Introduction

Non-alcoholic steatohepatitis (NASH) is a chronic and progressive form of non-alcoholic fatty liver disease (NAFLD). It can progress to cirrhosis and end-stage liver disease, with resource implications for health care systems [1]. It is estimated to affect 6% of adults worldwide [2]. Patients with NASH have higher liver-related and cardiovascular morbidity and mortality compared with patients with early-stage NAFLD and the general population [3–5].

Many pharmacological agents are currently under development and testing in randomized clinical trials, but there is currently no medication licensed for the treatment of NASH. In these trials, assessment of changes in disease activity requires a liver biopsy at baseline and end of treatment, because there are no validated non-invasive biomarkers [6]. Based on the liver biopsy, regulators, such as the Food and Drug Administration and the European Medical Agency, currently consider specific histological changes in the liver after at least one year as acceptable intermediary endpoints in phase 2b-3 trials that are likely to predict progression to clinical outcomes [7, 8].

However, liver biopsies are invasive, and carry risks for patients, including pain, haemorrhage, and even death, and, thus, are unpopular with patients [9]. Achieving high follow-up is crucial to valid interpretation of data from clinical trials, so assessing the proportion of patients that undergo biopsy is imperative for trialists. Trials to date report variable follow-up and it is unclear what factors affect follow-up proportions or how best to estimate likely completion in future trials [10–13]. Moreover, it is crucial to understand whether patients are willing to undergo biopsy solely for the benefit of medical science, particularly for patients in control groups who have not received therapy. Some trials, such as those providing behavioural treatments such as weight loss, cannot be blinded and people in the control group could be deterred from a second biopsy by virtue of being offered no or inferior treatment. This review, therefore, aims to quantify the proportion of participants in NASH trials with a valid follow-up biopsy to inform future trial sample size estimates and assess why biopsy data is not obtained.

Methods

This was a prospectively registered systematic review (PROSPERO ID: CRD42020189488) reported using the PRISMA guidelines. We followed the protocol with no changes except a minor revision described in the next section.

Results

We screened 839 references and included 41 studies with a total of 6,695 participants (PRISMA Flowchart in Fig 1) [10–13, 19–55]. Seven studies were conducted in middle-income countries with the remaining run in high-income countries. Thirty-six studies tested medication as monotherapy or polytherapy with other medication or dietary supplements, four tested dietary supplements, and one tested a behavioural weight loss programme. Thirty-one trials had a placebo control arm, five trials had diet and exercise advice as control arms, and five trials compared two or more different drugs or supplements. Twenty-four trials were two-arm, 15 were three-arm, and two were four-arm. Among the 3-arm and 4-arm trials, twelve trials investigated various doses of the same medication whereas five trials compared multiple medications as monotherapy or polytherapy. S1 Table in S1 File presents the detailed study characteristics. At baseline, participants were on average 53.8 (SD: 11.4) years and 51% were female. They had mean NAFLD activity score and fibrosis stage of 5.11 (SD: 1.27) and 2.30 (SD: 1.15), respectively.

Fig 1

PRISMA flowchart.

Across all studies, the percentage of participants with a valid follow-up biopsy was 82% (95% CI: 78%–86%, I² = 92%, prediction interval: 50–96%) (Fig 2). Where it was reported, 9% (95% CI: 4–18%, I² = 92%, n = 13) of participants declined a biopsy (Fig 3). Among all studies, the main reasons for not having a valid follow-up biopsy were participant-related occurring in 95 per 1,000 participants (95% CI: 69–129, I² = 92%) (S1 Fig in S1 File).

Fig 2

Proportion of participants (95% CI) with a valid follow-up biopsy.

Fig 3

Proportions of participants (95% CI) reporting to specifically decline a follow-up biopsy.

Other reasons leading to a lack of a follow-up biopsy were medical-related [16 per 1,000 participants (95% CI: 8–29, I² = 91%)], protocol-related [3 per 1,000 participants (95% CI: 1–8, I² = 86%)], trial conduct-related [1 per 1,000 participants (95% CI: 0–5, I² = 91%)], and other/unclear [3 per 1,000 participants (95% CI: 1–10, I² = 94%)] (S1-S5 Figs in S1 File).

There was no evidence that the location (high-income vs. middle-income counties) influenced the proportion of valid follow-up biopsies (S6 Fig in S1 File). In the analysis by geographical region, there was some limited evidence that this proportion was lower in two small studies conducted in Eastern Mediterranean but not in any other region (S7 Fig in S1 File). Moreover, there was no evidence that the interval to the follow-up biopsy (1 year vs. more than 1 year) affected this proportion (S8 Fig in S1 File). However, among the three studies that conducted an additional follow-up liver biopsy within 3 years, the proportion of participants with a second follow-up valid biopsy was 68% (95% CI: 61–75%, I² = 48%) when the proportion for the first follow-up biopsy was 89% (95% CI: 80–94%, I² = 74%) in these trials (S9 Fig in S1 File). Twenty-five trials reported the proportion of a follow-up biopsy by trial arm. Among these, there was no evidence of a difference in the proportion of valid follow-up biopsies between the active intervention and placebo arms (OR: 1.03, 95% CI: 0.87–1.22, I² = 0%) (S10 Fig in S1 File). Only one study with a high retention rate reported reimbursing participants for completing the study [55].

There were 32 studies that blinded personnel and participants and where it is reasonable to assume participants expected equal benefit between arms (risk of bias due to blinding in S2 Table in S1 File). There were eight open-label medication studies where expectations of benefit may or may not have been equal, and one open-label trial assessing a behavioural weight loss programme where blinding is by default not possible and where participants in the control arm presumably expected a second biopsy would not show large changes in histology. Among the open-label trials, there was no evidence that the proportion of valid follow-up biopsies differed between arms (OR: 1.11, 95% CI: 0.72–1.70, I² = 0%) (S11 Fig in S1 File). There was no evidence from a post-hoc meta-regression that the size of the study was associated with retention (b = 0.0009, 95% CI: -0.0001–0.0020, p = 0.08). The funnel plot showed no evidence of publication bias (S12 Fig in S1 File).

Biopsy-related serious adverse events occurred in 16 per 1,000 participants (95% CI: 8–33, I² = 54%) among the seven studies that specifically reported this outcome (Fig 4). Among the 17 serious adverse events, 4 were pain, 3 were hematoma, 1 was bile leak, and 9 were not reported. In 4 studies (n = 618) reporting a total of 27 non-serious adverse events, 25 were pain and 2 were vasovagal syncope. No biopsy-related deaths were reported.

Fig 4

Biopsy-related serious adverse events reported for N per 1,000 participants (95% CI).

Discussion

This systematic review and meta-analysis of 41 randomized controlled trials in NASH shows that the proportion of participants with a valid follow-up biopsy is on average 82% (78–86%), but with unexplained heterogeneity between trials. On average, nearly one in 10 participants declined a second biopsy. About 3 out of 10 did not have a further follow-up biopsy. There was no evidence that the proportion having a follow-up biopsy differed by location, length of follow-up, between intervention and placebo groups in blinded trials, or between groups in trials with open allocation.

These findings have implications for trial design of therapeutic trials in NASH, as the retention is essential for a sample size calculation. Firstly, many large-scale trials calculated their sample size based on a retention of 85–90%, but on average did not achieve this [11, 12, 39, 42, 47]. This can lead to underpowered studies that might not be able to answer their research questions, therefore, wasting resources and exposing participants to risky treatments and procedures. Future trials should allow for a lower retention to increase the likelihood of adequately powered studies. Secondly, the lack of evidence that the proportion of follow-up biopsy differ between arms in open label trials (i.e., where the risk of bias due to blinding was high) suggests that knowledge of the received treatment does not influence participants’ commitment to the second biopsy. This might have been expected if participants perceived they had not received an effective treatment. Thirdly, the sample size should be based on expected histological changes in the first follow-up biopsy given the lower retention in subsequent follow-up biopsies.

Knowledge of the proportion of biopsy-related adverse events specifically in this population could be used as part of the consent process in future trials. Participants can be informed that about 1 in 60 participants may experience a serious adverse event, with the possibility of serious pain occurring in about 1 in 270 and of hematoma in about 1 in 350. These are generally in line with previous estimates from two previous audits totalling 3,482 biopsies in diverse populations [56, 57]. These audits reported either no deaths or 3 deaths in inpatients with major comorbidities. No study in this review reported a biopsy-related death. This is reassuring and might reflect the strict trial eligibility criteria that exclude people with major comorbidities. Non-invasive biomarkers, such as magnetic resonance imaging and elastography, are emerging as alternatives to the liver biopsy that will minimise such adverse events [58, 59]. The new proposed definition of metabolic dysfunction-associated fatty liver disease [60] might reinforce the use of such biomarkers for diagnosis and as trial endpoints [58], although the optimal terminology remains to be determined [61].

Strengths of the study include being guided by Cochrane methods and including randomized trials of diverse interventions with a minimum follow-up of 1 year. The estimates remained robust irrespective of time to the first follow-up or location indicating that they should be applicable to studies worldwide. However, the study also has some limitations. Most studies did not report on biopsy-related adverse events and, thus, it remains unclear if participants in these studies did not experience these or if studies only reported on treatment-related adverse events. Our estimates include only studies specifically reporting adverse events and, thus, assume that the decision to report biopsy-related events is unrelated to their occurrence in these trials. Future studies should present detailed analyses of all adverse events.

Conclusions

The proportion of participants with a valid follow-up biopsy in therapeutic trials in NASH is on average 82%, with around 1 in 10 participants declining a follow-up biopsy. This provides a realistic assessment of likely follow-up to inform sample size calculations in future trials.