Who Should Not Be Surveilled for HCC Development after Successful Therapy with DAAS in Advanced Chronic Hepatitis C? Results of a Long-Term Prospective Study

Background and aims: The identification of patients with Hepatitis C Virus (HCV)-positive advanced chronic liver disease (aCLD) successfully treated by Direct Acting Antiviral Agents (DAAs) who really benefit from Hepatocellular Carcinoma (HCC) surveillance programs is still a matter of debate. We performed a long-term prospective cohort study on F3-F4 HCV-positive patients achieving Sustained Virologic Response (SVR) after DAAs treatment in order to identify patients who can safely suspend surveillance. Methods: 1000 patients with HCV-positive aCLD obtaining SVR by DAAs from January 2015 to December 2017 were divided into four groups according to baseline elastographic, ultrasonographic, clinical and biochemical features: (1) Group 1: 324 patients with Liver Stiffness Measurement (LSM) ≥ 9.5 ≤ 14.5 kPa, FIB-4 < 3.25 and APRI < 1.5 (2) Group 2: 133 patients with LSM ≥ 9.5 ≤ 14.5 kPa, FIB-4 ≥ 3.25 and/or APRI ≥ 1.5 (3) Group 3: 158 patients with LSM > 14.5 kPa, FIB-4 < 3.25 and APRI < 1.5 (4) Group 4: 385 patients with LSM > 14.5 kPa, FIB-4 ≥ 3.25 and/or APRI ≥ 1.5. FIB-4 and APRI scores were calculated at baseline and at SVR achievement. Each patient was surveiled twice-yearly by ultrasound for a median follow-up of 48 months. Results: among Group 1 patients, 1/324 (0.3%) developed HCC (0.09/100 patients/year [PY]), compared to 6/133 (4.5%) Group 2 patients (1.22/100 PY, p = 0.0009), 10/158 (6.3%) Group 3 patients (1.68/100 PY, p = 0.0001), 54/385 (14.0%) Group 4 patients (4.01/100 PY, p < 0.0001). HCC incidence was significantly lower in Group 2 compared to Group 3 (p = 0.004) and in Group 3 compared to Group 4 (p = 0.009). HCC risk fell in patients showing a decrease of FIB-4/APRI scores. Conclusions: the risk of HCC occurrence is negligible in about 90% of HCV-positive patients with baseline LSM ≥ 9.5 ≤ 14.5 kPa plus FIB-4 < 3.25 and APRI < 1.5 achieving SVR. Among this particular subset of patients, FIB-4/APRI scores may represent an accurate and inexpensive tool to distinguish patients not needing long-term HCC surveillance.


Lay Summary
International guidelines are divergent regarding the long-term HCC surveillance in F3 patients cured from HCV infection. The European Association for the Study of the Liver (EASL) recommends twice yearly abdominal ultrasounds in both F3 and F4 patients, while the American Association for the Study of the Liver Diseases (AASLD) recommends surveillance only in those with cirrhosis.
According to our results, the vast majority of F3 patients obtaining SVR can be safely suspended from HCC surveillance if well defined at baseline for clinical, biochemical and ultrasonographic parameters.
A not negligible absolute HCC risk persists in cirrhotic patients even if cured from HCV infection, and more data are needed before establishing definitive algorithms able to drive cost-effective strategies of HCC surveillance on an individual basis.
Due to Italian National Health System rules, since the introduction of DAAs in Italy from 2015 to 2017, only patients with advanced liver fibrosis (F3-F4 according to METAVIR Score [12]) could be reimbursed for treatment with DAAs; for this reason, all consecutive patients with HCV-positive aCLD referred to the Gastrohepatologic Clinic of Molinette Hospital, Turin, Italy, for DAAs therapy (sofosbuvir-based 87%) according to the EASL guidelines [13] between 1 January 2015 and 31 December 2017 were considered.
Inclusion criteria were as follows: >18 years; positive HCV-RNA by polymerase chain reaction (PCR); F3 and F4 liver fibrosis assessed by Transient Elastography (TE) within 3 months prior to inclusion into the study. Exclusion criteria were lack of written informed consent, TE not feasible or unreliable measurement, patients on waiting list for orthotopic liver transplant (OLT), post-OLT patients, past or current history of HCC, concomitant liver diseases such as haemochromatosis, Wilson's disease, drug-related liver disease, autoimmune hepatitis, HBsAg carriership, Human immunodeficiency Virus (HIV) infection, primary biliary cholangitis, and alpha-1-antitrypsin deficiency.
Patients were considered cirrhotic if showing LSM > 14.5 kPa by TE; stage 3 liver fibrosis was established by LSM values ranging from 9.5 to 14.5 kPa [14,15], but patients with a nodular liver surface, splenomegaly, porto-systemic collaterals detected by abdominal ultrasound (US), platelets < 120 × 10 9 /L, previous or current history of ascites, encephalopathy and variceal bleeding, or esophageal varices detected by endoscopy were considered cirrhotic [16], irrespective of their baseline LSM value. The reliability criteria for LSM were as follows: 10 valid measurements achieved with a success number ≥60% and an interquartile range-to-median ratio ≤30%. A minimum of 3 h fasting was required.
Out of 1188 consecutive patients treated with DAAs, 146 (12.2%) were excluded (HBV and/or HIV carriers, past or current history of HCC, pre/post-OLT patients, TE not feasible, lack of informed consent). Of the remaining 1042 patients, 23 (2.2%) did not achieve SVR (negative viremia 12 weeks after the end of treatment) and were excluded from the study. Out of 1019 sustained responders, 19 (1.9%) were lost to follow up. Therefore, the final analysis was performed in 1000 patients with SVR. The patient flow is reported in Figure 1.
considered cirrhotic [16], irrespective of their baseline LSM value. The reliability criteria for LSM were as follows: 10 valid measurements achieved with a success number ≥60% and an interquartile range-to-median ratio ≤30%. A minimum of 3 h fasting was required.
Out of 1188 consecutive patients treated with DAAs, 146 (12.2%) were excluded (HBV and/or HIV carriers, past or current history of HCC, pre/post-OLT patients, TE not feasible, lack of informed consent). Of the remaining 1042 patients, 23 (2.2%) did not achieve SVR (negative viremia 12 weeks after the end of treatment) and were excluded from the study. Out of 1019 sustained responders, 19 (1.9%) were lost to follow up. Therefore, the final analysis was performed in 1000 patients with SVR. The patient flow is reported in Figure 1. At baseline, a complete medical history and physical examination was undertaken and the following data were obtained from each patient: age, gender, ethnicity, smoking habits, alcohol intake, body mass index (BMI), waist circumference (WC), duration of At baseline, a complete medical history and physical examination was undertaken and the following data were obtained from each patient: age, gender, ethnicity, smoking habits, alcohol intake, body mass index (BMI), waist circumference (WC), duration of HCV infection, and relevant co-morbidities. The following data on laboratory parameters were also recorded to define baseline characteristics: complete blood count, routine liver biochemistry (alanine aminotransferase [ . We adopted an algorithm based on BMI, waist circumference, triglycerides and GGT to detect and graduate baseline liver steatosis by the fatty liver index [17]: a significant liver steatosis was associated with patients showing a cut-off ≥ 60. For each patient, we calculated FIB-4 [(Age (years)) × AST (U/L)/(Platelets (10 9 /L)\1 × \2 √ ALT (U/L))] [18] and APRI [(AST/AST upper limit normal)/Platelets (10 9 /L) × 100] [19] scores at baseline and at SVR achievement. An abdominal ultrasound (US) was performed on each patient at baseline in order to exclude HCC. Cirrhotic patients were stratified according to the Child-Turcotte-Pugh classification and Model for End-Stage Liver Disease [MELD] and underwent endoscopy to verify the presence of esophageal varices. Patients were divided into 4 groups according to their baseline characteristics: (1) Group 1: patients with LSM ≥ 9.5 ≤ 14.5 kPa and FIB-4 < 3.25 and APRI < 1.5 (2) Group 2: patients with LSM ≥ 9.5 ≤ 14.5 kPa and FIB-4 ≥ 3.25 or APRI ≥ 1.5 (3) Group 3: patients with LSM > 14.5 kPa or clinical/biochemical/US signs of cirrhosis and FIB-4 < 3.25 and APRI < 1.5 (4) Group 4: patients with LSM > 14.5 kPa or clinical/biochemical/US signs of cirrhosis and FIB-4 ≥ 3.25 or APRI ≥ 1. 5 We used the FIB-4 cut-off of ≥ 3.25 due to its positive predictive value of 82.1% for advanced fibrosis [20]; in order to better classify patients showing an indeterminate range (1.45-3.25), we used the APRI cut-off ≥ 1.5 to individuate carriers of significant fibrosis [21].
An abdominal US was planned at 6-month intervals and patients were followed until OLT, death or until the end of December 2021. Patients were also censored at the moment of HCC diagnosis, performed according to international guidelines [22,23] by histological examination or by contrast-enhanced imaging methods showing hypervascularity in late arterial phase and washout on portal venous and/or delayed phases. They were followedup for a median of 48 (IQR: 36-60) months after achieving SVR.
The study was performed in accordance with the principles of the Declaration of Helsinki and approved by the local ethics committee (Comitato Etico Interaziendale Città della Salute e della Scienza di Torino, Turin, Italy, n 452), and written informed consent was obtained from all patients.

Statistical Analysis
Continuous variables were reported as median (inter-quartile range (IQR)). Normality was checked by the D'Agostino-Pearson test. Categorical variables were reported as number and percentage. Comparison of continuous variables between independent groups was performed by the Mann-Whitney test. Regarding the dichotomous categorical variable, a Chi-squared test was performed for unpaired analysis. Comparison of Kaplan-Meier survival curves was performed using the Logrank test. The association between variables was assessed by Cox proportional hazards, the strength of association was reported as hazards ratio (H.R.) and 95% CI, respectively.

Impact of Change in FIB-4/APRI on HCC Risk
Due to the very small proportion (0.8%) of patients showing an increase in FIB-4/APRI scores after achieving SVR, we focused on Group 2 and 4 patients and we observed that the risk of incident HCC was reduced among patients who experienced a decrease of FIB-4/APRI scores below 3.25 and 1.5, respectively. In Group 2, two out of 85 (2.4%) patients showing a decline in FIB-4/APRI developed HCC compared with 4/48 (8.3%) patients with scores above the cut-offs, corresponding to an IR of 0.6/100 PY vs. 2.8/100 PY and a CIR at 12, 36 and 60 months of 1,2%, 2.6% and 2.6%, respectively, compared to 4.7%, 4.7% and 14.3%, p = 0.049 (Figure 4), respectively. In Group 4, 12/167 (7.2%) patients with a significant decrease in FIB-4/APRI scores showed "de novo" HCC compared to 42/218 (19.3%) patients with no relevant changes; HCC IR was 1.7/100 PY in the first sub-group versus 6.4/100 PY in the second one, with a CIR of 0.6%, 5.8% and 8.1% compared with 7.2%, 15.9%, 26.8%, respectively, p < 0.0001 ( Figure 5). Baseline characteristics of patients stratified according to their improvement in FIB-4/APRI scores are reported in Table 4

Impact of Change in FIB-4/APRI on HCC Risk
Due to the very small proportion (0.8%) of patients showing an increase in FIB-4/APRI scores after achieving SVR, we focused on Group 2 and 4 patients and we observed

Discussion
The current International Guidelines [2,3] diverge on the surveillance strategy regarding patients with aCLD achieving SVR, in particular those ones with baseline F3 fibrosis. The EASL [3] recommends long-term US surveillance in F3 patients, whereas

Discussion
The current International Guidelines [2,3] diverge on the surveillance strategy regarding patients with aCLD achieving SVR, in particular those ones with baseline F3 fibrosis. The EASL [3] recommends long-term US surveillance in F3 patients, whereas AASLD does not [2]. This divergence is mainly based upon the difficulty in establishing an accurate diagnosis of F3 fibrosis: liver biopsy can underestimate the presence of cirrhosis in patients histologically labeled as F3 [24], while TE is unable to distinguish F3 fibrosis from cirrhosis with certainty [25]. In order to better stratify the neoplastic risk in this particular subset of patients, recent studies [9][10][11] have tried to associate algorithms composed by demographic, clinical, biochemical risk factors to the histological or elastographic assessment, but to date none of them have been officially recommended.
According to our data, the HCC incidence in patients with LSM ≥ 9.5 ≤ 14.5 kPa achieving SVR is negligible (0.09/100 PY) in the vast majority of them after a median follow-up of 4 years; the identification of this low-risk group can be performed easily before treatment by associating clinical, biochemical variables and US imaging to LSM. Moreover, a significant decline in FIB-4/APRI scores after SVR achievement in this particular subset of patients with high baseline values is associated with a decreased risk of HCC (0.6/100 PY), enabling the individuation of additional patients at low risk of HCC. Overall, 90% of patients with LSM ≥ 9.5 ≤ 14.5 kPa were identified as having an HCC risk < 1%/year, well below the cost-effectiveness cut-off for HCC surveillance. On the other hand, the remaining 10% of patients with persistently high FIB-4/APRI scores were probably carriers of undiagnosed cirrhosis, therefore needing an HCC surveillance program due to the relevant HCC incidence found (2.8/100 PY).
Our results confirmed what was already found by Kanwal et al. [10], who showed that the HCC risk remained above the accepted threshold for surveillance in cirrhotic patients, even in the case of significant decline in FIB-4/APRI scores after therapy. However, at variance with our data, a recent study [11] showed that a post-treatment algorithm combining age/albumin/TE and, optionally, alfafetoprotein and alcohol intake enables a reliable risk stratification for the development of HCC, even among cirrhotic patients, identifying a large proportion of patients with an HCC risk < 1%/year, below the threshold considered to be cost-effective. Cost-effectiveness analyses regarding HCC surveillance changed over time; Cucchetti et al. [39] quoted an annual HCC risk threshold > 1.5%, above which HCC screening was cost-effective, but recent studies [40,41] focusing on patients achieving SVR suggested lower thresholds, from 1.32/100 PY up to 0.5/100 PY. According to Mueller et al. [42], HCC surveillance is cost-effective until the age of 70 for cirrhotic patients cured of hepatitis C and until the age of 60 for patients with non-cirrhotic advanced fibrosis. Irrespective of the thresholds considered, we acknowledge that FIB-4/APRI-based algo-rithms may not be the best method to stratify the HCC risk in cirrhotic patients achieving SVR; we have recently compared [43] different non-invasive scoring systems (the Forns index, FIB-4, albumin-bilirubin score [ALBI], age/gender/albumin-bilirubin/platelets score [aMAP]) in cirrhotics successfully treated by DAAs, and we found that ALBI showed the highest diagnostic accuracy for the detection of HCC, confirming previous data [44][45][46]. However, given the controversies regarding the markers and algorithms which should be adopted, we think that results from large prospective studies with longer follow-up are still needed before incorporating definite algorithms into official recommendations regarding the optimal HCC surveillance strategy in a cirrhotic population cured from HCV infection.
Very similar to what was previously reported [47], death occurred in about 6% of our patients due to liver-related causes in the majority of them (70%); however, the mortality rate was significantly lower among Group 1 patients compared with Group 3-4 patients, confirming the excellent clinical outcome of F3 patients with low FIB-4/APRI scores obtaining SVR. The strengths of our study are the prospective design, the large sample size with long-term follow-up, and the very low rate of patients lost to follow-up. However, our study was conducted in a single academic centre mainly recruiting Caucasian patients without a validation cohort, therefore limiting the reproducibility of our conclusions, and multicenter prospective studies are needed in order to make a solid recommendation.
Moreover, we acknowledge the possibility that some cirrhotic patients could have been included in Group 1 and 2, due to the wide range of kPa adopted to define F3 patients and despite the accurate clinical and ultrasonographic screening, however, according to our results, our approach would still work for these patients.
In conclusion, the great majority of patients with LSM ≥ 9.5 ≤ 14.5 kPa achieving SVR can be safely suspended from HCC surveillance if well defined at baseline for clinical, biochemical and US parameters, which are all currently used, before starting antiviral therapy with no cost increases. On the other hand, a word of caution is still necessary regarding the withdrawal or deferral of HCC surveillance in cirrhotic patients cured from HCV infection, as a late diagnosis of HCC implies severe consequences for an individual patient.

Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Ethics Committee of the Città della Salute e della Scienza-University Hospital of Turin, Italy (2CEI/452).

Informed Consent Statement:
Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.