1. Haploidentical Hematopoietic Stem Cell Transplantation for Hematological Malignancies Xiao-Jun Huang M.D. Ph.D. Peking University Institute of Hematology, Peking University People’s Hospital & Beijing Key Laboratory of HSCT, Beijing, P.R.China

2. 1 1 Education Clinic Research

3. The Cumulative Hematopoietic Stem Cell Transplantation (HSCT) Cases of PUIH PUIH  The Largest HSCT center in Asia  Now >400 cases of HSCT per year  Now >60% Allo-HSCT cases are Unmanipulated Haploidentical HSCT 24%

4. Current Clinical Results Strategy to Improve the Clinical Results Haploidentical Hematopoietic Stem Cell Transplantation for Hematological Malignancies 1 1 In vitro T-cell-depleted HSCT 2 2 Non-Myeloablative Haploidentical HSCT 3 3 Unmanipulated Myeloablative Haploidentical HSCT

5. GIAC protocol G: donor treatment with rhG-CSF I: intensified immunological suppression A: anti-human thymocyte immunoglobulin (ATG) for the prevention of GVHD C: combination of G-PB and G-BM Huang XJ, et al. Blood, 2006, 107(8):3065-3073 Huang XJ, et al. Annals of Medicine, 2008, 40,444-455 Huang XJ, et al. Clin Cancer Res. 2009;15:4777-4783 Huang XJ, et al. BBMT. 2011 Jun;17(6):821-30. 3. Unmanipulated Haploidentical HSCT

6. Effects of G-CSF on Bone Marrow in Healthy Donors HuangXJ, et al. Clin Transplant 2011: 25: 13–23 3. Unmanipulated Haploidentical HSCT

7. Immunoregulatroy Effects after G-CSF Administration to Healthy Donors Huang XJ, et al. Biol Blood Marrow Transplant.2011;17(2):197-204 3. Unmanipulated Haploidentical HSCT

8. Engraftment Huang XJ, et al. Biol Blood Marrow Transplant,2009, 15(5):632-8 n=348 P=0.008 n=348 CD34+ cells≥2.19×10 6 /kg CD34+ cells<2.19×10 6 /kg P<0.0001 Early stage Advanced stage 3. Unmanipulated Haploidentical HSCT

9. Characteristics of the Allo-Grafts Summary Statistics TNC 10 8 /kg CD34+ ×10 6 /kg CD3+ ×10 8 /kg CD4+ ×10 7 /kg CD8+ ×10 7 /kg CFU-GM ×10 5 /kg BM Range 1.2 ～ 8.30.2 ～ 8.80.1 ～ 1.10.4 ～ 7.30.3 ～ 3.7 1.3~9.4 Median3.531.390.201.101.082.62 PB Range 1.9 ～ 9.20.6 ～ 6.6 1.9 ～ 39.02.9 ～ 29.7 1.4~10.5 Median4.021.581.468.767.013.03 T Range5.2~14.20.8 ~ 13.40.8 ~ 6.73.3~39.63.7~29.62.2 ~ 19.9 Median7.562.651.7710.397.875.21 Huang XJ, et al. Bone Marrow Transplant, 2006, 38:291 3. Unmanipulated Haploidentical HSCT

10. Huang XJ, et al. Biol Blood Marrow Transplant 2009, 15(2) Huang XJ, et al. Biol Blood Marrow Transplant 2011; 17(6) Cumulative incidence of aGVHD after HLA-mismatched allo-HSCT Haplo=81 Identical=36 P=0.11 3. Unmanipulated Haploidentical HSCT

11. Probability of aGVHD with locus disparity Huang XJ, et al. Bone Marrow Transplant. 2006;38(4):291-7. 3. Unmanipulated Haploidentical HSCT

12. DFS & OS compared with HLA Matched Donor Huang XJ, et al. Blood, 2006, 107(8):3065-3073 3. Unmanipulated Haploidentical HSCT

13. Relapse compared with Unrelated Donor （ URD ） Huang XJ, et al. Clin Cancer Res, 2009, 15:4777-4783 PMRD=219 URD=78 PMRD=160 URD=60 3. Unmanipulated Haploidentical HSCT

14. Relapse compared with Identical Sibling （ ISD ） HuangXJ, et al. Biol Blood Marrow Transplant. 2011;17(6) Haplo=81 Identical=36 3. Unmanipulated Haploidentical HSCT

15. OS & DFS compared with ISD PMRD=81 ISD=36 P = 0.048 P = 0.029 HuangXJ, et al. Biol Blood Marrow Transplant. 2011;17(6) Haplo=81 Identical=36 3. Unmanipulated Haploidentical HSCT

16. Superior Graft-versus-Leukemia effect Haploidentical HLA-identical sibling High risk acute leukemia High risk acute leukemia HuangXJ, et al. Biol Blood Marrow Transplant. 2011 ;17(6):821-30 3. Unmanipulated Haploidentical HSCT

17. No. of Haploidentical HSCT accumulated in PUIH PUIH data 3. Unmanipulated Haploidentical HSCT

18. The changing of Composition of Haploidentical allo-HSCT in PUIH from 2007 to 2009 PUIH data 3. Unmanipulated Haploidentical HSCT

19. Studies on HLA-mismatched/haploidentical stem cell transplantation (GIAC) Patie nts (n) DiseaseConditioning GVHD prophylaxis aGHVDcGVHDTRMRelapseLFSReference 35 AML/ALL/CML /DLBCL/ ATL Standard intensity±TBI Tacrolimus based 56%19%11 pt9 pt40% Ichinohe et al. (2004) 171 ALL/AML/CML /MDS Bu/Cy/Ara- C/MeCCNU+ CsA/MTX/M MF 55%21.3% 19% SR @ 2yrs SR 12% SR 68% @ 2yrs Huang et al. (2006) 135 ALL/AML/CML /MDS Bu/Cy/Ara- C/MeCCNU+ATG CsA/MTX/M MF (II-IV) 40% 55%22%18% 64% @ yrs Lu et al. (2006) 68 AML/ALL/CML /MDS/ TBI/Cy/FluCy/MMF/(II-IV)5% * 4% @ 100 days 51% @ 1 yr 34% @ 1yr Luznik et al. (2008) 29 AML/ALL/CML /NHL/ Flu/Mel/OKT3/thio tepa CD3/CD19 depletion (II-IV) 48% 3 pt 20% @ 100 days 12 pt 35% @ 1yr Bethge et al. (2008) 42AML/ALL/CML Bu/Cy/Ara- C/MeCCNU+ATG CsA/MTX/M MF 57.2%27.2% 20.4±6.5% @ 1yr 21.43% 57.3±8% @ 3yrs Liu et al. (2008) 93CML Bu/Cy/Ara- C/MeCCNU+ CsA/MTX/M MF 64.25%27.16%28.3% @ 1yrCP1 3.77% 76.5% @ 1yr Huang et al. (2008) 45 AML/ALL/CML /NHL TBI/Cy/Ara-C/ATG CsA/MTX/M MF/ (II-IV)9 pt3 pt11 pt24 pt Wang et al. (2009) 46AML/CML/ALLTBI/Cy/Ara-C/ATG CsA/MTX/M MF (I-II)10.9%8.7% @ 2yrs 23.9% @ 2yr 70.6% @ 2yrs Chen et al. (2009) 250AML/ALL Bu/Cy/Ara- C/MeCCNU+ ATG CsA/MTX/M MF 45.8%31.3% AML 11.9% @ 3yrs AML 19.4% @ 3yrs AML70.7 %3yrs Huang et al. (2009)

20. Composition of HSCT Donor Types in 24 Transplant Units in China PUIH collected Mis %29.9%30.0%33.6%30.8%30.3%26.5%29.7%29.3% 3. Unmanipulated Haploidentical HSCT

21. Huang XJ, et al. BMT, 2006, 38:291 Huang XJ, et al. Blood, 2006, 107(8):3065-3073 Huang XJ, et al. Clin Cancer Res, 2009, 15: 4777-4783 Huang XJ, et al. BBMT. 2011 Feb;17(2):197-204 Part I Conclusions G-BM combined with PBSC from haploidentical family donors, without in vitro TCD, may be used as a good source of stem cells for allo-HSCT There is no difference in OS and LFS between patients receiving allografts from PMRD and URD 3. Unmanipulated Haploidentical HSCT

22. Huang XJ, et al Unpublished ， Blood Reversed

23. Part II ： Strategy to Improve the Clinical Results 1 1 Modified Donor Lymphocyte Infusion(DLI) 2 2 Manipulating the Graft 3 3 Optimize KIR ligand match/mismatch 4 4 Improve Immune Reconstitution 3. Unmanipulated Haploidentical HSCT

24. Relapse Remains a Problem after HSCT High risk leukemia Huang XJ et al, Biol Blood Marrow Transplant. 2009 Feb;15(2) Especially for advanced leukemia (58%- 74%) 3. Unmanipulated Haploidentical HSCT

25. Strategy-1 Our modified DLI G-CSF primed peripheral blood progenitor cells instead of steady donor lymphocyte harvests G-CSF primed peripheral blood progenitor cells instead of steady donor lymphocyte harvests Short-term CsA/MTX for prevention of DLI-associated GVHD Short-term CsA/MTX for prevention of DLI-associated GVHD GPBSCI Huang XJ et al, LEUKEMIA, 2006 ； 20 ， 365-368 Huang XJ et al, Bone Marrow Transplant. 2009;44(5):309-16 3. Unmanipulated Haploidentical HSCT

26. GVHD prophylaxis Reduced GVHD occurrence Huang XJ, et al. Hematologica, 2007,92:414-417 None MTX 3. Unmanipulated Haploidentical HSCT

27. Prevention of relapse using modified DLI can significantly increase survival following HLA- mismatched/Haplo-identical HSCT in patients with advanced-stage, acute leukemia 3. Unmanipulated Haploidentical HSCT Huang XJ,et al. Bone Marrow Transplant. 2011 Sep accepted

28. DiagnosisN=75 Jan,2003 - Sep,2010 AML N=42 >CR27 NR+REL35 ALL N=33 >CR28 NR+REL25 Patients Characteristic 3. Unmanipulated Haploidentical HSCT

29. Prophylactic GPBPCI Performed at 70 (20 ~ 314) d after HSCT MNC 1.0 (0.5-2.0)  10 8 /kg CD3+ 0.93 (0.2-2.12)  10 8 /kg No patients had profound and lasting pancytopenia after the prophylactic infusion 3. Unmanipulated Haploidentical HSCT

30. Cumulative incidence of grade Ⅲ to Ⅳ acute GVHD GVHD prophylaxis < 2w: 49.5% GVHD prophylaxis 2 ～ 4w: 31.6% GVHD prophylaxis 4 ～ 6w: 14.4% GVHD prophylaxis >6w: 9.3% The risk factor of DLI-associated acute GVHD Huang XJ,et al. Bone Marrow Transplant. 2011 Sep accepted 3. Unmanipulated Haploidentical HSCT

31. Cumulative incidence of aGVHD Huang XJ,et al. Bone Marrow Transplant. 2011 Sep accepted P=0.55 3. Unmanipulated Haploidentical HSCT

32. Cumulative incidence of cGVHD Huang XJ,et al. Bone Marrow Transplant. 2011 Sep accepted P=0.42 3. Unmanipulated Haploidentical HSCT

33. Cumulative incidence of TRM Huang XJ,et al. Bone Marrow Transplant. 2011 Sep accepted P=0.95 3. Unmanipulated Haploidentical HSCT

34. Cumulative incidence of relapse Huang XJ,et al. Bone Marrow Transplant. 2011 Sep accepted P=0.018 3. Unmanipulated Haploidentical HSCT

35. Probability of OS Huang XJ,et al. Bone Marrow Transplant. 2011 Sep accepted (P=0.013) 3. Unmanipulated Haploidentical HSCT

36. Lower relapse rate, a similar NRM, and a higher survival probability compared with non-DLI Can significantly increase the survival of patients with advanced-stage, acute leukemia even after HLA-mismatched, T-cell-replete HSCT Modified prophylactic DLI after HLA-mismatched/Haplo-identical HSCT Huang XJ,et al. Bone Marrow Transplant. 2011 Sep accepted 3. Unmanipulated Haploidentical HSCT

37. Risk stratification-directed DLI could reduce relapse of standard-risk acute leukemia after allo-HSCT Institute of Hematology Peking University Beijing, China ASH 2111 Oral Presentation

38. Efficacy of intervention Groups 3yr-Relapse TRMOSLFS A 18.1% 19.7% 66.0% 61.6% B 68.0% 11.2% 23.9% 20.8% C 29.8% 15.6% 55.4% 52.5% ASH 2111 Oral Presentation

39. Strategy-1 Conclusion m-DLI can be used for the treatment and prophylaxis of relapse after haplo-identical HSCT 3. Unmanipulated Haploidentical HSCT

40. p=0.005 p=0.00017 p=0.001 （ n=12 ） （ n=17 ） （ n=12 ） HuangXJ ， et al, Eur J Immunol. 2011 Feb;41(2):514-26 Predictive value of Th17 cells and Tc17 cells in allo-graft on acute GVHD

41. Treating donor mice with rhIL-11 and rhG-CSF promotes transplant- tolerance and preserves the effects of GVL after allogeneic bone marrow transplantation HuangXJ, et al. Leuk Res. 2009 Jan;33(1):123-8 Effects of different cytokines treatment on the recipients’ T cells proliferation activity in response to host alloantigens +14 d after BMT.

42. Strategy-2 Conclusion We may decrease the incidence of GVHD by manipulating the cell contents or function of graft? Mobilization with IL- 11 plus G-CSF ? 3. Unmanipulated Haploidentical HSCT

43. Strategy-3 KIR ligand match/mismatch to outcome on pretransplantation category aGVHD TRM Relapse OS KIR mismatch KIR match Huang XJ, et al. Biol Bone Marrow Transplant, 2008,14(3)

44. Strategy-3 Conclusion KIR ligand mismatch is associated with higher aGVHD, a greater relapse rate, and inferior survival in our haploidentical GIAC protocol---Donor Slection ? 3. Unmanipulated Haploidentical HSCT

45. ALC-30 ＞ 300/ul ALC- 30≤300/ul P<0.001 n=206 Strategy-4 Immune Reconstitution Huang XJ, et al. Bone Marrow Transplant, 2009,43: 29-36 TRM 3. Unmanipulated Haploidentical HSCT ALC-30>300/ul ALC-30≤300/ul

46. ** The counts of reconstituted CD3+ cells (cells/μl ) were significantly lower in HLA- mismatched patients at days 30 than those in HLA-matched patients, which reached normal level at days 60 in both HLA-matched and -mismatched patients. ** P ＜ 0.001 HuangXJ, J Cli Imm Online Publication Comparison of Reconstituted T cells subgroup between HLA match and mismatch

47. ** ** The counts of reconstituted CD4+ cells (cells/μl ) were significantly lower in HLA- mismatched patients at days 30, 60, 90, and 120 than those in HLA-matched patients, which did not reached normal level until 360 in both HLA-matched and mismatched patients, respectively. * P ＜ 0.05, ** P ＜ 0.001 HuangXJ, J Cli Imm Online Publication Comparison of Reconstituted T cells subgroup between HLA match and mismatch

48. Strategy-4 Conclusion Novel approach to improve the recovery of immune reconstitution are greatly required. IL-2 after HSCT ? A Randomized Clinical Trial Is Undergoing For Evaluing IL-2 After Haplo-identical HSCT In PUIH 3. Unmanipulated Haploidentical HSCT

49. Acknowledgements Stem cell collection center Hai-Yin Zheng Hong Xu Qing Zhao Su Wang Department of Bone Marrow Transplant Dai-Hong Liu Feng-Rong Wang Huan Chen Jing-Zhi Wang Kai-Yan Liu Lan-Ping Xu Wei Han Xiao-Hui Zhang Yu-Hong Chen Yu Wang Laboratory of PUIH Dan Li Ya-Zhen Qin Yan-Rong Liu Yue-Yun Lai