Normalization of serum alpha fetoprotein after direct acting antivirals in hepatitis C patients lowers hepatocellular carcinoma risk

Abstract

The elevation of serum alpha-fetoprotein (AFP) is frequently observed in patients with chronic hepatitis C (CHC). In most cases, the level decreased after antiviral treatment. This study investigated the relationship between post-treatment AFP normalization and the risk of hepatocellular carcinoma (HCC) in CHC patients without baseline HCC. A total of 483 patients treated with sofosbuvir-based direct-acting antivirals (DAAs) were enrolled and followed for a mean duration of 38.8 months. The mean age was 59.6 years, and 27.1% of patients had liver cirrhosis. The sustained virological response (SVR) rate was 98.6%. The pre-treatment AFP levels > 7 ng/mL, > 10 ng/mL, and > 15 ng/mL were observed in 25.4%, 18.8%, and 9.7% of patients, respectively. By 12 weeks post-treatment, 86.2% of patients had AFP levels < 7 ng/mL. During follow-up, 5.4% of patients developed HCC. The independent risk factors for HCC included age ≥ 65 years, liver cirrhosis, and post-treatment AFP > 7 ng/mL. The hazard ratio of HCC was 0.03 (95% CI: 0.01–0.3, P < 0.01) in the “Normalization” group, compared to the “Persistent elevation” group. The hazard ratio was 0.28 (95% CI: 0.1–0.73, P = 0.01) in the “Stable normal” group, compared to the “Elevation” group. The findings suggest that normalization of AFP levels or stable normal levels after treatment are associated with a reduced risk of HCC.

Introduction

Chronic hepatitis C (CHC) infection is a well-established risk factor for the development of hepatocellular carcinoma (HCC). Serum alpha-fetoprotein (AFP) is an oncofetal protein and is commonly used as a biomarker for HCC screening. However, AFP is non-specific and may be elevated in several other conditions, including pregnancy, germ cell tumors, chronic hepatitis with hepatic inflammation, recovery from hepatic necrosis, and hepatocyte regeneration after hepatic resection. The prevalence of elevated AFP was reported up to 43% among CHC patients^{1, 2, 3, 4–5}. Elevated AFP levels were associated with advanced stages of liver fibrosis, which increased the risk of HCC⁶. Previous studies had shown that AFP levels decrease following antiviral treatment with interferon or direct-acting antivirals (DAAs)^{7, 8, 9–10}. Despite achieving sustained virological response (SVR), the risk of HCC is not entirely diminished^11,12. Normalization of AFP levels after interferon therapy has been associated with a reduced risk of HCC^13,14. The role of post-treatment AFP dynamics may serve as a valuable tool for risk stratification. This study aimed to evaluate the association between normalization of serum AFP levels following DAA therapy and the risk of developing HCC.

Results

Patient characteristics

Over six years, 552 patients with CHC completed sofosbuvir-based DAA therapy. Of these, 25 patients with active HCC, 18 patients with inactive HCC before treatment, and 26 patients who lacked blood test results at 12 weeks post-treatment were excluded. A total of 483 patients who completed a 12-week follow-up after therapy were included in the analysis. The baseline characteristics are summarized (Table 1).

Table 1. Baseline characteristics of CHC patients treated with DAA therapy (N = 483).

Variable	Value	Percentages
Age (years)	59.6 ± 7.8
Sex (male/female)	230/253	47.6%/52.4%
BMI	24.9 ± 4.1
AST (IU/l)	71.5 ± 77.1
ALT (IU/l)	91.1 ± 62.2
Total bilirubin (mg/dl)	0.95 ± 0.35
Albumin (mg/dl)	4.1 ± 0.6
Alfa-fetoprotein (ng/l)	17.3 ± 121.2
HCV viral load (U/ml)	3,384,478 ± 596,481
HCV genotype (1/2/3/6/mixed)	240/186/5/44/8	49.7%/38.5%/1%/9.1%/1.7%
DAA regimen:
Sofosbuvir + velpatasvir	300	62.1%
Sofosbuvir + ledipasvir	136	28.2%
Sofosbuvir	43	8.9%
Sofosbuvir + daclatasvir	4	0.8%
Combined ribavirin	74	15.3%
Interferon-experienced	51	10.6%
Hepatic steatosis	153	31.7%
Liver cirrhosis	131	27.1%
FIB-4 score
≥ 3.25	178	36.9%
1.3–3.25	205	42.4%
< 1.3	100	20.7%
Underlying diseases
Diabetes mellitus	90	18.6%
Hypertension	143	29.6%
Hyperlipidemia	183	27.1%
Uremia	5	1%
IV drug abuser	12	2.5%
HBV carrier	28	5.8%
Lymphoma	4	0.8%
Hemophilia	1	0.2%

The mean age was 59.6 ± 11.9 years (range: 18–89), and 52.4% of patients were female. Prior interferon therapy had been administered to 51 patients (10.6%). Liver cirrhosis was diagnosed in 131 patients (27.1%), and 178 (36.9%) had advanced liver fibrosis, defined as a FIB-4 score ≥ 3.25. Hepatic steatosis was observed in 153 patients (31.7%), hepatitis B coinfection in 28 patients (5.8%), and diabetes mellitus in 90 patients (18.6%).

The mean pre-treatment HCV RNA level was 3,384,478 ± 6,140,069 IU/mL (range: 56–46,100,000). Genotype 1 was predominant (49.7%), with subtype 1b accounting for 80.4% of the total. The most commonly used regimen was sofosbuvir plus velpatasvir (62.1%). Ribavirin was added in selected cases for genotype 2 infection or decompensated liver cirrhosis. All patients received a 12-week treatment course, achieving a SVR rate of 98.6%.

HCC development after DAA therapy

During a mean follow-up period of 38.8 ± 18.8 months, 19 patients (3.9%) died. De novo HCC occurred in 26 patients (5.4%). Time to HCC diagnosis was distributed as follows: 3–6 months in 5 patients (19.2%), 6–12 months in 6 patients (23.1%), 1–2 years in 5 patients (19.2%), 2–3 years in 4 patients (15.4%), and more than 3 years in 6 patients (23.1%). The median time to HCC diagnosis was 20.7 months (range, 3.4–58.7) after treatment.

The mean age of patients who developed HCC was 67.9 ± 11.9 years, and 61.5% of the patients were male. Most had liver cirrhosis (88.5%), and only two patients (7.6%) had a FIB-4 score < 3.25. Tumor staging revealed stages I or II in 11 patients each (42.3%), and stages III or IV in 2 patients each (7.7%). Curative treatment was performed in 14 patients(53.8%) by surgery and in 5 patients (19.2%) by radiofrequency ablation.

Serum transaminase and AFP levels before and after DAA therapy

The mean pre-treatment alanine aminotransferase (ALT) level was 91.1 ± 93.4 IU/mL (range: 7–1041), with 382 patients (79.1%) exceeding the upper limit of normal (ULN: male > 41 IU/L, female > 31 IU/L). The post-treatment ALT level at 12 weeks post-treatment was 23.4 ± 20.4 IU/mL (range: 4-194). At 12 weeks post-treatment, the ALT level had decreased to 23.4 ± 20.4 IU/mL (range: 4–194), with 99 patients (20.5%) remaining above the ULN.

The mean pre-treatment AFP level was 17.28 ± 94.46 ng/mL (range: 0.4–1927). Elevated AFP levels were seen in 123 patients (25.4%) at > 7 ng/mL, 91 patients (18.8%) at > 10 ng/mL, 47 patients (9.7%) at > 15 ng/mL, and 39 patients (8.1%) at > 20 ng/mL. Patients with pre-treatment AFP > 15 ng/mL were older and exhibited higher aspartate aminotransferase (AST), ALT, international normalized ratio (INR), and the proportion of FIB-4 scores ≥ 3.25, along with lower albumin levels, compared with those < 15 ng/mL.

Among patients with pre-treatment AFP > 7 ng/mL, post-treatment levels decreased to < 7 ng/mL in 106 patients (86.2%). In non-HCC patients, AFP declined from 13.21 ± 36.7 to 4.09 ± 4.07 ng/mL (P < 0.01). In contrast, the HCC subgroup showed an increase from 88.8 ± 376.7 to 2180.3 ± 8904.6 ng/mL, though not statistically significant (P = 0.12). Overall, the post-treatment AFP level increased significantly from 17.28 ± 94.46 to 121.24 ± 2086.73 ng/mL (P < 0.01). Among the patients with an FIB-4 score < 1.3, the AFP level decreased significantly from 17.05 ± 55.56 ng/ml to 2.95 ± 1.89 ng/ml after therapy (P = 0.01) (Table 2).

Table 2. Serum AFP levels before and 12 weeks after DAA therapy.

Group	Pre-treatment AFP (ng/ml)	Post-treatment AFP (ng/ml)	P value
All patients	17.28 ± 94.46	121.24 ± 2086.73	< 0.01*
De novo HCC	88.77 ± 376.65	2180.3 ± 8904.68	0.12
Non-HCC	13.21 ± 36.7	4.09 ± 4.07	< 0.01*
FIB-4 score ≥ 3.25	22.84 ± 145.83	255.97 ± 3318.94	0.35
FIB-4 score 1.3 –3.25	12.56 ± 32.94	61.96 ± 939.49	0.40
FIB-4 score < 1.3	17.05 ± 55.56	2.95 ± 1.9	0.01*
Liver cirrhosis	30 ± 195.28	460.91 ± 4442.78	0.20
Non-liver cirrhosis	14.07 ± 26.16	4.91 ± 3.65	0.04*
Hepatic steatosis	15.88 ± 40.12	4.28 ± 3.41	< 0.01*
Non-steatosis	17.93 ± 111.03	175.47 ± 2523.91	< 0.01*

Predicted risk factors for HCC development after DAA

In univariate analysis by using Cox proportional hazards regression, age   ≥ 65 years, elevated AST level (pre- and post-treatment), low albumin level, low platelet count, absence of hepatic steatosis, FIB-4 score  ≥ 3.25, liver cirrhosis, and post-treatment AFP level > 7ng/ml were the risk factors for de novo HCC. In multivariate analysis, only age ≥ 65 years, liver cirrhosis, and post-treatment AFP > 7 ng/mL remained as independent predictors of de novo HCC (Table 3). No specific risk factors distinguished the patients who developed HCC earlier (< 3 years) from those diagnosed later (> 3 years) during follow-up.

Table 3. Risk factors for de Novo HCC after DAA therapy (Cox proportional hazards model).

Variable	Crude HR	95% CI	P value	Adjusted HR	95% CI	P value
Age ≥ 65	2.89	1.32–6.38	0.01*	2.83	1.25–6.43	0.01*
Male sex	1.92	0.87–4.22	0.11
Pre-Tx AST	1.01	1-1.01	< 0.01*	1	0.99–1.01	0.67
Post-Tx AST	1.02	1-1.03	0.03*	1	0.99–1.01	0.91
Pre-Tx ALT	1	1-1.01	0.23
Post-Tx ALT	1	1-1.02	0.32
Pre-Tx albumin	0.36	0.23–0.56	< 0.01*	0.88	0.35–2.19	0.78
Post-Tx albumin	0.21	0.1–0.45	< 0.01*	1	0.35–2.19	0.99
Pre-Tx platelet	0.98	0.98–0.99	< 0.01*	0.99	0.98-1	0.13
Post-Tx platelet	0.99	0.98–0.99	< 0.01*	1	0.3–3.35	0.99
Fib-4 ≥ 3.25	14.8	3.5-62.68	< 0.01*	0.8	0.1–6.3	0.83
Cirrhosis	20	5.98–66.54	< 0.01*	9.98	1.93–51.54	0.01*
Steatosis	0.09	0.12–0.63	0.02*	0.19	0.02–1.42	0.1
Diabetes mellitus	1.5	0.63–3.57	0.36
HBsAg positive	0.71	0.08–4.88	0.73
Pre-Tx AFP > 15	1.94	0.67–5.66	0.22
Post-Tx AFP > 7	6.25	2.88–13.6	< 0.01*	2.74	1.16–6.46	0.02*

*Pre-Tx: pre-treatment; Post-Tx: post-treatment; HR: hazard ratio; CI: confidence interval.

AFP trajectory patterns and HCC risk

The pattens of AFP dynamic change were categorized into four groups: (1) “Normalization (N)” group: pre-treatment AFP level > 7 ng/ml decreasing to post-treatment level < 7 ng/ml; (2) “Persistent elevation (PE)” group: pre- and post-treatment AFP level > 7 ng/ml”; (3) “Stable normal (SN)” group: pre- and post-treatment AFP level < 7 ng/ml”; (4) “Elevation (E)” group: pre-treatment AFP level < 7ng/ml, then increasing to post-treatment AFP level > 7 ng/ml.

During the follow-up period, there was one patient (0.9%) developed de novo HCC in the “N” group, 6 patients (35.3%) in the “PE” group, 13 patients (4%) in the “SN” group, and 6 patients (15.8%) in the “E” group (Fig. 1).

Fig. 1 [Images not available. See PDF.]

Patterns of AFP dynamic changes after DAA therapy and the corresponding incidence of HCC.

The risk of de novo HCC development across the four AFP trajectory groups is presented (Table 4). The high HCC risk was observed in the “PE” group, while the low risk was seen in the “N” and “SN” groups. Kaplan–Meier analysis of the patients with pre-treatment AFP levels > 7 ng/ml showed that the development of de novo HCC in the “PE” group was significantly higher than in the “N” group (Fig. 2A). The hazard ratio of HCC development in the “N” group versus the “PE” group was 0.03 (95% C.I.: 0.01–0.3, P < 0.01). For the patients with pretreatment AFP < 7ng/ml, the development of de novo HCC in the “E” group was significantly higher than in the “SN” group (Fig. 2B). The hazard ratio in the “E” group versus the “SN” group was 0.28 (95% C.I.: 0.1–0.73, P = 0.01).

Table 4. Risk of de Novo HCC according to AFP trajectory patterns.

AFP trajectory category	Hazard ratio	95% confidence interval	P value
Stable normal (SN)	Reference
Normalization (N)	0.26	0.03–2.02	0.2
Elevation (E)	1.9	1.17–3.09	0.01*
Persistent elevation (PN)	2.26	1.63–3.13	< 0.01*

*The four AFP trajectory categories were modeled as a categorical variable, using “Stable Normal” as the reference group.

Fig. 2 [Images not available. See PDF.]

Kaplan–Meier curves of cumulative HCC incidence following DAA therapy. (A) Comparison between the “Normalization (N)” group and the “Persistent Elevation (PE)” group. (B) Comparison between the “Stable Normal (SN)” group and the “Elevation (E)” group.

Among patients with elevated pre-treatment AFP levels, normalization of post-treatment AFP was associated with a 97% reduction in HCC risk compared to those with persistently elevated AFP levels. Similarly, among patients with normal pre-treatment AFP levels, those who maintained stable normal AFP levels post-treatment had a 72% lower risk of HCC compared to those who developed persistent elevation.

Due to the limited predictive role of AFP in the “SN” group, a subgroup analysis was conducted. The HCC risk was significantly higher in the patients aged ≥ 65 years (HR = 3.48, 95% CI: 1.03–11.7, P = 0.04), those with low post-treatment albumin levels (HR = 0.28, 95% CI: 0.09–0.92, P = 0.04), and those with liver cirrhosis (HR = 14.12, 95% CI: 2.31–86.43, P < 0.01).

Discussion

This retrospective observational study evaluated 483 CHC patients without baseline HCC who received sofosbuvir-based DAA therapy to investigate the incidence and predictors of de novo HCC. We found that age ≥ 65 years, liver cirrhosis, and serum AFP > 7 ng/mL at 12 weeks post-treatment were independent risk factors for HCC development. Patients with normalized or persistently normal AFP levels after therapy had a lower risk of HCC.

An elevated serum AFP level is frequently observed in CHC patients, even in the absence of HCC. The prevalence was reported variously due to differences in population selection, ethnicity, sample size, and AFP cut-off definitions. Previous studies reported that 23–38% of CHC patients had AFP ≥ 10 ng/mL^3.5 and 43% of patients with compensated cirrhosis had AFP ≥ 10 ng/mL¹. Others reported that 29% of patients had AFP > 12 ng/mL, 16.6% had AFP > 20 ng/mL, and 14% had AFP > 30 ng/mL^2,4,7. Elevated AFP is often associated with advanced fibrosis, with an AFP level of ≥ 15 ng/mL predictive of stage III/IV fibrosis and ≥ 17.8 ng/mL predictive of cirrhosis. In our cohort, AFP > 7 ng/mL, > 10 ng/mL, and > 15 ng/mL were observed in 25.4%, 18.8%, and 9.7% of patients, respectively. The proportion of patients with advanced liver fibrosis (defined as a FIB-4 score  ≥ 3.25) was significantly higher in those with AFP > 15 ng/ml than in those with AFP < 15 ng/ml (61.7% versus 41.1%, P = 0.01). The patients with higher AFP levels were more likely to have advanced liver fibrosis.

The increase in AFP in CHC is likely due to HCV-related hepatic inflammation. A possible mechanism is that the HCV core protein upregulates AFP production by activating transcription in the hepatocytes. After achieving SVR with antiviral therapy, hepatic inflammation improves and leads to a significant decrease in AFP levels. AFP levels decreased in a time-dependent manner, and this improvement continued after achieving SVR.

After interferon-based therapy, 86.7% of patients achieved normalization of AFP levels (< 10 ng/mL). With DAA therapy, 52% of patients had AFP < 5.5 ng/mL at the end of treatment and 73.1% by the end of follow-up. In our study, 86.2% of patients with pre-treatment AFP > 7 ng/mL normalized to < 7 ng/mL at 12 weeks post-treatment. Patients who failed to normalize AFP often had low serum albumin levels and were at greater risk for HCC.

The risk of HCC development significantly decreased in CHC patients after antiviral therapy. SVR has been associated with a 71% reduction in HCC risk^11,12and the risk appears to decrease over time¹⁵. Older age, diabetes mellitus, high AFP level, low albumin level, platelet count, and advanced liver fibrosis were reported as the risk factors for HCC occurrence in CHC patients with SVR after antiviral therapy^{14, 15, 16, 17–18}.

Several studies have proposed various post-treatment AFP cut-off values for HCC prediction^{19, 20, 21, 22, 23, 24, 25, 26, 27, 28–29}(Table 5). Several scoring systems, including post-treatment AFP levels, have been proposed to predict the development of HCC. Four studies identified AFP ≥ 6 ng/mL at the end of treatment as an independent predictor of HCC. In our study, we adopted a cut-off of 7 ng/mL based on prior literature and our laboratory’s reference range. Since serum AFP levels usually decrease after DAA therapy, the cut-off value for detecting HCC would be reduced after SVR. The time point of AFP at 12 weeks after treatment was most accurately predicted for HCC development compared to those at baseline and the end of treatment²⁶.

Table 5. Summary of studies evaluating serum APF cut-offs for HCC risk post-HCV DAA therapy.

Study (author, year)	Population	Treatment	AFP Cut-off (ng/mL)	Timing of AFP Measurement	Outcome
Kuwano et al., 202219	HCV patients post-SVR	DAA	> 3.8	End of treatment (EOT)	Independent predictor of HCC > 1 year post-SVR
Chow et al., 202420	HCV patients, 47% cirrhosis	DAA	> 10.5 > 5.6	Start of therapy End of therapy	Predictor of HCC
Mawatari et al., 202221	HCV patients, 23.5% cirrhosis	DAA	≥ 5.3	End of treatment	Independent predictor of HCC
Tani et al., 202022	HCV patients, 11.6% cirrhosis	DAA	≥ 6	End of treatment	Included in the scoring system for HCC prediction
Masetti et al., 201823	Cirrhotic HCV patients	DAA	≥ 6	Lack of AFP reduction during treatment	Associated with HCC development
Watanabe et al., 201924	HCV patients	DAA	≥ 6	End of treatment	AFP included as predictors of HCC
Ogawa et al., 201825	Cirrhotic HCV patients	DAA	≥ 9	End of treatment	Predictor of short-term HCC risk
Watanabe et al., 202326	HCV patients post-SVR	DAA	≥ 4	12 weeks after treatment	Most accurate predictor of HCC
Hiraoka et al., 201927	HCV patients	DAA	> 5	24 weeks after treatment	Included in the HCC surveillance score
Hamada et al., 201828	HCV patients	DAA & IFN-based	≥ 6	Post-treatment	Independent risk factor for HCC
Minami et al., 201529	HCV patients	DAA & IFN-based	> 5 (SVR), > 17 (non-SVR)	After treatment	Higher specificity for early HCC detection post-SVR

Dynamic changes in the AFP pattern have been explored as a means of predicting the risk of HCC. After interferon therapy, a decline in AFP levels was associated with a reduced incidence of HCC. Following DAA therapy, one study demonstrated that any reduction in AFP in cirrhotic patients lowered the risk of HCC, and a lack of AFP decline was an independent predictor of HCC²³. Those with AFP < 6 ng/ml had the lowest risk. In our analysis, normalization of elevated pre-treatment AFP was associated with a significantly reduced risk of HCC compared to persistent elevation of AFP.

Previous studies have proposed various AFP cut-off values or scoring systems to predict HCC risk, which may not be straightforward for simple clinical interpretation. Evaluating the AFP change from pre-treatment to 12 weeks post-treatment is a practical and easily applicable approach. The age ≥ 65 years, liver cirrhosis, and a post-treatment AFP level > 7 ng/ml at 12 weeks after DAA therapy were identified as independent risk factors for HCC development.

Our series found that 5 of the 26 (19.2%) HCC patients were diagnosed within 3 to 6 months after DAA therapy. It raises the possibility of occult HCC present at baseline, but undetected by imaging. Despite liver ultrasonography or triphasic CT performed before treatment, the sensitivity of imaging for early microscopic HCC is limited. Therefore, lack of AFP normalization or post-treatment AFP elevation may serve as a warning sign for subclinical or emerging HCC.

Our study also found that 23.1% of HCC cases were diagnosed after more than 3 years of follow-up, suggesting a subset of patients may develop delayed-onset HCC. Older age, liver cirrhosis, and elevated AFP were not significant predictors in this subgroup. Future studies are warranted to define risk factors for late HCC occurrence better.

In recent years, DAA therapy has replaced interferon for CHC therapy with high SVR rates and few side effects. More elderly or cirrhotic patients can be safely treated with protease inhibitor-free DAAs. This study has several strengths. The use of a uniform sofosbuvir-based regimen with a fixed 12-week duration eliminates treatment-related heterogeneity. Our cohort also included cirrhotic and elderly patients who were often excluded from prior interferon-based studies due to contraindications. However, this study still has some limitations. First, the findings may not be generalizable across ethnic groups. Larger, multi-center cohorts are needed to validate our results. Second, although the mean follow-up was 38.8 months, longer-term surveillance is essential to assess the prediction of AFP dynamics and HCC risk after DAA therapy.

In conclusion, age ≥ 65 years, liver cirrhosis, and elevated AFP levels (> 7 ng/mL) at 12 weeks after DAA therapy were independent risk factors for de novo HCC in CHC patients. Among those with elevated pre-treatment AFP, normalization after therapy was associated with a significantly reduced HCC risk. Similarly, patients with normal pre-treatment AFP who maintained stable AFP levels after treatment had a lower risk of HCC than those with post-treatment elevation. Dynamic monitoring of AFP provides a simple and clinically useful tool for stratifying HCC risk after DAA therapy.

Methods

Patient enrollment and data collection

From January 2017 to December 2022, patients aged ≥ 18 years with CHC who received sofosbuvir-based DAA therapy at the hepatology outpatient clinic of Far Eastern Memorial Hospital, Taiwan, were retrospectively enrolled. Patients were excluded if they had human immunodeficiency virus (HIV) infection, a history of alcoholism, or HCC before treatment.

Baseline demographic and clinical data were collected, including comorbidities such as hypertension, diabetes mellitus, hyperlipidemia, uremia, hepatitis B virus (HBV) carriage, lymphoma, hemophilia, and intravenous drug use history. Laboratory parameters, including complete blood count, serum albumin, total bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and prothrombin time measured by international normalized ratio (INR), were recorded at baseline and 12 weeks post-treatment.

Serum AFP levels were measured using the Roche Elecsys kit by electrochemiluminescence immunoassay (ECLIA), with a reference range of < 7 ng/mL. Liver fibrosis was assessed using the FIB-4 index: [(AST x age) / (√ALT x platelet count)]. A FIB-4 score ≥ 3.25 was used to identify advanced fibrosis or cirrhosis, while a score < 1.3 was considered to have a low likelihood of significant fibrosis³⁰. The evidence of liver cirrhosis and steatosis was diagnosed by abdominal ultrasound or computed tomography (CT) reports. HCV viral load and genotype were assessed before treatment. SVR was defined as undetectable serum HCV RNA 12 weeks after completion of therapy.

HCC surveillance

Patients underwent HCC surveillance at 3- to 6-month intervals after completing DAA therapy. Surveillance included abdominal ultrasound and serum AFP measurement at each hepatology clinic visit. The outcome follow-up was conducted in December 2023. During the follow-up, new diagnoses of hepatocellular carcinoma (de novo HCC) and mortality events were recorded.

The diagnosis of HCC was confirmed by histopathology, or according to the guidelines of the American Association for the Study of Liver Disease (AASLD) in 2005: (1) if a tumor larger than 2 cm in a cirrhotic liver and showing typical features of HCC (hypervascularity in the arterial phase and washout in the venous phase) at triphasic liver computed tomography (CT), or dynamic magnetic resonance MR imaging (MRI), or serum AFP > 200 ng/ml; (2) If a tumor measuring 1–2 cm in a cirrhotic liver showing typical features of HCC in two dynamic image modalities. The staging of HCC followed the American Joint Committee on Cancer (AJCC) 8th edition TNM system. “Active HCC” was defined as ongoing treatment for HCC, while “inactive HCC” meant cases with prior HCC that had been cured or controlled before initiation of DAA therapy.

Statistical analysis

Continuous variables were expressed as mean ± standard deviation and range. Categorical variables were presented as frequencies and percentages. The Mann–Whitney U test or Wilcoxon signed-rank test was used to compare continuous variables, and the Chi-square test was used for categorical variables. Cox proportional hazards regression was applied to identify risk factors associated with de novo HCC after antiviral therapy. Univariate analysis was first performed, and variables with significant associations (P < 0.05) were included in the multivariate model to determine adjusted hazard ratios (HR) and 95% confidence intervals (CI). Kaplan–Meier analysis and the log-rank test were used to compare the cumulative incidence of HCC among groups. All statistical tests were two-tailed, with significance set at P < 0.05. Analyses were conducted using Stata software (version 13.0; StataCorp, College Station, TX, USA).

Author contributions

C.K.L., S.H.W. and T.H.L. contributed to the study conception and design. C.K.L. contributed to the material preparation, data collection, and analysis. All authors contributed to drafting the manuscript, revising it, and approving the final manuscript.

Data availability

The data supporting this study’s findings will be made available from the corresponding author to researchers who provide a reasonable request. The data are not publicly available due to privacy and ethical restrictions.

Declarations

Ethical statement

This study was conducted according to institutional and international ethical guidelines. The study protocol was reviewed and approved by the Institutional Review Board of Far Eastern Memorial Hospital (Approval No. 111125-E). Written informed consent for DAA therapy was obtained from all participants.

Competing interests

The authors declare no competing interests.

Abbreviations

CHC

Chronic hepatitis C

HCC

Hepatocellular carcinoma

DAA

Direct-acting antivirals

SVR

Sustained virological response

AFP

Alfa-fetoprotein

CT

Computed tomography

MRI

Magnetic resonance imaging

PT

Prothrombin time

AST

Aspartate aminotransferase

ALT

Alanine aminotransferase

INR

International normalized ratio

ULN

Upper limit of the normal range

HR

Hazard ratio

CI

Confidence interval

FIB-4

Fibrosis-4

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