Efficacy and Mechanism Evaluation (Jul 2024)
Efficacy and safety of eculizumab in children with Shiga-toxin-producing Escherichia coli haemolytic uraemic syndrome: the ECUSTEC RCT
Abstract
Background Shiga-toxin-producing Escherichia coli haemolytic uraemic syndrome affects ~100 United Kingdom children each year. Around half need dialysis, a quarter develop serious complications with long-term consequences and ~3% die. No effective intervention is known; however, some studies report eculizumab, effective in atypical haemolytic uraemic syndrome, may be effective. Objectives To determine whether the severity of Shiga-toxin-producing Escherichia coli haemolytic uraemic syndrome is less in those given eculizumab. Design Randomised, double-blind, placebo-controlled, parallel-group trial with internal pilot phase and nested mechanistic laboratory studies. Setting Paediatric nephrology units in 12 United Kingdom hospitals. Participants Children aged 6 months to 150 × 109/l). Duration of haemolysis (number of days until lactate dehydrogenase within local centre reference range). Number of packed red blood cell transfusions required and volume (ml/kg). Duration markers of inflammation present (number of days until neutrophil cell count and C-reactive protein are in normal range for that centre). Persistent neurological defect at day 60 measured by structured expert assessment to include central nervous system examination, vision, hearing and neuropsychological assessment. CKD at 52 weeks (a composite end point of the presence of hypertension, albuminuria or estimated glomerular filtration rate (eGFR) < 90 ml/minute/1.73 m2 at 52 weeks). eGFR measurement using a centralised cystatin C assay at 52 weeks. Mechanistic studies Urine CFH levels. Urine VEGF levels. Presence of urine markers of podocyte damage (nephrin and Wilms tumour-1). Plasma VEGF and factor H levels. Plasma complement activation products (Bb, C3a, C4a and sC5b9 by ELISA and C3 and C4 activation markers by degradomics). Glomerular endothelial surface levels of factor H and C3d (a marker of complement activation) in a co-culture models of human conditionally immortalised podocytes and glomerular endothelial cells exposed to Shiga toxin. Whole exome sequencing and serum anti-factor H antibody levels. Results The target sample size was 134 participants, but recruitment was stopped early due to low recruitment and the impact of the COVID-19 pandemic. At the point the trial was stopped, 108 children had been screened for participation, of whom 87 were deemed eligible to participate. Thirty-six children were consented and randomised; 17 were randomised to eculizumab and 19 were randomised to placebo. One participant withdrew from the trial and one participant died. The majority of baseline data of the participants were comparable across the two groups; however, the participants in the placebo group were slightly older and consequently heavier than those in the eculizumab group. Reasons for slow recruitment included a fall in the incidence of Shiga-toxin-producing E. coli haemolytic uraemic syndrome during the trial period (up to a 37% reduction) and a lack of out-of-hours infrastructure for undertaking acute interventional clinical trials in children. The mean CSS at day 60 for participants randomised to eculizumab was 11.5 [standard deviation (SD) 8.4] compared to 14.6 (SD 7.7) for participants randomised to placebo [adjusted mean difference: −2.5, 95% confidence interval (CI) −7.8 to 2.8, p = 0.3]. Five participants (three eculizumab, two placebo) experienced an adverse event, and there were seven serious adverse events (SAEs) in six participants (five eculizumab, one placebo). None of the SAEs were considered related to the trial treatment. Mechanistic substudies Of the 36 participants recruited to the main trial, 32 consented to take part in the mechanistic studies and provided blood and/or urine samples. In anuric patients, only blood samples were collected. Urine factor H and vascular endothelial growth factor levels in serial samples The highest urine factor H levels were at day 1 (150 ng/ml), diminishing by day 4 (30 ng/ml), and completely normalising by day 30 (undetectable). The highest urine VEGF levels were at day 1 (average 1300 ng/ml) and by day 30 the levels were below 20 ng/ml. Markers of podocyte damage Western blots of urine cell pellets showed acute podocyte loss during active disease, which recovered by day 8. Plasma factor H and vascular endothelial growth factor levels No difference was seen for plasma levels of either factor H or VEGF at day 1 or day 30. Plasma degradomics analysis In a sample of five patients, N-termini consistent with complement C3 and C4 activation were much more abundant at day 1 compared with day 3. In vitro cell co-culture In response to Shiga toxin, there was a reduction in glomerular endothelial factor H levels, accompanied by evidence of complement activation (increased C3d levels) and this was critically dependent on the presence of podocytes. Shiga toxin had no effect when added to endothelial cells alone. Plasma complement activation products Mean plasma levels of Bb were elevated in both groups at baseline (4.38 mcg/ml in the eculizumab group and 10.38 mcg/ml in the placebo group, normal range 0.48–1.62 mcg/ml). They were also elevated at day 2 (5.91 mcg/ml in the eculizumab group and 4.09 mcg/ml in the placebo group) and day 4 (4.90 mcg/ml in the eculizumab group and 3.16 mcg/ml in the placebo group). At day 6 and day 8, Bb levels remained elevated in the placebo group (6.21 and 3.47 mcg/ml respectively) but were normal in the eculizumab group. In both groups, mean Bb levels were in the normal range at day 30. Mean plasma levels of C3a were elevated in both groups at baseline and at days 2 and 4. At days 6 and 8, mean levels remained elevated in the placebo group while mean levels in the eculizumab group were in the normal range. Levels were in the normal range for both groups by day 30. Mean plasma levels of C4a were elevated at all time points in both groups but fell significantly at day 30. Mean levels were 3852, 3026, 3423, 3067, 3425 and 1623 ng/ml at days 1, 2, 4, 6, 8 and 30 in the eculizumab group (normal range 110–699 ng/ml) and 3970, 3573, 2673, 4348, 2844 and 1992 ng/ml at the same respective time points in the placebo group. Mean plasma levels of sC5b9 were normal in both groups at all time points with the exception of day 4 and day 6 in the placebo group, which were elevated (487 and 514 ng/ml respectively, normal range 95–467 ng/ml). In the placebo group, a linear relationship was not established between CSS and baseline Bb (r = 0.43, p = 0.2); C3a (r = −0.16, p = 0.7); C4a (r = 0.15, p = 0.7) or sC5b9 (r = −0.17, p = 0.7). Similarly, a linear relationship was not established between CSS and the maximum value of Bb (r = 0.45, p = 0.1); C3a (r = 0.15, p = 0.6); C4a (r = 0.23, p = 0.4) or sC5b9 (r = −0.22, p = 0.5). Delivery of Shiga toxin to podocytes from patient-derived neutrophils Insufficient patient samples were obtained to complete this part of the work. Genetic variations in patients with Shiga-toxin-producing Escherichia coli haemolytic uraemic syndrome Data from whole exome sequencing have been obtained and analysis is ongoing. Conclusions In children with Shiga-toxin-producing E. coli haemolytic uraemic syndrome, the mean CSSs at day 60 were similar between those randomised to eculizumab and those randomised to placebo. However, since the trial was stopped early and did not recruit to the planned sample size, this cannot be interpreted as evidence of no effect. In order to deliver successful clinical trials of investigational medicinal products in acutely unwell children, a review of out-of-hours paediatric research infrastructure may be required. In the mechanistic substudies, we have established that urine factor H and VEGF levels are sensitive measures of early disease activity, and have demonstrated complement activation in patient serum using both ELISA and sophisticated proteomics technology. Urine factor H and VEGF levels and plasma degradomics for C3 and C4 proteins could all be further explored as new biomarkers of acute Shiga-toxin-producing E. coli haemolytic uraemic syndrome. Our co-culture cell work has demonstrated that podocyte cross-talk is responsible for factor H and complement activation levels on endothelial cells. Collectively this strongly supports the mechanistic hypothesis of a complement-mediated disease driven via the podocyte as the target cell. Trial registration This trial is registered as EudraCT2016-000997-39 and ISRCTN89553116. Funding This award was funded by the National Institute for Health and Care Research (NIHR) Efficacy and Mechanism Evaluation (EME) programme (NIHR award ref: 14/48/43) and is published in full in Efficacy and Mechanism Evaluation; Vol. 11, No. 11. See the NIHR Funding and Awards website for further award information.
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