2. Peking University People’s Hospital & Institute of Hematology Beijing Key Laboratory of HSCT, Beijing, P.R.China Xiao-Jun Huang M.D Donor Lymphocyte Infusion in Patients with Hematological Malignancies after Transplantation: Past, Present and Future

3. DLI is the most effective methods for relapse Kolb HJ, et al. Blood,1995,86:2041-50 Schmid, et al. J Clin Oncol,2007,25:4938-45 Background Patients receiving DLI (n=228) Patients not receiving DLI (n=171) P ＜ 0.0001

4. Limitation of traditional DLI  High occurrence of acute GVHD (49-91%)  Pancytopenia  Less effective for acute leukemia  Can not be used successfully for prophylaxis of relapse after allo-HSCT Kolb HJ, et al. Blood,1995,86:2041-50 Schmid, et al. J Clin Oncol,2007,25:4938-45 Deol A, et al. Cancer Treatment Reviews,2010,36:528 Background

5. New strategies for DLI  Count control of infused lymphocyte  Infusion of allo-depleted donor T cells  Infusion of mHAg-specific CTLs  Infusion of in vivo G-CSF primed lymphocyte Kolb HJ, et al. Blood,2008,112:4371-4383 Cicei F, et al. Lancet Oncol,2009,10:489-500 Deol A, et al. Cancer Treatment Reviews,2010,36:528 Huang XJ. et al. Blood Rev. 2013 Jan;27(1):55-62. Background

6. Effects of G-CSF on immune and hematopoietic cells in healthy donors Background HuangXJ, et al. Clin Transplant 2011: 25: 13–23

7. Modified Donor Lymphocyte Infusion(mDLI) Huang XJ, et al. Haematologica. 2007 Mar;92(3):414-7. Huang XJ, et al. Bone Marrow Transplant. 2009,44(5) Huang XJ, et al. BBMT. 2011 ;17(2):197-204 mDLI Safety G-CSF mobilized PB Short course of Immunosuppressive Agents Modified DLI Modified DLI Advantage of mDLI ⑴ Safety ： Lower aGVHD rate ⑵ Feasibility: Keeping GVL Effect mDLI Feasibility Background

8. Short-term MTX/CSA prophylaxis reduces incidence of GVHD Huang XJ, et al, Leukemia, 2006,20:365-368 Huang XJ, et al. Haematologica 2007,92:414-417 Huang XJ, et al, Bone Marrow Transplant. 2009;44(5):309-16 Background 49.5% 31.6% 14.4% 9.3% Matched-DLI Haplo-DLI

9. mDLI for the treatment of leukemia relapse after unmanipulated Haplo-HSCT Retrospective study (n=20) Patient charactersitics Male/Female: 15/5 Median age: 23 (6-50) years Diagnosis: AML (7 cases), ALL (10 cases), and CML (3 cases) Transplant protocol: unmanipulated HBMT using modified Bu/Cy+ATG conditioning regimens Follow-up: 1118 (754-1468) days after transplant and 808 (627- 1388) days after DLI Huang, et al. Haematologica,2007,92(3):414-417 1. Therapeutic mDLI

10. Huang, et al. Haematologica,2007,92(3):414-417 Results Median counts of MNCs in PBPCs 1.55 (0.8-11.02) ×10 8 /kg Median counts of CD3+ cells in PBPCs 0.61 (0.23-4.56) ×10 8 /kg Grades III-IV acute GVHD 30% Cumulative incidence of cGVHD 64% Two-year probability of LFS 40% Our results suggest that mDLI is a potentially effective therapeutic option for patients who relapsed after unmanipulated haploidentical HSCT. 1. Therapeutic mDLI

11. mDLI for the treatment of leukemia relapse after unmanipulated HSCT—Update data Leukemia patients relapsed after HSCT (n=84) Leukemia patients relapsed after HSCT (n=84) Chemotherapy alone (n=34) Chemotherapy alone (n=34) Chemotherapy plus DLI (n=50) Chemotherapy plus DLI (n=50) No differences in patient characteristics, except for patients in chemotherapy plus DLI group had a higher number of BM blast than chemotherapy group. Huang, et al. PUIH, unpublished data 1. Therapeutic mDLI

12. A B C D Chemotherapy + DLI Chemotherapy alone P=0.016 P=0.000 2-4 acute GVHD Chronic GVHD Relapse Leukemia free survival Huang, et al. PUIH, unpublished data 1. Therapeutic mDLI

13. Conclusion of Part one 1.mDLI was a potentially effective therapeutic option for patients who relapsed after HSCT 2.Chemotherapy plus DLI is superior to chemotherapy alone for treatment of patients who relapsed after transplantation Huang XJ, et al, Leukemia, 2006,20:365-368 Huang XJ, et al. Haematologica 2007,92:414-417 Huang XJ, et al, Bone Marrow Transplant. 2009;44(5):309-16 Huang XJ, et al. PUIH, unpublished data 1. Therapeutic mDLI

14. 2.Prophylactic DLI Relapse prevention using mDLI after HLA-identical transplant A multi-center study Wang Y, Huang XJ, et al. Clin Transplant, 2012 DOI: 10.1111/j.13990012.2012.01626.x Retrospective study (n=123) Patient characteristics Male/Female: 88/35 Median age: 37 (range, 11-56) years Diagnosis: AML (86 cases), ALL (37 cases Transplant protocol: HLA-identical sibling transplant using modified Bu/Cy conditioning regimens Follow-up: As of December 31, 2010

15. P=0.35 P=0.021 Patients who received prophylactic DLI had a higher incidence of chronic GVHD 2.Prophylactic DLI Wang Y, Huang XJ, et al. Clin Transplant, 2012 DOI: 10.1111/j.13990012.2012.01626.x

16. Prophylactic mDLI significantly decrease relapse rate and increase the survival of patients with advanced-stage 66% 46% P=0.02 P=0.001 36% 11% 2.Prophylactic DLI Wang Y, Huang XJ, et al. Clin Transplant, 2012 DOI: 10.1111/j.13990012.2012.01626.x

17. mDLI for the prophylaxis of relapse after Haplo-HSCT in patients with advance leukemia—Risk-factor analysis Retrospective study (n=88) Patient characteristics Male/Female: 53/35 Median age: 30 (range, 8-57) years Diagnosis: AML (54 cases), ALL (34 cases ） Transplant protocol: unmanipulated HBMT using modified Bu/Cy+ATG conditioning regimens Follow-up: A median time of 248 (34-2777) days after transplantation Wang Y, Huang XJ, et al. BMT, 2012.213 2.Prophylactic DLI

18. Our results suggest that higher OS was associated with use of prophylactic GPBSCI, AML and female sex in patients who underwent umanipulated HBMT Wang Y, Huang XJ, et al. BMT, 2012.213 2.Prophylactic DLI

19. Conclusion of Part Two Huang, et al. J Clin Immunol,2008,28:276-283 Wang Y, Huang XJ, et al. BMT, doi:10.1038/bmt.2012.213  Prophylactic mDLI can significantly decrease the relapse rate and increase the survival of patients with advanced stage acute leukemia  It can be recommended as a routine therapy choice after either HLA-identical sibling or Haplo-HSCT 2.Prophylactic DLI

20. Therapeutic Prophylactic High-risk mDLI Decrease relapse Improve survival Decrease relapse Improve survival Patients with Leukemia Standard-risk MRD(-) MRD(+) Prophylactic mDLI ? Decrease relapse ? Improve survival ? Decrease relapse ? Improve survival ? ? Hypothesis ? ? 3. Risk directed mDLI

21. The levels of WT1 could predict relapse of acute leukemia Zhao XS, et al. Ann Hematol. 2012;91:183-192. Zhao XS, et al. BMT. 2012;47:499-507 3. Risk directed mDLI

22. Reduce relapse and improve survival? Our objects- reduce relapse? Monitoring of MRD Risk-stratification based on MRD state Intervention with DLI or IL-2 3. Risk directed mDLI

23. Patients Subgroups 814 patients MRD (-): 709 patients (Group A ) MRD (+): 105 patients IL-2: 49 patients (Group B) DLI: 56 patients (Group C) Huang XJ, et al. Blood,2012,119(14):3256-62 3. Risk directed mDLI

24. Relapse Total: 22.0% (95% CI 18.4-25.6%) Group A: 18.1% (95% CI 14.6-21.6%) Group B: 64.4%(95% CI 44.8-84.0%) Group C 27.8% (95% CI 12.1-43.5%) Yan CH, Huang XJ, et al. Blood,2012,119(14):3256-62 3. Risk directed mDLI

25. Risk factors of relapse Univariate analysisP Disease type0.016 Remission state0.001 MRD-state posttransplant0.001 Intervention for MRD0.001 Multivariate analysisPOR MRD-negative post-transplant0.0000.255 Receiving DLI0.0000.269 Huang XJ, et al. Blood,2012,119(14):3256-62 3. Risk directed mDLI

26. GVHD and TRM Group A: Acute GvHD of any grade posttransplant: 30.2% 2-4 acute GvHD: 15.2% 3-4 acute GvHD: 4.9%. Chronic GvHD posttransplants: 38.8% Extensive chronic GvHD posttransplants: 32.9% VariableMRD- negativeMRD- positiveP*P* P#P# IL-2DLI Acute GvHD (%) post-intervention10.230.80.017 ≥Grade-28.227.90.017 ≥Grade-34.18.40.471 Chronic GvHD (%) post-intervention37.342.90.982 Extensive chronic GvHD30.534.20.858 Incidence of TRM at 3 years post-HSCT (%) 19.712.70.0811.414.40.897 Huang XJ, et al. Blood,2012,119(14):3256-62 3. Risk directed mDLI

27. Disease-free survival Huang XJ, et al. Blood,2012,119(14):3256-62 3. Risk directed mDLI

28. Risk factors of DFS Univariate analysisP Disease type0.017 Remission state0.002 MRD-state posttransplant0.003 Intervention for MRD0.005 Multivariate analysisPOR MRD-negative posttransplant0.0010.511 Receiving DLI0.0060.436 Huang XJ, et al. Blood,2012,119(14):3256-62 3. Risk directed mDLI

29. Overall survival Huang XJ, et al. Blood,2012,119(14):3256-62 3. Risk directed mDLI

30. Risk factors of OS Univariate analysisP Disease type0.025 Remission state0.002 MRD-state posttransplant0.003 Intervention for MRD0.001 Multivariate analysisPOR MRD-negative post transplant0.0070.644 Receiving DLI0.0020.553 Huang XJ, et al. Blood,2012,119(14):3256-62 3. Risk directed mDLI

31. Conclusion of Part Three Risk stratification-directed modified DLI could reduce relapse and improve survival of patients with standard risk acute leukemia patients after HSCT Huang XJ, et al. Ann Hematol. 2012;91:183-192. Huang XJ, et al. BMT. 2012;47:499-507 Huang XJ, et al. Blood,2012,119(14):3256-62 3. Risk directed mDLI

32. Future of DLI  G-CSF-mobilized peripheral blood progenitor cell,  in vitro generated donor T cells against leukemia-antigens,  Positive selected T cells, NK cells,  Suicide gene-transduced donor T cells Which graft for DLI is better ? What is the optimal dose ? What is the optimal schedule to maintain GVL effect? Huang XJ. et al. Blood Rev. 2013 Jan;27(1):55-62.

33. Acknowledgements Stem cell collection center Hai-Yin Zheng Hong Xu Qing Zhao Su Wang Department of bone marrow transplant Xiao-Jun Huang Kai-Yan Liu Dai-Hong Liu Lan-Ping Xu Huan Chen Wei Han Xiao-Hui Zhang Yu-Hong Chen Feng-Rong Wang Jing-Zhi Wang Yu Wang Chen-Hua Yan Yuan-Yuan Zhang Yu Ji Yu-Qian Sun Laboratory of PUIH Dan Li Ya-Zhen Qin Yan-Rong Liu Yue-Yun Lai