructed by the weighted gene coexpression network analysis and the Cox regression analysis and then verified by the Kaplan-Meier survival curve.IMH demonstrated in HBV-related HCC tissues was comparably lower than that in chronic hepatitis tissues. Two microbiome-based HCC molecular subtypes, defined as bacteria- and virus-dominant subtypes, were established and significantly correlated with discrepant clinical-pathologic features. Higher infiltration of M2 macrophage was detected in the bacteria-dominant subtype with to the virus-dominant subtype, accompanied by multiple upregulated metabolism pathways. Furthermore, a 3-gene risk signature containing CSAG4 , PIP4P2 , and TOMM5 was filtered out, which could predict the clinical prognosis of HCC patients accurately using the Cancer Genome Atlas data. CONCLUSIONS: Microbiome-based molecular subtyping demonstrated IMH of HBV-related HCC was correlated with a disparity in clinical-pathologic features and tumor microenvironment (TME), which might be proposed as a biomarker for prognosis prediction of HCC.

Development and validation of the nomogram based on INR and eGFR for estimation of mortality in patients with acute-on-chronic hepatitis B liver failure.

AIMS AND OBJECTIVES: Acute-on-chronic hepatitis B liver failure (ACHBLF) is a critical clinical syndrome with a high short-term mortality evolved from chronic hepatitis B (CHB)-related liver disease. Prediction of mortality risk and early intervention can improve the prognosis of patients. This study aimed to develop and validate the nomogram for short-time mortality estimation in ACHBLF patients defined according to Asian Pacific Association for the Study of the Liver (APASL). METHODS: A study of 105 ACHBLF patients with 90-day follow up was performed to develop the nomogram. Patients were randomly assigned to derivation cohort (n = 75) and validation cohort (n = 35) according to 7:3. Concordance index (C-index), calibration curve and decision curve analysis (DCA) were used to evaluate the nomogram. We also compared the nomogram with APASL ACLF research consortium (AARC) score, model for end-stage liver disease (MELD) score, MELD with serum sodium (MELD-Na) score and albumin-bilirubin (ALBI) score. The nomogram was validated using an external cohort including 40 patients. RESULTS: The 28-day and 90-day mortality of 105 patients were respectively 49.52% and 55.24%. Albumin (ALB), international normalized ratio (INR) and estimated glomerular filtration rate (eGFR) were independent predictors for 28-day mortality; INR and eGFR were independent predictors for 90-day mortality. C-index of Nomogram-1 for 28-day mortality and Nomogram-2 for 90-day mortality were respectively 0.82 and 0.81. Calibration curve and Hosmer-Lemeshow test (Nomogram-1, 0.323; Nomogram-2, 0.231) showed optimal agreement between observed and predicted death. Areas under receiver operator characteristic curve(AUROC) of Nomogram-1(0.772) and Nomogram-2(0.771) were larger compared with AARC, MELD, MELD-Na and ALBI score. The results were well estimated in the external validation cohort. CONCLUSIONS: This study highlighted the predictive value of eGFR, and the nomogram based on INR and eGFR could effectively estimate individualized risk for short-term mortality of ACHBLF patients defined according to APASL.

Low expression and Hypermethylation of ATP2B1 in Intrahepatic Cholangiocarcinoma Correlated With Cold Tumor Microenvironment.

Background: The efficacy of current therapeutic schedule is limited owing to fibroproliferative tumor microenvironment (TME) of cholangiocarcinoma, compelling a search for new therapeutic targets. Methods: Gene expression profiles and methylation profiles were obtained from UCSC Xena. Consensus clustering was performed on the transcriptome data of cholangiocarcinoma to determine the different immune subtypes. The differentially expressed genes (DEGs) between hot tumor and cold tumors were identified. ESTIMATE was used to assess immune score, and the cases were separated into relatively superior and inferior immune score groups. Single-sample gene set enrichment analysis was applied to assess 28 immune cells in the cholangiocarcinoma microenvironment. Unsupervised consensus was applied for methylation profiling to distribute the high and low methylation groups. The correlation between DNA methylation and mRNA expression was investigated, and the relationship between the ATP2B1 gene and the immune microenvironment was explored. Finally, 77 cases of intrahepatic cholangiocarcinoma (ICC) were collected for verification. Results: Seven subtypes were related to patient outcomes (P=0.005). The proportions of CD8+ T cells in the "hot" immune type was significantly greater than that in the "cold" immune type (P<0.05). Next, DEGs and DNA methylation-governed genes were intersected, and ATP2B1 was identified as a prognosis factor in ICC (P=0.035). ATP2B1 expression was positively correlated with immune scores (P=0.005, r=0.458), the levels of infiltrating CD8+ T cells (P=0.004, r=0.47), and CD4+ T cells (P=0.027, r=0.37). Immunohistochemistry confirmed that the amounts of CD8+ and CD4+ T cells were significantly higher in ICC tissue samples than in tissues with ATP2B1 overexpression (P<0.05). Conclusions: ATP2B1 overexpression can activate immune signals and prompt cold tumor response.

Mechanism of miR-424-5p promoter methylation in promoting epithelial-mesenchymal transition of hepatocellular carcinoma cells.

The current study set out to clarify the role of miR-424-5p promoter methylation in epithelial mesenchymal transition (EMT) of hepatocellular carcinoma (HCC) cells. The findings of quantitative real-time-polymerase chain reaction and methylation-sensitive high-resolution melting assays elicited that miR-424-5p was poorly expressed in HCC tissues and cells while highly methylated. Meanwhile, upon demethylation, miR-424-5p expression levels were partly recovered in HCC cells. In addition, miR-424-5p upregulation reduced cell viability and elevated apoptosis of HCC cells, in parallel with increased N-cadherin and decreased E-cadherin levels. Dual-luciferase reporter assay further validated that miR-424-5p bound to the kinesin family member 2A (KIF2A), and miR-424-5p overexpression downregulated KIF2A. In addition, KIF2A overexpression reversed the miR-424-5p-driven changes in terms of cell viability, apoptosis and EMT-related protein levels. Furthermore, xenograft tumors were established via injection of Huh7 cells, followed by miR-424-5p overexpression in vivo, which inhabited KIF2A downregulation and attenuated tumor growth along with decreased Ki67 positive expression, diminished N-cadherin and elevated E-cadherin levels. Overall, our findings supported the conclusion that miR-424-5p promoter methylation reduced miR-424-5p expression and upregulated KIF2A, thereby promoting HCC EMT.

Esophageal variceal ligation plus sclerotherapy vs. ligation alone for the treatment of esophageal varices.

Background: This study aimed to evaluate the efficacy and adverse events of esophageal variceal ligation (EVL) vs. EVL combined with endoscopic injection sclerosis (EIS) in the therapy of esophageal varices. Methods: Patients from January 2017 to August 2021 who received EVL alone (control group) or EVL plus EIS (intervention group) were enrolled in this retrospective study. Efficacy, including rebleeding (clinically hematemesis or melena, confirmed by endoscopy as esophagogastric varices bleeding), variceal recurrence rate (the presence of esophagogastric varices which is needed to be treated again) the number of sessions performed to complete eradication of varices, and safety (adverse events) were compared. The variceal recurrence-associated factors were derived by univariate and multivariate logistic regression analyses. Results: The variceal recurrence and rebleeding rate in the intervention group showed significantly lower than the control group (2.6% vs 10.3%, P = 0.006 and 20.7% vs 37.5%, P = 0.029, P = 0.006, respectively, in the 12-month follow-up). The adverse events (fever, chest pain, swallowing, and esophageal stricture) showed no significant difference between the two groups (P > 0.05). Further research showed that the efficacy of the intervention group was better than the control group only achieved in prophylactically endoscopic treatment patients. The diameter of esophageal varices and gastric varices co-exist showed significant effects on variceal recurrence in intervention group [odds ratio (OR) = 15.856; 95% confidence interval (CI), 1.709-160.143; P = 0.016 and OR = 4.5; 95% CI, 1.42-20.028; P = 0.021; respectively]. Conclusions: The intervention group may obtain lower recurrence, rebleeding rate, and fewer sessions performed to complete eradication of varices (number of sessions) and similar incidence of adverse events, especially for prophylactically treatment. Among the intervention group, the diameter of esophageal varices and gastric varices were closely associated with variceal recurrence.

Diagnostic value of 5 serum biomarkers for hepatocellular carcinoma with different epidemiological backgrounds: A large-scale, retrospective study.

Objective: Hepatocellular carcinoma (HCC) is a lethal global disease that requires an accurate diagnosis. We assessed the potential of 5 serum biomarkers (AFP, AFU, GGT-II, GPC3, and HGF) in the diagnosis of HCC. Methods: In this retrospective study, we measured the serum levels of each biomarker using ELISAs in 921 participants, including 298 patients with HCC, 154 patients with chronic hepatitis (CH), 122 patients with liver cirrhosis (LC), and 347 healthy controls from 3 hospitals. Patients negative for hepatitis B surface antigen and hepatitis C antibody (called "NBNC-HCC") and patients positive for the above indices (called "HBV-HCC and HCV-HCC") were enrolled. The selected diagnostic model was constructed using a training cohort (n = 468), and a validation cohort (n = 453) was used to validate our results. Receiver operating characteristic analysis was used to evaluate the diagnostic accuracy. Results: The α-L-fucosidase (AFU)/α-fetoprotein (AFP) combination was best able to distinguish NBNC-HCC [area under the curve: 0.986 (95% confidence interval: 0.958-0.997), sensitivity: 92.6%, specificity: 98.9%] from healthy controls in the test cohort. For screening populations at risk of developing HCC (CH and LC), the AFP/AFU combination improved the diagnostic specificity for early-stage HCC [area under the curve: 0.776 (0.712-0.831), sensitivity: 52.5%, specificity: 91.6% in the test group]. In all-stage HBV-HCC and HCV-HCC, AFU was also the best candidate biomarker combined with AFP [area under the curve: 0.835 (0.784-0.877), sensitivity 69.1%, specificity: 87.4% in the test group]. All results were verified in the validation group. Conclusions: The AFP/AFU combination could be used to identify NBNC-HCC from healthy controls and hepatitis-related HCC from at-risk patients.