Pediatric thrombotic thrombocytopenic purpura. EA3518 Recherche clinique en hématologie, immunologie et transplantation, équipe

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1 PROF. AGNÈS VEYRADIER (Orcid ID : ) Article type : Review Article Running title: Pediatric TTP Pediatric thrombotic thrombocytopenic purpura Bérangère S. Joly a,b,c, Paul Coppo c,d, Agnès Veyradier a,b,c a Service d hématologie biologique, groupe hospitalier Saint-Louis-Lariboisière, Assistance Publique-Hôpitaux de Paris, Université Paris-Diderot, Paris, France b EA3518 Recherche clinique en hématologie, immunologie et transplantation, équipe microangiopathies thrombotiques, ADAMTS13 et facteur Willebrand, Centre Hayem, hôpital Saint- Louis, Université Paris-Diderot, Paris, France c Centre National de Référence Maladies Rares des MicroAngiopathies Thrombotiques (CNR-MAT), Assistance Publique-Hôpitaux de Paris, Paris, France d Service d hématologie, hôpital Saint-Antoine, Assistance Publique-Hôpitaux de Paris, Université Pierre et Marie Curie, Paris, France Corresponding author: Professor Agnès Veyradier, MD, PhD Service d hématologie biologique Hôpital Lariboisière 2, rue Ambroise Paré Paris, France Tel: This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: /ejh.13107

2 Fax: agnes.veyradier@aphp.fr Abstract Child-onset thrombotic thrombocytopenic purpura (TTP) is a rare entity of thrombotic microangiopathy (TMA). The pathophysiology of the disease is based on a severe functional deficiency of ADAMTS13 (activity <10%), the specific von Willebrand factor (VWF)-cleavage protease. This deficiency may be either acquired (associated anti-adamts13 autoantibodies) or congenital (resulting from biallelic mutations of ADAMTS13 gene). ADAMTS13 deficiency is responsible for the accumulation of high molecular weight multimers of VWF and the formation of platelet thrombi in the microcirculation. Consequently, microangiopathic hemolytic anemia and consumption thrombocytopenia are associated with organ ischemia. The differential diagnosis with other TMAs, autoimmune cytopenias or hematological malignancies may be challenging. The exploration of ADAMTS13 (activity, antibodies, antigen, ADAMTS13 gene) supports the diagnosis of TTP. The first-line treatment of the acute phase of TTP is based on plasmatherapy. In congenital TTP, patients with a chronic disease benefit from a prophylactic plasmatherapy. In autoimmune TTP, steroids and B-cells depleting therapies increasingly are used together with plasma exchange. Long-term follow-up including the monitoring of ADAMTS13 activity is mandatory. A severe decrease in ADAMTS13 activity (< 10%) may predict relapses and preemptive B-cell depletion with rituximab can be used to prevent relapses. Keywords ADAMTS13, von Willebrand factor, thrombotic thrombocytopenic purpura, pediatrics, plasmatherapy. Manuscript 1. Introduction Child-onset thrombotic thrombocytopenic purpura (TTP) is a rare entity of thrombotic microangiopathy (TMA), a rare and life-threatening disease, referring to pathological features of vascular damage (incidence is estimated at <1 case per million children per year) (1,2). TMA syndromes are defined by a microangiopathic hemolytic anemia (presence of schistocytes on the blood smear), a consumption

3 thrombocytopenia and organ damage related to disseminated thrombi in the microcirculation (3). The pathophysiology of TTP is based on a severe functional deficiency of ADAMTS13 (A Disintegrin and Metalloprotease with ThromboSpondin type 1 repeats, member 13), the specific von Willebrand factor- (VWF) cleaving protease (4). Child-onset TTP may be either idiopathic or associated with miscellaneous clinical contexts (infections, autoimmune diseases, neoplasia, hematopoietic stem cells transplantation, organ graft ). Other TMA syndromes are reported in children (hemolytic uremic syndrome, HUS, associated with Shiga toxin producing Escherichia coli enteric infection affecting predominantly the kidney and atypical HUS related to a genetic or acquired dysregulation of the complement alternative pathway (5)) and their differential diagnosis with TTP may be challenging in children. 2. Pathophysiology of thrombotic thrombocytopenic purpura TTP occurs in adulthood or less frequently in childhood (about 10% of all TTP cases) (6). Indeed, the first case of TTP was described in 1924, in a 16-year-old girl (7). ADAMTS13 regulates the biological function of VWF cleaving ultralarge and hyperadhesive VWF multimers. ADAMTS13-VWF interaction is crucial in the pathophysiology of TTP von Willebrand factor VWF is an adhesive and multimeric glycoprotein containing several functional domains (A, B, C and D-domains) (Figure 1). Pre-pro-VWF contains a signal peptide, a propeptide (D1-D2 domains) and the mature VWF subunit (2050 aminoacids). VWF gene, located on chromosome 12 (12p13.3), contains 52 exons and spans 178 kb. VWF, synthetized in vascular endothelial cells and megakaryocytes, is stored in intracellular organelles: endothelial cells (Weibel-Palade bodies), platelets granules and megakaryocytes (αgranules). Plasma concentration of VWF is ~10 µg/ml, the half-life of VWF antigen is ~16 hours, and VWF multimers are cleared with a half-life of ~12 hours, in humans. VWF circulates as a globular protein. High shear stress of blood flow in the microcirculation induce conformational changes in multimeric VWF, unfolding of VWF and exposure of its interactive sites (8). VWF has a central role in hemostasis, through platelet adhesion and aggregation at sites of vascular

4 injury, and carrying factor VIII in the circulation (8). ADAMTS13 regulates VWF multimeric size by cleaving the Tyr Met 1606 bond in the VWF A2 domain (Figure 1). In 1982, Moake reported the presence of circulating ultralarge VWF multimers in TTP patients, usually absent from plasma of healthy persons. Thus, he identified the link between VWF and the pathogenesis of TTP (9) ADAMTS13 VWF-cleaving protease was purified in 1996 (10,11), cloned (12 14) and identified as the 13 th member of the family of metalloprotease ADAMTS in 2001 (15,16). ADAMTS13 is synthetized in the liver (hepatic stellate cells) and vascular endothelial cells. Structurally, ADAMTS13 shares similar domains with the ADAMTS family proteases. ADAMTS13 domain structure comprises a signal peptide, a propeptide, a metalloprotease, a disintegrin-like domain, a first thrombospondin type 1 repeat (TSP1), Cys-rich and spacer domains in the N-terminal region and seven additional TSP1 repeats and two CUB domains in the C-terminal region (Figure 1) (17). ADAMTS13 gene is localized in chromosome 9q34 and spans 29 exons encompassing ~37 kb in the human genome (12). ADAMTS13 is the specific VWF-cleaving protease. Arterial shear-stress induces conformational change in VWF and the Tyr Met 1606 resulting in the fragmentation of VWF strings Thrombotic thrombocytopenic purpura bond becomes accessible by ADAMTS13 for cleavage, TTP pathophysiology is specifically related to a severe functional deficiency of ADAMTS13 (ADAMTS13 activity <10% of normal). This deficiency leads to the accumulation of hyperadhesive ultralarge VWF multimers inducing the spontaneous formation of platelet-rich microthrombi within arterioles and capillaries and subsequent widespread microvascular ischemia (18 20). TTP is characterized by a microangiopathic hemolytic anemia, a consumption thrombocytopenia, with or without multivisceral ischemia (Figure 2). Clinically, the acute phase of the disease may include fever, neurological features (headache, confusion, coma, seizure, stroke, transient focal defect), cardiac and/or renal disorders (1). TTP is also characterized by relapses resulting from persistent or recurrent deficiencies in ADAMTS13 activity.

5 The mechanism for ADAMTS13 severe deficiency is either inherited (~5% of all TTP cases) or acquired (~95% of all TTP cases) (Figure 3) (2,6,18,19). In children, TTP represents less than 10% of all TTP cases (~1/3 of congenital TTP and ~2/3 of acquired TTP) (6). 3. Clinical and biological features of TTP The first case of TTP was described in 1924 by Moschcowitz (7). A 16-year-old girl suddenly developed weakness, pain, pallor, fever and petechiae (no platelet count available). Few days later, she developed neurological disorders (hemiparesis and paralysis), became comatose and died. Based on the autopsy reporting the presence of disseminated hyaline thrombi in the microcirculation (heart, kidney, spleen, liver), this patient was the first published case of TTP. Child-onset TTP (initial episode before age 18) is very rare and represents only ~10% of all TTP cases (Figure 3). In children, the first episode of TTP may be either idiopathic or associated with a clinical context (autoimmune disease, infection, hematopoietic stem cell transplantation, organ transplantation, neoplasia...) (2). In the absence of an appropriate treatment, TTP remains lifethreatening. In children, the diagnosis of TTP should be suspected when a bicytopenia (microangiopathic haemolytic anemia and consumption thrombocytopenia) is associated with organ failure or a previous diagnosis of autoimmune cytopenia (idiopathic thrombocytopenic purpura, anemia autoimmune hemolytic or Evans syndrome) not responding to specific treatments. In the classical form of childonset TTP, symptoms related to organ ischemia mostly concern the brain, and less frequently the heart and the kidney. TTP associates with fever, neurological features (headache, confusion, coma, seizures, strokes, or transient focal defects) and sometimes impaired renal function (proteinuria, moderate acute kidney injury). Abdominal (abdominal pain and vomiting) and cardiac features are associated to a disseminated disease Acquired TTP The prevalence of child-onset acquired TTP is ~1 case per million children. An analysis of the literature (21) and an original study conducted in the French cohort of 45 child-onset TTP (2) are in agreement and show the following characteristics: in children, the first TTP episode occured preferentially in girls (sex ratio: 2.5F/1M) and the age distribution ranged from 4 months to 17 years

6 old (median: 13 years). The inaugural presentation of the disease was idiopathic in 55% of acquired forms of TTP (median age of onset: 15 years) or associated with a clinical context in 45% of cases (median age of onset: 8 years). Child-onset TTP was characterized by hemolytic anemia (median hemoglobin levels: 6.7 g/dl), profound thrombocytopenia (median platelet count: /L) and a severe ADAMTS13 deficiency (ADAMTS13 activity <10%). Fever was reported in 36% of children and ischemic disorders affecting the brain, the kidney and/or the heart were reported in 64% of cases (40%, 42% and 7%, respectively). Impaired kidney function and acute kidney injuries were more frequent in non-idiopathic TTP (80%) than in idiopathic forms of the disease. More generally, acquired forms of child-onset TTP were mostly related to anti-adamts13 autoantibodies (82%) (2). The anti- ADAMTS13 autoantibodies are mainly IgG with a neutralizing action (inhibition of the catalytic activity of ADAMTS13) and/or a non-neutralizing action (complexation of ADAMTS13 and acceleration of its clearance). The anti-adamts13 antibodies are mainly directed against the spacer domain of the protease (95% of auto-antibodies) (Figure 1) (22). However, in some acquired TTP cases with ADAMTS13 deficiency, antibodies may be undetectable which could be related to the lack of sensitivity of available methods (23), to anti-adamts13 antibodies of IgM or IgA type (24), to an altered synthesis or secretion of ADAMTS13 (25), to consumption or further enzymatic degradation of the protease (26 28). The differential diagnosis between TTP and other cytopenias in children can be challenging and 31% of initial misdiagnosis (HUS, atypical HUS, autoimmune cytopenia, hematological malignancy) have been reported (2). Consequently, the specific treatment of TTP is delayed, and organ sequelae may occur. Some patients with impaired renal function at presentation were misdiagnosed as HUS and some of them developed an end stage renal disease in the absence of appropriate treatment (29). The mortality rate of the acquired form of the disease is 9% in children (2). During clinical and biological remission, anti-adamts13 antibodies disappear, in parallel with the normalization of ADAMTS13 activity. The disease evolves by acute phase interspersed with remission periods, and 24% of clinical relapses were reported in the French TTP pediatric cohort (2). Moreover, the appearance of biological features of autoimmunity (antinuclear antibodies, anti-double stranded DNA antibodies) and/or connective tissue diseases (systemic lupus erythematosus mainly) were reported in several patients, emphasizing the recommendation of the long-term follow-up of childonset TTP patients to rapidly diagnose these conditions and prevent tissue injury. HLA-DRB1*11 is

7 well established as a predisposing factor for idiopathic acquired TTP, whereas HLA-DRB1*04 is protective in Caucasian adult patients (30). Further investigations are in progress in children Congenital TTP Congenital TTP (Upshaw-Schulman syndrome) was described by Schulman in 1960 and Upshaw in Of all pediatric TMA, child-onset congenital TTP seems especially rare (~1/3 of all child-onset TTP cases, prevalence estimated at ~0.8 cases per million children) (Figure 3). Congenital TTP usually starts in the neonatal period with hematological features and severe jaundice (blood exchange transfusion), requiring an appropriate management in emergency, with potentially severe consequences (31). The phenotype of congenital TTP is also variable in terms of clinical severity (32), organ injuries and frequency of TTP episodes. The first congenital TTP episode occurs usually before 5 years old and the sex ratio is of 1F/1M. Congenital TTP shows clinical heterogeneity with approximately 50% of severe and chronic forms (requiring prophylactic plasmatherapy monthly) and 50% less severe forms associated with periods of remission of several years, not requiring prophylactic plasmatherapy. TTP episodes may be triggered by vaccinations, infections and surgical procedures, for example (29,33,34). Since the first report of ADAMTS13 sequence variations on chromosome 9q34 (12), and thanks to national registries for genetic rare diseases (29,35 38), congenital TTP is actually well described. Near 180 distinct sequence variations (60% missense; 40% non-sense, deletion and frameshift) of ADAMTS13 gene have been described in the international literature. The transmission of the disease is autosomal recessive. Two specific sequence variations of ADAMTS13 gene have been reported in European patients. The c.4143_4144dupa has exclusively been reported in patients with an ancestry from central and northern Europe (38,39). The sequence variation p.arg1060trp (c.3178c>t) has been reported with a high prevalence in pregnancy-onset congenital TTP patients from Europe, Scandinavia, North America and Turkey (33,35). The overlap between ADAMTS13 sequence variations found in Asian and European patients (40,41) is weak. Family consanguinity should be investigated in congenital TTP patients. Moreover, in vitro (36,40,42 45) and in silico (46) studies of ADAMTS13 sequence variations have confirmed the deleterious effect of some mutations on the function of ADAMTS13 or on the impairment of its secretion (42,44,45).

8 4. Diagnosis of thrombotic thrombocytopenic purpura in children ADAMTS13 is the unique biological marker able to definitely differentiate TTP from other TMA syndromes (HUS, atypical HUS) and hematological cytopenias Suspicion of TMA: initial investigations Among all TMA suspicion in children, standard biological investigations are needed to comfort the diagnosis (Table 1) (6). Firstly, standard hematological analysis should comprise blood cells count (erythrocytes, hemoglobin, leukocytes, platelets, reticulocytes), repeated blood smear (presence of schistocytes) and direct antiglobulin test. Secondly, additional biochemical investigations include hemolysis parameters (bilirubin, serum haptoglobin, lactate dehydrogenase), serum electrolytes, urine electrolytes, renal function (plasma urea and creatinine levels, glomerular filtration rate, proteinuria, hematuria), liver function, β-hcg (adolescent girls patients), and cardiac troponin Ic levels (of prognostic value) (47). Finally, other standard biological parameters are investigated: coagulation parameters, bacteriological analysis (cytobacteriological examination of the urine, blood culture, stool culture ), viral serology (HIV, HCV, HBV) and autoimmune investigations. However, a direct positive antiglobulin test or the absence of schistocytes on blood smears should not exclude the diagnosis of TTP, and 10% of TTP patients could display such atypical features, exposing them to diagnostic wandering (2,48). All these standard investigations are not specific for TTP in children and only a biological exploration of ADAMTS13 is able to confirm or to exclude the diagnosis ADAMTS13 investigations ADAMTS13 is the unique sensitive and specific marker for TTP. The biological documentation of TMA syndrome requires ADAMTS13 investigations based on ADAMTS13 activity measurement in first intention. In the absence of rapid turnaround assay for ADAMTS13 activity, ADAMTS13 investigations are performed retrospectively, to document the clinical diagnosis of TMA. In other words, the result of ADAMTS13 investigations should not delay the first-line treatment of TTP when the diagnosis is highly suspected (6).

9 ADAMTS13 activity ADAMTS13 activity is the most sensitive and specific parameter for TTP diagnosis. Screening for ADAMTS13 activity remains the first assay to perform when TMA is suspected. ADAMTS13 activity is measured using reference methods (FRETS-VWF73, full-length VWF ELISA) (49 51), limited to expert laboratories. Rapid turnaround assays also exist but they do not have the reliability of the reference methods (52,53). A severe functional deficiency in ADAMTS13 definitely confirms the diagnosis of TTP. Moreover, long-term follow-up of pediatric TTP patients is crucial. Biological monitoring of ADAMTS13 activity of child-onset acquired TTP in remission is justified, as in adults, to prevent relapses of the disease (prognostic value of the risk of relapse) ADAMTS13 autoantibodies When ADAMTS13 activity is undetectable (<10%), screening and titration of anti-adamts13 autoantibodies are performed to identify the mechanism for ADAMTS13 deficiency (autoimmune, other possible mechanism) (22,24). When anti-adamts13 autoantibodies are positive (IgGs mainly), they confirm the diagnosis of autoimmune TTP. However, the absence of anti-adamts13 autoantibodies is not sufficient to rule out the diagnosis of autoimmune TTP and further investigations are needed (ADAMTS13 monitoring and follow-up). In congenital TTP, some patients benefit from regular prophylactic plasmatherapy. Anti-ADAMTS13 antibodies are also screened and titrated in these patients, to identify the potential occurrence of an alloimmunization directed against ADAMTS13. However, no case of alloimmunization against ADAMTS13 after plasmatherapy has been reported yet ADAMTS13 antigen In congenital TTP, ADAMTS13 antigen levels (reference values: ng/ml) are measured to characterize a quantitative or qualitative deficiency in ADAMTS13 (6) ADAMTS13 gene ADAMTS13 gene analysis (NM_ ) is based on the sequencing of the 29 exons of ADAMTS13 gene, the exon / intron boundaries, the 3'UTR and 5'UTR regions and the promoter.

10 When the clinical history and the biological phenotype match with congenital TTP diagnosis, this analysis definitely confirms the diagnosis (12) Additional explorations When a TMA syndrome is suspected, some complementary explorations may be performed according to the clinical context: a bone marrow aspiration to rule out a central etiology for cytopenias, and in patients with organ involvement, chest X-ray, cerebral magnetic resonance imaging, cardiac explorations (electrocardiogram, echocardiography), and a renal biopsy (atypical HUS, chronic renal failure, and evaluation of renal prognostic) Differential diagnosis HUS is the most frequent child-onset TMA syndrome. HUS is usually associated with renal impairement and includes several distinct entities (5,54). An infection-associated HUS caused by Shiga-toxin-producing Escherichia coli strain (STEC-HUS, serotype O157: H7, O103, 026, mainly) is the most common child-onset TMA syndrome, particularly in children under 3-years-old (consisting of 80-90% of HUS) (55). In comparison with TTP, STEC-HUS is usually associated with bloody prodromal diarrhea, a severe renal impairment and a moderate thrombocytopenia. An infectionassociated HUS caused by Streptococcus pneumoniae (pneumonia, meningitis) is also reported in 5% of child-onset HUS. An atypical HUS (consisting of 5-10% of HUS) is caused by dysregulation of the complement alternative pathway, mutation in DGKE (diacyglycerol kinase ε), or cobalamin C deficiency (5,54,56). Autoimmune cytopenias (Evans syndrome, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia), hematological malignancies and autoimmune diseases (lupus, antiphospholipid syndrome, ) are also differential diagnosis of TTP (2,34). Moreover, partial ADAMTS13 deficiency (ADAMTS13 activity between 20 and 50%) are not specific and may be associated with miscellaneous clinical contexts (autoimmune diseases, severe infections, hepatocellular insufficiency, neoplasia or other TMA syndromes). These partial deficiencies are due to a consumption of ADAMTS13, a decrease in synthesis and/or secretion of ADAMTS13, an inhibition of ADAMTS13 by pro-inflammatory cytokines (interleukin 6), or a degradation of ADAMTS13 by proteolytic enzymes (elastases, thrombin).

11 5. Treatment of TTP Child-onset TTP is an emergency and requires rapid diagnosis and management in intensive care unit. Several strategies, based on our knowledge on the pathophysiology of the disease, are used in TTP (Figure 4) (57) Plasmatherapy The first-line treatment of the acute phase of child-onset TTP remains an emergency and is based on daily plasma replacement therapy (plasma infusion or plasma exchange), allowing an exogenous supply of ADAMTS13 (cleavage of multimeric high molecular weight multimers of VWF and saturation of anti-adamts13 autoantibodies in acquired forms) (Figure 4, Figure 5). Plasmatherapy is performed to achieve a complete response defined by normal platelet count ( 150x10 9 /L) for 2 consecutive days, normalization of LDH and clinical improvement (2,58). Some groups decrease plasmatherapy gradually to prevent exacerbations of the disease. In the congenital form of TTP, prophylactic plasmatherapy is the unique therapeutic option currently available (usually 10 ml/kg) and the frequency of plasma infusions is guided by the tolerance and chronicity of the disease. Anti-ADAMTS13 IgG titration should be performed regularly in these patients in order to detect the potential occurrence of anti-adamts13 alloantibodies (29,32,37,38) Immunomodulation In acquired TTP, steroids are usually used as an adjunctive treatment to curative first-line plasmatherapy (Figure 4, Figure 5). Patients refractory to the first-line treatment (platelet count which does not double after 4 days of intensive treatment and lactate dehydrogenase above normal value), or in case of exacerbation of the disease (absence of clinical improvement and/or thrombocytopenia <100x10 9 /L for 2 days), immunomodulation with rituximab (monoclonal anti-cd20 antibody) may be considered (Figure 4, Figure 5) (2,59). Rituximab is typically effective after 2 weeks following the first infusion, and therefore it may not prevent early death; moreover, rituximab administration needs to be associated with the pursue of daily therapeutic plasma exchange. Other immunosuppressive strategies (vincristine, pulses of cyclophosphamide, ciclosporin A and splenectomy) should be considered in the more severe forms of the disease (Figure 5) (2).

12 5.3. Other therapeutics Transfusions of red blood cell concentrates can be used in case of profound anemia, depending on clinical tolerance. Platelet concentrates should be avoided in the absence of life-threatening hemorrhagic symptomatology, because they may exacerbate the spontaneous formation of plateletrich and VWF-rich microthrombi (60) Innovating drugs Several novel therapies based on recombinant ADAMTS13 and anti-vwf molecules (caplacizumab, N-acetylcystein) emerge in TTP (Figure 5) (57). Recombinant ADAMTS13 (radamts13) is scheduled to be evaluated in a phase III therapeutic trial in the pediatric congenital form of the disease (ClinicalTrials.gov NCT ). Preliminary studies are encouraging and report a good tolerance and a good efficacy of the treatment (decrease of high molecular weight multimers of VWF, increase of platelets and decrease of lactate dehydrogenase) (61). Caplacizumab, a nanobody directed against the A1 domain of VWF, inhibits the interactions between platelet GPIb and VWF and has shown safety and efficacy in a phase II clinical trial devoted to adult patients (62). The randomized, doubleblind, placebo-controlled, Phase III HERCULES study of caplacizumab in adults with acquired TTP confirm that caplacizumab results in faster resolution of acquired TTP, reducing the time to platelet count response. Caplacizumab reduces TTP-related death, recurrence of TTP and has a favorable safety profile in adults acquired TTP patients (63). Thanks to mucolytic properties, N-acetylcystein limits the polymerization and the adhesion of VWF multimers (64). Promising results were reported in ADAMTS13 -/- mice, in prophylaxis treatment (64). A Phase I clinical trial is also in progress in children (ClinicalTrials.gov NCT ). 6. Long-term follow-up of TTP A regular long-term follow-up of all patients is recommended. Our use is to propose a monitoring of ADAMTS13 activity every 3 months during the first years following the initial episode of acquired TTP. Connective tissue diseases and biological features of autoimmunity may appear during follow-up (6% and 17% respectively in the French pediatric cohort) and should be anticipated (2).

13 6.1. Prevention of relapses TTP is a pathology evolving by acute phase and remission periods. The prevention of TTP relapses is therefore a major challenge. In acquired forms of TTP, the relapse rate is ~25% (2,21). Biological monitoring of ADAMTS13 activity is justified by the relapsing tendency of the disease (40% of clinical relapse in the current year when ADAMTS13 <10%, and 5% of relapse when ADAMTS13 is normal), and is aimed to detect biological relapse. Preemptive treatment with rituximab may be considered in autoimmune TTP, for patients in clinical remission when ADAMTS13 activity becomes undetectable (<10%) to avoid clinical relapse (2,65). In the congenital form of the disease, prophylactic plasmatherapy may be considered in chronic forms associated with organ ischemia (approximately 50% of patients). The delay between two plasma infusions (usually every 3 to 4 weeks) depends on each specific patient, as a function of the tolerance of the disease and the occurrence of clinical events Clinical and psychological follow-up Clinical (height, weight, body mass index, autoimmune disease) and psychological follow-up are recommended to evaluate the emergence of autoimmune disease and physical and/or psychological sequelae of the disease (2). Long-term follow-up (medical consultation, standard biology, ADAMTS13 activity monitoring) is highly recommended in TTP children. 7. Conclusion and perspectives Pediatric TTP is a rare entity within a rare disease, and may initially be misdiagnosed as other TMA syndrome, autoimmune cytopenia, malignant hemopathy. Congenital and acquired forms of childonset TTP have distinct biological and demographic features. Clinical, biological and therapeutic management of children has been significantly improved in the last few years, but international recommendations are still needed. Long-term follow-up of patients with a first TTP episode in childhood is highly recommended. Clinical studies of innovative drugs (recombinant ADAMTS13, N- acetylcystein) are currently in progress in pediatric TTP patients. In the next coming months, innovative therapeutic drugs, as recombinant ADAMTS13 and caplacizumab, should be validated and will improve TTP management. One of the perspectives of these clinical trials is to validate a

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17 29. Veyradier A, Lavergne J-M, Ribba A-S, Obert B, Loirat C, Meyer D, et al. Ten candidate ADAMTS13 mutations in six French families with congenital thrombotic thrombocytopenic purpura (Upshaw-Schulman syndrome). J Thromb Haemost 2004; 2: Coppo P, Busson M, Veyradier A, Wynckel A, Poullin P, Azoulay E, et al. HLA-DRB1*11: a strong risk factor for acquired severe ADAMTS13 deficiency-related idiopathic thrombotic thrombocytopenic purpura in Caucasians. J Thromb Haemost 2010; 8: Fujimura Y, Matsumoto M, Yagi H, Yoshioka A, Matsui T, Titani K. Von Willebrand factorcleaving protease and Upshaw-Schulman syndrome. Int J Hematol 2002; 75: Lotta LA, Wu HM, Mackie IJ, Noris M, Veyradier A, Scully MA, et al. Residual plasmatic activity of ADAMTS13 is correlated with phenotype severity in congenital thrombotic thrombocytopenic purpura. Blood 2012; 120: Moatti-Cohen M, Garrec C, Wolf M, Boisseau P, Galicier L, Azoulay E, et al. Unexpected frequency of Upshaw-Schulman syndrome in pregnancy-onset thrombotic thrombocytopenic purpura. Blood 2012; 119: Mariotte E, Azoulay E, Galicier L, Rondeau E, Zouiti F, Boisseau P, et al. Epidemiology and pathophysiology of adulthood-onset thrombotic microangiopathy with severe ADAMTS13 deficiency (thrombotic thrombocytopenic purpura): a cross-sectional analysis of the French national registry for thrombotic microangiopathy. Lancet Haematol 2016; 3: e von Krogh AS, Quist-Paulsen P, Waage A, Langseth ØO, Thorstensen K, Brudevold R, et al. High prevalence of hereditary thrombotic thrombocytopenic purpura in central Norway: from clinical observation to evidence. J Thromb Haemost 2016; 14: Camilleri RS, Scully M, Thomas M, Mackie IJ, Liesner R, Chen WJ, et al. A phenotype-genotype correlation of ADAMTS13 mutations in congenital thrombotic thrombocytopenic purpura patients treated in the United Kingdom. J Thromb Haemost 2012; 10: Fujimura Y, Matsumoto M, Isonishi A, Yagi H, Kokame K, Soejima K, et al. Natural history of Upshaw-Schulman syndrome based on ADAMTS13 gene analysis in Japan. J Thromb Haemost 2011; 9 Suppl 1:

18 38. Schneppenheim R, Budde U, Oyen F, Angerhaus D, Aumann V, Drewke E, et al. von Willebrand factor cleaving protease and ADAMTS13 mutations in childhood TTP. Blood 2003; 101: Schneppenheim R, Kremer Hovinga JA, Becker T, Budde U, Karpman D, Brockhaus W, et al. A common origin of the 4143insA ADAMTS13 mutation. Thromb Haemost 2006; 96: Lotta LA, Garagiola I, Palla R, Cairo A, Peyvandi F. ADAMTS13 mutations and polymorphisms in congenital thrombotic thrombocytopenic purpura. Hum Mutat 2010; 31: Miyata T, Kokame K, Matsumoto M, Fujimura Y. ADAMTS13 activity and genetic mutations in Japan. Hamostaseologie 2013; 33: Hommais A, Rayes J, Houllier A, Obert B, Legendre P, Veyradier A, et al. Molecular characterization of four ADAMTS13 mutations responsible for congenital thrombotic thrombocytopenic purpura (Upshaw-Schulman syndrome). Thromb Haemost 2007; 98: Donadelli R, Banterla F, Galbusera M, Capoferri C, Bucchioni S, Gastoldi S, et al. In-vitro and in-vivo consequences of mutations in the von Willebrand factor cleaving protease ADAMTS13 in thrombotic thrombocytopenic purpura. Thromb Haemost 2006; 96: Matsumoto M, Kokame K, Soejima K, Miura M, Hayashi S, Fujii Y, et al. Molecular characterization of ADAMTS13 gene mutations in Japanese patients with Upshaw-Schulman syndrome. Blood 2004; 103: Kokame K, Matsumoto M, Soejima K, Yagi H, Ishizashi H, Funato M, et al. Mutations and common polymorphisms in ADAMTS13 gene responsible for von Willebrand factor-cleaving protease activity. Proc Natl Acad Sci 2002; 99: Hing ZA, Schiller T, Wu A, Hamasaki-Katagiri N, Struble EB, Russek-Cohen E, et al. Multiple in silico tools predict phenotypic manifestations in congenital thrombotic thrombocytopenic purpura. Br J Haematol 2013; 160: Benhamou Y, Boelle P-Y, Baudin B, Ederhy S, Gras J, Galicier L, et al. Cardiac troponin-i on diagnosis predicts early death and refractoriness in acquired thrombotic thrombocytopenic purpura. Experience of the French Thrombotic Microangiopathies Reference Center. J Thromb Haemost 2015; 13:

19 48. Grall M, Azoulay E, Galicier L, Provôt F, Wynckel A, Poullin P, et al. Thrombotic thrombocytopenic purpura misdiagnosed as autoimmune cytopenia: Causes of diagnostic errors and consequence on outcome. Experience of the French thrombotic microangiopathies reference centre. Am J Hematol 2017; 92: Kokame K, Nobe Y, Kokubo Y, Okayama A, Miyata T. FRETS-VWF73, a first fluorogenic substrate for ADAMTS13 assay. Br J Haematol 2005; 129: Obert B, Tout H, Veyradier A, Fressinaud E, Meyer D, Girma JP. Estimation of the von Willebrand factor-cleaving protease in plasma using monoclonal antibodies to vwf. Thromb Haemost 1999; 82: Gerritsen HE, Turecek PL, Schwarz HP, Lämmle B, Furlan M. Assay of von Willebrand factor (vwf)-cleaving protease based on decreased collagen binding affinity of degraded vwf: a tool for the diagnosis of thrombotic thrombocytopenic purpura (TTP). Thromb Haemost 1999; 82: Thouzeau S, Capdenat S, Stépanian A, Coppo P, Veyradier A. Evaluation of a commercial assay for ADAMTS13 activity measurement. Thromb Haemost. oct 2013;110(4): Joly B, Barbay V, Borg J-Y, Le Cam-Duchez V. Comparison of markers of coagulation activation and thrombin generation test in uncomplicated pregnancies. Thromb Res 2013; 132: Jokiranta TS. HUS and atypical HUS. Blood 2017; 129: Joly BS, Zheng XL, Veyradier A. Understanding thrombotic microangiopathies in children. Intensive Care Med 2018; doi: /s Noris M, Remuzzi G. Atypical hemolytic-uremic syndrome. N Engl J Med 2009; 361: Joly BS, Vanhoorelbeke K, Veyradier A. Understanding therapeutic targets in thrombotic thrombocytopenic purpura. Intensive Care Med 2017; 43(9): Rock GA, Shumak KH, Buskard NA, Blanchette VS, Kelton JG, Nair RC, et al. Comparison of plasma exchange with plasma infusion in the treatment of thrombotic thrombocytopenic purpura. Canadian Apheresis Study Group. N Engl J Med 1991; 325: Scully M, McDonald V, Cavenagh J, Hunt BJ, Longair I, Cohen H, et al. A phase 2 study of the safety and efficacy of rituximab with plasma exchange in acute acquired thrombotic thrombocytopenic purpura. Blood 2011; 118:

20 60. Benhamou Y, Baudel J-L, Wynckel A, Galicier L, Azoulay E, Provôt F, et al. Are platelet transfusions harmful in acquired thrombotic thrombocytopenic purpura at the acute phase? Experience of the French thrombotic microangiopathies reference center. Am J Hematol 2015; 90: E Mannucci PM, Kempton C, Millar C, Romond E, Shapiro A, Birschmann I, et al. Pharmacokinetics and safety of a novel recombinant human von Willebrand factor manufactured with a plasma-free method: a prospective clinical trial. Blood 2013; 122: Peyvandi F, Scully M, Kremer Hovinga JA, Cataland S, Knöbl P, Wu H, et al. Caplacizumab for Acquired Thrombotic Thrombocytopenic Purpura. N Engl J Med 2016; 374: Scully M, Knöbl P, Kentouche K, Rice L, Windyga J, Schneppenheim R, et al. Recombinant ADAMTS-13: first-in-human pharmacokinetics and safety in congenital thrombotic thrombocytopenic purpura. Blood 2017; 130: Chen J, Reheman A, Gushiken FC, Nolasco L, Fu X, Moake JL, et al. N-acetylcysteine reduces the size and activity of von Willebrand factor in human plasma and mice. J Clin Invest 2011; 121: Hie M, Gay J, Galicier L, Provôt F, Presne C, Poullin P, et al. Preemptive rituximab infusions after remission efficiently prevent relapses in acquired thrombotic thrombocytopenic purpura. Blood 2014; 124:

21 Tables Table 1. Biological investigations in thrombotic microangiopathy syndromes. Hematological parameters Hemolysis parameters Direct antiglobulin test Biochemical parameters Coagulation parameters Bacteriological analysis Viral serology Autoimmune investigations Blood cells count (erythrocytes, hemoglobin, leukocytes, platelets, reticulocytes) Repeated blood smear analysis: schistocytes Bilirubin Serum haptoglobin Lactate dehydrogenase Serum and urine electrolytes Renal function (plasma urea and creatinine levels, proteinuria, hematuria) Liver function Cardiac troponin Ic levels Prothrombine time Activated partial thromboplastin time Fibrinogen levels Cytobacteriological examination of the urine Blood culture Stool culture HIV, HCV, HBV Antinuclear antibodies ± anti-dsdna antibodies Antiphospholipid antibodies Complement investigations Abbreviations: HIV: human immunodeficiency virus; HCV: hepatitis C virus; HBV: hepatitis B virus; dsdna: double stranded DNA

22 Legends to figures Figure 1. Structure-function of ADAMTS13 and von Willebrand factor. The structure of ADAMTS13 and von Willebrand factor are characterized by the presence of distinct domain structures. ADAMTS13 regulates von Willebrand factor multimeric size by cleaving the Tyr Met 1606 bond in the von Willebrand factor A2 domain. Figure 2. Pathophysiology of thrombotic thrombocytopenic purpura. Thrombotic thrombocytopenic purpura (TTP) pathophysiology is based on ADAMTS13 deficiency (congenital or acquired deficiency, mainly related to specific autoantibodies). In the absence of ADAMTS13, ultralarge von Willebrand factor multimers are released into the blood and bind spontaneously to platelets to form aggregates within the microcirculation. The von Willebrand factorplatelet aggregates are large enough to form microthrombi inducing microangiopathic hemolytic anemia, platelet consumption and tissue ischemia. Figure 3. Thrombotic thrombocytopenic purpura as a function of age of onset and mechanism for ADAMTS13 deficiency. Among thrombotic thrombocytopenic purpura (TTP) patients, the acquired form of the disease represents 90% of all TTP cases and the congenital form only 10%. The first episode of TTP occurred during adulthood in 95% of cases and during childhood (age <18) in 5% of cases. Interestingly, the proportion of congenital TTP is very low in adults (2%), whereas it is as high as 35% in children. Figure 4. Treatment of child-onset acquired thrombotic thrombocytopenic purpura. The standard treatment of the acute phase of child-onset acquired thrombotic thrombocytopenic purpura (TTP) is based on daily therapeutical plasmatherapy (and steroids) initiated in emergency until remission. Some TTP children may also be unresponsive, or they may exhibit an exacerbation of the disease. Additional rituximab remains the second-line treatment usually leading to a complete remission. Follow-up of TTP children in remission (medical consultation and ADAMTS13 monitoring) may show either a durable remission or relapses requiring standard treatment. ADAMTS13

23 monitoring is needed to identify a decrease of ADAMTS13 activity less than 10%, in the absence of clinical relapse and preemptive treatment with rituximab should be consider. Figure 5. Therapeutic targets in thrombotic thrombocytopenic purpura. The first therapeutic target in thrombotic thrombocytopenic purpura is ADAMTS13. Replacement therapies include plasma exchange and recombinant ADAMTS13 (radamts13). The second therapeutic target is anti-adamts13 autoantibodies. Immunomodulation drugs are based on steroids and rituximab, and less frequently on other immunosuppressive drugs. The third therapeutic target is von Willebrand factor. Caplacizumab has been recently evaluated in a phase III trial devoted to adult TTP patients (63). N-acetylcystein is currently in progress in pediatric TTP.

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