1. Service d ’ H é matologie On behalf of the « Western Algerian Group of Bone Marrow Transplant » WAG-BMT Haematology and Cell Therapy Depatment Hemobiology Department Viability of non cryopreserved CD34+Stem Cells

2. Service d ’ H é matologie Introduction  Autologous stem cell transplantation (ASCT) after a high dose conditioning is an established treatment modality with definitive indications for many hematological disorders.  However, this line of treatment requires many expensive resources, such as freezing of the harvest product in order to maintain cell viability until stem- cell reinfusion and the use of growth factors for the management of neutropenia.

3. Service d ’ H é matologie Aim of the study  To study the viability of CD34+ stem cells stored at +4°C  To demonstrate the feasibility and the safety of the ASCT without any cryopreservation neither the use of growth factors.

4. Service d ’ H é matologie Study design  STEP 1: Short Conditioning regiments ASCT in MM (MEL200) in 75 patients ASCT in HL (CBV) in 17 patients  STEP 2: Long conditioning regiments ASCT in HL (BEAM) in 02 patients ASCT in HL and NHL (EAM) in 15 patients

5. Service d ’ H é matologie Figure 1. Schema of procedure. STEP 1 Material and methods 75 patients with Multiple Myeloma

6. Service d ’ H é matologie 17 patients with Hodgkin Lymphoma STEP 1 Material and methods

7. Service d ’ H é matologie Wannesson L et al. Annals of Oncology 18: 623–632, 2007 STEP 1 Material and methods

8. Service d ’ H é matologie STEP 1 Material and methods  A 2ml sample was taken from 10 collection bags and then was sent to the flow cytometry laboratory where it was stored in the refrigerator at +4°C.  A CD34+ stem cells count were performed using a “Lyse no wash double platform CD34+ flow cytometry assay”  The viability of CD34+ cells was assessed with 7'AAD immediately after the end of apherisis (H0) then at (H24), (H48) and finally at (H72).

9. Service d ’ H é matologie STEP 1 Results H0H24H48H72 Viability (median)98%97.6%96%95% Viability (range)97.4 – 10092.4 – 99.491.6-9891-96

10. Service d ’ H é matologie STEP 1 Results y = -1,06x + 99,3 R 2 = 0,9571 p=0.05 CD34+ stem cells viability kenetic curve

11. Service d ’ H é matologie STEP 1 Results Brahimi M et al. Revue Algerienne D’hematologie 2011 ; 5 :46-48.

12. Service d ’ H é matologie STEP 1 Results Time of storage of the Harvest bags at +4°C n=200

13. Service d ’ H é matologie STEP 1 Results CBV (HODGKIN) (n = 17) Mel200 (MYELOMA) (n=75) CD34+ cells/Kg 3,66 (2,3-13,22)3,66 (1,5 - 9,7) Duration of neutropenia 13(9-24)10(06-17) Duration of thrombocytopenia, 12(9-37)13(9-24) No. of platelet transfusions, 1(0-2)1(0-3) No. of red blood cell transfusions, 2(0-8)2(0-9) Duration of intravenous antibiotics, 9(0-15)4(0-13) Duration of hospitalization, 21(15-37)17(12-30) Transplant Related Mortality 0100

14. Service d ’ H é matologie Mel200 protocol Bekadja MA et al. Hematol Oncol Stem Cell Ther 2012; 5(1): 49-53 Bekadja MA et al. EBMT 38 th meetting: Geneva, Switzerland 1-4 april 2012.page 140 (Abstract 905)

15. Service d ’ H é matologie Bekadja MA et al. Revue Algerienne D’hematologie 2011 ; 5 :50-53. CBV protocol

16. Service d ’ H é matologie STEP 2 «BEAM» VS «-EAM» BEAM (n=2)-EAM (n=15) BCNU (mg/m 2 )300 (D-2)--- VP16 (mg/m 2 )200 (D-5 to D-2) ARAC (mg/m 2 )400 (D-5 to D-2)2000 (D-5 to D-2) MELPHALAN (mg/m 2 )140 (D-1)

17. Service d ’ H é matologie STEP2 Material and methods 15 Patients with Hodgkin Lymphoma Talhi S et al. Hématologie, Vol. 1, Supplement 1, Mars 2012: pp 213 (Abstract) Bekadja MA et al. EBMT 38 th meetting: Geneva, Switzerland 1-4 april 2012.page 162 (Abstract 1140)

18. Service d ’ H é matologie STEP2 RESULTS n=30 Time of storage of the Harvest bags

19. Service d ’ H é matologie STEP2 RESULTS BEAM (HODGKIN) (n = 2) -EAM (HODGKIN) (n=15) CD34+ cells/Kg 6,98 (4,42-9,53)3,68 (2,71 - 6,15) Duration of neutropenia 12(11-13)13(10-32) Duration of thrombocytopenia, 11(11-12)13(9-24) No. of platelet transfusions, 2(1-4)3(2-4) No. of red blood cell transfusions 2(2-3)4(2-9) Transplant Related Mortality 00

20. Service d ’ H é matologie DAY : 0 (n=10)DAY : 6 (n=10) Viability ( % ) Per Bag 93,77 ( 70-100 )82,18 ( 54,24 – 94 ) MNC ( % ) Per Bag 51,24 ( 25,85 – 71,21 ) 57,05 ( 39,71 – 75 ) CD34 (% ) Per Bag 0,73 ( 0,16 – 1,94 )0,34 ( 0,19 – 0,54 ) STEP2 RESULTS Brahimi M et al. Unpublished data.

21. Service d ’ H é matologie Conclusion  We conclude that high-dose chemotherapy with non-frozen peripheral stem cells is safe in terms of haematopoietic reconstitution even without using growth factors.

22. Service d ’ H é matologie Department of Hemobiology EHU 1 st November, Oran, Algeria

23. Service d ’ H é matologie 23 Department of Hematology and Stem Cell Therapy EHU 1 st November Oran, Algeria

24. Service d ’ H é matologie Professor Mohamed Amine BEKADJA Bekadja MA et al. Hematol Oncol Stem Cell Ther 2012; 5(1): 49-53 Bekadja MA et al. EBMT 38 th meetting: Geneva, Switzerland 1-4 april 2012., (Abstract 905) Bekadja MA et al. Revue Algérienne d’Hématologie 2011 ; 5 :50-53. Bekadja MA et al. EBMT 38 th meetting: Geneva, Switzerland 1-4 april 2012, (Abstract 1140) Chief of Biology Pôle in EHU 1 st November