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. 2022 Jan 12;17(1):e0262267. doi: 10.1371/journal.pone.0262267

Features of patients who developed hepatocellular carcinoma after direct-acting antiviral treatment for hepatitis C Virus

Seiichi Mawatari 1,*, Kotaro Kumagai 1, Kohei Oda 1, Kazuaki Tabu 1, Sho Ijuin 1, Kunio Fujisaki 2, Shuzo Tashima 1,2, Yukiko Inada 3, Hirofumi Uto 1,3, Akiko Saisyoji 1,4, Yasunari Hiramine 4, Masafumi Hashiguchi 1,5, Tsutomu Tamai 1,5, Takeshi Hori 5, Ohki Taniyama 1, Ai Toyodome 1, Haruka Sakae 1, Takeshi Kure 1,6, Kazuhiro Sakurai 7, Akihiro Moriuchi 1,7, Shuji Kanmura 1, Akio Ido 1
Editor: Birke Bartosch8
PMCID: PMC8754290  PMID: 35020772

Abstract

Background

The features of hepatitis C virus patients with a sustained virologic response (SVR) who developed hepatocellular carcinoma (HCC) after direct-acting antiviral (DAA) therapy are unclear.

Methods

The study population included 1494 DAA-SVR patients without a history of HCC. The cumulative carcinogenesis rate after the end of treatment (EOT) and factors related to HCC were analyzed.

Results

Sixty (4.0%) patients developed HCC during a median observation period of 47.6 months. At four years, the cumulative carcinogenesis rate was 4.7%. A Cox proportional hazards analysis showed that age ≥73 years (hazard ratio [HR]: 2.148), male sex (HR: 3.060), hyaluronic acid (HA) ≥75 ng/mL (HR: 3.996), alpha-fetoprotein at EOT (EOT-AFP) ≥5.3 ng/mL (HR: 4.773), and albumin at EOT (EOT-Alb) <3.9 g/dL (HR: 2.305) were associated with HCC development. Especially, EOT-AFP ≥5.3 ng/mL was associated with HCC development after 3 years from EOT (HR: 6.237). Among patients who developed HCC, AFP did not increase in patients with EOT-AFP <5.3 ng/mL at the onset of HCC. Of these 5 factors, EOT-AFP ≥5.3 ng/mL was scored as 2 points; the others were scored as 1 point. The 4-year cumulative carcinogenesis rate for patients with total scores of 0–2, 3–4, and 5–6 points were 0.6%, 11.9%, and 27.1%, respectively (p<0.001).

Conclusions

EOT-AFP ≥5.3 ng/mL is useful for predicting HCC development after an SVR. However, AFP does not increase in patients with EOT-AFP <5.3 ng/mL at the onset of HCC. The combination of EOT-AFP, age, sex, HA, and EOT-Alb is important for predicting carcinogenesis.

Introduction

Chronic hepatitis C virus (HCV) infection affects 71 million people worldwide and approximately 399,000 people die each year from hepatitis C-related liver disease [1, 2]. In Japan, approximately 30,000 people died of hepatocellular carcinoma (HCC) in 2016. In 2007, the major etiology was persistent HCV infection, which accounted for 65% of all HCC deaths [3]. In 2011, it is estimated that the population of people with HCV infection in Japan was 0.98–1.6 million [3].

One of the goals of therapy is to cure HCV infection in order to prevent the complications of HCV-related liver diseases, including hepatic necroinflammation, fibrosis, cirrhosis, decompensated cirrhosis, HCC, and death [1].

In recent years, direct-acting antivirals (DAAs) have been approved, and IFN-free therapy with DAAs has achieved very high sustained virologic response (SVR) rates of ≥95% [2]. DAA treatment has been frequently administered to elderly or cirrhotic patients, and achieved high SVR rates [4–7]. However, the characteristics of patients who develop HCC during long-term observation are unclear. In the present study, we aimed to clarify the features of patients who developed HCC after a DAA-SVR.

Materials and methods

Study population

This was a prospective observational study conducted at 21 facilities belonging to the Kagoshima Liver Study Group in Japan. Study population enrollment is shown in Fig 1. In brief, a total of 1521 patients with chronic HCV infection and no history of HCC therapy were treated with DAAs, and achieved an SVR between October 2014 and December 2019, and was observed until July 2021. We excluded patients who had HBV co-infection, those who were confirmed to have tumors in the liver or other organs, and those who developed HCC during DAA treatment. Liver tumors included hypovascular tumors, such as dysplastic nodules or well-differentiated HCC diagnosed by contrast-enhanced (CE) computed tomography (CT) or magnetic resonance imaging (MRI) before therapy. Ultimately, 1494 patients were analyzed in this study. Written informed consent was obtained from the enrolled patients. The study protocol conformed to the ethical guidelines of the Declaration of Helsinki and was approved by the Kagoshima University Hospital Clinical Research Ethics Committee and the research ethics committee of each participating facility (approval numbers: 150138, 170199, 190297).

Fig 1. Flow chart of enrollment of the study population.

Fig 1

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HCC, hepatocellular carcinoma; DAA, direct acting antiviral.

The HCV RNA concentration was measured by TaqMan PCR, which has a lower quantitation limit of 1.2 log IU/mL. The Fib-4 index, a surrogate marker of liver fibrosis, was calculated based on the methods of previous studies [8]. Liver cirrhosis was comprehensively judged by hepatologists at each institution according to the platelet count, imaging, fibrosis markers, transient elastography, or varices formation.

Treatment protocol

Patients were treated with daclatasvir (DCV) and asunaprevir (ASV) for 24 weeks, sofosbuvir (SOF) and ledipasvir (LDV) for 12 weeks, ombitasvir (OBV) and paritaprevir (PTV) and ritonavir (r) for 12 weeks, SOF and ribavirin (RBV) for 12 weeks, elbasvir (EBR) and grazoprevir (GZR) for 12 weeks, DCV and ASV and beclabuvir (BCV) for 12 weeks, and glecaprevir (GLE) and pibrentasvir (PIB) for 8 or 12 weeks. All patients were treated according to Japanese guidelines for chronic HCV infection [9]. The initiation of the observation period was defined as the end of treatment (EOT) with DAAs.

Surveillance of HCC

The Japanese guidelines state that cirrhotic patients have an extremely high risk of developing HCC and should be monitored every 3–4 months, and that non-cirrhotic patients have a high risk of developing HCC and be monitored every 6 months by ultrasonography (US), CT or MRI [10]. HCC was diagnosed when typical vascular findings were observed by contrast-enhanced CT or MRI, which showed hyper-enhancement in the arterial phase and a washout pattern in the portal, or delayed phases. Abdominal US, CE-CT or CE-MRI were performed at 3–6-month intervals.

Statistical analyses

Statistical analyses were performed using the IBM Statistical Package for Social Sciences (SPSS) software program (version 26 IBM SPSS Statistics, Armonk, NY, USA). Categorical data were compared using the chi-squared test and Fisher’s exact test, as appropriate. Continuous variables were analyzed using the Mann-Whitney U test. The Kaplan–Meier method and log rank test were used to analyze the cumulative rates of HCC development. Correlation coefficients were obtained using Spearman’s rank correlation coefficient. P values of <0.05 were considered to indicate statistical significance. Factors associated with HCC development were determined using a Cox proportional hazards analysis with forward selection using p<0.10 as a cutoff for inclusion in the model. For the categorical data, we determined the cut-off values at which the optimal sensitivity and specificity were achieved using receiver operating characteristic (ROC) curves.

Results

Baseline characteristics

Table 1 shows the baseline characteristics of the patients. The average age was 67.1 years, 603 of patients were men (40.4%), 357 patients had cirrhosis (23.9%), 644 patients had a Fib-4 index of ≥3.25, which suggested advanced fibrosis (23.9%) [8], and 59 patients had a history of DAA treatment for HCV (3.9%). The median observation period was 47.6 months.

Table 1. Patient characteristics.

Characteristics Total n = 1494 HCC development (-) n = 1434 HCC development (+) n = 60 P value
Age, years (range) 67.1±10.8 (26–90) 66.9±10.8 (26–90) 71.5±9.2 (48–87) 0.001
Male, n (%) 603 (40.4) 566 (39.5) 37 (61.7) <0.001
Liver cirrhosis, n (%) 357 (23.9) 324 (22.6) 33 (55.0) <0.001
Body Mass Index, kg/m2 (n = 1179) 22.8±3.5 22.9±3.5 22.0±2.5 0.059
Prior DAA therapy, none/ experience, n (%) 1435/ 59 1377/ 57 58/ 2 0.574
Diabetes Mellitus, n (%) 249 (16.7) 240 (16.7) 9 (15.0) 0.444
Genotype 1/ 2/ 1+2 1196/ 297/ 1 1140/ 293/ 1 56/ 4/ 0 0.032
DCV+ASV/ SOF/LDV/ OBV/PTV/r/ SOF+RBV/ 362/ 480/ 108/ 198 340/ 455/ 106/ 194 22/ 25/ 2/ 4 0.015
GZR+EBR/ DCV/ASV/BCV/ GLE/PIB, n 103/ 12/ 231 98/ 12/ 229 5/ 0/ 2
HCV-RNA, logIU/mL 6.0±0.9 6.0±0.9 5.9±0.7 0.171
Platelet counts, ×104/μL 15.8±5.8 15.9±5.8 12.3±6.3 <0.001
Total bilirubin, mg/dL (n = 1491) 0.8±0.4 0.8±0.4 1.0±0.6 0.002
AST, U/L 49±34 49±34 58±37 <0.001
ALT, U/L 49±45 49±46 50±35 0.276
GGT, U/L 45±53 45±52 61±73 0.003
Alb, g/dL (n = 1457) 4.1±0.4 4.1±0.4 3.8±0.5 <0.001
Fib-4 index 3.82±2.98 3.72±2.88 6.31±4.16 <0.001
Fib-4 index >3.25, n (%) 644 (43.1) 599 (41.8) 45 (75.0) <0.001
Hyaluronic acid, (n = 1415) 166.2±271.5 162.4±274.0 252.2±190.2 <0.001
AFP, ng/mL (n = 1475) 9.5±31.5 9.3±31.8 15.8±25.1 <0.001
DCP, mAU/mL (n = 1059) 22.3±23.1 22.3±23.2 26.5±20.8 0.462
EOT-ALT, non WNL 170 (11.4) 159 (11.1) 11 (18.3) 0.070
EOT-Alb, g/dL (n = 1413) 4.1±0.4 4.1±0.4 3.8±0.4 <0.001
EOT-AFP, ng/mL (n = 1385) 4.6±7.6 4.4±6.8 9.3±17.6 <0.001
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Data are shown as the mean ± standard deviation, HCC, hepatocellular carcinoma; DAA, direct-acting antivirals; DCV, daclatasvir; ASV, asunaprevir; SOF, sofosbuvir; LDV, ledipasvir; OBV, ombitasvir; PTV, paritaprevir; r, ritonavir; RBV, ribavirin; GZR, grazoprevir; EBR, elbasvir; BCV, beclabuvir; GLE, glecaprevir; PIB, pibrentasvir; HCV, hepatitis C virus; AST, aspartate transaminase; ALT, alanine transaminase; GGT, γ-glutamyltransferase; Alb, albumin; AFP, α-fetoprotein; DCP, des-γ-carboxy prothrombin; EOT, end of treatment; WNL, within normal limit.

Comparison of the baseline characteristics between the patients who developed HCC and non-HCC patients

Sixty (4.0%) of 1494 patients developed HCC (Table 1). The cumulative rates of HCC development were 1.1% at 1 year, 1.8% at 2 years, 3.5% at 3 years, 4.7% at 4 years, and 5.6% at 5 years (Fig 2). The comparison of the baseline characteristics between patients who developed HCC and non-HCC revealed that the patients who developed HCC were older, were more frequently male, and had a higher incidence of cirrhosis in comparison to the non-HCC patients (Table 1). Regarding the blood test results of the HCC patients, the total bilirubin, aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), hyaluronic acid (HA), Fib-4 index, and alpha-fetoprotein (AFP) values were significantly higher, and the albumin level, and platelet count were significantly lower (Table 1). In addition, the AFP level at the EOT (EOT-AFP) was significantly as higher, and albumin at the EOT (EOT-Alb) were significantly lower (Table 1). However, the des-γ-carboxy prothrombin (DCP) level did not differ to a statistically significant extent.

Fig 2. Cumulative rates of HCC development in patients with DAA-SVR.

Fig 2

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HCC, hepatocellular carcinoma; DAA, direct-acting antiviral; SVR, sustained virologic response.

Setting the cut-off values for continuous variables associated with hepatocarcinogenesis

For the categorical data, we determined the cut-off values at which the optimal sensitivity and specificity were achieved using ROC curves (Table 2). The EOT-AFP had the higher area under ROC and positive likelihood ratio (Table 2).

Table 2. Setting cutoff values for continuous variables associated with hepatocarcinogenesis.

Cut off value Sensitivity (%) Specificity (%) PPV (%) NPV (%) Likelihood ratio odds ratio AUC
Age 73 years 53.3 69.9 6.9 97.3 1.774 2.660 0.624
AST 47 U/L 60.0 65.6 6.8 97.5 1.742 2.854 0.631
ALT 36 U/L 60.0 51.9 5.0 96.9 1.247 1.617 0.541
GGT 31 U/L 63.3 54.4 5.5 97.3 1.389 2.060 0.614
Total bilirubin 0.8 mg/dL 58.3 60.3 5.8 97.1 1.467 2.121 0.620
Platelet 11.3×104 /μL 53.3 78.1 9.2 97.6 2.428 4.060 0.683
Alb 3.9 g/dL 63.3 74.2 9.5 97.9 2.451 4.957 0.697
Hyaluronic acid 75 ng/mL 88.1 47.8 6.8 98.9 1.688 6.799 0.714
Fib-4 index 4.03 68.3 69.4 8.4 98.0 2.193 4.578 0.732
AFP 7.1 ng/mL 56.7 73.9 8.4 97.6 2.173 3.707 0.696
EOT-Alb 3.9 g/dL 55.9 77.9 9.9 97.6 2.533 4.478 0.693
EOT-AFP 5.3 ng/mL 65.0 78.3 11.9 98.0 2.980 6.657 0.732
5 risk factors† 2 81.0 76.0 13.7 98.8 3.373 13.513 0.838
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† The sum of scores when EOT-AFP ≥5.3 ng/mL was scored as 2 points; the others (i.e., age ≥73 years, male sex, hyaluronic acid ≥75 ng/mL, and EOT-Alb <3.9 g/dL) were scored as 1 point.

PPV, positive predictive value; NPV, negative predictive value; AUC, area under the receiver operator characteristic curve; AST, aspartate transaminase; ALT, alanine transaminase; GGT, γ-glutamyltransferase; Alb, albumin; AFP, α-fetoprotein; EOT, end of treatment.

Factors associated with the development of HCC in DAA-treated patients who achieved an SVR

The Cox proportional hazard analysis showed that the following factors were associated with the development of HCC: age ≥73 years (hazard ratio [HR]: 2.148), male sex (HR: 3.060), HA ≥75 ng/mL (HR: 3.996), EOT-AFP ≥5.3 ng/mL (HR: 4.773), and EOT-Alb <3.9 g/dL (HR: 2.305) (Table 3). In the analysis of 564 patients with Fib-4 index ≥3.25 which suggested advanced fibrosis [8], 45 patients developed HCC, and male sex (HR: 2.986), EOT-AFP ≥5.3 ng/mL (HR: 8.352), and EOT-Alb <3.9 g/dL (HR: 2.803) were associated with the development of HCC (Table 3). In the analysis of 1245 patients who did not develop HCC within 6 months and who were observed for more than 6 months, 51 patients developed HCC, and age ≥73 years (HR: 1.782), male sex (HR: 2.835), HA ≥75 ng/mL (HR: 3.481), EOT-AFP ≥5.3 ng/mL (HR: 4.257), and Alb <3.9 g/dL (HR: 2.084) were associated with the development of HCC (Table 4). In the analysis of 1195 patients who did not develop HCC within 1 year and who were observed for more than 1 year, 43 patients developed HCC, and male sex (HR: 3.088), HA ≥75 ng/mL (HR: 3.300), EOT-AFP ≥5.3 ng/mL (HR: 3.288), and EOT-Alb <3.9 g/dL (HR: 2.289) were associated with the development of HCC (Table 4).

Table 3. Factors associated with the development of hepatocellular carcinoma (HCC) in all patients and severe fibrosis.

Category Cut off Univariate Multivariate (All patients, n = 1284) Multivariate (Fib-4 index >3.25, n = 564)
P value P value Hazard Ratio 95%CI P value Hazard Ratio 95%CI
Age ≥73 years 0.001 0.005 2.148 1.264–3.651 0.068 1.774 0.958–3.288
Sex Male <0.001 <0.001 3.060 1.788–5.237 <0.001 2.986 1.588–5.615
Etiology Cirrhosis present <0.001 0.072 1.885 0.946–3.758
Prior DAA therapy DAA experience 0.574
Platelet counts <11.3×104 /μL <0.001
Total bilirubin ≥0.8 mg/dL 0.006
AST ≥47 U/L <0.001
ALT ≥36 U/L 0.078
GGT ≥31 U/L 0.006
Alb <3.9 g/dL <0.001
Hyaluronic acid ≥75 ng/mL <0.001 0.002 3.996 1.668–9.571
Fib-4 index ≥4.03 <0.001
AFP ≥7.1 ng/mL <0.001 0.064 0.457 0.199–1.047
EOT-ALT Not WNL 0.070
EOT-Alb <3.9 g/dL <0.001 0.003 2.305 1.336–3.977 0.003 2.803 1.425–5.517
EOT-AFP ≥5.3 ng/mL <0.001 <0.001 4.773 2.718–8.383 <0.001 8.352 3.314–21.047
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95%CI, 95% confidence interval; DAA, direct-acting antivirals; AST, aspartate transaminase; ALT, alanine transaminase; GGT, γ-glutamyltransferase; Alb, albumin; AFP, α-fetoprotein; EOT, end of treatment; WNL, within normal limit.

Table 4. Factors associated with the development of hepatocellular carcinoma (HCC) in over 6-month or 1-year follow up model.

Category Cut off Univariate Multivariate (after 6 month, n = 1245) Multivariate (after 1 year, n = 1195)
P value P value Hazard Ratio 95%CI P value Hazard Ratio 95%CI
Age ≥73 years 0.001 0.045 1.782 1.014–3.132 0.057 1.816 0.983–3.355
Sex Male <0.001 <0.001 2.835 1.605–5.007 <0.001 3.088 1.652–5.772
Etiology Cirrhosis present <0.001
Prior DAA therapy DAA experience 0.574
Platelet counts <11.3×104 /μL <0.001
Total bilirubin ≥0.8 mg/dL 0.006
AST ≥47 U/L <0.001
ALT ≥36 U/L 0.078
GGT ≥31 U/L 0.006
Alb <3.9 g/dL <0.001 0.019 2.084 1.127–3.856
Hyaluronic acid ≥75 ng/mL <0.001 0.007 3.481 1.411–8.583 0.009 3.300 1.343–8.105
Fib-4 index ≥4.03 <0.001
AFP ≥7.1 ng/mL <0.001
EOT-ALT Not WNL 0.070
EOT-Alb <3.9 g/dL <0.001 0.007 2.289 1.218–4.303
EOT-AFP ≥5.3 ng/mL <0.001 <0.001 4.257 2.352–7.704 <0.001 3.288 1.754–6.163
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95%CI, 95% confidence interval; DAA, direct-acting antivirals; AST, aspartate transaminase; ALT, alanine transaminase; GGT, γ-glutamyltransferase; Alb, albumin; AFP, α-fetoprotein; EOT, end of treatment; WNL, within normal limit.

Similarly, in the analysis of 1031 patients who did not develop HCC within 2 years and who were observed for more than 2 years, 34 patients developed HCC, and male sex (HR: 2.326), HA ≥75 ng/mL (HR: 4.085), EOT-AFP ≥5.3 ng/mL (HR: 4.272), and EOT-Alb <3.9 g/dL (HR: 2.352) were associated with HCC development (Table 5). In addition, in the analysis of 814 patients who did not develop HCC within 3 years and who were observed for more than 3 years, 16 patients developed HCC, and cirrhosis (HR: 5.775) and EOT-AFP ≥5.3 ng/mL (HR: 6.237) were associated with the development of HCC (Table 5).

Table 5. Factors associated with the development of hepatocellular carcinoma (HCC) in over 2-year or 3-year follow up model.

Category Cut off Univariate Multivariate (after 2 years, n = 1031) Multivariate (after 3 years, n = 814)
P value P value Hazard Ratio 95%CI P value Hazard Ratio 95%CI
Age ≥73 years 0.001
Sex Male <0.001 0.017 2.326 1.163–4.652
Etiology Cirrhosis present <0.001 0.003 5.775 1.792–18.613
Prior DAA therapy DAA experience 0.574
Platelet counts <11.3×104 /μL <0.001
Total bilirubin ≥0.8 mg/dL 0.006
AST ≥47 U/L <0.001
ALT ≥36 U/L 0.078
GGT ≥31 U/L 0.006
Alb <3.9 g/dL <0.001
Hyaluronic acid ≥75 ng/mL <0.001 0.011 4.085 1.389–12.015
Fib-4 index ≥4.03 <0.001
AFP ≥7.1 ng/mL <0.001
EOT-ALT Not WNL 0.070
EOT-Alb <3.9 g/dL <0.001 0.019 2.352 1.151–4.807
EOT-AFP ≥5.3 ng/mL <0.001 <0.001 4.272 2.061–8.858 0.002 6.237 1.933–20.123
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95%CI, 95% confidence interval; DAA, direct-acting antivirals; AST, aspartate transaminase; ALT, alanine transaminase; GGT, γ-glutamyltransferase; Alb, albumin; AFP, α-fetoprotein; EOT, end of treatment; WNL, within normal limit.

Comparison of the cumulative rates of HCC development

The cumulative rates of HCC development according to each cutoff value were compared. The cumulative rates of HCC development of patients who were ≥73 and <73 years of age were 2.2% and 0.5%, respectively, at one year, 3.9% and 0.7% at two years, 6.1% and 2.1% at three years, 7.6% and 3.3% at four years (Fig 3A). The cumulative rates of HCC development in male and female patients were 1.7% and 0.7%, respectively, at one year, 3.2% and 0.9% at two years, 6.2% and 1.7% at three years, and 7.2% and 3.2% at four years (Fig 3B). The cumulative rates of HCC development in patients with cirrhosis and non-cirrhosis were 3.2% and 0.5%, respectively, at one year, 4.4% and 1.0% at two years, 6.8% and 2.5% at three years, and 10.6% and 2.9% at four years (Fig 4A). The cumulative rates of HCC development in patients with HA ≥75 and <75 ng/mL were 2.0% and 0.2%, respectively, at one year, 3.1% and 0.5% at two years, 6.3% and 0.7% at three years, and 8.1% and 1.2% at four years (Fig 4B). The cumulative rates of HCC development in patients with EOT-Alb <3.9 and ≥3.9 g/dL were 3.1% and 0.5%, respectively, at one year, 4.7% and 1.0% at two years, 9.3% and 1.8% at three years, and 11.9% and 2.7% at four years (Fig 5A). Finally, the cumulative rates of HCC development in patients with EOT-AFP ≥5.3 and <5.3 ng/mL were 4.3% and 0.2%, respectively, at one year, 5.2% and 0.9% at two years, 9.4% and 1.9% at three years, and 12.8% and 2.4% at four years (Fig 5B).

Fig 3. The cumulative rates of HCC development by age and sex.

Fig 3

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The comparison of the cumulative rates of HCC development (a) between ≥73 years of age and <73 years of age, (b) between male and female. HCC, hepatocellular carcinoma; DAA, direct-acting antiviral.

Fig 4. The cumulative rates of HCC development by etiology and hyaluronic acid.

Fig 4

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(a) between cirrhosis and non-cirrhosis, (b) between HA ≥75 ng/mL and <75 ng/mL. HCC, hepatocellular carcinoma; DAA, direct-acting antiviral; LC, liver cirrhosis; HA, hyaluronic acid.

Fig 5. The cumulative rates of HCC development by albumin and alpha-fetoprotein at the end of treatment.

Fig 5

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(a) between EOT-Alb ≥3.9 g/dL and <3.9g/dL, (b) between EOT-AFP ≥5.3 ng/mL and <5.3 ng/mL. HCC, hepatocellular carcinoma; DAA, direct-acting antiviral; EOT, end of treatment; Alb, albumin; AFP, alpha-fetoprotein.

Relationship between changes in AFP and the development of HCC

Since EOT-AFP was strongly related to the development of HCC, we investigated the relationship between the transition of AFP and HCC development. The patients were classified into four groups according to their AFP before treatment and at the EOT, and the cumulative rates of HCC development were examined in each group. The four groups according to the AFP levels were as follows, (a) <5.3 ng/mL before treatment and <5.3 ng/mL at the EOT, (b) ≥5.3 ng/mL, <5.3 ng/mL, (c) <5.3 ng/mL, ≥ 5.3 ng/mL, and (d) ≥5.3 ng/mL, ≥5.3 ng/mL. The cumulative rates of HCC development at 1, 2, 3, and 4 years were as follows: (a) 0.2%, 1.0%, 1.7%, 2.1%, (b) 0%, 0.5%, 2.7%, 3.5%, (c) 3.8%, 8.0%, 18.8%, 30.6%, (d) 4.3%, 5.0%, 8.6%, 11.8%, respectively (Fig 6). There were statistically significant differences in the rates of HCC development between groups (a) and (c), (a) and (d), (b) and (c), and (b) and (d) (p<0.001); however, there were no significant differences between groups (a) and (b), or (c) and (d) (Fig 6).

Fig 6. The cumulative rates of HCC development for each change of AFP.

Fig 6

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AFP was classified into 4 groups before treatment and at the EOT: (a) <5.3 ng/mL before treatment, and <5.3 ng/mL at the EOT, (b) ≥5.3 ng/mL, <5.3 ng/mL, (c) <5.3 ng/mL, ≥5.3 ng/mL, and (d) ≥5.3 ng/mL, ≥5.3 ng/mL. HCC, hepatocellular carcinoma; DAA, direct-acting antiviral; EOT, end of treatment; AFP, α-fetoprotein.

Collinearity between hyaluronic acid and cirrhosis or advanced fibrosis

HA was related to the development of HCC. We therefore investigated the relationship between HA and cirrhosis, or advanced fibrosis. Patients with liver cirrhosis or severe fibrosis had significantly higher HA values than non-cirrhotic patients or patients with non-severe fibrosis (p<0.001, respectively, Fig 7A and 7B). Patients of >73 years of age had significantly higher HA values than who were <73 years of age (p<0.001, Fig 7C). However, there was no sex difference in the HA values (p = 0.649) (Fig 7D). HA showed a significant positive correlation with the Fib-4 index (correlation coefficient [rs] = 0.615, p<0.001) (Fig 8).

Fig 7. Collinearity between hyaluronic acid and cirrhosis or advanced fibrosis.

Fig 7

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(a) Comparison of HA in patients with and without cirrhosis. (b) Comparison of HA in patients with Fib4-index values of ≥3.25 and <3.25, (c) Comparison of HA in patients of ≥73 years of age and <73 years of age, (d) Comparison of HA in males and females. HA, hyaluronic acid.

Fig 8. Correlation between HA and the Fib-4 index.

Fig 8

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rs, correlation coefficient.

Development of HCC stratified by the score combining EOT-AFP, age, sex, hyaluronic acid, and EOT-Alb

A scoring system was developed based on the results of the multivariate analysis that included the EOT-AFP, age, sex, HA, and EOT-Alb. Of these five factors, EOT-AFP ≥5.3 ng/mL was associated with the development of HCC from 3 years after the EOT; thus, EOT-AFP ≥5.3 ng/mL was scored as 2 points, and others, such as age ≥73 years, male sex, HA ≥75 ng/mL, and EOT-Alb <3.9 g/dL were scored as 1 point. When the cutoff value of the new score was 2, the area under ROC, positive likelihood ratio, and odds ratio all increased (Table 2). The patients were divided into 3 groups based on the sum of these scores, as follows: 0–2 points (low-risk), 3–4 points (moderate-risk), and 5–6 points (high-risk). The cumulative carcinogenesis rate of each group was examined. The cumulative rates of HCC development in the low-, moderate-, and high-risk groups were 0%, 1.5%, and 12.4% respectively, at one year, 0.3%, 3.3%, and 15.0% at two years, 0.4%, 7.8% and 25.3% at three years, and 0.6%, 11.9% and 27.2% at four years (Fig 9). There were statistically significant differences between the groups in the rate of HCC development (p<0.001).

Fig 9. Development of HCC stratified by the score combining EOT-AFP, age, sex, hyaluronic acid, and EOT-Alb.

Fig 9

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EOT-AFP ≥5.3 ng/mL was scored as 2 points; the others (i.e., age ≥73 years, male sex, HA ≥75 ng/mL, and EOT-Alb <3.9 g/dL) were scored as 1 point. The patients were divided into the following 3 groups according to the sum of these scores: of 0–2 points (low-risk), 3–4 points (moderate-risk), and 5–6 points (high-risk). HCC, hepatocellular carcinoma; EOT, end of treatment; AFP, α-fetoprotein; HA, hyaluronic acid; Alb, albumin.

Alpha-fetoprotein and des-γ-carboxy prothrombin levels at the onset of HCC

AFP and des-γ-carboxy prothrombin (DCP) are tumor markers for HCC. In 60 patients who developed HCC, we confirmed the AFP and DCP levels at the onset of HCC. The details are summarized in Table 6. The median tumor size and median number of tumors was 1.8 cm and 1 nodule, respectively, 93.1% of patients developed HCC with vascularity. The comparison of the characteristics between patients with EOT-AFP ≥5.3 ng/mL and <5.3 ng/mL revealed that there was no significant difference in tumor size, number, or vascularity, the patients with EOT-AFP ≥5.3 ng/mL were more frequently male, and higher AFP and DCP levels. Especially, twenty-three patients (38.3%) had AFP levels above the upper limit (10 ng/mL), and 27 patients (45.8%) had DCP levels above the upper limit (40 mAU/mL). Twenty-two of 39 patients (56.4%) with EOT-AFP ≥5.3 ng/mL had levels above the upper limit at the onset of HCC. However, only 1 of 21 patients (4.8%) with EOT-AFP <5.3 ng/mL had a level above the upper limit (p<0.001). Similarly, AFP increased more than 30% at the onset of HCC in 19 of 39 patients (48.7%) with EOT-AFP ≥5.3 ng/mL, but only increased in two of 21 patients (9.5%) with EOT-AFP <5.3 ng/mL (p<0.001). On the other hand, among patients with EOT-AFP <5.3 ng/mL, the percentage of patients with DCP levels above the upper limit at the onset of HCC was similar to that of patients with EOT-AFP ≥5.3 ng/mL (p = 0.477). In addition, 22 patients (37.3%) had AFP and DCP levels within the normal limits at the onset of HCC. Among patients with EOT-AFP <5.3 ng/mL, a significantly higher percentage of patients had normal limits of AFP and DCP in comparison to those with EOT-AFP ≥5.3 ng/mL at the onset of HCC (p = 0.020).

Table 6. Comparison of the data at the onset of hepatocellular carcinoma (HCC).

At the onset of HCC ALL EOT-AFP <5.3ng/mL EOT-AFP ≥5.3ng/mL P value
n = 60 n = 21 n = 39
Age, years 73.9±8.8 75.8±8.8 72.9±8.8 0.201
Male, n (%) 37 (61.7) 17 (77.3) 20 (51.3) 0.022
Tumor size, cm (median) 2.2±1.8 (1.8) 2.6±2.8 (1.8) 2.0±0.8 (1.8) 0.738
Number of nodules, n (median) 1.3±0.7 (1) 1.4±0.9 (1) 1.3±0.6 (1) 0.467
Presence of vascularity, n (%) a (n = 58) 54 (93.1) 19 (95.0) 35 (92.1) 0.572
Platelet counts, ×104/μL 13.3±6.1 15.6±6.9 12.1±5.4 0.045
Total bilirubin, mg/dL 1.0±0.6 1.0±0.9 1.0±0.5 0.269
AST, U/L 29.5±10.6 27.5±9.7 30.6±11.1 0.212
ALT, U/L 20.8±9.2 18.4±7.1 22.1±10.0 0.209
Albumin, g/dL 4.0±0.5 3.9±0.6 4.0±0.5 0.735
AFP, ng/mL 114.7±453.0 4.34±8.0 174.0±555.1 <0.001
DCP, mAU/mL (n = 59) 276.1±924.3 126.6±362.0 358.6±1117.4 0.027
AFP ≥10ng/mL, n (%) 23 (38.3) 1 (4.8) 22 (56.4) <0.001
AFP ≥30% increase, n (%) 21 (35.0) 2 (9.5) 19 (48.7) 0.002
DCP ≥40 mAU/mL, n (%) (n = 59) 27 (45.8) 9 (40.9) 18 (47.4) 0.477
AFP and DCP within normal limit, n (%) (n = 59) b 22 (37.3) 12 (57.1) 10 (26.3) 0.020
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a One patient had hypovascular and hypervascular nodules. In one patient, the presence of vascularity was not evaluated due to renal failure.

b The normal limit was defined as AFP < 10 ng/mL, and DCP <40 mAU/mL

Data are shown as the mean ± standard deviation, EOT, end of treatment; AFP, α-fetoprotein; DCP, des-γ-carboxy prothrombin; EOT, end of treatment.

Discussion

In the present study, we revealed that age, sex, HA, EOT-AFP, and EOT-Alb were useful markers for predicting the development of HCC in patients who achieved an SVR with DAA treatment for HCV. By combining these factors, the risk of developing HCC could be significantly stratified (Fig 9). The median observation period was 47.6 months, with approximately 60% patients observed for more than 3 years. The advantage of our study is that the predictors were derived from a long observation period.

Since the EOT-AFP was associated with the development of HCC from 3 years after the EOT (Table 5), we investigated the relationship between the transition of AFP and the development of HCC. First, we focused on the transition of AFP before treatment and at the EOT. We revealed that there was no significant difference in the rate of HCC development between patients in whom AFP decreased to <5.3 ng/mL and those in whom AFP remained <5.3 ng/mL at the EOT (Fig 6). Next, we focused on the transition of AFP in patients who developed HCC. In the present study, 56.4% of patients who developed HCC with EOT-AFP ≥5.3 ng/mL had AFP levels above the upper limits at the onset of HCC (Table 6). However, among patients with EOT-AFP <5.3 ng/mL, the percentage of patients with levels above the upper limit or with a >30% increase in AFP at the onset of HCC were lower in comparison to patients with EOT-AFP ≥5.3 ng/mL (Table 6). In other words, in patients with EOT-AFP <5.3 ng/mL, AFP did not increase, even if they developed HCC; thus the measurement of AFP after an SVR is unlikely to predict the development of HCC. In fact, 35% of patients who developed HCC had EOT-AFP values of <5.3 ng/mL. There were no significant differences between the two groups with respect to tumor size, number and vascularity.　In these cases, by combining HA, EOT-Alb, sex, and age, it is possible to identify patients with a high risk of HCC development (Fig 9). To the best of our knowledge, no other studies have focused on the relationship between the AFP transition and carcinogenesis. In addition, in 40.9% of patients with EOT-AFP <5.3 ng/mL, DCP is above the upper limit at the onset of HCC (Table 6). The comparison of the baseline characteristics of patients who developed HCC and patients who did not develop HCC revealed was no significant difference in the DCP level; thus, it is considered useful to measure DCP in the clinical course. However, it should be noted that in 37.3% of patients who developed HCC, the AFP and DCP values were both within the normal limits (Table 6). It seems difficult to predict carcinogenesis in patients who have achieved a DAA-SVR from tumor markers alone.

AFP is a glycoprotein with a molecular weight of 67 kDa, which was found in human fetal serum by Bergstrand in 1956 [11]. AFP is not only a tumor marker of HCC, but also a marker of the induction of hepatic progenitor cells [12–14]. In addition, the serum AFP level reflects the function of the liver stem or progenitor cells in patients with acute liver failure [15]. as well as inflammation and fibrosis in patients with chronic hepatitis B [16]. In the present study, the AFP level before DAA therapy was not associated with the development of HCC. Additionally, there was no significant difference in the rate of HCC development between decrease to AFP <5.3 ng/mL and remain AFP <5.3 ng/mL at the EOT (Fig 6). The AFP levels before treatment may reflect liver regeneration, inflammation, and fibrosis, as well as microscopic HCC. DAA treatment may reduce inflammation and may reveal tumor-derived AFP. Alternatively, the proliferative activity of hepatocytes may cause hepatocarcinogenesis [17].

HA is an acidic mucopolysaccharide that is widely distributed in the connective tissue of the body, and is produced in the liver by activated hepatic stellate cells. During chronic liver inflammation, there is continuous hepatic stellate cell activation and therefore increased HA synthesis [18]. HA is related to liver fibrosis. It is a part of many non-invasive algorithms that are used to asses liver fibrosis (e.g., ELF score [19] and Hepascore [20]). In this study, the HA levels of patients with liver cirrhosis were higher than those of non-cirrhotic patients and showed a positive correlation with the Fib-4 index (Fig 8). A previous report showed that the initiation of liver cancer requires the inhibition of p53 by CD44 (a receptor of HA)-enhanced growth factor signaling [21]. HA can be a predictive marker for the development of HCC after DAA-SVR.

Several reports have shown that the elimination of the HCV by interferon (IFN)-based therapy suppresses the development of HCC; the cumulative rate of carcinogenesis after an SVR is reported to be 2.3–8.8% at 5 years and 3.1–11.1% at 10 years, depending on patient characteristics [22]. As is observed with IFN-based therapy, the elimination of HCV induced by DAA treatment reduces the risk of HCC and mortality [23–28]. On the other hand, some reports have indicated that DAA treatment promotes the development of HCC [29–31]. In the present study, the cumulative rates or HCC development were 4.7% at 4 years, with a median observation period of 47.6 months (Fig 2), and 10.6% at 4 years in cirrhotic patients (Fig 4A). In addition, risk factors for hepatocarcinogenesis after an SVR in patients who receive IFN-based therapy include older age, advanced liver fibrosis, male sex, the AFP level after treatment, glucose metabolism disorders, lipid metabolism disorders and alcohol intake, and other factors [17, 22, 32]. Similar risk factors are expected with DAA treatment [33–35]. Wisteria floribunda agglutinin-positive Mac-2 binding protein (M2BPGi), a marker of liver fibrosis, predicts the early occurrence of HCC after an SVR in DAA-treated patients [36, 37]. In the present study, EOT-AFP and HA had a high predictive ability for HCC development. Even if EOT-AFP was <5.3 ng/mL, it is possible to identify patients with a high risk of HCC development with the combination of HA, EOT-Alb, sex, and age (Fig 9). Actually, new score system according to the sum of these risk factors showed the higher area under ROC, positive likelihood ratio, and odds ratio (Table 2). When the patients were divided into the low-risk (0–2 points), moderate-risk (3–4 points), and high-risk (5–6 points) groups, in comparison to the low-risk group, the 4-year cumulative carcinogenesis rate was 45.3 times higher in the high-risk group and 19.8 times higher in the moderate-risk group (Fig 9). Only 5 patients in the low-risk group developed HCC, there was only one patient with 0 points, while 4 patients who had 2 points. The factors were as follows: age and HA, male and HA, age and male, and EOT-AFP (n = 1 each). In the future, long-term follow-up is necessary for patients with these 5 risk factors, and the usefulness of these cut-off values will need to be validated in other cohorts.

This study performed HCC screening based on the Japanese guidelines. The American Association for the Study of Liver Diseases (AASLD) [38], The European Association for the Study of the Liver (EASL) [39], and The Asian Pacific Association for the Study of the Liver (APASL) [40] guidelines recommend ultrasound screening every 6 months. In particular, the AASLD guideline shows that the risk of HCC is significantly lower in patients without cirrhosis in comparison to those with cirrhosis, and surveillance is not recommended for these patients [38]. However, in this study, out of 60 patients who developed HCC, 27 patients (45%) were non-cirrhotic and 15 (25%) had a Fib-4 index of <3.25 (Table 1); thus, HCC surveillance after DAA-SVR should be considered important, even for non-cirrhotic patients. The EASL Clinical Practice Guidelines show that non-cirrhotic F3 patients, regardless of etiology, may be considered for surveillance based on an individual risk assessment, and in patients who were treated viral chronic hepatitis, there was no evidence for a timing or stiffness threshold to stop surveillance in patients who were included in surveillance programs [39]. In this study, a few patients in the low and moderate-risk groups developed HCC. In the future, it will be necessary to consider the cases in which surveillance can be stopped after DAA-SVR.

An early high incidence of HCC was observed in DAA-treated patients with hypovascular tumors, such as dysplastic nodules [41–43]. We also reported that hypovascular tumors developed into hepatocellular carcinoma at a high rate despite the elimination of HCV by DAA treatment [44]. In the present study, we examined the occurrence of de novo HCC, and therefore excluded patients with hypovascular tumors diagnosed by CT or MRI before treatment. However, the doubling time of HCC is reported to be 100 days, and it theoretically takes approximately 9 years for a 10-μm HCC to become a 10-mm lesion that can be detected by diagnostic imaging [45]. In other words, the involvement of AFP in hepatocarcinogenesis several years later may indicate the presence of microscopic HCC.

HIV seropositivity accelerates the progression of fibrosis related to chronic hepatitis C [46]. A nationwide survey in Japan revealed that nearly one-fifth of HIV-positive patients are co-infected with HCV. The determination of HCV genotypes revealed that genotype 3 or 4, which is rarely seen in HCV mono-infected patients in Japan, was found in a substantial fraction of HIV-infected patients [47]. In this cohort, there were no patients who received the therapy for HIV, and with HCV genotype 3 or 4. We therefore considered that there were no HIV-HCV co-infected patients in this cohort.

HCC occurs as a result of hepatic inflammation and/or changes in the tumor microenvironment. HCV clearance can suppress fibrosis and reduces the incidence of HCC [48]. It is assumed that microRNAs (e.g., miRNA-122) and inflammatory cytokines (e.g., transforming growth factor [TGF]-beta, vascular endothelial growth factor [VEGF], and interleukin-6) are involved in this inflammation, and microRNAs are involved in HCV clearance and HCC development [49–51]. HCV clearance limits fibrosis and reduces the incidence of HCC by switching TGF-beta signaling from fibro-carcinogenesis to tumor suppression [52]. Otherwise, the administration of DAAs induces an early increase in serum VEGF and a change in the inflammatory pattern, which coincides with HCV clearance [53]. In this study, we have not been able to investigate the onset of HCC with microRNAs and inflammatory cytokines; however, we consider this to be an issue for future study.

The present study was associated with several limitations. First, various factors (alcohol intake, obesity, metabolic syndrome, and aspirin use, etc.) were not examined after treatment. Second, we could not diagnose fibrosis by histological or non-invasive methods, such as transient elastography; thus, cirrhosis was likely to have been underdiagnosed. Therefore, we adopted fibrosis markers, such as the Fib-4 index and HA before DAA treatments. Third, other fibrosis markers, such as type IV collagen or M2BPGi, could not be measured.

In conclusion, EOT-AFP ≥5.3 ng/mL is a useful marker for predicting the development of HCC after an SVR; however, the AFP level did not increase in patients with EOT-AFP <5.3 ng/mL at the onset of HCC. The combination of EOT-AFP, age, sex, HA, and EOT-Alb is important for predicting carcinogenesis.

Supporting information

S1 File. Analysis data set.

All patients’ data sets were included in the following file.

(XLSX)

Click here for additional data file. (216.1KB, xlsx)

Acknowledgments

The present study was carried out in the following 21 facilities (Kagoshima Liver Study Group): Kagoshima University Hospital, Kirishima Medical Center, Miyazaki Medical Center Hospital, Kagoshima Kouseiren Hospital, Kagoshima City Hospital, Saiseikai Sendai Hospital, Kohshinkai Ogura Hospital, Ikeda Hospital, Izumi General Medical Center, Oshima Hospital, Ibusuki Medical Center, Kagoshima medical center, Hirono Clinic, Kagoshima Teishin Hospital, Satsunan Hospital, Nagaki Clinic, Dr. NAKANISHI’s office, Southern Region Hospital, Tanegashima Medical Center, Fujimoto General Hospital, and Nakayama Clinic. We thank the following investigators: Yasushi Imamura (Kagoshima Kouseiren Hospital), Dai Imanaka (Ikeda Hospital), Toshihiro Fujita (Oshima Hospital), Kengo Tsuneyoshi (Oshima Hospital), Akihiko Oshige (Ibusuki Medical Center), Shuichi Hirono (Hirono Clinic), Masahito Nagaki (Nagaki Clinic), Chihiro Nakanishi (Dr. NAKANISHI’s office), and Toshihiro Nakayama (Nakayama Clinic). We also thank Ms. Hiromi Eguchi, Ms. Yuko Morinaga, and Ms. Eriko Koreeda for their technical assistance and data management (belonging to Digestive and Lifestyle Diseases, Department of Human and Environmental Sciences, Kagoshima University Graduate School of Medical and Dental Sciences).

Data Availability

All relevant data are within the manuscript and its Supporting Information files (S1 File).

Funding Statement

This work was supported in part by a grant-in-aid from the Ministry of Health, Labour and Welfare of Japan (grant number: 18K15821). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Birke Bartosch

21 Oct 2021

PONE-D-21-28792Features of patients who developed hepatocellular carcinoma after direct-acting antiviral treatment for hepatitis C VirusPLOS ONE

Dear Dr. Mawatari,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In the current study, Mawatari et aimed to shed light on the features of HCV-infected patients developing HCC upon DAA treatment. After studying a cohort of 1494 DAA-SVR patients, they found that 60 of them (=4%) developed HCC within 47.6 months and in these patients. The main conclusion is that AFP>5.3 ng/mL at the end of the DAA treatment (EOT) can be useful for predicting HCC development after an SVR. Eventually, the combination of the following markers is associated for predicting HCC: EOT-AFP>5.3 ng/mL, males aged >73 years, hyaluronic acid >75 ng/mL, EOT-albumin <3.9 g/dL.

The study is essentially descriptive and statistically sound. However, I have some concerns about the usefulness of these markers in predicting HCC in DAA treated patients:

1. These markers have not been evaluated in a cohort without DAA treatments (par ex. NASH, NAFLD)

2. I have doubts about the age and sex considered as risk factors in this study since higher HCC rates are encountered in older males irrespectively of HCC etiology.

3. The authors state that EOT-AFP is an important indicator for HCC development, but at the same time they state in table 5 that in 37.3% of the patients who developed HCC, the AFP (and DCP) values were within the normal limits. This is confusing. Also, DCP has not been explained and commented.

4. Other markers that could be of interest upon HCV clearance such as miRNA-122 have not been studied and discussed. Immune markers such as TGF-beta, VEGF, IL-6, etc. have not been addressed neither.

Reviewer #2: In this manuscript, Mawatari and colleagues analyzed the features of patients with chronic C Hepatitis successfully treated with DAAs who developed HCC over time.

He conducted a well-designed prospective study, with a large study population and considerable follow-up.

Although the parameters (including AFP levels) associated with HCC development after DAAs therapy have been already analyzed and described by several authors, changes in AFP levels over time after DAAs were not considered before and represent therefore a novel parameter of interest.

The study has a rationale and has been carried out with an adequate statistical approach.

The article address a an important topic, considering that HCV infection is now easily cured with IFN-free therapies, but HCC risk still remains after achieving SVR. The authors analyzed the risk factors for HCC and created a 5- factors score that could be use to estimate the cumulative HCC risk over time, categorizing the patients in low-moderate-high risk. The laboratory parameters used in this score system are routinely tested in clinics, therefore the score, if further validated, could be suggested as tool in clinical practice.

The fact that liver fibrosis was not diagnosed by biopsy or transient elastography represents a limitation, but the authors acknowledged it. A validation of this score will be important in order to understand its potential role in clinical practice in earlier identify subjects at higher risk.

Major comments:

• In the introduction, the authors explain the concept of RASs in relationship to non-response to DAAs. I don’t find this particular topic related to the main subject of the manuscript since only the patients who achieved SVR were enrolled. This paragraph should be removed.

• The authors do not mention HIV-coinfected patients, who have overall a higher risk for cancer. Were they excluded from the study population? If so, the authors should mention that in the Methods. Otherwise, they should consider this variable.

• Is the study still ongoing? It is well specified when the observation started but it’s not clear to me if the observation period is over.

• Patients with EOT-AFT<5.3 ng/ml did not show any changes in AFP levels at HCC onset. Did the author observe any peculiar characteristics (size, number of nodules, vascularization?) in HCC occurred in this subgroup of patients?

Minor comments:

Material and Methods:

-“observation” should be replaced by “observational”

- Instead of “The study population is shown in Figure 1”, I would rather write “Study population enrollment is shown…” I would suggest also to modify Figure 1 title consistently with that, specifying that it represents a flow chart of the study population enrollment.

- Titles of Table 3 and 4 are the same. Authors should be differentiate.

Reviewer #3: This is a Japanese multicentre cohort study of n=1494 post-SVR patients following DAA treatment. The study followed the cohort (median 47-months) and described the factors associated with the development of HCC (N=60). They identify: age, males, hyaluronic acid, AFP and albumin as factors associated with HCC. They then developed a risk stratification score based on these factors.

The manuscript is well structured and clear. A strength of the study is the exploration of AFP dynamics. This explores the impact of change in AFP and then relationship to DCP. This supports the data that AFP is not a great screening tool.

Comments:

•The major issue with this study is the categorisation of cirrhosis. It is clear from multiple studies that cirrhosis is a major aetiological factor in HCC development and the vast majority of HCCs occur in the context of cirrhosis. In this study only 55% of HCC was associated with cirrhosis which suggests that cirrhosis was underdiagnosed. It is likely the result of suboptimal assessment of liver fibrosis. This would impact any analysis of factors associated with HCC, especially if they are factors that are also related to fibrosis/cirrhosis. Key to this is hyaluronic acid which is a strong marker of fibrosis. The authors were not clear about their definition of cirrhosis and this needs to be stated clearly in the methods. Specifically, cut-offs for FIB4, clinical and biochemical criteria etc. It is not acceptable to state “other factors”.

•Hyaluronic acid (HA) is related to liver fibrosis. It is a part of many non-invasive algorithms of liver fibrosis. Given this, it is likely closely related to cirrhosis. Potentially it is collinear with cirrhosis. In the 2-year follow-up and 3-year follow-up models HA and cirrhosis are alternatively significant. Collinearity between HA and cirrhosis and advanced fibrosis must be explored.

•Gender and age differences in HA may also have an impact. Could this be analysed in the cohort?

•HA should also form a part of the discussion.

•Usually, to analyse de novo HCC, any HCC exclusion that developed within 3-6 months post treatment should be excluded. Could this be done to see if this changes the analysis of factors.

•A weakness is co-factors which could not be addressed including: alcohol, metabolic syndrome, and aspirin use. This would lead to confounding of results.

•This is a Japanese population with different screening protocols compared with other international guidelines. Here, even non-cirrhotic patients were surveyed and cirrhotics were evaluated at a 3-4 month interval when most international guidelines are 6-months US. This should be made clear in the discussion as it will impact generalisability.

Minor:

•P15 – The comparison is cirrhosis to no cirrhosis (not chronic hepatitis as the HCV has been cleared).

•Table 3: Aetiology should be cirrhosis (present or not)

•Table 3: In the Advanced fibrosis multivariable analysis, why are insignificant outcomes presented?

•Figure 2 should change the y-axis to small scale.

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Reviewer #1: No

Reviewer #2: Yes: Viola Guardigni

Reviewer #3: Yes: mark danta

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PLoS One. 2022 Jan 12;17(1):e0262267. doi: 10.1371/journal.pone.0262267.r002

Author response to Decision Letter 0

20 Nov 2021

Thank you for your valuable feedback. We have revised the manuscript based on the comments.

Response to reviewer 1

1. These markers have not been evaluated in a cohort without DAA treatments (par ex. NASH, NAFLD)

Response: In the present study, we aimed to clarify the features of patients who developed HCC after an SVR with DAA treatment. Therefore, we could not evaluate the carcinogenic features in a cohort without DAA treatment (e.g., NASH, NAFLD). In addition, various factors (alcohol intake, obesity, etc.) were not examined after treatment in this cohort. We have mentioned this in the Discussion section.

2. I have doubts about the age and sex considered as risk factors in this study since higher HCC rates are encountered in older males irrespectively of HCC etiology.

Response: As you pointed out, it is well-known that older males are at increased risk for the development of HCC. We believe that these factors should always be considered in a statistical analysis. In fact, this cohort demonstrates that “older male” is an independent risk factor for the development of HCC.

3. The authors state that EOT-AFP is an important indicator for HCC development, but at the same time they state in table 5 that in 37.3% of the patients who developed HCC, the AFP (and DCP) values were within the normal limits. This is confusing. Also, DCP has not been explained and commented.

Response: AFP and DCP are tumor markers for HCC. We therefore confirmed the AFP and DCP levels at the onset of HCC to consider whether these markers were useful for carcinogenesis monitoring. More details can be found in the following section (Alpha-fetoprotein and des-γ-carboxy prothrombin levels at the onset of HCC) on page 19. What we wanted to insist in this chapter is that in patients with EOT-AFP <5.3 ng/mL, AFP did not increase, even if they developed HCC. On the other hand, 27 patients (45.8%) had DCP levels above the upper limit (40 mAU/mL) at the onset of HCC. In brief, DCP at the onset of HCC was not affected by EOT-AFP, it is considered useful to measure DCP in the clinical course. However, 22 patients (37.3%) had AFP and DCP levels within the normal limits at the onset of HCC. In particular, 57.1% of patients with EOT-AFP<5.3 ng/mL had AFP and DCP levels within the normal limits at the onset of HCC. It seems difficult to predict carcinogenesis in patients who have achieved a DAA-SVR from tumor markers alone. We discussed these details on page 22–23.

We added the baseline data about DCP. There was no statistically significance in the des-γ-carboxy prothrombin (DCP) level; however, there were many missing data.

4. Other markers that could be of interest upon HCV clearance such as miRNA-122 have not been studied and discussed. Immune markers such as TGF-beta, VEGF, IL-6, etc. have not been addressed neither.

Response: Thank you for your suggestion. Unfortunately, we could not measure the miRNA, TGF-beta, VEGF, and IL-6 levels. We added a comment about these markers to the Discussion section. We added the following statement to the Discussion section:

HCC occurs as a result of hepatic inflammation and/or changes in the tumor microenvironment. HCV clearance can suppress fibrosis and reduces the incidence of HCC. It is assumed that microRNAs (e.g., miRNA-122) and inflammatory cytokines (e.g., transforming growth factor [TGF]-beta, vascular endothelial growth factor [VEGF], and interleukin-6) are involved in this inflammation, and microRNAs are involved in HCV clearance and HCC development. HCV clearance limits fibrosis and reduces the incidence of HCC by switching TGF-beta signaling from fibro-carcinogenesis to tumor suppression. Otherwise, the administration of DAAs induces an early increase in serum VEGF and a change in the inflammatory pattern, which coincides with HCV clearance. In this study, we have not been able to investigate the onset of HCC with microRNAs and inflammatory cytokines; however, we consider this to be an issue for future study.

Response to reviewer 2

Major comments:

• In the introduction, the authors explain the concept of RASs in relationship to non-response to DAAs. I don’t find this particular topic related to the main subject of the manuscript since only the patients who achieved SVR were enrolled. This paragraph should be removed.

Response: We have removed the concept of RASs.

• The authors do not mention HIV-coinfected patients, who have overall a higher risk for cancer. Were they excluded from the study population? If so, the authors should mention that in the Methods. Otherwise, they should consider this variable.

Response: Thank you for your suggestion. In this study, we did not mention HIV-co-infected patients. There were no patients who received therapy for HIV. A nationwide survey in Japan revealed that nearly one-fifth of HIV-positive patients are co-infected with HCV. The determination of HCV genotypes revealed that genotype 3 or 4, which is rarely seen in HCV mono-infected patients in Japan, was found in a substantial fraction of HIV-infected patients. This cohort did not include patients with HCV genotype 3 or 4. We therefore considered that there were no HIV-HCV co-infected patients in this cohort.

We added the following statement in the Discussion section.

HIV seropositivity accelerates the progression of fibrosis related to chronic hepatitis C. (Benhamou). A nationwide survey in Japan revealed that nearly one-fifth of HIV-positive patients are co-infected with HCV. The determination of HCV genotypes revealed that genotype 3 or 4, which is rarely seen in HCV mono-infected patients in Japan, was found in a substantial fraction of HIV-infected patients. In this cohort, there were no patients who received the therapy for HIV, and with HCV genotype 3 or 4. We therefore considered that there were no HIV-HCV co-infected patients in this cohort.

• Is the study still ongoing? It is well specified when the observation started but it’s not clear to me if the observation period is over.

Response: The study is still ongoing. The patients were observed until July 2021. We added this information to the Materials and Methods section.

• Patients with EOT-AFT<5.3 ng/ml did not show any changes in AFP levels at HCC onset. Did the author observe any peculiar characteristics (size, number of nodules, vascularization?) in HCC occurred in this subgroup of patients?

Response: We have added the characteristics of HCC size, number of nodules, and vascularization in Table 6. The tumor size, number, and vascularity did not differ between the two groups to a statistically significant extent.

Minor comments:

Material and Methods:-“observation” should be replaced by “observational”

Response: We changed “observation” to “observational”.

- Instead of “The study population is shown in Figure 1”, I would rather write “Study population enrollment is shown…” I would suggest also to modify Figure 1 title consistently with that, specifying that it represents a flow chart of the study population enrollment.

Response: We changed the sentence to “Study population enrollment is shown in Figure 1”, and modified the Figure 1 title accordingly.

- Titles of Table 3 and 4 are the same. Authors should be differentiate.

Response: We added subtitles to Tables 3, 4, and 5.

Response to reviewer 3

Comments:

•The major issue with this study is the categorisation of cirrhosis. It is clear from multiple studies that cirrhosis is a major aetiological factor in HCC development and the vast majority of HCCs occur in the context of cirrhosis. In this study only 55% of HCC was associated with cirrhosis which suggests that cirrhosis was underdiagnosed. It is likely the result of suboptimal assessment of liver fibrosis. This would impact any analysis of factors associated with HCC, especially if they are factors that are also related to fibrosis/cirrhosis. Key to this is hyaluronic acid which is a strong marker of fibrosis. The authors were not clear about their definition of cirrhosis and this needs to be stated clearly in the methods. Specifically, cut-offs for FIB4, clinical and biochemical criteria etc. It is not acceptable to state “other factors”.

Response: We stated the definition of cirrhosis as follows:

Liver cirrhosis was comprehensively judged by hepatologists at each institution according to the platelet count, imaging, fibrosis markers, transient elastography, or varices formation. Unfortunately, many facilities have not been able to investigate elastography. As you indicated, cirrhosis was likely to be underdiagnosed in this study. We added this to the limitations. Hyaluronic acid is a fibrosis marker and has been adopted in scoring systems for the diagnosis of liver cirrhosis (e.g., ELF score, Hepascore etc.); however, in this study, the parameters for calculating these scores were insufficient. We therefore added the cases with Fib-4 index ≥ 3.25, who were diagnosed with severe fibrosis, to the baseline data (Table 1).

•Hyaluronic acid (HA) is related to liver fibrosis. It is a part of many non-invasive algorithms of liver fibrosis. Given this, it is likely closely related to cirrhosis. Potentially it is collinear with cirrhosis. In the 2-year follow-up and 3-year follow-up models HA and cirrhosis are alternatively significant. Collinearity between HA and cirrhosis and advanced fibrosis must be explored.

Response: Thank you for your comments. We added the collinearity between HA and cirrhosis and advanced fibrosis (e.g., Fib-4 index ≥ 3.25).

•Gender and age differences in HA may also have an impact. Could this be analysed in the cohort?

Response: We analyzed the association between sex and age and HA in Fig.5. HA was associated with age; however, there was no significant association between HA and sex.

•HA should also form a part of the discussion.

Response: In the Discussion section, we discussed HA as follows:

HA is an acidic mucopolysaccharide that is widely distributed in the connective tissue of the body, and is produced in the liver by activated hepatic stellate cells. During chronic liver inflammation, there is continuous hepatic stellate cell activation and therefore increased HA synthesis. HA is related to liver fibrosis. It is a part of many non-invasive algorithms that are used to asses liver fibrosis (e.g., ELF score and Hepascore). In this study, the HA levels of patients with liver cirrhosis were higher than those of non-cirrhotic patients and showed a positive correlation with the Fib-4 index (Fig 5). A previous report showed that the initiation of liver cancer requires the inhibition of p53 by CD44 (a receptor of hyaluronic acid)-enhanced growth factor signaling. HA can be a predictive marker for the development of HCC after DAA-SVR.

•Usually, to analyse de novo HCC, any HCC exclusion that developed within 3-6 months post treatment should be excluded. Could this be done to see if this changes the analysis of factors.

Response: We added the model of HCC exclusion that developed within 6 months and 1 year in Table 4. With the exception of albumin, the factors associated with carcinogenesis were similar.

•A weakness is co-factors which could not be addressed including: alcohol, metabolic syndrome, and aspirin use. This would lead to confounding of results.

Response: As you pointed out, we could not address co-factors, including alcohol, metabolic syndrome, and aspirin use. We have described this as a limitation of this study.

•This is a Japanese population with different screening protocols compared with other international guidelines. Here, even non-cirrhotic patients were surveyed and cirrhotics were evaluated at a 3-4 month interval when most international guidelines are 6-months US. This should be made clear in the discussion as it will impact generalisability.

Response: Thank you for your comments. In the Discussion section, we added the following text:

This study performed HCC screening based on the Japanese guidelines. The American Association for the Study of Liver Diseases (AASLD), The European Association for the Study of the Liver (EASL), and The Asian Pacific Association for the Study of the Liver (APASL) guidelines recommend ultrasound screening every 6 months. In particular, the AASLD guideline shows that the risk of HCC is significantly lower in patients without cirrhosis in comparison to those with cirrhosis, and surveillance is not recommended for these patients. However, in this study, out of 60 patients who developed HCC, 27 patients (45%) were non-cirrhotic and 15 (25%) had a Fib-4 index of <3.25 (Table 1); thus, HCC surveillance after DAA-SVR should be considered important, even for non-cirrhotic patients. The EASL Clinical Practice Guidelines show that non-cirrhotic F3 patients, regardless of etiology, may be considered for surveillance based on an individual risk assessment, and in patients who were treated viral chronic hepatitis, there was no evidence for a timing or stiffness threshold to stop surveillance in patients who were included in surveillance programs. In this study, a few patients in the low and moderate-risk groups developed HCC. In the future, it will be necessary to consider the cases in which surveillance can be stopped after DAA-SVR.

Minor:

•P15 – The comparison is cirrhosis to no cirrhosis (not chronic hepatitis as the HCV has been cleared).

Response: We changed cirrhosis to no cirrhosis

•Table 3: Aetiology should be cirrhosis (present or not)

Response: We changed cirrhosis to cirrhosis present in Tables 3, 4 and 5.

•Table 3: In the Advanced fibrosis multivariable analysis, why are insignificant outcomes presented?

Response: Factors associated with HCC development were determined using a Cox proportional hazards analysis with forward selection using p<0.10 as a cutoff value for inclusion in the model. The above is described in the Statistical analyses section.

•Figure 2 should change the y-axis to small scale.

Response: We changed the y-axis to a smaller scale in Figure 2.

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Decision Letter 1

Birke Bartosch

21 Dec 2021

Features of patients who developed hepatocellular carcinoma after direct-acting antiviral treatment for hepatitis C Virus

PONE-D-21-28792R1

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Reviewer #2: The authors have addressed each of my previuos comments exhaustively. I have no further comment to add concerning the contents. I think that the manuscript could be accepted for publication on PLOS ONE.

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Acceptance letter

Birke Bartosch

26 Dec 2021

PONE-D-21-28792R1

Features of patients who developed hepatocellular carcinoma after direct-acting antiviral treatment for hepatitis C Virus

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