1. Pavan Kumar Bhamidipati 1, John F. DiPersio 1, Keith Stockerl-Goldstein 1, Geoffrey L. Uy 1, Peter Westervelt 1, Feng Gao 2, Ravi Vij 1, Mark A. Schroeder 1, Camille N. Abboud 1 and Rizwan Romee 1 T-Cell Replete Peripheral Blood Stem Cell Transplantation after Non-Myeloablative Conditioning with Post-Transplant High Dose Cyclophosphamide is Safe and is Associated with Acceptable Outcomes Bone Marrow Transplantation & Leukemia Section, Division of Oncology, Washington University School of Medicine, Saint Louis, MO Division of Biostatistics, Washington University School of Medicine, Saint Louis, MO

2. Conflicts of interest: None to declare

3. Background Allogeneic stem cell transplantation is still the most preferred option for aggressive hematological malignancies HLA matched donor availability continues to be a major hurdle Luznik and Fuchs colleagues from Johns Hopkins University - Non-Myeloablative regimen and post-transplant high dose cyclophosphamide (PT-Cy) with bone marrow as stem cell source Low GVHD rates But higher rates of relapse Luznik L et al. Biol Blood Marrow Transplant 2008, 14(6):641-650.

4. 10-6-5-4-3-20153 60 30100 180 365 Fludarabine 30 mg/m2/day I.V Cyclophosphamide 14.5 mg/Kg I.V TBI 200 cGy Bone marrow derived stem cells Cyclophosphamide 50 mg/Kg I.V 15 4 Filgrastim 5 µg/kg/day Tacrolimus IV ….  PO Luznik L et al. Biol Blood Marrow Transplant 2008, 14(6):641-650. MMF 15 mg/kg  PO

5. Alternative application of this protocol Bashey et al. and Bacigalupo et al. - Myeloablative regimen and bone marrow as stem cell source with PT-Cy Grosso et al. - 2-step approach MA with donor T-cells obtained from peripheral blood as graft source Followed by PT-Cy and CD34 selected product Non-myeloablative conditioning with T-replete peripheral blood as graft source and PT-Cy has not been well studied Bashey et al. Biol Blood Marrow Transplant 2012; 18(12): 1859-66. Bacigalupo et al. Biol Blood Marrow Transplant 2013; 19(1): 117-122. Grosso et al. Blood 2011; 118(17): 4732-9.

6. Rationale for this regimen More tolerable than myeloablative regimen Unmanipulated T-replete product Less labor intensive than cellular manipulation procedures Less expensive than ex vivo T-cell depletion Better graft versus leukemia effect

7. 10-6-5-4-3-20153 60 30100 180 365 Fludarabine 30 mg/m2/day I.V Cyclophosphamide 14.5 mg/Kg I.V TBI 200 cGy Cyclophosphamide 50 mg/Kg I.V 15 4 Filgrastim 5 µg/kg/day Tacrolimus IV ….  PO Luznik L et al. Biol Blood Marrow Transplant 2008, 14(6):641-650. MMF 15 mg/kg  PO G-CSF mobilized PBSC infusion

8. Patients and Methods Retrospective review- single institution (Washington University) N = 21 (1/2009-5/2013) Median follow up - 235 (17- 1174) days End points - Gray’s sub-distribution method to account for the presence of competing risks OS RFS NRM Relapse Acute GVHD (aGVHD) Chronic GVHD (cGVHD) Fine, J. P., & Gray, R. J. J Am Stat Assoc 1999, 94:496-509.

9. Baseline Characteristics Median Age (Range)45 years (22-74) Gender Male12 (57%) Female9 (43%) Diagnosis Aplastic Anemia1 Acute Myeloid Leukemia14 (67%) Favorable Risk1 Intermediate Risk6 Poor Risk7 Ph –ve ALL2 CML in blast crisis1 CLL with t-MDS1 Non-Hodgkin’s Lymphoma2 DLBCL with t-MDS1 Hepatosplenic γδ T-cell lymphoma1 Disease Status at allo-HSCT (n=20) CR12 CR27 CR31 Active Disease10 Prior Transplant History Autologous1 Syngeneic1 Allogeneic3

10. Transplant Characteristics Patient-Donor sex matching M/M5 F/F6 M/F5 F/M5 Patient-Donor relationship Matched unrelated donor (MUD)1 Mismatched unrelated donor (mMUD) 2 Mismatched related donor (mMRD) - Haplo-identical 18 HLA Match Grade 3 out of 66 4 out of 62 5 out of 107 6 out of 103 9 out of 102 10 out of 101 Patient-Donor CMV Status Negative-Negative4 Negative-Positive5 Positive-Negative4 Positive-Positive8 ABO Match Match9 Mismatch12 20/21 had no available HLA matched donors 8/21 (39%) are ethnic minorities

11. Graft Characteristics and Engraftment Stem cell dose – quartiles (range) 5x10 6 /kg (2.58-14.24) T-cell dose Median (range) 19.7x10 7 /kg (9.18-28.32) 1-5 5-10 X10 6 /Kg >10

12. Graft Characteristics and Engraftment Stem cell dose – quartiles (range) 5x10 6 /kg (2.58-14.24) T-cell dose Median (range) 19.7x10 7 /kg (9.18-28.32)

13. Graft Characteristics and Engraftment 20 patients (95%) had complete donor engraftment Day 30 - 19/21 ( Day 100 - Median time to neutrophil engraftment - 15 (12-28) days Median platelet engraftment - 16 (11-40) days 19.7x10 7 /kg (9.18-28.32)

14. OS and RFS Median overall survival At 1year - 57% At 2 years - 41% Median Relapse free survival At 1 year - 45% At 2 years - 36% * censored for competing risks

15. OS and RFS – Active vs. Remission * censored for competing risks

16. Non relapse mortality and relapse Non-relapse mortality 100 days - 9.5% 1 year - 14% Rate of relapse 180 days - 26% 1 year - 39% * censored for competing risks

17. Acute and chronic GVHD Cumulative incidence of acute GVHD at 180 days Grades II-IV – X pt (%) Grades III-IV - 4 patients [19%] Cumulative incidence of chronic GVHD 1 year - 12% Extensive - 1 patient Extensive * censored for competing risks

18. Cause of deathN = 10 Disease relapse5 NRM5 Septic shock2 Failed engraftment and septic shock1 Fungal infection and grade-IV GVHD of liver1 Acute MI1 Cause of death and infectious complications CMV reactivation occurred in 14/17 (82%) patients at risk None of the patients developed CMV disease BK virus - infection in – 5/21 (24%)

19. Conclusions Single institution study using T-replete G-CSF mobilized peripheral blood stem cells with Hopkins regimen Safe regimen, consistent multilineage donor engraftment Acceptable rates of GVHD – despite relatively high T-cell doses G-CSF mobilized peripheral blood is probably a reasonable alternative to bone marrow as source of stem cells in a haplo-identical setting The relative efficacy of this regimen need to confirmed in a prospective trial This combination serves as platform for further modification in the setting of haplo-identical, mismatched or unrelated donor setting