ATYPICAL HEMOLYTIC UREMIC SYNDROME : TREATMENT OPTIONS Chantal Loirat, Hopital Robert Debré, Paris, France

aHUS IN CHILDREN FRENCH PEDIATRIC REGISTRY 90 children (onset before age 16), 82 pedigrees 46 boys, 44 girls Sporadic HUS 68 (83 %), familial HUS 14 (17 %, 22 children) CFH mutation 17 (19 %) MCP mutation 13 (14 %) CFI mutation 8 (9 %) C3 mutation 6 (7 %) CFB mutation 1 (1 %) Combined mutations 7 (8 %) (CFI + either CFH, or MCP, or CBF, or C3(2); MCP + C3 ; CFH + CFH) Anti-CFH antibodies 10 (11 %) Complement – HUS 62 (69 %) Unexplained HUS 28 (31 %)

AGE AT ONSET 90 patients (French cohort) CFH J3 J12 CFI J1 J18 J28 J358 MCP CFB C3 Combined I + B I + C3 I + H I + C3 H + H I + MCP MCP + C3 Anti CFH Ab 8 m 3 y 6 m J1 Unexplained 52 % 3 m 1 y 4 y 12 y 16 y Heterozygous Homozygous

EXTRA-RENAL INVOLVEMENT 90 patients (French cohort) Central nervous system (seizures, coma, diffuse cerebral ischemia) 16 (18 %) Multivisceral failure 2 Cardiorespiratory arrest 3 Pancreatitis/hepatitis 5

OVERALL RISK OF DEATH AND EVOLUTION TO END STAGE RENAL DISEASE 90 patients (French cohort) DEATH 6 (7 %) (4 from CNS involvement, 1 from pulmonary hemorrhage/ CRA, 1 from sepsis) all during the first year after onset) ESRD 38 (42 %)

RELAPSING COURSE (from 1 to 9 relapses) N (%) of patients with relapses of HUS CFH homozygous mutation n=4 3 (75 %) CFH heterozygous mutation n=5 3 (60 %) not in ESRD MCP mutation n=13 10 (77%) CFI mutation n=8 1 (12 %) C3 mutation n=6 1 (17 %)* CFB mutation n=1 1 Combined mutations n=7 5 (71 %) * Anti-CFH antibodies n=10 5 (50 %) Unexplained HUS n=28 7 (25 %) * one additional child had chronic hemolysis/thrombocytopenia until bilateral nephrectomy

PROGNOSIS ACCORDING TO COMPLEMENT MUTATION French cohort CFH (n=13) C3 (n=6) CFI (n=8) MCP (n=13) p=ns CFI Free of ESRD or death C3 p=ns MCP CFH p=0.0003 Time (months) At 1y At 5y

PROGNOSIS OF aHUS WITH COMBINED MUTATIONS 7 patients French cohort MCP CFH CFI 3 CFB C3 Free of ESRD or death > 1mutation MCP CFH CFH CFH Time (months) C3 + MCP

PROGNOSIS IN THE « ORPHAN GROUP » COMPARED TO CFH AND MCP GROUPS French cohort CFH (n=13) MCP (n=13) No mutation (n=28) Free of ESRD or death CFH No mutation MCP p=0.02 p=0.04 Time (months) At 1y At 5y

PLASMATHERAPY IN aHUS WITH CFH MUTATION review in Loirat et al, Pediatr Nephrol 2008 + Lapeyraque, Pediatr Nephrol 2008 4 children with homozygous CFH mutation, 6 children + 1 adult with heterozygous CFH mutation benefited from rescue and preventive plasmatherapy (follow-ups : 7 m to 6 y) Example from Davin, Pediatr Nephrol 2008 2 monozygotic twin sisters, HUS at 4 and 4.5 y heterozygous S1191L mutation, SCR 20 First twin : PE with FFP, 40 ml/kg/day x 10 days → resolution of hemolysis Screatinine 166 → 137 µmol/l next 4 months : 5 FFP infusions,10 ml/kg, for each of 3 relapses → ESRD Second twin : PE with FFP, 40 ml/kg/day x 21 → resolution of hemolysis Screatinine 132 → 61 µmol/l → PE tapered to 40 ml/kg every 2 weeks 2 relapses treated successfully by daily PE x 7 days follow-up 6 years, Screat 66 µmol/l long-term PE therapy appears to have benefits over FFP infusions alone

HOMOZYGOUS CFH MUTATIONS RESPONSE TO PLASMATHERAPY 4 patients, French pediatric cohort Year of HUS Plasmatherapy at the acute phase Outcome 1994 – 1995 n = 2 (cousins) 1. no 2*. 15 ml/kg weekly relapse and ESRD at M9 successfull transplantation with FFP 10 ml/kg twice a month, x 7 y preserved RF with FFP 15 ml/kg weekly x 4 y plasma-resistant relapse at 4 y → ESRD 2003 and 2006 n = 2 3. FFP 10 ml/kg x 12 4. PE 60 ml/kg x 10 preserved RF with FFP 10 ml/kg every 2 to 3 weeks, plasma- dependent, x 5 y FFP 10 ml/kg x 3 times a week, x 2 y * published by Nathanson 2001, 2006 ; Larakeb 2008

Homozygous CFH mutation 3694-3697 del TAGA, SCR20 Hemofiltration Homozygous CFH mutation 3694-3697 del TAGA, SCR20 Girl born on Sept 8, 2006 3 times a week FFP infusion 10 ml/kg Daily PE 450 400 350 300 250 200 150 100 50 oct-06 nov-06 déc-06 janv-07 févr-07 mars-07 avr-07 mai-07 juin-07 juil-07 août-07 sept-07 oct-07 nov-07 déc-07 janv-08 févr-08 mars-08 avr-08 mai-08 juin-08 juil-08 août-08 sept-08 20 sept-06 Creatinine (µmol/l) multivisceral failure Hemoglobin (g/l) Platelets (103/ml)

COMPLEMENT DYSREGULATION AS A CAUSE OF VASCULAR COMPLICATION : RESCUE BY PE Larakeb et al, Pediatr Nephrol 2007 HUS at 7 m preserved renal function from age 4 to 9 y under weekly plasma infusions plasma exchange-resistant relapse at age 9 y → ESRD after 3 years on dialysis without plasmatherapy, sudden unilateral blindness with ischemic/hemorrhagic retinal lesions, rescued by daily plasma exchanges (60 mg/kg FFP) x 10 d, then 3 times a week → complete recovery after 4 weeks One child with homozygous CFH mutation (Y899X, SCR15)

HETEROZYGOUS CFH MUTATION OUTCOME ACCORDING TO PERIODS AND TREATMENT MODALITIES 13 patients, French pediatric cohort Year of HUS Plasmatherapy * at the acute phase Outcome 1984-2001 n=10 no 10/10 ESRD or death within < 1 year 9/10 2004-2007 n = 3 yes 3/3 1. FFP 10 ml/kg x 3 weekly 2. PE (FFP) 60 ml/kg daily x 15 3. FFP 10 ml/kg x 3 weekly no renal sequelae 3/3 FFP 10 ml/kg weekly (plasma-dependent) x 1 y PE (FFP) 60 ml/kg weekly (plasma-dependent) x 2.9 y FFP 10 ml/kg monthly (no relapses) x 4 y * defined as FFP ≥ 10 ml/kg or PE with FFP 30-60 ml/kg, x ≥ 5 days

Heterozygous CFH mutation W1189R, SCR20 Weekly PE, 60 ml/kg FFP Girl born on Aug 2005 Heterozygous CFH mutation W1189R, SCR20 Weekly PE, 60 ml/kg FFP Weekly FFP infusion, 10 ml/kg Daily PE 300 250 fever fever fever 200 150 100 50 déc-05 janv-06 févr-06 mars-06 avr-06 mai-06 juin-06 juil-06 août-06 sept-06 oct-06 nov-06 déc-06 janv-07 févr-07 mars-07 avr-07 mai-07 juin-07 juil-07 août-07 sept-07 oct-07 nov-07 déc-07 janv-08 avr-08 mai-08 juin-08 juil-08 févr-08 mars-08 Creatinine (µmol/l) Hemoglobin (g/l) Platelets (103/ml)

aHUS WITH MCP MUTATION EFFECT OF PLASMATHERAPY Italian Registry* (Caprioli et al, 2006) 14 patients French pediatric cohort** 13 patients N episodes Treated n=23 Untreated n=12 n=37 Remission 21 (91 %) 12 (100 %) 11 (92 %) 35 (95 %) * Italian cohort : FFP 10-20 ml/kg/day and/or PE 30-40 ml/kg, for a total of 2-36 treatments in 2-6 weeks ; hematological normalization with or without renal sequelae ** French cohort : PE 40-60 ml/kg x ≥ 4, hematological normalization and no renal sequelae

UNEXPLAINED HUS OUTCOME ACCORDING TO TREATMENT 28 patients (French cohort) treated* n = 12 not treated n = 16 Outcome** poor 7 (58 %) favourable 5 (42 %) poor 5 (42 %) favourable 8 (50 %) * FFP 10 ml/kg x ≥ 4 d (n = 4) PE with FFP 30-60 ml/kg x ≥ 4 d (n = 8) ** poor : death, ESRD, CRI ; favourable : no sequelae or mild proteinuria

aHUS : PLASMATHERAPY (1) In practice : what should be done at admission ? « Guideline for initial therapy », European Pediatric Study Group for HUS in press, Pediatr Nephrol 2008 When to start ? - as soon as possible (within 24 h) as soon as the patient’s condition allows it (BP, volemia, hydroelectrolyte equilibrium, anemia…) - question : even if serum creatinine not elevated ? yes, because : * approximately 50 % of HUS with CFH mutation go to ESRD at the 1st episode * delay in treatment initiation can be deleterious in HUS with anti-CFH antibodies

(2) (2) Which volume ? - Exchange 1.5 plasma volume (60-75 ml/kg) with FFP for restitution - If PE is impossible, infuse FFP 10-20 ml/kg (if BP and cardiac function OK) (3) Which frequency during the first month ? - daily x 5 days - 5/week x 2 weeks - 3/week x 2 weeks (4) What are the situations which allow not to do PE or to stop early ? - MCP mutation (PE only during relapses ?) (5) Which frequency after the 1st month ? empirical : try to find the threshold dose (PE or FFP infusion) and interval for each individual patient

aHUS TREATMENT THE TRIGGERING EFFECT OF INFECTIONS (upper respiratory tract infection, fever, gastroenteritis) CFH CFI MCP C3 No mutation mutation mutation mutation Caprioli 2006 18/26 3/5 12/12 46/66 (69 %) (60 %) (100 %) (70 %) French pediatric cohort 7/17 5/8 9/12 4/5 21/28 (41 %) (62 %) (75 %) (80 %) (75 %) Eradicate adenoïdal, tonsils, dental infections Triggering effect of vaccinations documented in some patients : benefit probably outweighs the risk → perform vaccinations, including influenza Intensify preventive plasmatherapy during infections and vaccinations (at least intensify biological monitoring) In patients with permanent activation of the alternative pathway and very low C3 : preventive antibiotics and vaccination against Neisseria meningitis and Streptococcus pneumoniae

aHUS ASSOCIATED WITH ANTI-CFH ANTIBODIES 10 patients (French pediatric cohort) Treatment at the acute phase none 2 (retrospective diagnosis) FFP infusions only 1 PE 7 + steroïds 5 + immunosuppressors 4 (aza 2, MMF 1, rituximab-cyclophosphamide 1) Relapses (2 to 3) 5 Outcome ESRD 2 (1 untreated, 1 delayed treatment) HT/proteinuria/GFR 70-80 5 (1 untreated, 4 treated) no sequelae 3 (treated)

PROGNOSIS IN PATIENTS WITH ANTI-CFH ANTIBODIES COMPARED TO PATIENTS WITH CFH MUTATION French Cohort CFH (n=13) Anti CFH Ab (n=10) Anti CFH Ab p=0.01 Free of ESRD or death CFH Time (months) At 1y At 5y

3 year-old boy Before PE After PE Anti-FH titer(AU/ml) PE Dialysis IvIg Corticotherapy Azathioprine D15 M2 Relapse 1 M6 M7 Relapse 2 M13 M15 M17 Relapse 3 M23 M27 M30 M44 M48 M54 D-15 Diarrhea Onset No renal sequelae at M54 H. Nivet, Tours

Corticosteroid therapy 11 year old boy Anti-FH titer(AU/ml) Plasma Exchanges Corticosteroid therapy + azathioprine FFP Dialysis RBC transf M2 M3 M6 M7 W3 M1 Onset J.L. André, Nancy B. Ranchin, Lyon

IgG anti-FH (AU) 7 y old girl HUS Apr 12, 2007 18 PE Steroïds May 2008 MMF Sept 2008 11 RBC transf Hematological normalization within 2 weeks Renal function normalization within 2 weeks J.L. André, Nancy

D-HUS WITH ANTI-CFH ANTIBODIES PREPARATION TO TRANSPLANTATION AND POST-TRANSPLANT COURSE Kwon T, 2008 500 1000 1500 2000 2500 C3 anti CFH Ab 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 months transplantation prednisone + azathioprine FFP PEX mg/l AU/ml Hemodialysis rituximab prednisone + cyclosporine + mycophenolate mofetil graft biopsy no TMA no rejection Creatinine 85 µmol/l

Number of patients transplanted within year after recurrence POST-TRANSPLANT RECURRENCE IN aHUS PATIENTS GENOTYPED FOR CFH, CFI AND MCP Retrospective studies Review in Loirat and Fremeaux-Bacchi, Pediatr Transplant 2008 Mutation Number of patients transplanted Recurrence (% patients) Graft failure for recurrence within year after recurrence (% of recurrences) CFH 34 81 % CFI 8 100 % MCP 10 1/2 No mutation in CFH, CFI or MCP 20 83 % 76 % 88 % 20 % 30 % 27

POST-TRANSPLANT RECURRENCE IN PATIENTS WITH NO MUTATION IN CFH, CFI, MCP, CFB AND C3 AND NO ANTI-CFH ANTIBODIES French pediatric cohort 7 transplantations in 5 children* → 2 graft loss due to thrombosis (1) and rejection (1) → no recurrence of HUS * including one familial form of HUS

PLASMATHERAPY TO PREVENT POST-TRANSPLANT RECURRENCE IN 2 CFH-MUTATED SIBLINGS EFFICACY OF PLASMA EXCHANGES WITH FFP STARTED BEFORE RENAL TRANSPLANTATION Olie, AJKD 2005; Davin, Pediatr Nephrol 2008 CFH heterozygous S1191L mutation, SCR 20) Older sister Tx1 Tx2 Twin 1 Prophylactic PE PE before surgery daily PE X 7 weekly PE X 2 months then PE 1 /2 weeks no yes Relapses day 2 no treatment Screat 80 to 200 µmol/L when PE 1/2w PE 5 days later M10 and M12(CMV) daily PE then weekly Outcome graft loss Screatinine 127µmol/L at 5 y

Graft loss within the year after recurrence THE RISK OF RECURRENCE AFTER RENAL TRANSPLANTATION IN ADULT PATIENTS French adult cohort Transplantations performed after year 2000 Mutation N patients transplanted N grafts Recurrence (% grafts) Trt of recurrence Graft loss within the year after recurrence CFH 17 18 (2 with preventive plasmatherapy) 14 (78 %) (1 treated preventively) PE 11/14 10/14 (71 %) CFI 8 9 (2 with preventive plasmatherapy) 4 (44 %) (no prevention) PE 4/4 (follow-up 1 to 4.2 y) C3 6 (1 with preventive plasmatherapy) 5 (62 %) PE 5/5 4/5 (80 %) 1 PE-dependent 1 y after recurrence B 1

aHUS WITH CFH MUTATION LIVER (n = 2) AND LIVER + KIDNEY (n = 10) TRANSPLANTATIONS INITIAL EXPERIENCE WITHOUT PREOPERATIVE PLASMATHERAPY (Remuzzi 2002, 2005 ; Cheong 2004) 3 children → 3 deaths (2 with diffuse thrombotic/ischemic liver lesions, 1 PTLD) SUCCESS OF COMBINED LIVER + KIDNEY TRANSPLANTATION UNDER PLASMATHERAPY BEFORE AND DURING SURGERY (Saland 2006 ; Jalanko 2008) 3 children PE with FFP, 1,25 to 2 plasma volume (50 to 100 ml/kg) just before surgery FFP infusion (20 to 36 ml/kg) during surgery or PE (70 ml/kg) between liver and kidney transplantation + post operative anticoagulation → excellent function of both grafts at 8 m, 15 m and 4 y follow-up + 5 children (unpublished) : 4 combined tx → 3 success,1 death (1/7 = 14 %) 1 liver tx → success (+ 1 adult, combined tx → success )

aHUS INDICATIONS OF COMBINED LIVER + KIDNEY OR LIVER TRANSPLANTATION IN 2008 Consensus Conference, Bergamo, Dec 13, 2007 CFH MUTATION Consensus + : - patient who lost a 1st graft due to recurrence - patient with a family member with the same mutation who lost a graft due to recurrence - patient with a mutation reported in the litterature or registries (www.fh-hus.org) as associated with recurrence after transplantation - in such cases, liver transplantation has to be considered when renal function is preserved by plasmatherapy CFI MUTATION Same recommendations as for HUS mutation (debatable for isolated CFI mutations) CFB OR C3 MUTATION Too early to give recommendations

PATIENTS FOR WHOM ISOLATED KIDNEY TRANSPLANTATION IS RECOMMENDED Bergamo Conference (2) Patients with MCP mutation Patients without identified mutation in CFH, CFI, CFB and C3 Patients with "low risk" CFH, CFI and C3 mutations : same mutation in a family member, a patient in the litterature or registries, without post-transplant recurrence Except for patients with MCP mutations, intensive plasmatherapy (PE) started just before surgery is recommended

aHUS Treatment for the future CFH concentrate (LFB Laboratory) - 1st trials : end of 2009 - 1st candidates : patients with total or partial CFH deficiency - respective place of PE and CFH concentrate to be evaluated in the other patients with functionnal CFH deficiency (2) COMPLEMENT BLOCKERS - near future : anti-C5 monoclonal antibodies : eculizimab - other options in development

ECULIZUMAB IN aHUS Preliminary data (with permission of physicians and Alexion) J. Nuernberger et al (Essen) - One adult, aHUS, Y475S CFH mutation * 1st transplant : loss after recurrence * 2 nd transplant : recurrence, resistant to 4 PE → 1 injection of 600 mg eculizumab → remission, normalization of graft function follow-up : 5 months B. Hurault de Ligny et al (Caen) - One woman, aHUS with C3 mutation (R570Q) * 2nd transplant, Jan 2004 : several recurrences, responsive to PE May 2008 : recurrence, PE-dependent. Switch to eculizumab (protocol as for PNH) → remission maintained. Follow-up : 2 months.

CONCLUSION Although evidence from therapeutic trials is lacking, early plasmatherapy, best by PE, remains first line treatment to rescue aHUS, especially in patients with CFH mutation or anti-CFH antibodies. Long-term plasmatherapy also appears efficient to prevent relapses in patients with CFH mutation. Liver transplantation, isolated or combined with kidney transplantation, has to be discussed on a case by case basis for patients with mutations of complement factors synthetized in the liver. The decision remains difficult. We are at the crossing of roads, as new therapies, such as eculizumab or other complement inhibitors, and CFH concentrate, will soon be evaluated. Hopefully, these new therapeutics will allow that aHUS will no longer end-up in ESRD and the discussion of liver or combined liver + kidney transplantation might become obsolete.

THANKS TO Veronique FREMEAUX-BACCHI and the group of the Laboratory of Immunology, Hopital Européen Georges Pompidou Marie-Agnès Dragon-Durey Jacques Blouin Lubka Roumenina Christophe Hue Arnaud GARNIER, Hopital Robert Debré The members of the French Society of Pediatric Nephrology The patients and their families The Association pour l'Information et la Recherche sur les Maladies Rénales Génétiques (AIRG) and the Programme Hospitalier de Recherche Clinique