Long-term Outcomes of Ledipasvir/sofosbuvir Treatment of Chronic Hepatitis C in Four Teenagers With Signicant Liver Fibrosis

Long-term outcomes of therapy with ledipasvir/sofosbuvir (LDV/SOF) in children with chronic hepatitis C (CHC) and signicant liver brosis were evaluated. Among 35 patients aged 12-17 years treated with LDV/SOF, there were four participants with signicant brosis: one with a brosis score of F2 in the METAVIR scale, and three with cirrhosis (F4) evaluated with transient elastography (TE) at baseline. Patients were followed every 4 weeks during the treatment, at the end of the therapy, week 12 posttreatment, and one year after the end of treatment. One year after the end of treatment, the hepatitis C viral load was undetectable in three patients. In 2/4 patients, a signicant regression of liver brosis was observed (from stage F4 and F2 to F0-F1 in the METAVIR scale). In one patient, the liver stiffness measurement median increased 12 weeks after the end of the treatment and then decreased but still correlated with stage F4. One patient was lost to follow-up after week 4. A one-year observation of teenagers with CHC and signicant brosis treated with LDV/SOF revealed that regression of liver brosis is possible, but not obligatory. Further observations in larger groups of patients are necessary to nd predictors of liver brosis regression. FIB-4 (56.5%) mild in LSM, in the APRI and FIB-4 2 of 4 patients, signicant improvement and regression of liver brosis revealed (from stage F4 and F2 to F0-F1 on the METAVIR scale) one year after the end of LDV/SOF therapy. In one patient, the LSM 12 weeks after the end of the treatment and then decreased, but it still correlated to stage F4. in the APRI observed.


Introduction
In the majority of cases, hepatitis C virus (HCV) infection in children leads to chronic hepatitis C (CHC) with a possible progression to serious liver disease. The risk of cirrhosis in HCV-infected children and adolescents was estimated at 1 to 2% [1][2][3][4][5] . However, a number of recent observational studies have demonstrated that a higher proportion of pediatric patients than had been previously thought may develop advanced liver disease resulting from HCV infection [6][7][8]  patients infected with HCV during childhood and found that serious liver disease developed in 32% of cases with a median of 33 years after infection, irrespective of the mode of infection 8 . Thus, children infected vertically developed cirrhosis at an earlier age compared to patients infected from other sources, which is consistent with other observations 4 . The risk of HCV-related hepatocellular carcinoma (HCC) was 5%, the incidence of liver transplant was 4%, and the risk of death was 3% 8 . New highly effective interferon-free therapies for HCV infection based on direct-acting antivirals (DAAs) may be helpful for the prevention of long-term liver disease progression related to HCV by eliminating the virus 8,9 . In a recent systematic review with meta-analysis on the e cacy and safety of DAAs in children and adolescents, Indol et al. demonstrated that among the patients receiving all doses of treatment, 100% of cases reached a sustained virologic response (SVR) 9 . Among the subjects receiving at least one dose of DAA, the lowest e cacy rates were observed among cirrhotic patients (83%) 9 . However, only a few outcomes of DAA treatment in cirrhotic pediatric patients have been reported, and there is scarce and limited data on the in uence of these therapies on liver brosis 9,10 . We recently reported that among our 35 patients aged 12-17 years qualifying for ledipasvir/sofosbuvir (LDV/SOF) treatment, 11% of cases presented with signi cant brosis (F ≥ 2 in METAVIR scale), including 9% with cirrhosis 6 . Thus, in this paper, we aim to present the long-term outcomes of therapy in this speci c group of patients. In particular, the in uence of LDV/SOF treatment on the extent of liver brosis was analyzed.

Study group
In our single tertiary health care pediatric infectious disease center, consecutive HCV-infected patients aged 12-17 years treated between August 2019 and February 2020 were quali ed for the real-life therapeutic program 'Treatment of Polish Adolescents with Chronic Hepatitis C Using Direct Acting Antivirals (POLAC PROJECT)'. Patients infected with genotypes 1 and 4 HCV were treated with sofosbuvir/ledipasvir (SOF/LDV), which was available courtesy of a donation by the pharmaceutical company. Patients with CHC (diagnosed in subjects with over a 6-month duration of disease con rmed with positive nucleic acid testing, HCV RNA, using a quantitative real-time polymerase chain reaction, RT-PCR, Abbott Real Time HCV, Abbott Laboratories, Abbott Park, Illinois, USA; measurement linearity range 12-1.0 x 10 8 IU/ml) were quali ed for treatment irrespective of the extent of liver brosis or previous ineffective treatment. The duration of treatment was established according to the recommendations of the European Society of Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN): patients received 12 weeks of therapy unless they were infected with HCV genotype 1 with a history of previous ineffective interferon-based treatment and presented with cirrhosis 11 . Patients in this study were followed every 4 weeks during the treatment, at the end of the therapy, week 12 posttreatment (to assess the e cacy of the treatment based on a sustained virologic response, SVR12), and one year (52 weeks) after the end of treatment to assess long-term outcomes. Body mass index standard deviation (SD) scores (BMI z-scores) were calculated according to the WHO Child Growth Standards and Growth reference data using the WHO Anthropometric calculator AnthroPlus v.1.0.4. Obesity was diagnosed in children with a BMI z-score > 2 SD and overweight > 1 SD.
In this prospective study, we analyzed only a group of patients presenting with signi cant brosis (F ≥ 2 in METAVIR scale) established by transient elastography (TE) at the start of the treatment.

Transient elastography (TE)
TE was performed by trained examiners certi ed by the manufacturer, using the FibroScan device (Echosens, Paris, France). Two different probes were used: medium (M) and XL (for obese patients). TE was performed in patients who had fasted for at least 2 hours. Liver stiffness measurement (LSM) and controlled attenuation parameter (CAP) were simultaneously obtained. The adequacy of the measurement was assessed by the FibroScan device. The examination was considered successful when 10 valid measurements were conducted with at least a 60% success rate and an interquartile range (IQR) of less than 30% of the median LSM value 12 . The nal LSM result was expressed as the median value of at least 10 valid measurements, and it was assessed in kilopascals (kPa). It corresponded to the liver brosis in the METAVIR scale according to the Castera TE cutoffs as follows: no to mild brosis (F0-F1), LSM up to 7.0 kPa; moderate brosis (F2), LSM 7.1 to 9.4 kPa; severe brosis (F3), LSM 9.5 to 12.4 kPa; and cirrhosis (F4), LSM ≥ 12.5 kPa 13 . Liver brosis was considered signi cant if the LSM median was > 7 kPa, corresponding to a METAVIR F score ≥ 2 points. The nal CAP values ranged between 100 and 400 decibels per meter (dB/m) and were assigned as follows: no steatosis (S0, CAP 0-238 dB/m); mild steatosis (S1, CAP 239-260 dB/m); moderate steatosis (S2, CAP 261-290 dB/m); and severe steatosis (S3, CAP > 290 dB/m) 10,14 . TE examination was performed three times: on the day the patient started treatment, week 12 posttreatment, and one year after the end of the treatment, simultaneously with the biomarker evaluation.

Biomarker evaluation
Biochemical serum testing was performed using commercially available laboratory kits. For both alanine and aspartate aminotransferase (ALT and AST) serum levels, 40 IU/L was considered the upper limit of normal (ULN). Two indirect brosis biomarkers were calculated, namely, the aspartate transaminase-to- According to previously published data, the following cutoffs for the biomarkers were considered: APRI > 0.5 and FIB-4 > 1.45, which suggests signi cant brosis, and APRI > 1.5, which suggests cirrhosis 15,17 .

Ethical Statement
Written informed consent was collected from all the patients and/or their parents/guardians before their inclusion in the study. The investigation was performed in accordance with the ethical standards in the 1964 Declaration of Helsinki and its later amendments. The local ethics committee by the Medical University of Warsaw approved this study.

Participants
Among the 35 patients included in this therapeutic program, there were four patients aged 12-17 years with signi cant brosis: one presenting with a brosis score of 2 on the METAVIR scale and three with cirrhosis (F4 on the METAVIR scale). Their Child-Pugh class was A for all three cirrhotic patients. All four patients were infected vertically from an HCV-infected mother. Three subjects were infected with genotype 1b HCV and one with genotype 4. Patient 2 was coinfected with human immunode ciency virus (HIV). All patients were treated with a xed dose of 90 mg/200 mg LDV/SOF. Two patients were quali ed for a 12-week treatment, and two for a 24-week therapy. The baseline characteristics of these patients are presented in Table 1.

Treatment outcomes
Four weeks after the start of treatment, HCV viral load was below the lower limit of detection in three patients and undetectable in one patient. A decrease in the ALT level was observed in all four patients. At the end of the treatment, two patients had an undetectable HCV viral load, and it was unavailable in the remaining two subjects because their visits had to be cancelled due to the coronavirus disease-19 (COVID-19) pandemic. A similar situation was observed at 12 weeks posttreatment (SVR assessment).
One year after the end of treatment, the viral load was undetectable in three patients, which con rmed the e cacy of the treatment. ALT levels in these patients were normal (  Fig. 1). In Patient 1, LSM improvement was accompanied by a decrease in the CAP from S1 to S0, whereas in Patient 2, an increase in the CAP was found (Table 1, Fig. 1). Due to the COVID-19 pandemic, the 12-week posttreatment visits for Patients 3 and 4 had to be cancelled. Thus, data for these two patients were unavailable.
At the nal visit, one year after completing the treatment, the LSM median corresponded to F0-F1 in Patients 1 and 4, whereas in Patient 2, the LSM was lower compared to the previous visit but still corresponded to an F4 score on the METAVIR scale. However, the APRI values decreased for all three of these patients (Table 1, Fig. 1). The CAP value for Patient 1 increased compared to the 12-week posttreatment visit, but it was lower compared to the initial visit. A decrease in the CAP was observed for Patient 2 (from stage S3 to stage S1). However, in both of these patients, their BMI z-scores were over 1.0, suggesting obesity (Patient 1) or overweight (Patient 2). In Patient 4, CAP correlated to no steatosis (Table 1, Fig. 1). Patient 3 did not show up for this visit and was consequently lost to follow-up.

Discussion
A xed-dose combination of LDV/SOF is approved by the European Medicines Agency and Food and Drug Administration for the treatment of chronic HCV infection with genotypes 1, 4, 5, and 6 in children and adolescents 3 years of age and older [18][19][20] . Its safety and e cacy have been con rmed in phase II and III clinical studies [18][19][20] . However, there are only limited data available on the LDV/SOF e cacy in children with signi cant brosis 9 . In addition, little is known about the long-term outcomes and the in uence of the antiviral treatment on liver brosis. Patients with con rmed liver brosis should be closely monitored even after effective antiviral treatment of CHC 21 . Interestingly, there is evidence that in adult patients, liver brosis may be to some extent reversed by DAA treatment [21][22][23][24]  Factors that could in uence the regression of liver brosis after successful DAA treatment remain unknown. In the study by Mogahed et al., comorbidities or previous ineffective treatment with interferon were not associated with increased LSM one year after SVR had been achieved 10 . Among our patients, the one subject in which the LSM increased after completing the LDV/SOF treatment was coinfected with HIV; he had been previously ineffectively treated with interferon with ribavirin and was older compared to patients who achieved regression of brosis.
The COVID-19 pandemic, which was announced in March 2020, has led to the disruption of the healthcare system and has had a signi cant negative impact on the care of patients with chronic diseases, including our therapeutic program for children with CHC. In March 2020, our department was transformed to a setting dedicated for COVID-19 patients. Consequently, all visits for non-COVID patients between March and July 2020 had to be cancelled or postponed. However, we have prepared and followed the new guidelines for management of children and adolescents with CHC during the COVID-19 pandemic 28 . Several efforts were made to prioritize patient care in our children with CHC. In the case of patients receiving DAA therapy, these efforts included using telemedicine for monitoring the patient's general condition, adherence to treatment, and the side effects of the therapy at least every four weeks; cooperating with general practitioners; using local laboratory testing for follow-up testing; and engaging in the home delivery of DAAs 28 . All these efforts resulted in completing the full treatment regimen by all four patients. However, several monitoring visits were unfortunately cancelled. This problem in particular appeared in Patient 3, who was the last patient included in the therapeutic program (in February 2020). He completed his visit at 4 weeks of treatment, but the next monitoring visits were cancelled and replaced by phone calls every 4 weeks. Home delivery of LDV/SOF was arranged for him until the end of treatment. After 12 weeks of treatment his ALT and AST were tested in the local laboratory, which revealed further improvement in their levels (120 and 44 IU/L, compared to 438 and 184 IU/L at the start of treatment, and 272 and 111 IU/L after 4 weeks). The patient was asked to attend follow-up visits to assess the SVR and long-term outcome of treatment, but due to family and social problems and the long distance from our center, he refused to come and was lost to follow-up.
The main limitation of this study was the low number of patients included in the nal analysis. However, to the best of our knowledge, there is only one report available on the long-term effects of DAA treatment in children including the in uence of the therapy on liver brosis 10 . Therefore, it is worthwhile to share our unique data. In most pediatric cohorts, cirrhotic patients are underrepresented; thus, nding larger groups of these speci c patients would be di cult. The second issue are the gaps in the available data due to the disruption caused by COVID-19 pandemic. However, it is worth noting, that DAA therapies are simple, short, and safe. Thus, even when close monitoring of patients is not possible, positive outcomes of treatment may be achieved.
Based on our experience, we conclude that treatment with a xed-dose LDV/SOF in children with signi cant brosis in the course of CHC is safe and effective. A one-year observation of these patients after the end of treatment revealed that regression of liver brosis is possible but not obligatory. Further observations in larger groups of patients are necessary to nd predictors of liver brosis regression in pediatric patients with CHC.