Anticomplement Treatment in Atypical and Typical Hemolytic Uremic Syndrome

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Anti-complement treatment in atypical and typical hemolytic uremic syndrome Fadi Fakhouri, Chantal Loirat

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S0037-1963(18)30035-0 https://doi.org/10.1053/j.seminhematol.2018.04.00910.1093/ndt/gfx19610.1128/microbiolspec.EHE 0013-2013 Reference: YSHEM50958 To appear Seminars in Hematology in: Cite this article as: Fadi Fakhouri and Chantal Loirat, Anti-complement treatment in atypical and typical hemolytic uremic syndrome, Seminars in Hematology,doi:10.1053/j.seminhematol.2018.04.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Seminars in Hematology Anti-complement treatment in atypical and typical hemolytic uremic syndrome

Fadi Fakhouri, MD, PhDa and Chantal Loirat, MDb

a. Centre de Recherche en Transplantation et Immunologie UMR 1064, INSERM, Université de Nantes and Department of Nephrology and Immunology, Centre Hospitalier Universitaire de Nantes, Nantes, France.

b. Assistance Publique–Hôpitaux de Paris, Department of Pediatric Nephrology, Hôpital Universitaire Robert Debré, Université Paris Diderot, Sorbonne Paris Cité, Paris, France

Corresponding author: Chantal Loirat, Service de Néphrologie, Hôpital Universitaire Robert Debré, 48 Boulevard Serurier, 75019 Paris, France. [email protected] Telephone: +33 1 40 03 21 46. Fax: +33 1 40 03 24 68. Word count: Abstract: 118; Text: 4506.

Financial disclosure: FF and CL served on advisory boards and in teaching courses for Alexion Pharmaceuticals. FF and CL served on advisory boards for Roche. FF serves as member of the scientific advisory board of Alexion M11-001 atypical Hemolytic Uremic Syndrome international registry, and CL as coordinator for France for this registry.

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Abstract The dissection of the pathogenic mechanisms of the various forms of the haemolytic uremic syndrome (HUS) has paved the way for the design of specific efficacious treatments. Such mechanistic approach led to a revolution in the management of atypical HUS with the use of the first-in class C5 blocker, eculizumab. The availability of this anti-complement drug has also raised unsettled questions regarding the cost/burden and optimal duration of therapy and its use in secondary HUS. The efficacy of eculizumab in Shiga toxin producing E coliassociated-HUS is not to date established and the results of ongoing prospective studies are eagerly awaited. Nevertheless, the emergence of anti-complement therapies (eculizumab and other drugs in development) has transformed our approach of HUS. 1. Introduction Hemolytic uremic syndrome (HUS) is defined by the triad of mechanical haemolytic anaemia, thrombocytopenia and acute kidney injury. It is one of the most impressive examples of how deciphering the specific mechanisms of the various forms of a syndrome can lead to a physiopathology-based efficient treatment. The demonstration in the late 1900s that postdiarrheal/typical HUS, the most common form of thrombotic microangiopathy (TMA) in children, was due to Shiga toxin (Stx) producing E coli (STEC), and that thrombotic thrombocytopenic purpura (TTP), one of the most frequent forms of TMA in adults, results from a severe deficiency in a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS 13), helped clinicians to distinguish TTP from HUS. Consequently, up to recent years, the term atypical HUS (aHUS) has been used to define any HUS related neither to STEC infection nor to severe ADAMTS13 deficiency.

Thus it

encompassed both primary forms of aHUS, and a variety of secondary aHUS related to infections (Streptococcus Pneumoniae, human immunodeficiency virus), malignancies, drugs (cancer chemotherapy, calcineurin inhibitors), autoimmune diseases (systemic lupus

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erythematosus (SLE), vasculitis), medical conditions (solid organ and hematopoietic stem cell transplantation (HSCT)) or metabolic disease (cobalamine C (cblC) defect). A major step forward was the demonstration over the 1998-2009 decade that primary aHUS was a disease of complement alternative pathway (CAP) dysregulation. Such dysregulation is linked to 1) Loss-of-function variants in the genes of CAP regulatory proteins, complement factor H (CFH), membrane cofactor protein (MCP or CD46), complement factor I (CFI) and thrombomodulin (THBD) or 2) gain-of-function variants in the genes of the C3 convertase components (complement factor B (CFB) and C3), or 3) anti-CFH inhibitory autoantibodies (review in [1]). CAP dysregulation impairs the physiological protection of endothelial cell surfaces against CAP activation-induced damage, leading to TMA (see review by R. Taylor of the mechanisms of aHUS in this issue and see [1]). More recently, young children with a hitherto unexplained aHUS were found to carry variants in the gene of diacylglycerol kinase ε (DGKE), an endothelial cell and podocyte protein with no direct link with the complement system, opening a new chapter of non-complement related forms of aHUS [2]. These breakthroughs led to various etiology-based classifications of HUS, proposed by nephrologists and haematologists (review in [1] and [3]). Anecdotically, the term aHUS is currently either restricted to aHUS cases not related to a coexisting disease/condition or specific infection [1, 4], or includes all TMAs except STEC-HUS and TTP [3]. More importantly, the demonstration that aHUS is a disease of CAP hyper activation was the starting point for the use of the first clinically available complement inhibitor, eculizumab. In turn, the demonstration of eculizumab efficiency in aHUS reactivated or initiated studies of complement activation in various forms of HUS, including STEC-HUS, and raised the hope that eculizumab might be also efficient in these forms of HUS. In this review, we describe how complement blockade therapy has revolutionized aHUS outcome and management, and

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discuss controversial issues of the optimal treatment duration in aHUS, and the potential efficacy of eculizumab in STEC-HUS and secondary aHUS.

2. Complement blockade treatment in atypical HUS 2.1. Rationale 2.1.1. Atypical HUS is a disease of complement hyper activation Even though primary aHUS is an ultra-rare disease (estimated incidence 0.23-0.42 cases per million population, with onset during childhood in 40-50% of patients) [5-7], approximately 1300 aHUS patients screened for complement genes variants and anti-CFH antibodies have been reported so far [1]. Variants in complement genes were identified in roughly 50 % of patients, mostly CFH variants in adults (up to 30%), and MCP variants (up to 20%) and antiCFH antibody-HUS (approximately 10%) in children. The high frequency of anti-CFH antibody in aHUS children in India (56%) [8] and South Corea (29%) [9] is unexplained. 2.1.2. Outcome of primary atypical HUS was poor in the pre-complement inhibitors era Two retrospective series from Italy [10] and France [6] have outlined the severe prognosis of aHUS in the pre-eculizumab era, when treatment relied on plasma therapy (Figure 1). The rate of death was higher in children than adults [6]. Conversely, renal outcome was better in children than adults, with progression to end stage renal disease (ESRD) in 16% of children versus 46% of adults at first episode, and 36-48% versus 64-67% at 3-5 years follow-up (Figure 1). Renal outcome in adults was severe whatever the genetic background, including in patients with no complement gene variant identified. Children and adults with a CFH variant had a similar poor outcome, and only children with MCP-HUS had a relatively favourable outcome, despite relapses (25% ESRD at a median follow-up of 17.8 years) [6]. In keeping with the persistence of complement activation, inflammation and endothelial injury in aHUS patients under plasma exchange/plasma infusion (PE/PI) [11], plasma therapy did not appear

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to improve renal outcome in the two series. Interestingly, the Italian series outlined the discrepancy between haematological and renal response to plasma therapy: half of children and two-thirds of adults progressed to ESRD or death at 3 years’ follow-up despite haematological remission under plasma therapy in 78% and 53% of cases, respectively [10]. In an international audit study of 71 children with aHUS, of whom 83% received plasma therapy (PE in 69%), 11 % of patients did not achieve hematological remission, 17 % remained dialysis-dependent and 31% had complications of central venous catheters at day 33 [5]. The only subgroup that significantly benefits from PE therapy in association with immunosuppressive treatment is anti-CFH antibody- HUS (See Chapter 2.4.2). Among adults and children with preserved renal function, approximately 30% had relapses during the first year, and 20% of adults to 50% of children in subsequent years [6]. Last, aHUS was associated with an overall risk of 60% recurrence after kidney transplantation and recurrence was a major risk of graft loss, particularly in patients with CFH, C3 and CFB variants (recurrence risk > 90%) (review in [1]). This often hopeless prognosis of aHUS explains why the eculizumab option was eagerly awaited for until it was approved for the treatment of aHUS by the US Food and Drug Administration and the European Medical Agency in 2011. 2.2. Eculizumab efficacy in aHUS Four industry-sponsored prospective trials of eculizumab have been performed in a total of 100 patients (78 adults, 22 children) (see [4] for trials’ design). Because of the rarity of the disease, these trials were not randomized between PE-treated and eculizumab-treated patients. In 80 patients (22 children) treated during full-blown episodes of HUS, median delay was 7-8 days for normalization of platelet count (≥150 G/L), and varied from 14 days [12] to 48-54 days [13, 14] for normalization of lactate-deshydrogenase level. Mean increase in estimated glomerular filtration rate (eGFR) at 1 or 2 years under eculizumab was 64 mL/min/1.73m2 in

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children [14, 15] compared to 30-35 mL/min/1.73m2 in adults [12, 13, 16, 17]. Shorter time from onset of HUS episode to eculizumab initiation was correlated with a significantly greater gain in eGFR [12, 16]. In 20 adults treated with eculizumab after a long period of plasma therapy (median 8.6 months), mean improvement of eGFR was only 6-8 mL/min/1.73m2 [12, 16]. The analysis of 18 patients treated in France with eculizumab since 2009, compared to 41 paired controls treated with PE between 2004 and 2008, indicated a significantly decreased risk of progression to ESRD within 3 months and one year [18]. Figure 1 summarizes the changes of aHUS outcome in patients treated with eculizumab in prospective trials and in the retrospective paired series, compared to patients from the precomplement inhibitor era treated with plasma therapy. A number of case reports indicate that eculizumab can also rescue central nervous system manifestations, ischemic cardiomyopathy, distal ischaemic manifestations, ophthalmologic involvement and ulcero-necrotic skin lesions (review in [1, 4]). All reports claimed in favour of early treatment initiation and the superiority of eculizumab compared to PE to rescue kidney or other organs injury. Last but not least, eculizumab rescues or prevents post-transplant recurrence of aHUS and thus allows successful kidney transplantation, transforming the life of many patients who lost prior kidney grafts from recurrence (review in [1] and [12, 19]). 2.3. Eculizumab in aHUS in practice Recommendations for clinical practice including eculizumab doses and regimen have been published recently [3, 4]. 2.3.1. Timing for eculizumab initiation The general consensus is that in children with a high suspicion of aHUS, eculizumab should be first line treatment (within 24 hours of onset whenever possible) (Figure 1). If eculizumab

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is not available, PE with fresh frozen plasma (FFP) (60ml/kg per session) are indicated (PI ≥10 mL/kg if PE not available), with switching to eculizumab as soon as possible. Eculizumab should also be first line treatment at any age in patients with relapse of HUS, a family history of HUS or HUS recurrence in the renal graft. Conversely, PE with FFP (1-1.5 plasma volume per session) remains the first-line treatment in adults requiring investigations to eliminate TTP and secondary forms of aHUS. aHUS adults started on PE should be switched to eculizumab as soon as the diagnosis of aHUS is established and not later than after 5-7 days under PE (Figure 1) At any age, absence of thrombocytopenia at onset does not rule out the diagnosis of aHUS. Conversely, hematological remission under PE does not guarantee renal function improvement. Results of genetic screening are not necessary for deciding to start eculizumab treatment. Patients with no complement gene variants identified may have a similar response to eculizumab as patients with a complement variant [12, 14], with the exception of DGKEand cblC-HUS (see Chapters 2.4.3 and 2.4.4). 2.3.2. Monitoring of eculizumab treatment Complement Haemolytic 50 (CH50) (or other hemolysis based assay or Wieslab Complement System) is the most commonly used test to check complement blockade. Optimal complement blockade is generally defined as CH50 < 10%. Measurement of trough eculizumab plasma level has been developed in various countries [20-22], with a recommended target range (Cmin) >100 µg/mL, a threshold higher than the minimal level of 50 µg/mL sufficient for complement blockade, in order to obviate complement activation triggered by incidental events such as seasonal infections or vaccination [3]. In practice, checking CH50 at day 7, just before the second dose, and after spacing doses to every 2 weeks (or more), to ensure complement blockade is maintained between doses, is reasonable [23]. The simultaneous checking of CH50 and eculizumab trough level is useful mainly in patients with persistent

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thrombocytopenia after the first or second dose, those without renal function improvement, and patients experiencing a relapse under eculizumab (Figure 1).

CH50 and eculizumab

trough levels both outside the therapeutic range (>20% and < 50 µg/ml, respectively) are indications for increased dose and/or shortened interval between doses. A rare explanation can be eculizumab urinary linkage in severely proteinuric patients [21].

Infection,

inflammation and surgery may induce a transient resistance to eculizumab through the generation of higher densities of C3b on target cells, as densely packed C3b molecules may compete with eculizumab for the recruitment of C5 molecules [24]. Patients resistant to complement inhibition by eculizumab (CH50 100% despite eculizumab trough level > 100 µg/ml) should be screened for a variant in eculizumab C5 binding site gene, mostly present in Asian individuals [25]. Finally, clinical resistance to eculizumab treatment also requires rechecking diagnosis and investigating non-complement related aHUS (DGKE- and cblCHUS) (Figure 1). Conversely, eculizumab high trough level (> 500-1000 µg/ml) associated with CH50<10% indicate over dosage of the drug, which allows spacing the interval between doses, thus reducing treatment burden and cost [20, 21, 26-28]. The monitoring of plasma sC5b9 level to assess the efficacy of complement blockade therapy at the endothelial level is not validated. Increased sC5b9 levels have been reported both in acute and remission phases of aHUS [29], and in about 50% of patients in remission under eculizumab [21, 30]. The utility of other biomarkers of complement activation (e.g. C3d, C5a) is not established either [21, 27, 30]. The clinical applicability of an ex vivo assay of seruminduced C5b9 deposition on endothelium to differentiate active disease from remission, monitor eculizumab effectiveness or predict relapses after eculizumab discontinuation requires further studies [30].

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2.3.3. Neisseria meningitis invasive infection under eculizumab Activated terminal complement pathway is required for efficient serum bactericidal activity (SBA) towards Neisseria species (mostly meningococci and gonococci). Thus, patients under eculizumab are at high risk of meningococcal disease, (estimated 1000-2000-fold increase [31]).

Quadrivalent A,C,W,Y meningococcal conjugate vaccine and B meningococcal

vaccine are required for patients under eculizumab [32], with the hope that vaccination might upregulate opsonophagocytic killing of meningococci, and thus compensate for the absence of efficient SBA. Unfortunately, opsonophagocytic killing of meningococci has recently been shown to be suppressed by eculizumab, due to inhibited release of C5a, the C5 split product needed for phagocytosis upregulation [33]. Two of the 100 aHUS patients who participated in the eculizumab trials had invasive meningococcal infection [13] and reports of meningococcal infection in aHUS patients under eculizumab therapy were in vaccinated patients, including the B vaccine [34]. Due to the uncertain efficacy of vaccines, long term antibioprophylaxis (methyl penicilline) is recommended in some countries (France, UK) for patients under eculizumab. Interestingly, 39% of meningococcal strains responsible of invasive infections in patients with congenital complement deficiencies have reduced susceptibility to penicillin G and the same can be expected in patients under complement-blockade therapy [35]. Patients and their care givers have to be informed that infectious symptoms require immediate medical advice, information card shown to medical staff, prompt blood culture and parenteral third generation cephalosporin treatment initiation whenever judged justified. 2.3.4. Eculizumab discontinuation The arguments pleading for or against treatment discontinuation are well known: the risk of meningococcal infection, the burden of repeated intravenous infusions, the cost on one side, and the risk of relapse with potentially irreversible renal damage on the other side. However,

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the main question is whether all aHUS patients have a continuous CAP hyperactivation and thus continuous complement-induced endothelial cells damage. To date, there is no definite proof that this assumption is true in all patients, particularly in those without documented complement gene variants. Data regarding eculizumab discontinuation in aHUS patients are accumulating and derive from case reports (see Supplemental data on eculizumab discontinuation), small series [7, 36, 37], and 3 main cases series [38-40] (Table 1). Eculizumab discontinuation is to be discussed after at least 6 months of treatment (3 months in MCP patients) and after normalization or stabilisation of renal function. Overall, the available data indicate that eculizumab discontinuation is a reasonable option in patients with no documented complement gene variants. In the largest retrospective series, none out of 17 patients with no rare variant detected relapsed [39], and in all other published cases combined, the risk of relapse in patients with no variant is below 10%. In contrast, in the aforementioned series [39], 8 out of 11 patients (72%) with CFH variants and 4 of 8 (50%) with MCP variants relapsed after treatment cessation. In all cases reports combined, the risk of relapse after eculizumab discontinuation is 60%, 37.5% and 43% in patients with CFH, MCP and C3 variants. Data regarding patients with CFB and CFI variants are scarce (Table 1). In the overwhelming majority of cases, rapid restart of eculizumab after relapse led to a rapid control of aHUS without additional renal damage. Follow-up after eculizumab discontinuation even in the two largest series is rather limited, (median 8.9 months [38] and 22 months [39]) and the impact of post-discontinuation relapses on long-term renal outcome remains to be assessed. A prospective observational study is ongoing in order to better define the outcome of aHUS patients after eculizumab discontinuation (NCT02574403). The optimal duration of retreatment of patients who experience a relapse after treatment discontinuation is unknown: short course (3 months) in patients with MCP variants versus more extended treatment in CFH, C3 and FB variants? Life-long or “on-demand” treatment?

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2.4. Eculizumab in specific forms of aHUS 2.4.1. Pregnancy-HUS Pregnancy-HUS is rather a frequent condition, as it represented 16-20% of HUS cases in women of bearing age. Two recent registry-based retrospective studies included 22 and 87 cases of pregnancy-HUS occurring predominantly in the post-partum (3/4 of cases) [41, 42]. In both studies, patients with pregnancy-HUS shared with aHUS patients the same severity at presentation (41-71% of patients required dialysis), the same high frequency of complement genes variants (40-56%), mostly in CFH gene, and the same risk of relapse (16-28%). In the largest study [41], patients were treated mainly with plasma therapy (PE in 78% of cases), only 4 (5%) received eculizumab, and 53% of them reached ESRD. In the smaller study [42], among eculizumab treated patients (n=10), none reached ESRD, whereas among patients (n=12) treated with plasma, 6 (50%) reached ESRD. The efficacy of eculizumab in pregnancy-HUS has also been documented in cases reports [43, 44]. These data, even though retrospective, indicate that pregnancy-HUS is an aHUS triggered by pregnancy and should be managed as such. Nevertheless, the differential diagnosis of HUS in the setting of pregnancy/postpartum is often difficult, as pre-eclampsia, HELLP syndrome, and delivery haemorrhage may mimic HUS. Data regarding pregnancy outcome in patients with a history of aHUS remain scarce. A recent study [45] of 27 pregnancies in 14 women with a history of aHUS (before, during or after pregnancy) and a high prevalence of complement gene variants (10/14 (71%)), challenges the long-held assumption that pregnancy has a catastrophic prognosis in these patients and thus should be formally contra-indicated. Only a quarter of the 27 pregnancies were complicated by episodes of HUS. Twenty-two (78%) pregnancies resulted in live birth, 2 preterm infants were stillborn and early spontaneous abortions occurred in 4 pregnancies. Similarly, in a recent study [41], 39/87 (44%) patients had had at least one uneventful pregnancy before

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presenting pregnancy-HUS. Obviously, the availability of a disease-specific efficacious treatment has helped improve the perspectives of pregnancy in aHUS patients. Besides, eculizumab use during pregnancy seems to be safe, with limited transplacental transfer and no major teratogenic or infectious complications reported to date [46]. However, pregnant women may require higher doses or shorter interval between eculizumab perfusions in order to achieve complement blockade [46, 47]. This is probably due to increased eculizumab distribution volume and C5 synthesis during pregnancy. Nevertheless, pregnancy in women with a history of aHUS remains a high-risk pregnancy. Patients should be informed of the potential risks and an early and close multidisciplinary monitoring of pregnancy (and up to 3 months post-partum) is mandatory. Currently, prophylactic eculizumab is not widely used, and the relevance of plasma infusions is far from certain [45]. Moreover, some patients may have chronic kidney disease (CKD) or infraclinical renal damage with inherent risks of renal function deterioration during pregnancy and hypertensive complications (preeclampsia, HELLP syndrome), against which eculizumab does not confer protection [47]. 2.4.2. Anti-CFH antibodies-associated HUS In this form of complement-dependent aHUS (see Supplemental material), early initiation of PE associated with immunosuppressive treatment (rituximab or cyclophophamide) and corticosteroids has drastically reduced the risk of severe CKD or death compared to PE without immunosuppression. In the large series from India [8], such treatment schedule allowed the decrease of mean anti-CFH antibody titer from 7053 arbitrary units (AU)/mL to ≤ 1000 AU/mL (normal <150 AU/mL). Subsequent maintenance immunosuppression (mycophenolate mofetil (MMF)) drastically reduced the risk of relapse compared to no maintenance immunosuppression [8, 48] (Figure 2). Eculizumab is efficacious in this form of aHUS as in other complement-aHUS [18, 38-40, 49-55]. The association of eculizumab with

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glucocorticoids and an immunosuppressive drug (MMF) is recommended, with the view to obtain the decrease of anti-CFH antibody titer, allowing eculizumab withdrawal [4, 51]. Yet, the proportion of patients under eculizumab as first line treatment for whom MMF suffices to decrease anti-CFH antibody titer, and which titer allows eculizumab discontinuation remains hardly documented. The other option is to continue eculizumab monotherapy indefinitely, though a spontaneous decrease of antibody titer seems exceptional (Figure 2c) [49]. Interestingly, 2 patients with high anti-CFH antibody titers remained asymptomatic during 9 and 12 years without treatment [49]. A prospective study is needed to establish guidelines for eculizumab discontinuation in anti-CFH antibody-HUS. 2.4.3. DGKE-HUS In this non-complement-dependent form of aHUS (see specific clinical course

in

Supplemental material), the benefit of PE/PI as well as of eculizumab is unproven [2, 56]. Although in vitro studies showed no C3 deposits on DGKE knockout endothelial cells [57], signs of complement activation (mildly depressed/fluctuating C3, increased sC5b9 levels) are observed in some patients (28%, 10/35) [56], suggesting secondary activation of complement on damaged endothelial cells. Among 6 patients treated with eculizumab, resolution of HUS was observed in 2, no clear benefit in 4, and relapses of HUS despite adequate complementblockade in 2. Thus the benefit of anti-complement therapy seems limited in DGKE-HUS [56]. 2.4.4. CblC defect-HUS HUS can reveal or complicate cblC defect (the most frequent inborn error of cobalamin (vitamine B12) metabolism) not only in very young children (< 1 year), but also in older children and young adults [58-61] (see Supplemental material and Supplemental Table 1). Noteworthy, prompt diagnosis and hydroxocobalamin treatment allow renal recovery, as opposed to catastrophic natural renal outcome. Eculizumab has been inefficient in 4 patients

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(see Supplemental material). This is the only genetic form of HUS which can be cured and prevented by a cheap treatment. 2.4.5. Secondary aHUS Secondary aHUS is a heterogeneous entity. Even though the initial endothelial injury may not be due to complement, current research is focused on assessing whether complement activation occurs as a second hit leading to the persistence of TMA. Complement involvement is highly suspected in aHUS associated to immune complexes-mediated diseases such as SLE. It is substantially documented in antiphospholipid syndrome (APS)-associated TMA [62], where animal and clinical data indicate that complement activation (C5a) is required for the induction of the thrombotic effect of antiphospholipid antibodies. In HSCT-HUS, a body of evidence has linked complement activation to TMA occurrence and severity (see review by S. Jodele in this issue). The role of complement activation is suggested in anti-vascular endothelial growth factor-[63] and pneumococcus-associated-HUS (see review in [1]) . The frequency of complement gene variants in secondary aHUS has not been extensively studied. A single series reported such variants in 7 out of 24 patients with de-novo TMA after renal transplantation [64], but these variants are of unknown clinical relevance. Thus to date, therapeutic complement blockade is used as a clinical test to assess whether a type of secondary aHUS is, at least partially, complement-dependent. However, management of secondary aHUS includes in priority the withdrawal or treatment of the triggering drug or condition, and this may be sufficient to control HUS. Experience with eculizumab derives mostly from cases reports and limited series, with the potential bias of preferential publication of positive results. Eculizumab has been used in SLE and/or APS (reviewed in [65]), gemcitabine [66, 67], cancer [68], solid organ transplantation-associated HUS [69], with mostly, a beneficial effect. The largest published series [70] included 29 adult patients mainly with drug-induced or systemic diseases-associated-HUS. Eculizumab was used after PE and

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treatment of the underlying cause failed to control TMA (median time from diagnosis to eculizumab use: 13 days (7-26)). HUS was severe (dialysis required in 48% of cases and neurological and cardiac present in 38%). A beneficial haematological and renal effect was observed in 20/29 (69%) cases, rapidly following the start of eculizumab (median time: 12 days (7-17)). Fifteen (75%) out of these 20 patients had a > 50% reduction in serum creatinine, mostly in the first month of treatment. Haematological remission without renal function improvement occurred in 6 (21%) patients and no beneficial effect was seen in 3 (10%). Only 2 (7%) patients had a pathogenic complement gene variant (CFH and CFH-related protein 1). Except these 2 patients, all remaining 27 patients discontinued eculizumab after a median treatment duration of 8 weeks (3-18) and a median number of eculizumab doses of 6 (3-11). No HUS relapse occurred after eculizumab discontinuation, with a rather short follow-up (5.2 months (4.2-14.1)). In all, eculizumab is used as empirical, salvage therapy in PE-resistant patients with secondary aHUS. Duration of treatment is limited to few weeks as potential haematological and renal benefit is evident rapidly after eculizumab initiation, and relapse risk after discontinuation appears to be extremely low. 3. Complement blockade treatment in STEC-HUS 3.1. Rationale 3.1.1. Complement activation in STEC-HUS The demonstration of eculizumab efficacy in aHUS renewed interest in a potential role of complement activation also in STEC-HUS. Indeed, while a decrease (generally slight) of C3 plasma levels is observed in only 20-25% of patients at the acute phase of postdiarrheal/STEC-HUS [71, 72], 60 to 100% of patients have elevated levels of soluble sC5b9 [71-75] (see review of the mechanism of Stx-induced complement activation in [1]) 3.1.2. Outcome of STEC-HUS in the pre-complement inhibitor era

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Prognosis of STEC-HUS in children is generally favourable, with low in-hospital death rate (1-3%) and no renal sequelae at 5 years follow-up in approximately 70% of children [76, 77]. . However, approximately 20% of children have neurological complications at the acute phase, with a 4% to 25% risk of permanent neurological impairment [76, 78] (Figure 3 and supplemental Figure 1). Ischemic cardiomyopathy (3-5% of children), severe hemorrhagic colitis or pancreatitis may also be life-threatening. Last, 30% of children have residual mild CKD, implying a risk of progression to more severe CKD in adulthood [76] (Figure 3) STEC-HUS has a poor prognosis in elderly patients. A 33% death rate was observed in 20002006 in patients older than 60 years [79]. During the O104 STEC outbreak in 2011, the overall mortality was only 3.3% (27/810) but median age of patients who died was 74 years compared to 43 years for the total cohort [80]. Prospective randomized trials in the 1990’s [81] and clinical experience with PE [78, 82-85] failed to prove a benefit from any treatment (Figure 3 and Supplemental Figure 1). Thus, novel therapies for extra renal complications or severe renal forms of STEC-HUS are needed. 3.2. Eculizumab in STEC-HUS The first report of eculizumab treatment in STEC-HUS was in 3 children with O157 STECHUS and neurological complications, who improved within 24 hours of first dose of eculizumab and had full recovery after 2 to 4 doses [86]. This publication led to eculizumab treatment in more than 250 patients during the 2011 outbreak of STEC O104-HUS in Germany and France, most of them after failure of PE [82, 84, 85, 87]. Figure 3 summarizes data from 3 retrospective analysis of eculizumab-treated compared to eculizumab-untreated patients during this outbreak. In two analysis, patients treated with eculizumab had more frequent neurological complications and more severe renal injury at baseline than untreated patients, but outcomes were similar in both groups [82-84], and similar in children to outcomes in historical series [76, 77, 82]. This can be interpreted as no marked benefit from

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eculizumab compared to PE or supportive treatment, but, to our opinion, also as a potential benefit from eculizumab in the most severe forms of the disease, whose final outcome was similar to that of milder forms. Yet, another analysis comparing patients treated with eculizumab to untreated controls matched for baseline severity, failed to show short term benefit of eculizumab treatment [85] (Figure 3). In subsequent smaller series and case reports, all retrospective and non-controlled, eculizumab treatment was associated with a rapid and full recovery of neurologic manifestations and ischemic cardiomyopathy in 66% (18/27) and 82% (9/11) of patients. However, it did not prevent death from neurological complications or cardiac failure in 15% (4/27) of patients, and neurological sequelae in 19% (Supplemental Figure 1). Early initiation of therapy seemed to be of primary importance for rescuing brain involvement. Yet, comparison of neurological outcomes in the pre-eculizumab and eculizumab era does not suggest a clear benefit from eculizumab treatment (Supplemental Figure 1). Ongoing randomized controlled trials (NCT02205541 in France; ECUSTEC, UK) will assess eculizumab benefit in STEC-HUS.

Until results are available, treating with

eculizumab patients with CNS involvement or other life threatening complications may be a reasonable option.

Conclusion In all, eculizumab has revolutionized the treatment of aHUS. It also raises still debated questions about the burden/cost and optimal duration of treatment and the place of C5 inhibition in secondary aHUS. The development of new C5 blockers as well as other complement modulators will undoubtedly impact the management of aHUS. To date, the benefit of eculizumab in STEC-HUS is not as clear as in aHUS. Ongoing studies, and potentially yet to be planned studies in countries with high incidence of STEC, may help settle the question of the relevance of eculizumab for STEC-HUS patients. Nevertheless, therapies that target the Stx itself remain an interesting approach [88]. 17

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[69] Commereuc M, Karras A, Amrein C, Boussaud V, Sberro-Soussan R, Guillemain R, et al. Successful treatment of acute thrombotic microangiopathy by eculizumab after combined lung and kidney transplantation. Transplantation. 2013;96:e58-9. [70] Cavero T, Rabasco C, Lopez A, Roman E, Avila A, Sevillano A, et al. Eculizumab in secondary atypical haemolytic uraemic syndrome. Nephrol Dial Transplant. 2017;32:466-74. [71] Ahlenstiel-Grunow T, Hachmeister S, Bange FC, Wehling C, Kirschfink M, Bergmann C, et al. Systemic complement activation and complement gene analysis in enterohaemorrhagic Escherichia coli-associated paediatric haemolytic uraemic syndrome. Nephrol Dial Transplant. 2016;31:1114-21. [72] Westra D, Volokhina EB, van der Molen RG, van der Velden TJ, Jeronimus-Klaasen A, Goertz J, et al. Serological and genetic complement alterations in infection-induced and complement-mediated hemolytic uremic syndrome. Pediatr Nephrol. 2017;32:297-309. [73] Thurman JM MR, Emlen W, Wood S, Smith C, Akana H, et al. Alternative pathway of complement in children with diarrhea-associated hemolytic uremic syndrome. . Clin J Am Soc Nephrol 2009;4:1920-4. [74] Ferraris JR, Ferraris V, Acquier AB, Sorroche PB, Saez MS, Ginaca A, et al. Activation of the alternative pathway of complement during the acute phase of typical haemolytic uraemic syndrome. Clin Exp Immunol. 2015;181:118-25. [75] Brady TM, Pruette C, Loeffler LF, Weidemann D, Strouse JJ, Gavriilaki E, et al. Typical Hus: Evidence of Acute Phase Complement Activation from a Daycare Outbreak. J Clin Exp Nephrol. 2016;1. [76] Rosales A, Hofer J, Zimmerhackl LB, Jungraithmayr TC, Riedl M, Giner T, et al. Need for long-term follow-up in enterohemorrhagic Escherichia coli-associated hemolytic uremic syndrome due to late-emerging sequelae. Clin Infect Dis. 2012;54:1413-21. [77] Mody RK, Gu W, Griffin PM, Jones TF, Rounds J, Shiferaw B, et al. Postdiarrheal hemolytic uremic syndrome in United States children: clinical spectrum and predictors of inhospital death. J Pediatr. 2015;166:1022-9. [78] Nathanson S, Kwon T, Elmaleh M, Charbit M, Launay EA, Harambat J, et al. Acute neurological involvement in diarrhea-associated hemolytic uremic syndrome. Clin J Am Soc Nephrol. 2010;5:1218-28. [79] Gould LH, Demma L, Jones TF, Hurd S, Vugia DJ, Smith K, et al. Hemolytic uremic syndrome and death in persons with Escherichia coli O157:H7 infection, foodborne diseases active surveillance network sites, 2000-2006. Clin Infect Dis. 2009;49:1480-5. [80] Frank C, Werber D, Cramer JP, Askar M, Faber M, an der Heiden M, et al. Epidemic profile of Shiga-toxin-producing Escherichia coli O104:H4 outbreak in Germany. N Engl J Med. 2011;365:1771-80. [81] Michael M, Elliott EJ, Craig JC, Ridley G, Hodson EM. Interventions for hemolytic uremic syndrome and thrombotic thrombocytopenic purpura: a systematic review of randomized controlled trials. Am J Kidney Dis. 2009;53:259-72. [82] Loos S, Ahlenstiel T, Kranz B, Staude H, Pape L, Hartel C, et al. An outbreak of Shiga toxin-producing Escherichia coli O104:H4 hemolytic uremic syndrome in Germany: presentation and short-term outcome in children. Clin Infect Dis. 2012;55:753-9. [83] Loos S, Aulbert W, Hoppe B, Ahlenstiel-Grunow T, Kranz B, Wahl C, et al. Intermediate Follow-up of Pediatric Patients With Hemolytic Uremic Syndrome During the 2011 Outbreak Caused by E. coli O104:H4. Clin Infect Dis. 2017;64:1637-43. [84] Kielstein JT, Beutel G, Fleig S, Steinhoff J, Meyer TN, Hafer C, et al. Best supportive care and therapeutic plasma exchange with or without eculizumab in Shiga-toxin-producing E. coli O104:H4 induced haemolytic-uraemic syndrome: an analysis of the German STEC-HUS registry. Nephrol Dial Transplant. 2012;27:3807-15.

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[85] Menne J, Nitschke M, Stingele R, Abu-Tair M, Beneke J, Bramstedt J, et al. Validation of treatment strategies for enterohaemorrhagic Escherichia coli O104:H4 induced haemolytic uraemic syndrome: case-control study. Bmj. 2012;345:e4565. [86] Lapeyraque AL, Malina M, Fremeaux-Bacchi V, Boppel T, Kirschfink M, Oualha M, et al. Eculizumab in severe Shiga-toxin-associated HUS. N Engl J Med. 2011;364:2561-3. [87] Delmas Y, Vendrely B, Clouzeau B, Bachir H, Bui HN, Lacraz A, et al. Outbreak of Escherichia coli O104:H4 haemolytic uraemic syndrome in France: outcome with eculizumab. Nephrol Dial Transplant. 2014;29:565-72. [88] Melton-Celsa AR, O'Brien AD. New Therapeutic Developments against Shiga ToxinProducing Escherichia coli. Microbiol Spectr. 2014;2 (5). :doi: 10.1128/microbiolspec.EHEC0013-2013. Legends to Figures: Figure 1: Management and outcome of children and adults with atypical HUS before and after the introduction in clinical practice of the first complement inhibitor, eculizumab. a. Percentages are from 2 retrospective series, from Italy (149 children and 99 adults) [10] and France (89 children and 125 adults) [6] . Percentages of patients in ESRD include deceased patients. Percentages of relapses are from the French series [6]. b. Percentages are from prospective non controlled trials of eculizumab in adults (3 trials including 17, 20 and 41 patients) [12, 13, 16, 17] or children (1 trial including 22 children) [14, 15]. c. Percentages are from a retrospective series issued from the French registry of aHUS patients, comparing 18 adult patients treated with eculizumab since 2009 to 41 paired controls treated with PE between 2004 and 2008 [18] P values for eculizumab –treated patients versus PE – treated patients, * p=0.02; # p=0.04

Abbreviations: AID, autoimmune disease. aHUS, atypical hemolytic uremic syndrome. CblC, cobalamine C. ESRD, end-stage renal disease. F-up, follow-up. PCR, polymerase chain reaction. PE, plasma exchange. PI, plasma infusion. Plt, platelet count. STEC, Shiga toxinproducing E coli. Stx, shiga toxin. TMA, thrombotic microangiopathy. TTP, thrombotic thrombocytopenic purpura. y, year

23

Figure 2: Outcome of anti-CFH antibodies-associated HUS before and after the introduction in clinical practice of the first complement inhibitor, eculizumab. a. Percentages are from the review by Dragon-Durey et al [48] of outcomes in 101 patients with anti-CFH antibody-HUS treated with plasma exchange only or predominantly, compared to 112 treated with plasma exchange and immunosuppression, reported in series published between 2009 and 2014. b. Percentages are from the United Kingdom and Ireland series published by Brocklebank et al [49] of 17 patients with anti-CFH antibodies treated with PE without immunosuppression c. Summary of 18 patients with anti-CFH antibody-HUS treated with eculizumab (case reports or series). Eleven patients were PE/PI- resistant (8) or dependent (3) and therefore were switched to eculizumab, and 4 received first line eculizumab (pre-eculizumab treatment was not documented in 3 other patients). Eculizumab was given as monotherapy in 13 patients [38, 49, 50, 52-54] or with simultaneous immunosuppression ± glucocorticoids in 5 patients.[18, 39, 40, 49, 51, 55] (patient 4 in [18] is patient 23 in [39]). Anti-CFH antibodies titers increased to very high levels in 3 patients treated with eculizumab alone [49], while they decreased

below the positive threshold

in the 5 patients treated with eculizumab +

immunosuppression (rituximab in 1, MMF in 2, rituximab+MMF in 1, transplant immunosuppression in 1) [18, 39, 40, 49, 51, 55]. Eculizumab was stopped after 2, 8, 24 and 37 months in 4 of the latter patients [18, 39, 40, 51, 55]. They had normal serum creatinine and no relapse 3, 7, 11, and 12 months after eculizumab withdrawal, 2 of them under maintenance MMF treatment (maintenance treatment not documented in the 2 others). Only 3 of the 13 patients treated with eculizumab alone stopped treatment after 0.8, 2.6 and 5.5 months (Anti-CFH antibody titers not documented). One of them (eculizumab withdrawal after 0.8 month) had relapse of HUS 0.7 months after eculizumab withdrawal [38].

24

Abbreviations: CFH: complement factor H. CKD, chronic kidney disease. HUS, hemolytic uremic syndrome. IS, immunosuppression. MMF, mycophenolate mofetil. PE, plasma exchange. PI, plasma infusion.

Figure 3: Outcome of STEC-HUS before and after the introduction in clinical practice of the first complement inhibitor, eculizumab [76, 77, 81-85]. #

not significant; *p<0.05; ** p=0.001, patients treated with eculizumab or PE/eculizumab

compared to patients treated with PE only or with best standard of care only a. Eculizumab was given in combination with or after PE in 7 of the 13 patients. Indications for eculizumab were neurological symptoms in 10 patients and/or prolonged dialysis with slow recovery of kidney function. Patients received eculizumab after a median of 22 days (2 to 115) after diagnosis of HUS. Outcome at median follow-up 3 years was documented in 11 patients treated with eculizumab and 61 patients not treated. b. Patients receiving eculizumab after PE were compared with patients treated only with PE or who received only best standard of care. c. Patients receiving eculizumab after PE were compared with a matched control group with similar severity of HUS at baseline, treated only with PE. First dose of eculizumab was at mean day 10.2 (SD 4.6).

In hospital, 21% of eculizumab-treated patients had new

neurological complications and 40% required further PE. Mean number of dialysis was similar in eculizumab-treated and untreated patients (mean (SD) 7.6 (6.3) versus 10.3 (10.2)).

CKD

stages

were

defined

according

http://www.kdigo.org/clinical_practice_guidelines/

25

to

KDIGO

2012,

pdf/CKD/KDIGO_2012_CKD_GL.pdf.

Stage 2: Estimated glomerular filtration rate (eGFR) 60-89 ml/min/1.73m2; Stage 3: eGFR 30-59 ml/min/1.73m2; Stage 5 (ESRD): eGFR < 15 ml/min/1.73m2 or dialysis. Abbreviations: BCS, best standard of care. CKD, chronic kidney disease. ESRD, End stage renal disease. Ht, hematocrit. HUS, haemolytic uremic syndrome. PE, plasma exchange. p.o, per os. RBC, red blood cells. SCr, serum creatinine. SD, standard deviation. STEC, Shiga toxin producing E coli. WBC, white blood cells

Table 1. Risk of relapse of atypical HUS after eculizumab discontinuation according to complement genetics in 60 patients reported in the literature and 37 patients from a French retrospective series. Only patients with primary atypical HUS on native kidneys and screened for complement variants are included in this analysis. Patients with anti-CFH antibodies or kidney transplant are not included. Eculizumab treatment was reinitiated in all patients who relapsed but one, and outcome was favourable in all retreated patients. Published case reports and series a French retrospective studyb Patients with Patients with Patients who Patients who relapse after relapse after Variants discontinued discontinued discontinuation discontinuation N N N (%) N (%) CFH and CFH/CFHR1 15 9 (60) 11 8 (72.7) hybrid MCP 8 3 (37.5) 8 4 (50) C3 7 3 (42.8) 1 0 CFI 4 0 / / CFB 1 0 / / No variant identified 25 2 (8) 17 0 Total 60 17 (28.3) 37 12 (32.4) CFH: complement factor H; CFHR1: complement factor H related protein 1; CFI: complement factor I; CFB : complement factor B ; MCP : membrane cofactor protein ; a. 13 case reports (see references in Supplemental data on eculizumab discontinuation), and 5 cases series [7, 3638, 40] b. [39]

Figures

26

27

28