HBV-KMT2B integration drives hepatic oncogenic processes in a human gene-edited iPSC-derived model.

BACKGROUND & AIMS: Hepatitis B virus (HBV)-DNA integration into the host genome contributes to hepatocellular carcinoma (HCC) development. KMT2B is the second most frequent locus of HBV-DNA integration in HCC, however its role and function remain unclear. We aimed to clarify the impact of HBV-KMT2B integration in HCC development using a human genome-edited induced pluripotent stem cells (iPSCs) model. METHODS: Based on the genetic information on HBV-KMT2B integration in HCC, we determined its complete DNA sequence and transcript variants. To exclude the effect of other oncogenic mutations, we reproduced HBV integration in healthy donor iPSCs with an intact genome and analyzed its effects using iPSC-derived hepatic progenitor cells (HPCs) and hepatocytes (iPS-Heps). RESULTS: The reproduced HBV-KMT2B integration significantly upregulated the proliferation of hepatic cells. Comprehensive transcriptional and epigenetic analyses revealed enhanced expression of cell cycle-related genes in hepatic cells with HBV-KMT2B integration based on perturbation of histone 3 lysine 4 tri-methylation(H3K4me3), mimicking that in the original HCC sample. Long-read RNA-sequence detected the common KMT2B transcript variants in the HCC sample and HPCs. Overexpression of the truncated variant significantly enhanced proliferation of hepatic cells, whereas HBV-KMT2B fusion transcripts did not enhance proliferation. HBV-KMT2B-integrated HPCs exhibited replication stress and DNA damage, indicating that our model initiated the process of hepatocarcinogenesis due to abnormally promoted KMT2B function. CONCLUSIONS: Our disease model using genetically engineered iPSCs provides the first insight into both the KMT2B function in HCC development and the oncogenic processes by HBV-KMT2B integration. We clarified the novel oncogenic mechanism in HBV-related HCC due to aberrant KMT2B function.

High serum gamma-glutamyltransferase level after hepatitis C virus elimination is a risk factor for the development of hepatocellular carcinoma.

AIM: Gamma-glutamyltransferase (GGT) is known as an oxidative stress marker, induced by alcohol consumption and metabolic disorders, and is reported as a predictor of hepatocellular carcinoma (HCC) development after hepatitis C virus (HCV) elimination. However, it is not clear whether GGT serves simply as a surrogate marker for overlapping metabolic diseases or reflects HCV-specific carcinogenicity. We investigated the association between GGT and hepatocarcinogenesis after achieving a sustained viral response (SVR), accounting for drinking habits or diabetes, and examined predisposing factors associated with GGT levels after SVR. METHODS: This is a prospective, multicenter, and observational study using the database of 1001 patients after HCV eradication with direct-acting antiviral agents. The association of GGT at SVR with cumulative HCC development was examined in a multivariate analysis using Cox proportional hazard models after adjustment for covariates including alcohol and diabetes. The association between oxidative stress markers or genetic factors and GGT levels was analyzed. RESULTS: High GGT levels at SVR were associated with HCC development (HR] 2.38, 95% CI 1.10-5.17). This association was also significant when restricted to patients without alcohol consumption or diabetes (HR 8.38, 95% CI 2.87-24.47). GGT levels were correlated with serum growth differentiation factor 15 levels, a marker of mitochondrial dysfunction. Single-nucleotide polymorphisms of ZNF827 and GDF15 were associated with high GGT levels. CONCLUSIONS: High GGT levels at SVR were associated with HCC development after accounting for alcohol consumption and diabetes. GGT levels are influenced by genetic predisposition and may reflect mitochondrial dysfunction after HCV eradication.

A20 in hepatic stellate cells suppresses chronic hepatitis by inhibiting DCLK1-JNK pathway-dependent chemokines.

Hepatic stellate cells (HSCs) are responsible for liver fibrosis accompanied by its activation into myofibroblasts and the abundant production of extracellular matrix. However, the HSC contribution to progression of liver inflammation has been less known. We aimed to elucidate the mechanism in HSCs underlying the inflammatory response and the function of tumor necrosis factor α-related protein A20 (TNFAIP3). We established A20 conditional knockout (KO) mice crossing Twist2-Cre and A20 floxed mice. Using these mice, the effect of A20 was analyzed in mouse liver and HSCs. The human HSC line LX-2 was also used to examine the role and underlying molecular mechanism of A20. In this KO model, A20 was deficient in >80% of HSCs. Spontaneous inflammation with mild fibrosis was found in the liver of the mouse model without any exogenous agents, suggesting that A20 in HSCs suppresses chronic hepatitis. Comprehensive RNA sequence analysis revealed that A20-deficient HSCs exhibited an inflammatory phenotype and abnormally expressed chemokines. A20 suppressed JNK pathway activation in HSCs. Loss of A20 function in LX-2 cells also induced excessive chemokine expression, mimicking A20-deficient HSCs. A20 overexpression suppressed chemokine expression in LX-2. In addition, we identified DCLK1 in the genes regulated by A20. DCLK1 activated the JNK pathway and upregulates chemokine expression. DCLK1 inhibition significantly decreased chemokine induction by A20-silencing, suggesting that A20 controlled chemokine expression in HSCs via the DCLK1-JNK pathway. In conclusion, A20 suppresses chemokine induction dependent on the DCLK1-JNK signaling pathway. These findings demonstrate the therapeutic potential of A20 and the DCLK1-JNK pathway for the regulation of inflammation in chronic hepatitis.

Granulomatous hepatitis with Crohn's disease: a case report.

A 45-year-old man who was regularly followed up for Crohn's disease (CD) and maintained clinical remission with vedolizumab (VDZ). At 37 years old, he was diagnosed CD from longitudinal ulcers in the distal ileum by balloon-assisted enteroscopy (BAE). During the follow-up, liver enzyme elevation, splenomegaly and thrombocytopenia were in progress. Esophagogastric varices suggested chronic liver disease and portal hypertension. Magnetic resonance elastography (MRE) showed liver stiffness of 3.4 kPa and proton density fat fraction (PDFF) of 1.86%. He was diagnosed with granulomatous hepatitis based on a liver biopsy. The hepatic venous pressure gradient (HVPG) was mildly elevated at 7 mmHg, consistent with the pre-sinusoidal portal hypertension due to granulomatous hepatitis. We report a rare case with granulomatous hepatitis diagnosed from liver injury and portal hypertension, despite the stable intestinal symptoms of CD.

Analysis of prognosis and background liver disease in non-advanced hepatocellular carcinoma in two decades.

BACKGROUND/AIM: Antiviral hepatitis and systemic therapies for hepatocellular carcinoma (HCC) remarkably progressed in the recent 10 years. This study aimed to reveal the actual transition and changes in the prognosis and background liver disease in non-advanced HCC in the past 20 years. METHODS: This retrospectively recruited 566 patients who were diagnosed with non-advanced HCC from February 2002 to February 2022. The prognosis was analyzed by subdividing according to the diagnosis date (period I: February 2002-April 2009 and period Ⅱ: May 2009-February 2022). RESULTS: Patients in period II (n = 351) were significantly older, with lower albumin-bilirubin (ALBI) scores and alpha-fetoprotein (AFP) and more anti-viral therapy, systemic therapy, and hepatic arterial infusion chemotherapy as compared with those in period I (n = 215). The etiology ratio of the background liver disease revealed decreased hepatitis C virus from 70.6% to 49.0% and increased non-B, non-C from 17.7% to 39.9% from periods I to Ⅱ. The multivariate analysis revealed older age and higher ALBI score in Barcelona Clinic Liver Cancer (BCLC) 0/A stage, AFP of >20 ng/mL, and higher ALBI score in BCLC B stage as independent prognosis factors. Fine-Gray competing risk model analysis revealed that liver-related deaths significantly decreased in period II as compared to period I, especially for BCLC stage 0/A (HR: 0.656; 95%CI: 0.442-0.972, P = 0.036). CONCLUSION: The characteristics of patients with non-advanced HCC have changed over time. Appropriate background liver management led to better liver-related prognoses in BCLC 0/A.

Prognostic significance of C-reactive protein in unresectable hepatocellular carcinoma treated with atezolizumab and bevacizumab.

AIM: C-reactive protein (CRP) is both an inflammatory and prognostic marker in various cancers. This study aimed to elucidate the characteristics of CRP and the prognostic factors in patients who were administered with atezolizumab plus bevacizumab (ATZ + BEV) for unresectable hepatocellular carcinoma (HCC). METHODS: A total of 213 patients who received ATZ + BEV for HCC from November 2020 to March 2023 at 15 hospitals were enrolled in this retrospective study. The prognosis was analyzed by subdividing the patients based on baseline characteristics, radiologic response, and treatment lines. Accuracy of survival prediction was assessed using CRP, alpha fetoprotein (AFP), C-reactive protein and alpha fetoprotein in immunotherapy (CRAFITY), and Glasgow Prognostic Score. RESULTS: Compared with patients with baseline CRP <1 mg/dL, those with baseline CRP ≥1 mg/dL (n = 45) had a significantly higher baseline albumin-bilirubin score and AFP levels, significantly lower disease control rate (62.2%), and significantly shorter median overall survival (hazards ratios 2.292; 95% confidence interval 1.313-5.107; log-rank test, p < 0.001). Multivariate analysis identified CRP ≥1 mg/dL, AFP ≥100 ng/mL, and modified albumin-bilirubin grade as the significant prognostic factors. The baseline CRP, AFP, CRAFITY, and Glasgow Prognostic Score demonstrated higher discrimination for 1-year survival prediction after first-line ATZ + BEV administration, compared with beyond second line, with area under the receiver operating characteristic curves of 0.759, 0.761, 0.805, and 0.717, respectively. CONCLUSIONS: CRP was a significant biomarker in patients treated with ATZ + BEV for HCC. Elevated CRP levels may indicate aggressive cancer progression and potential resistance to ATZ + BEV therapy.

Wisteria floribunda Agglutinin-Positive Mac-2 Binding Protein but not α-fetoprotein as a Long-Term Hepatocellular Carcinoma Predictor.

Identification of high-risk patients for hepatocellular carcinoma (HCC) after sustained virological responses (SVR) is necessary to define candidates for long-term surveillance. In this study, we examined whether serum markers after 1 year of SVR could predict subsequent HCC development. Total 734 chronic hepatitis C patients without a history of HCC who achieved SVR with direct-acting antivirals were included. The regular surveillance for HCC started from 24 weeks after the end of treatment (SVR24). Factors at SVR24 and 1 year after SVR24 were analyzed for predicting HCC development. During the mean observation period of 19.7 ± 10 months, 24 patients developed HCC. At SVR24, Wisteria floribunda agglutinin-positive mac-2 binding protein (WFA±M2BP) ≥ 1.85 and α-fetoprotein (AFP) ≥ 6.0 ng/mL were independent factors of HCC development. However, at 1 year after SVR24, WFA±M2BP ≥ 1.85 was associated with subsequent HCC development (hazard ratio: 23.5, 95% confidence interval: 2.68-205) but not AFP. Among patients with WFA±M2BP ≥ 1.85 at SVR24, 42% had WFA±M2BP < 1.85 at 1 year after SVR24 (WFA±M2BP declined group). Subsequent HCC development was significantly lower in the declined group than in the non-declined group (1 year HCC rate: 0% vs. 9.4%, p = 0.04). In conclusion, WFA±M2BP but not AFP could identify high and no-risk cases of HCC at 1 year after SVR. Therefore, it was useful as a real-time monitoring tool to identify the candidates for continuous surveillance for HCC.

Validation of hepatocellular carcinoma risk scores in Japanese chronic hepatitis B cohort receiving nucleot(s)ide analog.

BACKGROUND AND AIM: The prediction of hepatocellular carcinoma (HCC) development during nucleotide/nucleoside analog (NA) therapy is clinically important in patients with chronic hepatitis B. Although several useful models for HCC prediction have been previously reported, their usefulness in the Japanese population is unclear. Therefore, this study examines the applicability of these models in Japanese patients. METHODS: Four hundred forty-three patients with no history of HCC who were treated with entecavir, tenofovir alafenamide, or tenofovir disoproxil fumarate were enrolled. PAGE-B, modified-PAGE-B, and REACH-B scores were calculated, and subsequent HCC development was investigated. RESULTS: The mean follow-up duration was 5.1 years, and a total of 33 patients (7.4%) developed HCC during the follow-up period. Multivariate analysis revealed that old age (hazard ratio [HR] 1.05, 95% confidence interval [CI] 1.01-1.09, P = 0.011), male gender (HR 2.62, 95% CI 1.06-6.49, P = 0.037), and low platelet count (HR 0.83, 95% CI 0.77-0.91, P < 0.001) were independent predictors of HCC development. These factors are the same as the factors identified in the PAGE-B model. Time-dependent area under the receiver operating characteristic (AUROC) curve revealed that the AUROCs for 3 and 7 years of PAGE-B (AUROC: 0.786 and 0.744 at 3 and 7 years, respectively) were continuously higher than those of REACH-B (0.658 and 0.543) and modified PAGE-B AUROC (0.772 and 0.731). CONCLUSIONS: PAGE-B, which can easily identify high-risk cases, can be useful for predicting HCC development in Japanese patients treated with NA therapy.

Three criteria for radiological response on survival in patients with hepatocellular carcinoma treated with lenvatinib.

AIM: Lenvatinib (LEN) is a newly approved, multikinase inhibitor for treating unresectable hepatocellular carcinoma. In the present study, we investigated the impact of three different criteria for evaluating radiological objective response (OR) on overall survival in real-world data. METHODS: Consent for LEN therapy was obtained from 51 patients from April 2018 to March 2019. A total of 40 patients who received a minimal cumulative duration of 4 weeks of LEN were included in the analysis. Enhanced computed tomography scan was performed at baseline and every 4-8 weeks after LEN administration. Overall survival and OR were assessed with three different evaluations, as follows: Response Evaluation Criteria in Solid Tumors 1.1, modified Response Evaluation Criteria in Solid Tumors, and Choi criteria. RESULTS: The average observation period for all participants after LEN introduction was 209.4 ± 77.5 days. The Response Evaluation Criteria in Solid Tumors 1.1, modified Response Evaluation Criteria in Solid Tumors, and Choi criteria identified 10 of 40 (25.0%), 15 of 40 (37.5%), and 18of 40 (45.0%) patients with OR, respectively. The median overall survival in progressive disease evaluated by each criterion was 227 days. This result was significantly shorter than OR. Furthermore, the cumulative duration of LEN administration (>150 days) represented a significant prognostic factor (HR 0.160. 95% CI 0.039-0.646, P = 0.001). CONCLUSION: The Response Evaluation Criteria in Solid Tumors 1.1, modified Response Evaluation Criteria in Solid Tumors, and Choi criteria were useful therapeutic evaluation methods in LEN therapy for unresectable hepatocellular carcinoma. LEN's appropriate effect evaluation and management might lead to a better prognosis.

Wisteria floribunda agglutinin-positive mac-2 binding protein as an age-independent fibrosis marker in nonalcoholic fatty liver disease.

The assessment of liver fibrosis is essential because it correlates with mortality risk in nonalcoholic fatty liver disease (NAFLD). This study aims to examine whether serum fibrosis markers could identify candidate patients likely to have advanced fibrosis. We enrolled 352 patients with NAFLD and performed liver biopsies in 97 patients. The area under the receiver operating characteristic curve (AUROC) of liver stiffness by magnetic resonance elastography for histological advanced fibrosis was 0.910, and the optimal cutoff value was 4.07 kPa. To predict severe liver stiffness (≥4.07 kPa), the AUROC for Wisteria floribunda agglutinin-positive mac-2 binding protein (WFA+-M2BP) and FIB-4 were 0.897 (cutoff value, 1.08) and 0.880 (cutoff value, 2.53), respectively. After stratification of patients into four age groups as quartile, the optimal cutoff values of WFA+-M2BP for predicting severe liver stiffness were similar in each group (1.09, 1.08, 1.10, and 1.12). On the other hand, those of FIB-4 increased in parallel with age (1.47, 2.19, 2.99, and 3.88). In conclusion, WFA+-M2BP was precise for estimating severe liver stiffness in NAFLD with single cutoff value independent of age. Hence, identifying high-risk cases using WFA+-M2BP from a large number of NAFLD patients is clinically significant.

Tenofovir alafenamide for hepatitis B virus infection including switching therapy from tenofovir disoproxil fumarate.

BACKGROUND AND AIM: Tenofovir alafenamide (TAF) is a new prodrug of tenofovir, enabling treatment of patients with hepatitis B virus (HBV) infection at a lower dose than tenofovir disoproxil fumarate (TDF), via more efficient delivery of tenofovir to the hepatocytes. We compared the efficacy and safety of TDF and TAF and investigated switching from TDF to TAF therapy. METHODS: Consent for TDF and TAF therapy was obtained from 117 and 67 patients from August 2014 to January 2018. In total, 45 and 14 patients were administered with TDF and TAF, respectively, as naïve therapy, and 36 patients were switched from TDF to TAF. The antiviral effects and renal function safety were assessed. RESULTS: At week 48, the antiviral effects on patients receiving TDF and TAF as naïve therapy were similar in terms of reduction of HBV DNA (-5.6 ± 1.8 logIU/ml vs -5.0 ± 1.7 log IU/ml; P = 0.34) and hepatitis B surface antigen (-0.29 ± 0.64 logIU/ml vs -0.15 ± 0.42 logIU/ml; P = 0.71) levels. A significant decrease in the estimated glomerular filtration rate (eGFR) was seen at 48-week TDF treatment (-5.34 ± 7.69 ml/min/1.73 m2 ; P < 0.001). Switching from TDF to TAF did not increase the HBV DNA or hepatitis B surface antigen at 24 weeks. Although the eGFR worsened during TDF therapy (-7.32 ± 4.87 ml/min/1.73 m2 ), it improved significantly at week 4 (+3.93 ± 6.18 ml/min/1.73 m2 ; P = 0.008) and week 24 (+2.89 ± 4.26 ml/min/1.73 m2 ; P = 0.020) after switching from TDF to TAF. CONCLUSION: Tenofovir disoproxil fumarate and TAF showed adequate antiviral effects as naïve therapies. Furthermore, switching from TDF to TAF therapy contributed to the maintenance of the antiviral effect and recovery of renal dysfunction.

Risk assessment of hepatocellular carcinoma development by magnetic resonance elastography in chronic hepatitis C patients who achieved sustained virological responses by direct-acting antivirals.

Prediction of hepatocellular carcinoma (HCC) development after sustained virological response (SVR) is clinically important, and the usefulness of noninvasive markers for prediction HCC have been reported. The aim of this study was to compare the prediction accuracy for HCC development by noninvasive markers. A total of 346 patients with chronic hepatitis C without history of HCC who achieved SVR through direct-acting antivirals were included. Magnetic resonance elastography (MRE) and serum fibrosis markers were measured 12 weeks after the end of treatment, and the subsequent HCC development was examined. The mean observation period was 26.4 ± 7.9 months, and 24 patients developed HCC. Area under the receiver operating characteristic curve of liver stiffness by MRE, Wisteria floribunda agglutinin-positive mac-2 binding protein and FIB-4 for predicting HCC within 3 years was 0.743, 0.697 and 0.647, respectively. The 1/2/3-year rates of HCC development in patients with liver stiffness ≥3.75 KPa were 6.6%, 11.9% and 14.5%, whereas they were 1.4%, 2.5% and 2.5% in patients with liver stiffness <3.75 KPa (P < 0.001). Multivariate analysis revealed that liver stiffness ≥3.75 was an independent predictive factor for HCC development (hazard ratio, 3.51; 95% confidence interval, 1.24-9.99). In subgroup analysis, there were 132 patients who were <73 years old and had liver stiffness <3.75 KPa, and no HCC development was observed in these patients. Diagnostic accuracy for predicting HCC development was higher in MRE than serum fibrosis markers and measurement of liver stiffness by MRE could identify patients with high and low risk of HCC development after SVR.

Real-life experience of lusutrombopag for cirrhotic patients with low platelet counts being prepared for invasive procedures.

BACKGROUND AND AIMS: The present study aimed to report our real-life experience of the TPO receptor agonist lusutrombopag for cirrhotic patients with low platelet counts. METHODS: We studied platelet counts in 1,760 cirrhotic patients undergoing invasive procedures at our hospital between January 2014 and December 2017. In addition, we studied 25 patients who were administered lusutrombopag before invasive procedures between June 2017 and January 2018. Effectiveness of lusutrombopag to raise platelet counts and to avoid transfusion and treatment-related adverse events were analyzed. RESULTS: In 1,760 cirrhotic patients without lusutrombopag prior to invasive procedures, proportion of patients whose platelet counts <50,000/µL and needed platelet transfusions were 66% (n = 27/41) for radiofrequency ablation, 43% (n = 21/49) for transarterial chemoembolization, and 55% (n = 21/38) for endoscopic injection sclerotherapy / endoscopic variceal ligation, respectively. In 25 cirrhotic patients treated by lusutrombopag prior to the invasive procedures, platelet counts significantly increased compared with baseline (82,000 ± 26,000 vs. 41,000 ± 11,000/µL) (p < 0.01). Out of 25 patients, only 4 patients (16%) needed platelet transfusion before the invasive procedures. The proportion of patients with low platelet count and who needed platelet transfusions was significantly low in patients treated with lusutrombopag compared to those not treated with lusutrombopag (16% (4/25) vs. 54% (69/128), p = 0.001). Platelet counts after lusutrombopag treatment and prior to invasive procedures were lower in patients with a baseline platelet count ≤30,000/µL (n = 8) compared with those with a baseline platelet count >30,000/µL (n = 17) (50,000 ± 20,000 vs 86,000 ± 26,000/µL, p = 0.002). Patients with a baseline platelet count ≤30,000/µL with spleen index (calculated by multiplying the transverse diameter by the vertical diameter measured by ultrasonography) ≥40 cm2 (n = 3) had a lower response rate to lusutrombopag compared to those with spleen index <40 cm2 (n = 5) (0% vs. 100%, p = 0.02). Hemorrhagic complication was not observed. Recurrence of portal thrombosis was observed and thrombolysis therapy was required in one patient who had prior history of thrombosis. CONCLUSIONS: Lusutrombopag is an effective and safe drug for thrombocytopenia in cirrhotic patients, and can reduce the frequency of platelet transfusions.

Non-invasive liver fibrosis assessment correlates with collagen and elastic fiber quantity in patients with hepatitis C virus infection.

AIM: Elastic fiber deposition is a cause of irreversibility of liver fibrosis. However, to date, its relevance to clinical features has not yet been clarified. This study aimed to clarify the correlation between non-invasive markers of fibrosis and fiber quantity, including elastic fiber, obtained from computational analysis. METHODS: This retrospective study included 270 patients evaluated by non-invasive liver fibrosis assessment prior to liver biopsy. Of these patients, 95 underwent magnetic resonance elastography (MRE) and 244 were assessed with Wisteria floribunda agglutinin-positive Mac-2 binding protein (WFA+ -M2BP). Using whole-slide imaging of Elastica van Gieson-stained liver biopsy sections, the quantity of collagen, elastin, and total fiber (elastin + collagen) was determined. RESULTS: The total fiber quantity showed significant linear correlation with fibrosis stage F0-F4. Collagen fiber quantity increased from stage F0 to F4, whereas elastic fiber quantity increased significantly only from stage F2 to F3. Spearman's rank correlation test revealed that non-invasive liver fibrosis assessment significantly correlates with each fiber quantity, including correlation between total fiber quantity and the Fibrosis-4 (FIB-4) index (r = 0.361, P < 0.001), WFA+ -M2BP values (r = 0.404, P < 0.001), and liver stiffness value by MRE (r = 0.615, P < 0.001). Receiver operating characteristic (ROC) curve analyses revealed that the area under ROC for predicting higher elastic fiber (>3.6%) is 0.731 by FIB-4 index, 0.716 by WFA+ -M2BP, and 0.822 by liver stiffness by MRE. CONCLUSION: Liver fibrosis correlates with fiber quantity through non-invasive assessment regardless of fiber type, including elastic fiber. Moreover, MRE is useful for predicting high amounts of elastic fiber.

Prediction of Hepatocellular Carcinoma After Sustained Virological Responses Using Magnetic Resonance Elastography.

Direct acting antivirals (DAAs) have made it possible for most patients to achieve sustained virologic responses (SVR).1 However, some patients developed hepatocellular carcinoma (HCC) even after the eradication of hepatitis C virus2; therefore, it is clinically important to identify patients with a high risk of HCC development after SVR.

Wisteria floribunda agglutinin-positive Mac-2 binding protein predicts early occurrence of hepatocellular carcinoma after sustained virologic response by direct-acting antivirals for hepatitis C virus.

AIM: The aim of this study is to clarify the value of serum Wisteria floribunda agglutinin-positive Mac-2 binding protein (WFA+ -M2BP) for predicting hepatocellular carcinoma (HCC) in chronic hepatitis C patients who achieved sustained virologic response (SVR) by therapy with interferon-free, direct-acting antivirals (DAAs). METHODS: This is a retrospective cohort study that included 567 patients who underwent antiviral therapy with an interferon-free DAA regimen and achieved SVR. Serum WFA+ -M2BP was measured after SVR. Factors predictive of HCC occurrence and recurrence were analyzed in the patients after stratification by previous treatment history of HCC. RESULTS: Among 518 patients who had no history of HCC, 13 developed HCC. Post-SVR WFA+ -M2BP ≥1.75 cut-off index (C.O.I., P < 0.001) and α-fetoprotein (AFP) level ≥6 ng/mL (P = 0.01) were significant predictors of HCC development. Multivariate analysis showed that post-SVR WFA+ -M2BP ≥1.75 C.O.I. was an independent factor significantly associated with the development of HCC (hazard ratio [HR] 6.0; 95% confidence interval (CI), 1.8-19.4; P = 0.003). Among 49 patients who had a previous history of HCC, 22 had recurrence after SVR. Post-SVR AFP ≥6 ng/mL was the only factor associated with recurrence-free survival (HR 3.1; 95% CI, 1.3-7.5; P = 0.01). CONCLUSIONS: Post-SVR WFA+ -M2BP is a predictive factor for the development of HCC in patients with no previous HCC history and treated with DAAs. Post-SVR AFP was predictive for HCC recurrence after DAA therapy.