1. Cord Blood (CB) Hematopoietic Progenitor Cell (HPC) Characterization and Correlation with Ethnicity: A Report From The COBLT STUDY 1 M S Cairo, 2 G Cohen, 2 E.L. Wagner, 3 J Fraser, 4 L Jensen, 2 S Carter, 5 N.A. Kernan, 6 J Kurtzberg 1 Georgetown University, Washington DC, 2 The Emmes Corporation, Rockville MD, 3 University of California Los Angeles, Los Angeles CA, 4 National Heart, Lung and Blood Institute, Bethesda MD, 5 Memorial Sloan Kettering Cancer Center, New York NY, 6 Duke University Medical Center, Durham NC

2. Introduction Advantages of CB and CBT Ease and safe procurementEnriched HPC Rapid availabilityImmaturity of T cell immunity Decreased viral transmissionDecreased severe AGVHD Multi-ethnic representationReduced chronic GVHD Disadvantages of CB and CBT Decreased supplyDelay in hematopoietic recovery Expensive to developProlonged immune reconstitution Limited cell doseLack of available cells for ACI Genetic/infectious transmissionIncreased infectious morbidity

3. Introduction CB CD34 + /kg graft content has been significantly correlated with engraftment and survival following UCBT Wagner et al, Blood, 2002 Styczynski et al, BMT, 2004 CB vs BM CD34 + /38 - has a higher cloning efficiency, proliferate more rapidly and generate more HPC Cardoso et al, PNAS, 1993 Hao et al, Blood, 1995 CB CD34 + /CD90 + (Thy-1 + ) possess a high clonogenic cell expansion capacity and increased HPP-CFC Mayani et al, Blood, 1994 PB CD34 + /CD41 + (CD61 + ) subsets are significantly correlated with platelet recovery following Auto PBSCT Derecksen et al, JCO, 1998

4. Objectives To characterize processed CB HPC subsets including CD34 +, CD34 + /CD38 -, CD34 + /CD90 + and CD34 + /CD61 + To correlate processed CB HPC subsets with in vitro CB HPC colony formation including total CFU, CFU-GEMM, CFU-GM and BFU-E To determine the relationship of birth weight, gestational age, ethnicity, gender and type of delivery with processed CB HPC

5. Cord Blood Banks, Data Coordinating Center and NHLBI Cord Blood Bank Duke University J. Kurtzberg PI COBLT Study Committee N.A.Kernan, Chair/L Jensen, NHLBI Data Coordinating Center (Emmes) S. Carter & E.L. Wagner Cord Blood Bank Georgetown University M.Cairo, PI Cord Blood Bank University of California, LA J Fraser, PI

6. Methods Cord blood collection and processing were performed as described by COBLT Fraser,Cairo, Kurtzberg J. Hematotherapy, 1998 Cord Blood CD34 + and CD34 + subset characterization (300-1200 events) ProCount ® /FACSort™ — Duke and UCLA IgG 1 FITC isotype control ISHAGE/Epics XL-MCL —Georgetown University IgG 1 PE isotype control

7. Methods PhenotypeMonoclonal Antibodies CD34 + /CD61 + CD34 PE/CD61 FITC/CD45 PerCP CD34 + /CD90 + CD34 PE/CD90 FITC/CD45 PerCP CD34 + /CD38 - CD34 PE/CD38 FITC/CD45 PerCP CD34 + /Isotype controlCD34 PE/IgG 1 FITC/CD45 PerCP *ProCount/Isotype controlNucleic Acid Dye/IgG 1 FITC/CD45 PerCP * Duke & UCLA

8. Methods In vitro HPC Colony Assays Processed CB (1 x 10 5 nucleated cells /mL) in IMDM, 2% FBS in Methocult ® media Incubated at 37°C, 5% for 11-14 days in 24 well tissue culture plates. BFU-E Bright red ≥ 50 cells/burst, tightly packed, multicentered CFU-GM Colorless, granular, ≥ 30 cells/colony, dispersed formation CFU-GEMM “Fried egg” appearance, ≥ 40 cells/colony, compact, spherical hemoglobinized area at center or one side.

9. Results Maternal Donor Screened 34,799 (100%) Maternal Donor Consented 20,710 (60%) Total Collections 16,764 (81%) 45% Pregnancy related 29% Refused 9% Medical History 6% Risk Behavior 11% Other 44% No Staff 41% Exclusions 15% Other Long Term Storage 8,731 53% of Collections Low Counts Poor Viability Positive Culture No HPC Positive HGB Screen Cryopreserved 11,077 (66%) 64% CB Volume 11% +ID 7% History 18% Other

10. Ethnic Distribution of Long Term Stored Cryopreserved UCB White363242% Hispanic184121% African-American121314% Mixed101812% Asian98711%

11. Delivery, Gender, Gestational Age, Birth Weight Type of DeliveryGender Vaginal74%Males52% C-Section26%Females48% Gestational Age (wks)Birth Weight (gms) < 378%< 25001% 3817%2501-300011% 3930%3001-350037% 4031%3501-400036% 4114%>400015%

12. Cord Blood Volume, TNC, TMNC, CD34 + Pre-ProcessingPost Processing Volume80 mL25 mL Total Nucleated cells1.8 x 10 9 9.2 x 10 8 (-22%) Total Mononuclear cells4.7 x 10 8 3.8 x 10 8 (-19%) Total CD34 + NA3.4 x 10 6

13. Representative Flow Cytometry Dot Plots of CD34 +, CD34 + /CD38 -, CD34 + /CD61 + and CD34 + /CD90 + CD34 + CD34 + /CD38 - CD34 + /CD61 + CD34 + /CD90 +

14. Cord Blood Hematopoietic Progenitor Cells (HPC) CD34 + Subsets (x 10 5 )In vitro HPC (x 10 5 ) CD34 + 34 ± 42Total CFU23.2 ± 19.8 CD34 + /CD38 - 3.4 ± 4.8BFU-E13.3 ±13.1 CD34 + /CD61 + 7.3 ± 9.5CFU-GM9.3 ± 8.6 CD34 + /CD90 + 0.3 ± 1.9CFU-GEMM0.5 ± 1.1

15. Significant Correlation of CB Log CD34 + vs Log Total CFU Log CD34 + (x 10 4 ) Log All CFU (x 10 4 )

16. Significant* Correlations of CB Log CD34 + vs CFU-GM, CFU-GEMM & BFU-E CD34 + vs CFU-GMCD34 + vs CFU-GEMMCD34 + vs BFU-E r = 0.68 P = 0.000 n = 8696 r = 0.52 p = 0.000 n = 4189 r = 0.61 p = 0.000 n = 8689 * All p<0.001

17. Positive Correlation of Log Total Nucleated Cell Count (TNCC) with Log CD34 + Count in COBLT Processed Cord Blood

18. Significant* Correlations of CB Log CD34 + subsets with Log CFUs Log CD34 + /CD61 + Log CD34 + /CD38 - Log CD34 + /CD90 + NRNRNR Log Total CFU50730.5550480.3336000.30 Log Total BFU-E50690.4950450.3135970.27 Log Total CFU-GM 50750.5150500.3536000.27 Log Total CFU-GEMM 26890.4126840.2722810.26 * All p<0.001

19. Significant* Correlations of CB Log CD34 + Subsets with Each Other Log CD34 + Log CD34 + /CD38 - Log CD34 + /CD61 + Log CD34 + /CD90 + Log CD34 + 1.000.690.640.47 Log CD34 + /CD38 - 0.691.000.350.40 Log CD34 + /CD61 + 0.640.351.000.47 Log CD34 + /CD90 + 0.470.400.471.00 * All p<0.0001

20. Significant* Reduction in CB CD34 + /CD38 - and CD34 + /CD61 + in African Americans & Asians vs Caucasians & Hispanics * All p<0.001 CD34 + /CD38 - CD34 + /CD61 + White BlackAsianHispanic Other White BlackAsianHispanic Other 53 44 WhiteAfrican AmericanAsianHispanicOther 63 44 21 14

21. Correlation of CB HPC with Birth Weight and Gestational Age Birth WeightGestational Age CD34 + /CD61 + CD34 + /CD38 - CD34 + /CD61 + CD34 + /CD38 -

22. Significant Correlation of CB HPC with Delivery and Gender DeliveryGender C-Section vs VaginalMale vs Female Total CFU + 8.7% (5.8-11.7) CD34+/CD61+ + 6.5% (0.4-13.0) p <0.001p <0.035

23. Summary Post processing CB log CD34 + was significantly correlated with log total CFU, BFU-E, CFU-GM and CFU-GEMM Post processing CB log CD34 + subsets (CD38 -, CD61 +, CD90 + ) were significantly correlated with log total CFU, BFU-E, CFU-GM and CFU-GEMM Post processing CB log CD34 + subsets were significantly correlated with each other (CD34 + /CD38 -, CD34 + /CD61 +, CD34 + /CD90 + )

24. Summary African-American and Asian donors were associated with significantly less CD34 + /CD38 - and CD34 + /CD61 + processed CB HPC subsets C-section delivery was significantly associated with increased total processed CB CFU Male gender was significantly associated with increased CB CD34 + /CD61 + HPC subset Birth weight was significantly correlated with both CD34 + /CD38 - and CD34 + /CD61 + and inversely correlated with gestational age.

25. Speculation Post processing CB HPC CD34 + subsets and/or in vitro CB HPC CFUs may potentially correlate with the kinetics of lineage specific CB engraftment post CBT Ethnicity, gender, delivery, birth weight and /or gestational age may have a significant effect on CB HPC and therefore, may be a factor in the future selection of a processed CB unit for CBT The current COBLT UCBT study will determine the significance of these observations.

26. Acknowledgments NHLBI Support N01-HB-67136, N01-HB-67142, N01-HB-67132 and N01-HB-67141 Cord Blood collection, processing and flow cytometry personnel at Carolina, UCLA and Georgetown University Cord Blood Banks Maternal donors and newborns for their public generosity