Advances in HLA Matching for Unrelated Donor Transplantation Dennis L. Confer, M.D. Chief Medical Officer National Marrow Donor Program Minneapolis, Minnesota

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4 Van Rood JJ, et al. Reprinted by permission from Macmillan Publishers Ltd.

5 Reprinted by permission from Macmillan Publishers Ltd. Van Rood JJ, et al, Nature 1958;181:

6 Milestones in Allogeneic Transplantation First allogeneic transplant – 6 related bone marrow donors 1968 – First single donor related transplants III AIAIIA BIBIIB Mom Dad

7 Likelihood of a Sibling Match Match likelihood1 – no match “n” siblings1 – (0.75) n 1 sibling1 – (0.75) 1 = siblings1 – (0.75) 2 = siblings1 – (0.75) 4 = siblings1 – (0.75) 7 = siblings1 – (0.75) 1.3 = 0.30

8 More Milestones 1973 – First unrelated donor transplants 1974 – Anthony Nolan Registry started 1986 – NMDP started in St. Paul, Minn.

9 NMDP’s Early History Dr. Bob Graves and daughter, Laura 1984 – National Organ Transplant Act 1985 – NIH Technical Assessment Panel $1.2 million Congressional Appropriation Founders – Bob Graves, Jeff McCullough, John Hansen, Herb Perkins Vice-chair -Adm. E.R. “Bud” Zumwalt, Jr.

10 National Marrow Donor Program Adult Donors & Cord Blood Units – Nov, 2008 Adult Donors 7,357,370 CBUs 91,758

11 NMDP Transplants Facilitated by Fiscal Year 1987–2008 Cord blood Peripheral blood stem cells Bone marrow

12 NMDP Transplant Recipients by Diagnosis – Selected Malignancies

13 NMDP Recipients >55 Years of Age

14 Worldwide Transplants Facilitated with Unrelated Donors by Calendar Year 1997–2007 Recipients Bone marrow Peripheral blood stem cells Cord blood (approximate)

17 National Marrow Donor Program Adult Donors & Cord Blood Units – Nov, 2008 Adult Donors 7,357,370 CBUs 91,758

18 HLA Typing by Serology Known antigens A = ~25 B = ~55 DR = ~20 Total = ~100 Known problems –Sera from donors –Many donors Caucasians –Requires live lymphocytes –Most typing for A, B only –High rate of incorrect assignments in 1990’s (>20%)

19 HLA Typing Methods Serology Identifies HLA molecules on the cell surface using antigen specific anti-sera DNA Identifies HLA molecules by defining the DNA code in the cell nucleus Antigen Protein DNA

20 HLA Nomenclature Serology A2 ….. A2 DNA A*0201 A*0202 A*0203 A*0204 A*0205 ….. A*0273 Serology DNA

21 Human MHC Region HLA-A HLA-C & B DRB1 DQA1 & B1 Chromosome 6 Regions HLA class II region loci HLA class III region loci HLA class I region loci Class IIClass IIIClass I Centromere Telomere p q 6p21.31 PSMBB9 (LMP2)TAPBPDPB2DPB1DPA1DOADMADMBTAP1TAP2PSMBB8 (LMP1)DOBDQB1DQA1DRB1DRB2DRB3DRA LTA TNF-  LTBHSPA1BHSPA1AHSPA1LC2BFC4AC4BP450, C21B MICBMICAHLA-BHLA-CHLA-EHLA-AHLA-GHLA-FHFE

22 HLA Class I and II Groove-Domain Cytoplasmic TM Class I Class II Exon 4 Exon 3 Exon Exon 3 Exon 2 Class IClass II Peptide-binding groove 11 22 33 2m2m 11 22 11 22

23 Resolution of DNA Typing Low Resolution Similar to serology e.g., A*02XX IntermediateGroups of alleles e.g., A*0201/02/04 = A*02MS High ResolutionNearly allele-level Allele levelAllele level

24 Transition to DNA-based Typing DRB1 typing –1992 –A, B typed donors A, B typing –1997 demonstration projects A, B & DR at recruitment –2000 Current File

25 DNA Issues: Explosion of Alleles Known Alleles November 2008 A = 697 B =1010 C =382 DRB1 =603

26 DNA Issues: Managing a Mixed Registry B*1501,*4001 B*15AD B62,60 B38,60 B*1502,*4001 Patient B15,40 B8,56 B*15AD,40AB >7 million in NMDP Registry

27 DNA Issues: Data Compression B*1801, B*5101 B*1807, B*5107 B*1811, B*5112 B*1801/07/11, B*5101/07/12 or B*18DFT, B*51AEH or B*18XX, B*51XX Laboratory Report 1 GAGGAGGTTAAGTTTGAG 2 GAGCTGCTTAAGTCTGAG 3 GAGCAGGTTAAACATGAG 4 GAGTACTCTACGGGTGAG 5 GAAGCAGGATAAGTTTGA 6 CAGCAGGATAAGTATGAG 7 TGGCAGCTTAAGTTTGAA 8 GTACTCTACGTCTGAGTG 9 TGGCAGGGTAAGTATAAG Etc. Laboratory Data Laboratory Interpretation

28 DNA Issues: Data Compression B*1801, B*5101 B*1807, B*5107 B*1811, B*5112 B*1801/07/11, B*5101/07/12 or B*18DFT, B*51AEH or B*18XX, B*51XX Laboratory Report 1 GAGGAGGTTAAGTTTGAG 2 GAGCTGCTTAAGTCTGAG 3 GAGCAGGTTAAACATGAG 4 GAGTACTCTACGGGTGAG 5 GAAGCAGGATAAGTTTGA 6 CAGCAGGATAAGTATGAG 7 TGGCAGCTTAAGTTTGAA 8 GTACTCTACGTCTGAGTG 9 TGGCAGGGTAAGTATAAG Etc. Laboratory Data B*1801, B*5101B*1811, B*5101 B*1801, B*5107B*1811, B*5107 B*1801, B*5112B*1811, B*5112 B*1807, B*5101 B*1807, B*5107 B*1807, B*5112 Laboratory Interpretation User’s Interpretation

29 Searching in a Mixed Registry Old Search Algorithm –Convert all typing to “Serology” –Match on serology –Examine DNA-typed donors for more New Search Algorithm – HapLogic sm –Convert serology to “DNA” –Convert low resolution DNA to high resolution

30 Understanding Haplotypes ABDRDQ C An individual’s HLA phenotype is determined by the HLA genes on the chromosome #6 inherited from each parent The phenotype is dictated by two haplotypes Haplotype from Mom Haplotype from Dad

This is the demo footer 31 Patient: A*0222 B*1504 DRB1*0901 A*2402 B*3501DRB1*1402 Sibling 1: A*02XXB*35XXDRB1*07XX A*68XX B*44XX DRB1*14XX Sibling 2: A*24XXB*15XXDRB1*07XX A*29XX B*44XX DRB1*09XX

32 HLA B-HLA C B*0801 is found with Cw*0701 about 95% of the time HLA DRB1-HLA-DQB1 DRB1*0101 is found with DQB1*0501 about 97% of the time Linkage disequilibrium

33 Strategy for Haplotype Generation Calculate high-resolution allele frequencies by race Build up estimations of extended high-resolution haplotypes Run successive 2-3 locus estimations of tightly-linked loci using modified EM (Expectation Maximization) algorithm [B-C] [DRB3/DRB4/DRB5-DRB1] [DQA1-DQB1] Consider the resulting haplotypes as a “locus” and run estimations for the loosely-linked loci A - [B-C] B - [DRB3/DRB4/DRB5-DRB1] A - [B-C] - [DRB3/DRB4/DRB5-DRB1/DQA1-DQB1]

34 bioinformatics.nmdp.org

35 Top 100 Caucasian A,B,C & DRB1 High-Resolution Haplotypes Frequency Haplotype Rank

36 Searching Patients — Haplotype Frequencies Haplotype 2 Frequency Haplotype 1 Frequency

1. Calculation for each individual locus at A, B, DRB1, DQB1 and C

2. Calculation for the overall (composite) likelihood of a 6/6, 5/6, or 4/6 allele level match across all loci - Pr(6), Pr(5)

41 DNA Issues: Data Compression B*1801, B*5101 B*1807, B*5107 B*1811, B*5112 B*1801/07/11, B*5101/07/12 or B*18DFT, B*51AEH or B*18XX, B*51XX Laboratory Report 1 GAGGAGGTTAAGTTTGAG 2 GAGCTGCTTAAGTCTGAG 3 GAGCAGGTTAAACATGAG 4 GAGTACTCTACGGGTGAG 5 GAAGCAGGATAAGTTTGA 6 CAGCAGGATAAGTATGAG 7 TGGCAGCTTAAGTTTGAA 8 GTACTCTACGTCTGAGTG 9 TGGCAGGGTAAGTATAAG Etc. Laboratory Data B*1801, B*5101B*1811, B*5101 B*1801, B*5107B*1811, B*5107 B*1801, B*5112B*1811, B*5112 B*1807, B*5101 B*1807, B*5107 B*1807, B*5112 Laboratory Interpretation User’s Interpretation

42 Primary data HLA Typing Lab NMDP Primary data HLA typing information

43 Primary data A listing of sequences that are present or absent in a donor/cord HLA typing

44 Symbol shows that primary data exists and the donor: +may be an allele match (A+, P+) ―will not be an allele match (M-, L-) #excludes at least one allele of the patient’s pair (P#) Primary data indicators

45 Primary data available No primary data available Primary data indicators Still has a low probability to match the patient at the B locus!

46 P# always describes a pair of alleles Patient’s allele pair is not present in the donor’s HLA typing P#P# = A/L, L/A or L/L The primary data does not provide enough information to tell where the allele mismatch will be located What is that P# all about? Primary data indicators

47 +–#+–# Primary data indicators

48 SUMMARY HLA has come a long way in 50 years HCT has come a long way in 22 years Advanced bioinformatics techniques are needed to: –Address the ever-changing field of HLA –Keep HLA data current and usable –Simplify and speed the process of adult donor and cord blood unit identification

49 Acknowledgments NMDP Staff Martin Maiers Neil Smeby Michelle Setterholm Histocompatibility Advisory Group Leaders and Scientists Bob Hartzman Bob Baitty Carolyn Hurley Jennifer Ng Bill Klitz Craig Kollman

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