1. Donor lymphocyte infusion as a treatment of a complete DiGeorge syndrome K.Zdráhalová 2, E.Mejstříková 1,2,T.Kalina 1,2,P.Sedláček 2, A. Janda 1, H.Žižková 3, Z.Sieglová 3,A.Šedivá 1, J.Bartůňková 1, J.Starý 2, P.Kobylka 3, P.Hubáček 2,4, O.Hrušák 1,2 2 Department of Pediatric Hematology and Oncology, 1 Department of Immunology, 3 Institute of Hematology and Blood Transfusion, 4 Department of Pediatrics, Prague, Czech Republic

2. Introduction - DiGeorge syndrome the most common deletion syndrome in humans – the most common deletion syndrome in humans – monoallelic microdeletion of 22q11.2 (DiGeorge monoallelic microdeletion of 22q11.2 (DiGeorge and velo-cardio-facial sy, conotruncal anomaly) variable phenotype even in pts with the same deletion variable phenotype even in pts with the same deletion manifestation: ("CATCH22") manifestation: ("CATCH22") mainly heart defects; hypoparathyroidism mainly heart defects; hypoparathyroidism thymic hypoplasia  immunodeficiency, facial dysmorphism, developmental and behavioral problems thymic hypoplasia  immunodeficiency, facial dysmorphism, developmental and behavioral problems Deletion or interstitial deletion of 10p13 - other rare cause of DiGeorge sy (type II) Deletion or interstitial deletion of 10p13 - other rare cause of DiGeorge sy (type II)

3. Patient: male, born June 18 th 2004, now 10 months Family history: healthy parents, 0 siblings Personal history: 1 st pregnancy, polyhydramnios  amniocentesis  normal karyotype 46, XY term delivery, fetal hypoxia  Cesarian section, resuscitation, intubation, artificial ventilation resuscitation, intubation, artificial ventilation esophageal atresia + tracheoesophageal fistula: esophageal atresia + tracheoesophageal fistula: D+2 operation D+2 operation bilateral choanal atresia: D+5 operation bilateral choanal atresia: D+5 operation congenital heart defects: D+13 operation congenital heart defects: D+13 operation

4. Stigmatisation: Stigmatisation: * face * genitals * eyes * CNS CHARGE association CHARGE association (Coloboma, Heart disease, Atresia choanae, Retarded growth + (Coloboma, Heart disease, Atresia choanae, Retarded growth + development and/or CNS anomalies, Genital anomalies and/or development and/or CNS anomalies, Genital anomalies and/or hypogonadism, Ear anomalies and/or deafness) hypogonadism, Ear anomalies and/or deafness)Problems: recurrent infections, septicemias recurrent infections, septicemias recurrent respiratory distress  ventilation recurrent respiratory distress  ventilation apneas, irritability, states of altered consciousness apneas, irritability, states of altered consciousness

5. Immunology: lymphocyte subsets in 2 months of age: lymphocyte subsets in 2 months of age: CD3+ 0% CD3+ 0% NK 40% NK 40% CD4+ 0% CD4+ 0% CD8+ 0% CD8+ 0% CD19+ 58% CD19+ 58%  absent T cells  absent T cells response to mitogens: absent response to mitogens: absent MRI - absent thymus MRI - absent thymus 0,0001 0,001 0,01 0,1 1 10 CD4 (10^9/L) CD8 (10^9/L) CD3 (10^9/L) CD19 (10^9/L) NK (10^9/L) prior DLI 16.12.05 at 12.8.04

6. Complete DiGeorge Syndrome (diagnosis at 2 months) (diagnosis at 2 months) microdeletion 22q11 not found microdeletion 22q11 not found prophylaxis started : prophylaxis started : cotrimoxazole + itraconazole + IVIG cotrimoxazole + itraconazole + IVIG

7. 2 months of age

8. 1st donor lymphocyte infusion age 6 months unrelated donor from register, 8/10 (B, Cw) no conditioning no GVHD prevention 1x 10 6 /kg CD3+; 0.2x 10 6 /kg CD34+ due to mistake non irradiated blood products administered (7 times prior 1 st DLI, 1 time after 1 st DLI)

9. 1st donorlymphocyteinfusion(cont.) 1st donor lymphocyte infusion (cont.) Complications: D+10: isolated skin isolated skin aGVHD aGVHD (stage 3, grade II) (stage 3, grade II) sepsis sepsis cardiopulmonary cardiopulmonary instability instability capillary leak sy capillary leak sy Chimerism: D+10 donor detected 0,0001 0,001 0,01 0,1 1 10 CD4 (10^9/L) CD8 (10^9/L) CD3 (10^9/L) Between 1st and 2nd DLI after ATG 0 10 days post 1 st DLI 8 after ATG

10. aGVHD aGVHD, Capillary leak sy

11. Immunosupressive therapy: rATG Fresenius (25mg/kg 3x D+10, D+12, D+14) rATG Fresenius (25mg/kg 3x D+10, D+12, D+14) CsA CsA corticosteroids - MP (2mg/kg) corticosteroids - MP (2mg/kg) GVHD resolved GVHD resolved corticosteroids - 2 weeks 2 mg/kg, 1 week 1 mg/kg, corticosteroids - 2 weeks 2 mg/kg, 1 week 1 mg/kg, 1 week 0.5 mg/kg, then tapered (D+35) 1 week 0.5 mg/kg, then tapered (D+35) CsA continued CsA continued***** D+33 last extubation!- aged 7 months D+33 last extubation!- aged 7 months

12. 2nd donor lymphocyte infusion age 7 months, D+36 after 1 st DLI the same donor no conditioning prevention of GVHD: CsA (continued) 0.89x 10 6 /kg CD3+

13. 2nd donor lymphocyte infusion (cont.) Complications: D+27 EBV infection: (B cell proliferation, oligoclonality,  IgM; no clinical manifestation) withdrawal of CsA Rituximab (375 mg/m 2 ) proliferation of CD8+ activated T cells started CD19 0 0,001 0,01 0,1 1 10 CD4 CD8 EBV rituximab 0 days post 2 nd DLI 27 10^9/L CD3

14. 1st DLI acute GVHD d 0 d 8 d 10d 19 d 34 2nd DLI d 10 d 25 EBV infection and prior rituximab d 41 d 55 proliferation of activated T cells, severe liver GVHD last non irradiated trf No proof of engraftment of non irradiated blood transfusions chimerism in non separated blood: recipient mainly, donor detected chimerism in FACSorted T lymphocytes CD3+ (D+41 and D+55 CD4+ and CD8+): mainly donor Chimerism after 1st and 2nd DLI

15. D+34: jaundice - bilirubin 4 mg/dL dif.dg.: hepatic GVHD hepatic GVHD EBV lymphoproliferation (EBV in blood 0; in organs?) EBV lymphoproliferation (EBV in blood 0; in organs?) hepatic infection - not found hepatic infection - not found********* D+35 2nd Rituximab (375 mg/m 2 ) D+41 corticosteroids (MP 1 mg/kg) hepatic aGVHD neutropenia - granulo 215! neutropenia - granulo 215! D+55 preventive ATB, antimycotics

16. D+45 - D+49: agranulocytosis (0 granulo)  corticosteroids (MP 2 mg/kg)  corticosteroids (MP 2 mg/kg) CsA CsA G-CSF 5x G-CSF 5x D+52: granulo 3000  bilirubin 13.7 mg/dL  isolated liver GVHD stage 3, grade III  isolated liver GVHD stage 3, grade III

17. D+56: rATG Fresenius 1 dose, 25mg/kg D+57:  bilirubin 23.8 mg/dL  isolated liver GVHD stage 4, grade IV  isolated liver GVHD stage 4, grade IV*********  corticosteroids (D+63 1.5 mg/kg, D+83 1 mg/kg)  corticosteroids (D+63 1.5 mg/kg, D+83 1 mg/kg) CsA continues CsA continues Current status: D+108 after 2 nd DLI, age 10 months  bilirubin 7.6 mg/dL CD8 activated T cells absolutely decreased slight gradual psychomotor development

18. Patient aged 10 months Thank you.