1. Atypical HUS – Updates 2016 Christoph Licht The Hospital for Sick Children Division of Nephrology 21.6.2016 aHUS RareConnect Webinar

2. Thrombotic microangiopathy (TMA) TMA is a pathological entity, which is defined by endothelial cell injury with subsequent thrombosis in small capillaries and arterioles.

3. Frequency of TMA subtypes French experience in a population of about 65 million STEC-HUS 100/year 23/10 6 children <2 y 8/10 6 children <15 y Pneumococcal HUS 2-4/year aHUS 10/year 0.10-0.20/ 10 6 population/year From Chantal Loirat – with permission Immune TTP 3/year 0.09/10 6 children <18 y Congenital TTP 2/year Unknown incidence Cbl-C defect-HUS ≤1/year 30 published cases

4. Clinical symptoms Hemolytic uremic syndrome (HUS) - Hemolytic anemia (94%) - Thrombocytopenia (84%) - Renal failure (83%) Extrarenal manifestation - GI tract (40%) - CNS (25%) - Cardiac (10%) - GI / pancreas (5%) - Lungs (5%) - Eyes (1-2%) Gasser et al, Schweiz Med Wochenschr 1955) ; Noris et al, J Am Soc Nephrol 2011; Fremeaux-Bacchi et al, Clin J Am Soc Nephrol 2013

5. TMA manifestation Triggering factors / conditions Threshold for TMA manifestation C5 inhibition Predisposition nonemildsevere Riedl et al, Semin Thromb Hemost 2014

6. The complement system - activation Lectin pathway Bacteria Lectin pathway Bacteria Classical pathway Antibodies, immune complexes Classical pathway Antibodies, immune complexes Alternative pathway Spontaneously active (“tick over”) Alternative pathway Spontaneously active (“tick over”) C3 Convertase C3bBb C5 Convertase C4bC2aC3b C5 Convertase C3bBbC3b C3 C3a C3b C5 C5b C5a C6/C7/C8/C9 C3 Convertase C4bC2a

7. Complement activation and regulation on ECs C3 C3b MCP C3b CFH iC3b C3b DAF Bb CD59 -Inactivation of C3b -Decay acceleration of C3bBb -Blockade of MAC / C5b-9 formation FI

8. aHUS-associated complement mutations Loss of function mutations - Factor H (CFH) - Factor I (CFI) - Membrane cofactor protein (MCP/CD46) - Thrombomodulin (THBD/CD141) Autoantibodies - CFH (in combination with CFHR-3/CFHR-1 deletion: DEAP-HUS) Gain of function mutations - CFB - C3 Diacylglycerolkinase-  (DGKE) Plasminogen (PLG) + von Willebrand Factor (VWF) Noris and Remuzzi, N Engl J Med 2009; George and Nester, N Engl J Med 2014 C3bC3b C5b-9 CFH, CFI, MCP,THBD C3bC3b C5b-9C3 CFB Lemaire et al, Nat Genet 2013; Bu et al, J Am Soc Nephrol 2014

9. Age of disease onset in pediatric aHUS based on genetic / autoimmune background From Chantal Loirat – with permission 01246810121416 DGKE MCP CFH, CFI Anti-CFH Abs C3 No identified abnormality Age [years] French pediatric cohort (n=89): 28% of children had onset between birth and 6 months and 28% between 6 months and 2 years

10. Age at disease manifestation and genotype determine renal outcome Fremeaux-Bacchi et al, Clin J Am Soc Nephrol 2013

11. DGKE mutations – clinical course and outcome Lemaire et al, Nat Genet 2013

12. Treatment of aHUS Eculizumab Eculizumab (Soliris ® ) Humanized monoclonal anti-C5 antibody Leaves proximal complement intact Weak anaphylatoxin (C3a) Immune complex clearance (C3b) Microbial opsonization (C3b) Blocks terminal complement Binds with high affinity to C5 Blocks activation of C5 to generate C5a and C5b Blocks activation of terminal complement (C5b9) Eculizumab C5C5 Proximal Terminal C5aC5a Complement Cascade C5b-9C5b-9C5bC5b C3C3C3aC3a C3bC3b Rother et al, Nat Biotechnol 2007

13. Eculizumab treatment trials in aHUS C08-002 (acute aHUS) Legendre / Licht et al, N Engl J Med 2013; Licht et al, Kidney Int 2015 Weeks on eculizumab Patients achieving end points (%) (platelet count ≥150  10 9 /L)† (platelet count ≥150  10 9 /L and LDH ≤ ULN) †12% of pts at baseline ≥15 mL/min/1.73 m 2 Mean baseline values Serum creatinine 352 µmol/L eGFR 22.9 mL/min/1.73 m 2 Discontinuation of dialysis in 80% of patients on dialysis at baseline

14. Eculizumab treatment trials in aHUS C08-003 (chronic aHUS) Legendre / Licht et al, N Engl J Med 2013; Licht et al, Kidney Int 2015 (platelet count ≥150  10 9 /L) (platelet count ≥150  10 9 /L and LDH ≤ ULN) 17 / 20 of patients had normal platelets at baseline Median baseline values Serum creatinine 234 µmol/L eGFR 28 mL/min/1.73 m 2 Weeks on eculizumab Patients achieving end points (%)

15. The risk of aHUS recurrence after eculizumab discontinuation depends on the genetic background From Chantal Loirat – with permission Mutations Patients who discontinued N Patients who relapsed after discontinuation N (%) Mean duration of eculizumab treatment in relapsers months Mean delay from eculizumab discontinuation to relapse months CFH 9 (2 on dialysis) 5* (55)8 (5.5-14)2.7 (0.9-6) MCP61 (16)69 CFI30 C3 1 (on dialysis) 0 No mutation identified 17 (4 on dialysis) 1* (6)61.5 7/36 (19.4%) relapsed after discontinuation. Treatment was reinitiated and outcome favourable in all. * One patient with CFH/CFHR1 hybrid gene and one patient with no identified mutation, both on dialysis, were recommenced on eculizumab for significant hemolysis and hyperkaliemia.

16. TMA spectrum Loirat et al, Pediatr Nephrol 2015

17. Establishing the diagnosis of aHUS Loirat et al, Pediatr Nephrol 2015

18. Thrombotic microangiopathy: Hemolytic anemia, thrombocytopenia, renal failure ADAMTS13 activity HUS with coexisting disease / condition pos Stx testing neg >10%<10% STEC HUSTTP yes no aHUSTMA spectrum Genetic testing CFH antibodies Genetic testing CFH antibodies

19. Thrombotic microangiopathy: Hemolytic anemia, thrombocytopenia, renal failure Eculizumab TMA recurrence (no CFH Ab) TMA recurrence (no CFH Ab) TMA in children TMA in adults PE ADAMTS13 normal ADAMTS13 <10% PE Co-existing disease Treat underlying disease Eculizumab Patient very ill (renal/extrarenal) and Stx unlikely Eculizumab Supportive care Eculizumab Stx testing ADAMTS13 testing TMA spectrum w/u ADAMTS13 normal Stx negative/unlikely TMA post solid organ transplantation Eculizumab Loirat et al, Pediatr Nephrol 2015

20. Auotimmune (anti-CFH abs) HUS / DEAP HUS Treatment based outcome Sinha et al, Kidney Int 2014 Combined PE + IS No combined therapy Maintenance IS No maintenance therapy 76% 41% 69% 92% 71% 33% 87% 46% p < 0.0001 p = 0.010 Renal survival (GFR ≥30 ml/min/1.73m 2 ) based on induction with combined PE + IS (steroids ± cyclophosphamide or rituximab) Relapse free survival based on maintenance with IS (steroids ± MMF or azathioprine)

21. Anti-CFH abs indicate treatment efficacy and remission and predict risk of relapse and prognosis Sinha et al, Kidney Int 2014

22. Treatment for patients with CFH antibodies Loirat et al, Pediatr Nephrol 2015

23. EC C3 and C5b-9 deposition correlates with aHUS activity Noris et al, Blood 2014 Conclusion 1: Complement activation in serum causes complement activation on ECs. Conclusion 2: Both, the serum and the ECs contribute to complement fixation on ECs.

24. Noris et al, Blood 2014 EC complement deposition assay allows for adjustment of eculizumab dose (i.e. dose increase)

25. Noris et al, Blood 2014 EC complement deposition assay allows for adjustment of eculizumab dose (i.e. enhanced dose intervalls)

26. Managing a TMA patient in 2016 Establish clinical diagnosis of TMA Rule out / confirm clinically obvious “TMA with coexisting disease” If likely, initiate w/u for “TMA with coexisting disease”, including: - Vasculitis panel - Microbiology w/u for viral or bacterial infections - Plasma / serum / urine for metabolic disorder tests If unlikely, initiate work-up for most relevant differential diagnoses, i.e. Stx HUS, TTP and aHUS: - ADAMTS13 activity + abs - E. coli + Stx PCR + LPS antibodies - C3, C4, CH50 (extended complement activation panel) - Complement genetics (+ MLPA) - CFH abs - Complement proteins If possible, save extra samples, in particular if PI/PE is considered