Thrombotic thrombocytopenic Purpura Flora Peyvandi Angelo Bianchi Bonomi Hemophilia and Thrombosis Center IRCCS Ca’ Granda Ospedale Maggiore Policlinico University of Milan Milan, Italy Disclosures Research Support/P.I. No relevant conflicts of interest to declare Employee No relevant conflicts of interest to declare Consultant Kedrion, Octapharma Major Stockholder No relevant conflicts of interest to declare Speakers Bureau Shire, Alnylam Honoraria No relevant conflicts of interest to declare Scientific Advisory Ablynx, Shire, Roche Board Objectives • Advances in understanding the pathogenetic mechanisms and the resulting clinical implications in TTP • Which tests need to be done for diagnosis of congenital and acquired TTP • Standard and novel therapies for congenital and acquired TTP • Potential predictive markers of relapse and implications on patient management during remission Thrombotic Thrombocytopenic Purpura (TTP) First described in 1924 by Moschcowitz, TTP is a thrombotic microangiopathy characterized by: • Disseminated formation of platelet- rich thrombi in the microvasculature → Tissue ischemia with neurological, myocardial, renal signs & symptoms • Platelets consumption → Severe thrombocytopenia • Red blood cell fragmentation → Hemolytic anemia TTP epidemiology • Acute onset • Rare: 5-11 cases / million people / year • Two forms: congenital (<5%), acquired (>95%) • M:F ratio 1:3 • Peak of incidence: III-IV decades • Mortality reduced from 90% to 10-20% with appropriate therapy • Risk of recurrence: 30-35% Peyvandi et al, Haematologica 2010 TTP clinical features Bleeding 33 patients with ≥ 3 acute episodes + Thrombosis “Old” diagnostic pentad: • Microangiopathic hemolytic anemia • Thrombocytopenia • Fluctuating neurologic signs • Fever • Renal impairment ScullyLotta et et al, al, BJH BJH 20122010 TTP pathophysiology • Caused by ADAMTS13 deficiency (A Disintegrin And Metalloproteinase with ThromboSpondin type 1 motifs, member 13) • ADAMTS13 cleaves the VWF subunit at the Tyr1605–Met1606 peptide bond in the A2 domain Furlan M, et al. Blood 1996; Tsai HM. Blood 1996; Zheng XL, et al. JBC 2001; Levy GG, et al. Nature 2001; Fujikawa K, et al. Blood 2001, Kremer Hovinga et al, Nat Rev Dis Primers 2017 ADAMTS13 deficiency ADAMTS13 deficiency normal values 40-160% severe deficiency <10% Congenital Acquired (<5%) (>95%) ADAMTS13 gene Anti-ADAMTS13 mutations autoantibodies TTP diagnosis flowchart 1) ADAMTS13 activity to confirm TTP Clinical diagnosis of TTP clinical diagnosis 2) Anti-ADAMTS13 IgG to investigate ADAMTS13 severe the cause of ADAMTS13 deficiency deficiency (<10%) 3) Sequencing of ADAMTS13 gene in selected cases Anti-ADAMTS13 IgG Positive Negative Acquired TTP Congenital TTP (95-98%) (2-5%) ADAMTS13 assays • ADAMTS13 activity • ADAMTS13 antigen • Anti-ADAMTS13 antibodies – Total anti-ADAMTS13 IgG – Neutralizing ADAMTS13 activity (mixing assay) – ADAMTS13-specific immune complexes ADAMTS13 activity assays …and more Peyvandi et al, JTH 2010 FRETS-VWF73 assay • Gold standard • Based on FRET (fluorescence resonance energy transfer) mechanism • Relatively fast (although at least 2 hours) • Easy to perform (although not automated) ADAMTS13 absentpresentNOemission emissionat 440at 440 nm nm ADAMTS13 Kokame et al, BJH 2005 Anti-ADAMTS13 antibodies • ELISA-based methods detecting anti-ADAMTS13 IgG are the most used (developed in-house, commercial kits) HRP substrate Streptavidin HRP-conjugate 2 Patient’s plasma Biotinylated anti-human IgG Anti-ADAMTS13 antibody (in 1 patient’s plasma) Cell-colture conditioned media rADAMTS13-V5 containing ADAMTS13 Anti-V5 antibody Anti-mouse IgG In-house anti-ADAMTS13 IgG ELISA set-up (Bettoni et al, JTH 2012) TTP treament: rationale ADAMTS13 deficiency normal values 40-160% severe deficiency <10% Congenital Acquired (<5%) (>95%) Replace functional ADAMTS13 Remove anti-ADAMTS13 antibodies Replace functional ADAMTS13 Down-regulate immune system activation Current and novel therapies: congenital TTP Current therapies • Fresh frozen plasma (15-20 ml/Kg/die) • Virally inactivated factor VIII concentrate containing ADAMTS13 Novel therapies • Recombinant ADAMTS13 (rADAMTS13) • Gene therapy • Anfibatide Allford et al, BJH 2000; Scully et al, BJH 2006; Kopic et al, JTH 2016; Scully et al, Blood 2016 128:135, ASH CONGRESS 2016; Jin et al, Blood 2013; Laje et al, Blood 2009; Verhenne et al, ATVB 2017; Zheng et al, Blood Adv 2016 rADAMTS13 • In contrast to plasma infusions, rADAMTS13 can be infused at lower volumes and contains no additives of human or animal origin • SHP655 (former BAX930), Shire (former Baxalta) • Preclinical and phase I clinical trial (NCT02216084) completed: – 15 patients, three dosing cohorts – rADAMTS13 was safe and well tolerated – Immunogenicity tests performed at screening, pre-dose and upon study completion, were negative in all subjects • Phase 3 trial with SHP655 to be started (randomized, open-label, 2-period crossover study) rADAMTS13 may provide an important alternative replacement therapy for patients with congenital TTP Kopic et al, JTH 2016; Scully et al, Blood 2016 128:135, ASH CONGRESS 2016 Gene therapy in animal models • Transplantation of hematopoietic progenitor cells transduced with a lentiviral vector encoding a full-length ADAMTS13 (activity 26.3±14.3% of normal murine pooled plasma at 5 months after transplantation) resulted in elimination of ultralarge VWF multimers and offered systemic protection against ferric chloride–induced arterial thrombosis Laje et al, Blood 2009 • A single injection of AAV8-based vector expressing N-terminal ADAMTS13 domains (mMDTCS) resulted in long-term expression of functional ADAMTS13 at therapeutic levels (activity 0.6 U/mL at 20 weeks) and prevented TTP in a congenital mouse model of TTP ∼ Jin et al, Blood 2013 • Hydrodynamic tail vein injection of the nonviral sleeping beauty SB100X transposon system expressing murine ADAMTS13 resulted in long-term expression (25 weeks) of supraphysiological levels of ADAMTS13 transgene (184±17% of normal murine plasma pool) and prevented TTP in a congenital mouse model of TTP Verhenne et al, ATVB 2017 Anfibatide • Platelet glycoprotein 1b (GPIb) antagonist derived from snake venom • It inhibits platelet agglutination and thrombus formation in vitro and in vivo in murine models of thrombosis (Lei et al, TH 2014) Platelet counts in ADAMTS13 KO mice treated with PBS or anfibatide (60 ng/g body weight) after shigatoxin challenge • Blockade of VWF-GP1b interaction by injection of anfibatide twice daily (half-life 5-7 h) was efficacious for treating shigatoxin-induced TTP in ADAMTS13 KO mice Zheng L et al, Blood Advances 2016 Current and novel therapies: acquired TTP Current therapies • Plasma exchange • Immunosuppressors Novel therapies • Caplacizumab • Recombinant ADAMTS13 • N-acetylcysteine • Eculizumab • Bortezomib Scully et al, Br J Hematol 2012 (UK guidelines); Sarode et al, J Clin Apher 2013 (USA guidelines); Scully et al, J Blood Med 2014, Plaimauer et al, JTH 2011 & JTH 2015, Tersteeg et al, ATVB 2015; Jian et al, Blood 2012; Chapin et al, BJH 2012; Tsai et al, BJH 2013; Shortt et al, NEJM 2013; van Balen et al, EJH 2014; Mazepa et al, BJH 2014; Yates et al, Transfusion 2014; Patriquin et al, BJH 2016; Peyvandi et al, NEJM 2016; Peyvandi et al, JTH 2017 Mainstay of TTP treatment for acquired TTP Standard of care based on two pillars Daily PEX until confirmed Immunosuppression Treatement duration Treatement platelet normalization (corticosteroids and/or RTX) Remove ULVWF and anti- ADAMTS13 autoantibodies Replace functional ADAMTS13 Suppress over-activity of immune system PEX, plasma exchange; RTX, rituximab Scully et al, Br J Hematol 2012 (UK guidelines); Sarode et al, J Clin Apher 2013 (USA guidelines); Scully et al, J Blood Med 2014 Acute phase: plasma exchange • Gold standard therapy • Reduce mortality from 90% to 10-20% Rock et al, NEJM 1991 • Start PEX within 24 h • At least a daily plasma volume (PV) exchange (1.5 PV during 1-3 days) • Stop PEX when PLT count >150000/mmc for ≥ 2 consecutive days, and LDH and clinical symptoms recovered • Tapering of PEX may reduce exacerbation Scully et al, Br J Hematol 2012 (UK guidelines), Sarode et al, J Clin Apher 2013 (USA guidelines) Acute phase: corticosteroids • Based on clinical practice/case series (Vesely et al Blood 2003, Perotti et al Haematologica 1996, Coppo et al BJH 2006, Cataland et al BJH 2007) • Associated with PEX • Standard (1 mg/kg/die) versus high dose (10 mg/kg/die for 3 days) methylprednisolone IV • Tapering during 4-8 weeks from remission Balduini et al, Ann Hematol 2010 Refractory TTP • Persistent thrombocytopenia or LDH elevation after 7 days, despite daily PEX • Progression of clinical symptoms • Intensification of PEX every 12 hours, or double plasma volume (Shumak et al, 1995; Bobbio-Pallavicini et al, 1997; Bandarenko & Brecher, 1998; Kahwash & Lockwood, 2004; Nguyen et al, 2008) • Rescue therapy: CSA, VCR, CTX and splenectomy • Rituximab is the current treatment of choice (Scully et al, BJH 2007) Acute phase: rituximab • Genetically engineered chimeric murine/human monoclonal antibody directed against the CD20 Indications: • Refractory TTP • Exacerbation (< 30 days from platelet count normalization) • Relapsing chronic TTP (Fakhouri et al, 2005; Scully et al, 2007; Scully et al, 2011) Schedule: • Dosage: 375 mg/mq/week for 4 weeks • When dosed during PEX, postpone next PEX > 4 h later (Hull et al, 2006; Scully et al, 2007) • Intensified regimen (every 3-4 days) during PEX (McDonald et al, 2010) ACUTE PHASE