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Thrombotic Microangiopathy: A Focus on Atypical Hemolytic Uremic Syndrome Bradley P. Dixon, MD Division of Nephrology and Hypertension, Cincinnati Children’s Hospital Medical Center

Case Presentation 11 year old white male, previously healthy, presents to the emergency department with 3d of vomiting and abdominal pain, and developed fever and dark urine today. No diarrhea. ROS significant for headache, sore throat, bleeding gums PMHx and PSHx signficant for febrile seizures 1 year ago, dental extractions five days ago Family history unremarkable.

Case Presentation, continued Physical exam demonstrates BP 109/72 HR 87, with soft, nontender abdomen. No pedal edema noted. No scleral icterus or jaundice. Initial labs: – Platelet count of 18K, H/H of 12.8/36.5, nl WBC – LDH 2445 U/L, 1+ schistocytes. Normal CPK. – Mild/moderate renal dysfunction with SCr 1.42 – Large blood and >300 protein on urinalysis Diagnosis = TMA Differential Dx? Additional workup?

Thrombotic Microangiopathy (TMA) Convergence of many different pathomechanisms – Common link is endothelial injury – Platelet activation and aggregation in microvasculature – Fibrin deposition – Mechanical trauma to RBC – Occurs in many microvascular beds, but renal microvasculature especially susceptible to this process

Thrombotic microangiopathy (TMA): Differential Diagnosis Modified from Besbas et al. Kidney International 2006;70: Stem Cell Transplant STEC-HUS Shigatoxin ( E coli H0157:H7; H0104:H4) “Primary” or aHUS Acquired TTP (antibody induced ADAMTS13 deficiency) Others HELLP, Methyl malonic aciduria, antiphospholipid antibody syndrome Infections S pneumoniae, HIV, H1N1 influenza A Drugs (calcineurin inhibitors, ticlopidine, clopidogrel) TMA Thrombocytopenia Microangiopathic hemolytic anemia Thrombi in the microvasculature Organ dysfunction Congenital TTP (ADAMTS13 deficiency) Vasculitis (SLE & other Collagen vascular diseases) DIC with multi- organ failure

Moschcowitz described a new fatal disorder in a 16 year-old girl associated with acute fever, severe anemia, heart failure and stroke. Pathology showed thrombosis of the terminal arterioles and capillaries of multiple organs – Singer emphasized the role of platelets and coined the term “thrombotic thrombocytopenic purpura (TTP)” Gasser et al described the hemolytic uremic syndrome (HUS), noting platelet-fibrin thrombi in microvasculature – Hypocomplementemia and Factor H deficiency identified in a case of HUS without diarrheal prodrome – Moake described the association of chronic relapsing TTP with ultra-large VWF multimers (ULVWF) and hypothesized a defect in vWF processing leading to platelet aggregation – Link between HUS and Shigatoxin producing E coli discovered. The History of TTP & HUS

The Problem with Names Diarrheal/D + vs. Non-diarrheal/D - HUS – Diarrheal illness can serve as trigger for atypical HUS HUS/TTP – Both aHUS and TTP can occur in adults and children – Renal dysfunction can be either severe or mild in aHUS – CNS manifestations may occur in either HUS or TTP Current preferred terms are atypical HUS, TTP, and STEC-HUS

The Problem with Names: Why do we care anyway? Morbidity and mortality are significant in untreated patients Early effective therapy can minimize long-term morbidity and organ damage Therapeutic implications of diagnostic certainty – Plasmapheresis vs. eculizumab Prognostic implications of diagnostic certainty – aHUS and TTP likely to recur, whereas STEC-HUS is not

Clinically Distinguishing aHUS and TTP aHUSTTP Platelet CountMildly decreased or normalSeverely decreased Lung InvolvementOften seenAlmost never seen Renal InvolvementGenerally prominentGenerally mild CNS InvolvementUsually mildUsually prominent

Onset – Fulminant in 80% of patients – Indolent in 20% of patients – Most present with clinical triad – Microangiopathic hemolytic anemia, thrombocytopenia, acute kidney injury Extrarenal manifestations are common – CNS, cardiovascular morbidity in ~20% of patients – Diarrhea may be present in 30% of patients Very high recurrence rate in kidney transplant Atypical HUS: Clinical Features

Atypical HUS and Complement Biology Linked to uncontrolled activation of the alternative pathway of complement system – Up to 70% of patients have an identifiable defect in complement regulation Triggering event typically necessary – Infections, medications, surgery, pregnancy Uncontrolled complement activation on endothelium leads to injury, features of TMA

Primer on Complement: Alternative Pathway C3 C3a C3b C5C5aC5b Factor B C6-C9 Ba Bb Factor D C3 C3b C3a Properdin Endothelial Cell

Primer on Complement: Regulation of the Alternative Pathway Endothelial Cell Factor H C3b MCP iC3b Factor I Thrombomodulin C3b Bb DAFDAF

It’s Complement, Except When It’s Not… Diacylglycerol kinase ε (DGKE) – Intracellular enzyme expressed in endothelial cells, podocytes, and platelets – Phosphorylates arachidonic acid–containing diacylglycerol (AADAG), reducing activation of prothrombotic PKC – Not an integral component of the complement system Lemaire M et al, Nature Genetics 2013

It’s Complement, Except When It’s Not… Mutations in DGKE recently described in a small cohort of patients with atypical HUS – Autosomal recessive with nearly complete penetrance – Presented < 1 year of life, and had persistent urinary abnormalities (microscopic hematuria and proteinuria) – Does not demonstrate recurrence after transplantation Lemaire M et al, Nature Genetics 2013

Coagulation Pathway in aHUS Understanding of aHUS disease biology may be evolving as a crossroads between coagulation and complement pathways – THBD plays role in controlling both pathways – Mutations in DGKE confer a prothrombotic state in the microvasculature – Very recently, mutations in PLG found in cohort of 36 adult aHUS patients using targeted genomic enrichment and massively parallel sequencing (Bu et al, JASN 2014)

Diagnostic Evaluation of TMA Evaluation for secondary causes (pneumococcus, HIV, SLE) Assessment of complement system (aHUS) Assessment of ADAMTS13 (TTP) Assessment for Shigatoxin (STEC-HUS)

Evaluation of Complement in TMA Functional assessments – CH50 (Classical pathway) – AP50 (Alternative pathway) Pitfalls – Depends on systemic consumption – Interassay variability Beneficial in monitoring eculizumab therapy

Evaluation of Complement in TMA Quantitative assessments – Serum C3 and C4 Can help distinguish classical from alternative pathway activation – Dependent on systemic consumption – C3 variably decreased in atypical HUS – Serum Factor H, I, B Factor H decreased 15-70% in pts with CFH mutation Factor I occasionally decreased in pts with CFI mutation Factor B may be decreased with alternative pathway activation – May normalize with TPE before sample is drawn

Evaluation of Complement in TMA Quantitative assessments – Membrane cofactor protein expression by flow cytometry on PBMCs Expression typically ~50% decreased in heterozygous pts May be normal in qualitative defects – Factor H Autoantibody by ELISA Detected autoantibodies may not be biologically relevant Can be detected in normal individuals May normalize with TPE before sample is drawn

Most definitive method for assessing complement – CFH, CFI, CD46, CFB, C3, THBD, DGKE, CFHR5, CFHR1, CFHR3 Thrombomodulin, DGKE expressed in non- circulating cells or intracellularly Prognostic implications – Progression to ESRD Factor H > Factor I, B > ? DGKE > MCP – Transplant recurrence Factor H, I, B, C3 >> MCP, DGKE Evaluation of Complement in TMA: Mutational Analysis

Challenges – Time consuming Results in weeks to months – Does not inform acute management TAT improving with NextGen techniques and better bioinformatics – Expensive Testing is ~$6000 at University of Iowa, CCHMC, Blood Center of Wisconsin Insurance may not cover testing

Evaluation of Complement in TMA: Mutational Analysis Challenges – Mutations (variants) may not be identified Lack of identified mutation (~30-40% of patients with aHUS) does not exclude aHUS or indicate lack of efficacy of eculizumab

Evaluation of Complement in TMA: Mutational Analysis Challenges – Variants may not be biologically relevant “Variants of undetermined clinical significance” – Synonymous variants associated with disease in literature – Non-synonymous variants in which predictive algorithms disagree on pathogenicity – Non-synonymous variants that are common in the population (polymorphisms)

Evaluation of Complement in TMA: Complement Activation Biomarkers sC5b-9 and C5a – Markers of terminal pathway activation – Suppressed by adequate levels of eculizumab Useful for monitoring therapy Bb – Marker of alternative pathway activation C3a, iC3b, C3c and C3d – Markers of proximal pathway activation (C3 convertases)

Thrombotic Thrombocytopenic Purpura (TTP) TTP linked to presence of ultralarge multimers of von Willebrand Factor (Moake et al, NEJM 1982) – Multimers remain uncleaved and bound to endothelial cells, binding to platelets and leading to aggregation Defects in the vWF cleaving protease ADAMTS13 largely responsible for TTP – Deficient in congenital TTP (Levy et al, Nature 2001) – Inhibited by autoantibodies in acquired TTP (Tsai et al, NEJM 1998; Furlan et al, Blood 1998)

Diagnostic Evaluation of TTP Diagnosis primarily clinical Historical testing methods – Agarose gel electrophoresis of vWF multimers – Ristocetin cofactor assay – Collagen binding assay – Technically challenging with interassay variability More recently, ADAMTS13 biology exploited to standardize testing methodology

Evaluation of ADAMTS13 in TMA Enzymatic activity of ADAMTS13 – Most common method = cleavage of fluorogenic modified ADAMTS13 substrate (FRET-VWF73) – Normal result > 67% activity – TTP < 5-10% activity – Can be mildly to moderately decreased (10-40%) in a number of other diseases/conditions DIC, liver dysfunction, sepsis/severe systemic inflammation Pregnancy

Evaluation of Shigatoxin in TMA Stool culture using sorbitol MacConkey agar plates – 93% of E. coli isolates ferment sorbitol, whereas E. coli O157:H7 does not –Pitfalls Negative in up to 50% cases of STEC-HUS Negative with non-O157:H7 strains that produce Shigatoxin (German outbreak in 2011 due to O104:H4)

Evaluation of Shigatoxin in TMA Molecular Testing for Shigatoxin – ELISA/Immunoassay for Shigatoxin – PCR for stx1/stx2 genes Sensitivity and specificity > 95% for either method

Case Presentation, continued ADAMTS13 normal (82%) Stool culture, Stx testing (EIA and PCR) negative C3 48 (  ), C4 23 (nl), Factor H, I, B levels normal Factor H autoantibody negative CD46 FACS with ~50% expression Genetic analysis of CFH, CFI, CFB, THBD, C3 nl Genetic analysis of MCP reveals novel c.97+1G>A heterozygous mutation, predicted to cause disease Diagnosis = Atypical HUS.

How Do We Treat This Patient?

Plasma infusion, TPE – Long considered first-line therapy for aHUS First successful uses in reversing the disease reported nearly 30 years ago Mechanism of action – Provision of non-mutant complement regulatory proteins – In the case of TPE, also removal of mutant factors or autoantibodies Plasma Therapy in aHUS

Efficacy – Ample anectodal evidence of efficacy – No well-controlled prospective clinical trials showing efficacy in aHUS – Two early prospective trials in 1988 compared plasma therapy with supportive care alone No benefit in death, ESRD, proteinuria or hypertension Did not distinguish between STEC-HUS and aHUS – Likely has little to no role in aHUS caused by membrane bound or intracellular factors (MCP/CD46, thrombomodulin, DGKE) Plasma Therapy in aHUS

Pros – Widely accepted in treatment of aHUS – Also first-line therapy for TTP (clinical overlap) – Available at most pediatric and adult centers Cons – Complications include hyperproteinemia, catheter related central venous thrombosis and infection, anaphylaxis to plasma – Some patients demonstrate continued disease activity despite plasma therapy, or relapse after discontinuation – Technically challenging in small children Plasma Therapy in aHUS

Mechanism of action – Blockade of C5 conversion to C5a and C5b, preventing membrane attack complex C5b-9 formation – C3 convertase remains intact, therefore opsonization of pathogens is preserved Eculizumab in aHUS Eculizumab is a humanized monoclonal antibody against complement factor C5. –FDA approval in 2011 for treatment of aHUS

Efficacy – Two prospective open-label multicenter industry- sponsored trials with total of 37 patients Adults with plasma-dependent/responsive aHUS (20 pts) Adults with plasma-refractory aHUS (17 pts) – Prospective open-label phase II trial with 19 pediatric patients Eculizumab in aHUS

Efficacy – Prospective Trials – Hematologic normalization (platelets and LDH) on eculizumab 76-90% of adult patients at median of 37 weeks (NEJM, 2013) 82% of pediatric patients by 26 weeks (Kidney Int, 2016) – Renal improvement on eculizumab Mean eGFR improvement –  5mL/min/1.73m 2 in plasma-dependent adult patients –  33mL/min/1.73m 2 in plasma-refractory adult patients –  64mL/min/1.73m 2 in pediatric patients 4/5 adult patients and 9/11 pediatric patients on dialysis able to discontinue Eculizumab in aHUS

Pros – Very well tolerated (peripheral IV, infusion length 35 min, no premedication necessary) – Highly effective, even in patients on dialysis from aHUS – Highly effective in aHUS patients both with and without identified complement defect(s) Cons – Risk of meningococcal disease – Expensive! ($409,500/year according to Forbes) – Debate ongoing as to the optimal length of therapy – Discontinuation may lead to relapse of the disease Eculizumab in aHUS

May reconstitute complement defect in patients with secreted complement regulators – Factor H, Factor I, Factor B, C3 – Likely ineffectual in MCP, Thrombomodulin, DGKE Early reports with high perioperative morbidity and mortality (Remuzzi et al., Lancet 2002 and others) Later experiences have shown more promise (Saland et al., Am J Trans 2006; Saland et al., CJASN 2009) – Intensive perioperative therapy with TPE, anticoagulation Liver Transplantation in aHUS

Factor H concentrate and recombinant Factor H Mini-Factor H TT30 – Soluble recombinant fusion protein consisting of iC3b- binding region of CR2 and inhibitory domain of Factor H Mirococept (Membrane targeting sCR1) Recombinant soluble thrombomodulin RA (Peptide C5 inhibitor) ACH-4471 (Small molecule Factor D inhibitor) What’s on the Horizon for aHUS?

What a hematologist brings to the table – Greater experience with TTP – Greater depth of pathomechanisms/DiffDx of thrombocytopenia, MAHA – May have better understanding of diagnostic evaluation of TMA Who’s On First? What a nephrologist brings to the table – Greater experience with STEC-HUS – Greater depth of pathomechanisms/DiffDx of renal dysfunction – May have better understanding of complement biology Team-based approach at CCHMC – Hematology, Nephrology, Critical Care – Cooperative evaluation and development of treatment plans

Summary of Diagnostic Evaluation of TMA aHUS – Quantitation of serum complement proteins* C3, C4 Factor H, Factor I, Factor B – MCP FACS on PBMCs – Factor H AutoAb ELISA* – Mutational analysis Factor H, Factor I, MCP, C3, Factor B, Thrombomodulin, DGKE, CFHR5, CFHR1-CFHR3 deletion STEC-HUS – Stool culture – Stx ELISA or PCR TTP – ADAMTS13 activity +/- inhibitor assay & inhibitor Ab ELISA* *IMPORTANT! These tests should be obtained prior to the initiation of plasmapheresis!

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