Dynamic changes in non-invasive markers of liver fibrosis are predictors of liver events after SVR in HCV patients

Citation: Fernández-Alvarez, P.; Guerra-Veloz, M.F.; Vilches-Arenas, A.; Cordero-Ruíz, P.; Bellido-Muñoz, F.; Caunedo-Alvarez, A.; Carmona-Soria, I. Dynamic Changes in Non-Invasive Markers of Liver Fibrosis Are Predictors of Liver Events after SVR in HCV Patients. Viruses 2023,15, 1251. https:// doi.org/10.3390/v15061251 Academic Editors: Philippe Gallay and Yinzhong Shen Received: 20 April 2023 Revised: 22 May 2023 Accepted: 25 May 2023 Published: 26 May 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). viruses Article Dynamic Changes in Non-Invasive Markers of Liver Fibrosis Are Predictors of Liver Events after SVR in HCV Patients Paula Fernández-Alvarez 1, María Fernanda Guerra-Veloz 2, Angel Vilches-Arenas 3,* , Patricia Cordero-Ruíz1, Francisco Bellido-Muñoz 1, Angel Caunedo-Alvarez 1and Isabel Carmona-Soria 1 1Department of Gastroenterology and Hepatology, Hospital Universitario Virgen Macarena, 41009 Seville, Spain; [email protected] (P.F.-A.); patricia.corder[email protected] (P.C.-R.); [email protected] (F.B.-M.); [email protected] (A.C.-A.); [email protected] (I.C.-S.) 2Department of Gastroenterology and Hepatology, King’s College Hospital, London SE5 9RS, UK; [email protected] 3Department of Preventive Medicine and Public Health, Faculty of Medicine, University of Seville, 41009 Seville, Spain *Correspondence: [email protected]; Tel.: +34-662160100 Abstract: Objectives: The course of progressive liver damage after achieving sustained virological response (SVR) with direct-acting antivirals (DAAs) remains undetermined. We aimed to determine risk factors associated with the development of liver-related events (LREs) after SVR, focusing on the utility of non-invasive markers. Methods: An observational, retrospective study that included patients with advanced chronic liver disease (ACLD) caused by hepatitis C virus (HCV), who achieved SVR with DAAs between 2014 and 2017. Patients were followed-up until December 2020. LREs were defined as the development of portal hypertension decompensation and the occurrence of hepatocellular carcinoma (HCC). Serological markers of fibrosis were calculated before treatment and one and two years after SVR. Results: The study included 321 patients, with a median followup of 48 months. LREs occurred in 13.7% of patients (10% portal hypertension decompensation and 3.7% HCC). Child–Pugh [HR 4.13 (CI 95% 1.74; 9.81)], baseline FIB-4 [HR 1.12 (CI 95% 1.03; 1.21)], FIB-4 one year post-SVR [HR 1.31 (CI 95% 1.15; 1.48)] and FIB-4 two years post-SVR [HR 1.42 (CI 95% 1.23; 1.64)] were associated with portal hypertension decompensation. Older age, genotype 3, diabetes mellitus and FIB-4 before and after SVR were associated with the development of HCC. FIB-4 cut-off values one and two years post-SVR to predict portal hypertension decompensation were 2.03 and 2.21, respectively, and to predict HCC were 2.42 and 2.70, respectively. Conclusions: HCV patients with ACLD remain at risk of developing liver complications after having achieved SVR. FIB-4 evaluation before and after SVR may help to predict this risk, selecting patients who will benefit from surveillance. Keywords: hepatitis C virus; new direct-acting antivirals; sustained virological response; liver fibrosis; non-invasive serum fibrosis markers 1. Study Highlights What is known: • Achievement of SVR is associated with clinical benefits, including a reduction in hepatic and extrahepatic manifestations. • The use of non-invasive scores in patients with HCV infection prior to treatment is well established. Although they may be used to aid disease management after SVR, their application after SVR is uncertain, and is not currently recommended. •What is new here: • Determining risk factors for LRE after SVR will optimize patient surveillance after therapy. • Dynamic assessment of the FIB-4 index, before and after SVR, is an adequate predictor of LRE after SVR in HCV-ACLD patients. Viruses 2023,15, 1251. https://doi.org/10.3390/v15061251 https://www.mdpi.com/journal/viruses Viruses 2023,15, 1251 2 of 17 • This study provides cut-offs for the FIB-4 score one and two years after SVR to predict LRE (portal hypertension decompensation and hepatocellular carcinoma) in HCVACLD patients. 2. Introduction Hepatitis C virus (HCV) is one of the most common causes of chronic liver disease worldwide [ 1 ]. According to World Health Organization estimates, approximately 100 million people have been exposed to HCV and 71 million had chronic HCV infection [2]. In the last decade, the introduction of new direct-acting antivirals (DAAs) has revolutionized the natural history of chronic HCV, achieving sustained virological response (SVR) rates of over 95%, regardless of HCV genotype [ 3 ]. However, the residual liver damage that persists in patients with advanced chronic liver disease (ACLD) after SVR has not yet been defined, and the relatively short follow-up after DAA treatments has prevented any firm conclusions from being drawn. Post-SVR surveillance is guided by the presence of pre-therapeutic liver fibrosis and the patient’s comorbidities. Liver biopsy is the gold standard for evaluating fibrosis, but it is an invasive procedure with low applicability. Thus, non-invasive techniques, such as transient elastography (TE) and blood-based biomarkers, have replaced this procedure in recent years. While these methods are useful for estimating liver fibrosis before HCV treatment, they are not recommended in assessing liver fibrosis post-SVR [4]. Post-SVR surveillance is currently universally advocated in patients with cirrhosis [ 5 , 6 ], but it is controversial in patients with advanced fibrosis (F3) [6]. The aim of our study was to assess factors predicting the risk of liver-related events (LREs) in HCV-ACLD patients who achieved SVR with DAA therapy. Dynamic changes in liver fibrosis after SVR were determined by non-invasive methods to optimize patient follow-up. 3. Materials and Methods 3.1. Study Population This was an observational, retrospective, single-center study conducted in Hospital Universitario Virgen Macarena (Seville, Spain). Patients with ACLD who achieved SVR after DAA therapy between 1 November 2014 and 1 December 2017 were included. Followup was carried out until December 2020. Advanced fibrosis was defined as TE > 10 kPa. If these measures were not available, patients were selected by serological marker scores: Fibrosis-4 (FIB-4) > 3.25 and/or aspartate-to-platelet ratio index (APRI) > 1.5. Cirrhosis was defined by TE > 12.5 kPa and/or a combination of endoscopy and imaging criteria (presence of esophageal varices and imaging findings of portal hypertension). Patient history of hepatic decompensation (ascites, hepatic encephalopathy (HE), spontaneous bacterial peritonitis (SBP) or variceal bleeding (VB)) one year previously, or present at treatment initiation, was collected using electronic medical records. Exclusion criteria were (1) age under 18; (2) HCC diagnosis 5 years prior to DAA therapy; (3) Child–Pugh (CP) score > B8; (4) liver transplant; (5) hepatitis B virus (HBV) and/or human immunodeficiency virus (HIV); or (6) patient treated with interferon therapy. Baseline demographic variables, risk behaviors (past or current drug and alcohol use), medical history (comorbidities such as diabetes, hypertension, dyslipidemia and chronic kidney disease), HCV genotype, DAA therapy, CP class and MELD score were collected. SVR was defined as undetectable HCV RNA at 12 weeks after the end of therapy using the Cobas ® 4800 Systems HCV quantitative nucleic acid test (Roche Molecular Diagnostics). During follow-up, abdominal ultrasound and laboratory tests were performed every 6 months. 3.2. Non-Invasive Assessment of Fibrosis and Clinical Outcomes Non-invasive serological methods to assess liver fibrosis, the APRI ((aspartate aminotransferase [AST] [U/L]/upper limit of normal)/platelet count × 100) [ 7 ] and FIB-4 score Viruses 2023,15, 1251 3 of 17 (age [years] × AST [U/L]/(platelet count [10 9 /L] × alanine aminotransferase [ALT] 1/2 [U/L])) [ 7 ] were calculated within 60 days of prior HCV treatment initiation and during follow-up 1 and 2 years after achieving SVR. Liver stiffness measurement (LSM) (FibroScan ® , Echosens) performed within 60 days prior to HCV treatment initiation was recorded. To ensure the quality of measurements, the following criteria had to be fulfilled: (1) at least 10 valid shots; (2) a success ratio (ratio of valid shots to the total number of shots) above 60%; and (3) interquartile range (IQR) ≤30%, as recommended by current guidelines [7]. LREs were defined as portal hypertension decompensation (ascites, VB, HE) and the occurrence of HCC. Liver-related and non-liver-related mortality was also recorded. Patients were followed until the first occurrence of liver-related decompensation, transplantation or death. Only the first event was reported in a single subject. 3.3. Statistical Analysis For the descriptive analysis, qualitative variables are expressed as absolute frequency (N) and percentage (%). Quantitative variables are expressed as mean (standard deviation, SD) and median (interquartile range, IQR). The normality of quantitative variables was tested using the Shapiro–Wilk test. Comparisons between groups for variables with normal distribution were performed using the Student’s ttest (for independent samples), and for variables that do not follow a normal distribution using the Mann–Whitney U test. Additionally, the Chi-square test was used to determine the relationship between categorical variables. Continuity correction on 2 × 2 contingency tables was applied, using Fisher’s exact test. The Kaplan–Meier curve was used to estimate the cumulative individual survival probability over time. Cox regression analyses were performed to identify predictors of liver-related events (LREs). First, univariate analysis was performed to identify statistically significant variables associated with follow-up until the development of portal hypertension decompensation or hepatocellular carcinoma (HCC) (p< 0.15). Multivariate analysis was then performed to select the final Cox models to assess the association of each LRE and each variable included in the model controlled by the rest of the variables. The results were expressed as adjusted hazard ratios (HR) and their 95% confidence intervals (CI). To investigate the predictive ability of repeated measurement of serological markers to determine the risk of developing LRE, sensitivity and specificity were calculated using a receiver operating characteristics (ROC) curve. Analyses were performed using IBM SPSS Statistics 26. 4. Results 4.1. Study Population A total of 573 patients with HCV received treatment with DAAs between 1 November 2014 and 1 December 2017. Of these, 346 had advanced fibrosis or cirrhosis and 336 achieved SVR. Finally, 321 patients met the inclusion criteria and were enrolled in the study (Figure 1). The baseline characteristics of these patients are described in Table 1. Median age was 59.1 (10.6) years and 68.8% (221) were male. Among the main comorbidities, 39.3% (126) had hypertension (HT), 21.5% (69) diabetes mellitus (DM), 13.4% (43) heart disease (HD), 6.5% (21) dyslipidemia (DL) and 4.4% (14) chronic kidney disease (RCD). Past (22.9%) or current (15%) alcohol intake and history of drug use (24.3%) was reported. Most patients had HCV genotype 1 (77.6%). Viruses 2023,15, 1251 4 of 17 Viruses 2023, 15, x FOR PEER REVIEW 4 of 17 Figure 1. Study flowchart. CP: Child–Pugh; HD: hepatic decompensation; SVR: sustained virological response. Table 1. Baseline characteristics of the patients included in the study. Characteristics N (%)/Me CI (95%)/P25; P75 Sex Male Female 221 (68.8) 100 (31.2) 63.8; 73.9 26.1; 36.3 Age 57 52.0; 67.5 Alcohol user Yes Ex 48 (15.0) 73 (22.9) 11.1; 19.0 18.3; 27.5 IDUs Yes Ex-IDUs 5 (1.6) 77 (24.3) 0.2; 3.0 19.5; 29.0 HT 126 (39.3) 33.9; 44.6 DM 69 (21.5) 17.0; 26.0 DL 21 (6.5) 3.8; 9.3 CKD 14 (4.4) 2.1; 6.6 Figure 1. Study flowchart. CP: Child–Pugh; HD: hepatic decompensation; SVR: sustained virological response. 4.2. Follow Up and Liver-Related Events The median follow-up was 48 (37; 56.0) months. Twenty-two patients died during this period (6.85%), and liver-related mortality occurred in ten patients. Mean follow-up until death was 30 (17.5; 41.0) months. LREs occurred in 44 patients (13.7%). None of the patients with advanced fibrosis (F3) developed LREs during follow-up. The most frequent was portal hypertension decompensation, which was present in 32 patients (10%). The incidence rate for decompensation was 2.30/100 patient years. HCC occurred in 12 patients (3.7%), with an incidence rate of 0.86/100 patient years. Finally, four patients (1.25%) required liver transplantation. Viruses 2023,15, 1251 5 of 17 Table 1. Baseline characteristics of the patients included in the study. Characteristics N (%)/Me CI (95%)/P25; P75 Sex Male Female 221 (68.8) 100 (31.2) 63.8; 73.9 26.1; 36.3 Age 57 52.0; 67.5 Alcohol user Yes Ex 48 (15.0) 73 (22.9) 11.1; 19.0 18.3; 27.5 IDUs Yes Ex-IDUs 5 (1.6) 77 (24.3) 0.2; 3.0 19.5; 29.0 HT 126 (39.3) 33.9; 44.6 DM 69 (21.5) 17.0; 26.0 DL 21 (6.5) 3.8; 9.3 CKD 14 (4.4) 2.1; 6.6 HD 43 (13.4) 9.7; 17.1 Genotype 1 2 3 4 249 (77.6) 4 (1.2) 44 (13.7) 24 (7.5) 73.0; 82.2 0.03; 2.5 9.9; 17.5 4.6; 10.4 DAA treatment 1. Sofosbuvir + Daclatasvir 2. Simeprevir + Sofosbuvir 3. Sofosbuvir/Ledipasvir 4. Ombitasvir/Paritaprevir/Ritonavir + Dasabuvir 5. Sofosbuvir/Veltapasvir 6. Elbasvir/Grazoprevir 7. Glecaprevir/Pibrentasvir 8. Sofosbuvir/Velpatasvir/Voxilaprevir 19 (7.6) 30 (12.0) 144 (57.6) 24 (9.6) 9 (3.6) 23 (9.2) 1 (0.4) 0 4.3; 10.9 7.9; 16.1 51.4; 63.8 5.9; 13.3 1.3; 5.9 5.6; 12.8 0; 1.2 0 Child–Pugh A5 A6 B7 B8 262 (83.7) 15 (4.8) 28 (8.9) 8 (2.6) 79.6; 87.8 2.4; 7.2 5.8; 12.1 0.8; 4.3 MELD 7 7; 8 TE (kPa) 15.4 12.0; 23.0 FIB-4 2.9 1.8; 5.3 APRI 1.2 0.7; 23 Previous decompensation 32 (10.0) 7; 14 APRI: aspartate-to-platelet ratio index; CI: confidence interval; CKD: chronic kidney disease; CP: Child–Pugh; DAA: direct-acting antivirals; DL: dyslipemia; DM: diabetes mellitus; HD: heart disease; HT: hypertension; IDUs: intravenous drug users; Me: median; N: number of patients; TE: transient elastography. 4.3. Factors Associated with Portal Hypertension Decompensation The median follow-up until the onset of hepatic decompensation was 48 (35.5; 56.8) months. The baseline characteristics of these patients are shown in Table S1. The type and frequency of hepatic decompensation developed during follow-up are shown in Figure 2. Viruses 2023,15, 1251 6 of 17 Viruses 2023, 15, x FOR PEER REVIEW 6 of 17 Figure 2. Portal hypertension decompensations developed during follow-up. Univariate analysis revealed that patients who developed portal hypertension decompensations during follow-up had worse liver function according to the CP score, had presented hepatic decompensation before treatment, had higher FIB-4 at baseline and after SVR (1 year and 2 years post-SVR) and higher APRI at baseline and 2 years post-SVR (Table S2). According to multivariate analysis, at baseline, the CP score (hazard ratio (HR) 4.13 [1.74; 9.81]; p < 0.001) and FIB-4 (HR 1.12 [1.03; 1.21]; p = 0.006) were significantly associated with portal hypertension decompensation. At follow-up, only FIB-4 evaluated at any time (1 year and 2 years post-SVR) was associated with portal hypertension decompensation (Table 2). Table 2. Factors associated with risk of developing portal hypertension decompensation (multivariate analysis). Portal hypertension decompensation Characteristics HR CI (95%) p Model 1 Child–Pugh A B 1 4.13 1.74; 9.81 <0.001 FIB-4 (baseline) 1.12 1.03; 1.21 0.006 Model 2 Child–Pugh A B 1 3.14 1.24; 7.94 0.016 FIB-4 (1 year) 1.31 1.15; 1.48 <0.001 Model 3 Child–Pugh A B 1 2.84 1.16; 6.95 0.023 FIB-4 (2 years) 1.42 1.23; 1.64 <0.001 CI: confidence interval; DM: diabetes mellitus; GT: genotype; HR: hazard ratio. 4.4. Factors Associated with HCC The mean follow-up until HCC developed was 28.25 months (SD 4.33; 95% confidence interval (CI) 19.76–36.74). The baseline characteristics of these patients are shown in Table S2. In the univariate analysis, the development of HCC was associated with older age, genotype 3, DM, CP score, FIB-4 score at baseline and after SVR (1 year and 2 years post- SVR) and APRI index assessed 2 years post-SVR (Table S2). Figure 2. Portal hypertension decompensations developed during follow-up. Univariate analysis revealed that patients who developed portal hypertension decompensations during follow-up had worse liver function according to the CP score, had presented hepatic decompensation before treatment, had higher FIB-4 at baseline and after SVR (1 year and 2 years post-SVR) and higher APRI at baseline and 2 years post-SVR (Table S2). According to multivariate analysis, at baseline, the CP score (hazard ratio (HR) 4.13 [1.74; 9.81]; p< 0.001) and FIB-4 (HR 1.12 [1.03; 1.21]; p= 0.006) were significantly associated with portal hypertension decompensation. At follow-up, only FIB-4 evaluated at any time (1 year and 2 years post-SVR) was associated with portal hypertension decompensation (Table 2). Table 2. Factors associated with risk of developing portal hypertension decompensation (multivariate analysis). Portal Hypertension Decompensation Characteristics HR CI (95%) p Model 1 Child–Pugh A B 1 4.13 1.74; 9.81 <0.001 FIB-4 (baseline) 1.12 1.03; 1.21 0.006 Model 2 Child–Pugh A B 1 3.14 1.24; 7.94 0.016 FIB-4 (1 year) 1.31 1.15; 1.48 <0.001 Model 3 Child–Pugh A B 1 2.84 1.16; 6.95 0.023 FIB-4 (2 years) 1.42 1.23; 1.64 <0.001 CI: confidence interval; DM: diabetes mellitus; GT: genotype; HR: hazard ratio. 4.4. Factors Associated with HCC The mean follow-up until HCC developed was 28.25 months (SD 4.33; 95% confidence interval (CI) 19.76–36.74). The baseline characteristics of these patients are shown in Table S2. In the univariate analysis, the development of HCC was associated with older age, genotype 3, DM, CP score, FIB-4 score at baseline and after SVR (1 year and 2 years post-SVR) and APRI index assessed 2 years post-SVR (Table S2). According to the multivariate analysis, at baseline, older age (HR 1.13 [1.05; 1.22]; p= 0.001 ), genotype 3 (HR 33.18 [6.24; 176.50]; p< 0.001), DM (HR 4.92 [1.26; 19.22]; p= 0.022 ) and Viruses 2023,15, 1251 7 of 17 FIB-4 (HR 1.15 [1.04; 1.27]; p= 0.005) were significantly associated with the development of HCC. At follow-up, FIB-4 remained a good predictor of HCC, 1 year post-SVR (HR 1.38 [1.09; 1.75]; p= 0.026) and 2 years post-SVR (HR 1.53 [1.21; 1.95]; p< 0.001) (Table 3). Table 3. Factors associated with risk of developing HCC (multivariate analysis). HCC Characteristics HR CI (95%) p Model 1 Age 1.13 1.05; 1.22 0.001 Genotype Others GT 3 1 33.18 6.24; 176.50 <0.001 DM No Yes 1 4.92 1.26; 19.22 0.022 FIB-4 (baseline) 1.15 1.04; 1.27 0.005 Model 2 Age 1.14 1.06; 1.23 0.001 Genotype Others GT 3 1 34.86 6.29; 193.15 <0.001 DM No Yes 1 4.89 1.21; 19.78 0.026 FIB-4 (1 year) 1.38 1.09; 1.75 0.026 Model 3 Age 1.10 1.02; 1.18 0.010 Genotype Others GT 3 1 36.69 6.73; 199.90 <0.001 DM No Yes 1 6.84 1.59; 29.33 0.010 FIB-4 (2 years) 1.53 1.21; 1.95 <0.001 CI: confidence interval; DM: diabetes mellitus; GT: genotype; HCC: hepatocellular carcinoma; HR: hazard ratio. 4.5. Implications of Serological Marker Levels after SVR on the Risk of Developing LRE Dynamic changes in serological marker (FIB-4 and APRI) levels were observed in the assessment before and after SVR, but not on repeated measurements after SVR. This pattern appears both in patients who did not develop any LRE and in those who did (Figure 3). Additionally, the FIB-4 score assessed at any time during follow-up was associated with the risk of developing any LRE, and therefore we focused on this biomarker. Patients who developed portal hypertension decompensation during follow-up also showed a higher FIB-4 score after SVR than patients who did not (1 year post-SVR: 3.9 vs. 1.5; p< 0.001/2 years post-SVR: 3.8 vs. 1.5; p< 0.001) (Figure S2). Similarly, patients who developed HCC during follow-up had higher FIB-4 levels after SVR than patients who did not (1 year post-SVR: 3.47 vs. 1.56; p< 0.007/2 years post-SVR: 4.2 vs. 1.6; p< 0.001) (Figure 3). In a receiver operating characteristic (ROC) analysis, the FIB-4 cut-offs associated with the risk of developing portal hypertension decompensation were 2.03 (area under the curve (AUC) 0.824; 95% CI 0.754; 0.903) and 2.21 (AUC 0.819; 95% CI 0.733; 0.905) 1 and 2 years post-SVR, respectively. A significant difference in the progression rates of portal hypertension decompensation was demonstrated between patients with FIB-4 ≥ 2.03 1 year post-SVR and FIB-4 ≥ 2.21 2 years post-SVR than those with lower values ( p< 0.001 ) (Figure 4). Viruses 2023,15, 1251 8 of 17 Viruses 2023, 15, x FOR PEER REVIEW 8 of 17 Figure 3. Changes in serological markers during follow-up in patients who developed liver-related events and in those who did not develop them: portal hypertension decompensation (A), HCC (B). (A) (B) Figure 3. Changes in serological markers during follow-up in patients who developed liver-related events and in those who did not develop them: portal hypertension decompensation ( A ), HCC ( B ). HCC: hepatocellular carcinoma. The circles represent extreme values (>1.5 times the interquartile range). Asterisks (*) represent very extreme values (>3 times the interquartile range). Viruses 2023,15, 1251 9 of 17 Viruses 2023, 15, x FOR PEER REVIEW 9 of 17 HCC: hepatocellular carcinoma. The circles represent extreme values (>1.5 times the interquartile range). Asterisks (*) represent very extreme values (>3 times the interquartile range). In a receiver operating characteristic (ROC) analysis, the FIB-4 cut-offs associated with the risk of developing portal hypertension decompensation were 2.03 (area under the curve (AUC) 0.824; 95% CI 0.754; 0.903) and 2.21 (AUC 0.819; 95% CI 0.733; 0.905) 1 and 2 years post-SVR, respectively. A significant difference in the progression rates of portal hypertension decompensation was demonstrated between patients with FIB-4 ≥ 2.03 1 year post-SVR and FIB-4 ≥ 2.21 2 years post-SVR than those with lower values (p < 0.001) (Figure 4). Figure 4. Cumulative incidence of portal hypertension decompensation according to FIB-4 evaluated 1 year post-SVR (A) and 2 years post-SVR (B). 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