1. High Resolution HLA Matching in Bone Marrow Transplantation
Dr. John Harvey & Dr. John Moppett
We would like to thank the organisers for the invitation to deliver this presentation.
In this presentation we will review the evolution of and evidence for high resolution HLA matching for unrelated donor transplants, and highlight what we believe are the benefits of HR typing by reviewing the impact of prospective high resolution matching on patient outcome for paediatric ALL at the Bristol Royal Hospital for Children.
Filton
NHSBT - H & I Dept & Bristol Royal Hospital for Children 1

2. NHSBT - H & I Dept. & Bristol Royal Hospital for Children
Histocompatibility & Immunogenetics Dept. NHSBT to years of providing an HLA typing and Donor Selection Service to the Bristol Royal Hospital for Children
The H&I department at NHSBT in Bristol has been providing tissue typing and graft identification advisory services to the Bristol transplant Unit for 24 years now since it opened.
NHSBT - H & I Dept & Bristol Royal Hospital for Children
Bristol Royal Hospital for Children 2

3. NHSBT - H & I Dept. & Bristol Royal Hospital for Children
Resolution levels used By NHSBT, Bristol for donor selection at differing HLA typing epochs
Adapted from - BLOOD, VOL. 118, No.23
2000 – 2001 (HR - class II)
2002 – 2012
NHSBT - H & I Dept & Bristol Royal Hospital for Children
This onion diagram graphically describes the levels of resolution used by NHSBT, Bristol to match donors over time.
Resolution from 1988 to 1999 was to the first field of the nomenclature (serological equivalence)
From 2000 to 2001 the matching was high resolution at class II but intermediate at class I
From January 2002 onwards it was high resolution at class I And II 3

4. Evolution of HLA typing methods at NHSBT, Bristol from 1988 to 2012
Dates
Class I Method
Class II Method
1988 – 1992
Serology – low resolution
RFLP – low resolution
Serology – low resolution
SSO – low resolution
SSP/SSO – low resolution
SSP/SSO – intermediate resolution
SSP/SSO – high resolution
SSO/SBT/SSP – high resolution
This slide outlines the evolving HLA typing methods deployed at NHSBT Bristol for donor recipient matching.
Please note that from 1988 to 1999 all typing was to the low resolution level.
From 2000 to 2001 the resultant types were to intermediate level typing for class I and high resolution typing for class II
From Jan the typing and matching was to high resolution
NHSBT - H & I Dept & Bristol Royal Hospital for Children
Bristol Royal Hospital for Children 4

5. HLA High Resolution Typing at NHSBT
1. Intermediate type obtained using the Luminex platform Sequence Specific Oligonucleotide (SSO) method.
2. High resolution type obtained using group specific Sequence Based Typing (SBT).
In our typing regime we initiate our high resolution type by firstly intermediate typing to identify the groups of alleles present in the donor or recipient that need to be resolved. This we achieve by Luminex SSO typing where the luminex is the analytical platform used rather like a flow cytometer and we use oligonucleotide tagged fluorescent microbeads (differing fluorescent signal for each oligonucleotide) specific for the HLA loci investigated.
Using this type we then select flanking primers to amplify the allele groups identified for downstream DNA sequencing. By sequencing informative regions of the gene we are able to generate a high resolution HLA type. Any further ambiguities that remain are resolved using Sequence Specific Primers where required.
NHSBT - H & I Dept & Bristol Royal Hospital for Children
Bristol Royal Hospital for Children 5

6. Literature on influence of high resolution HLA matching
Morishima,Y.; Sasazuki,T.; Inoko,H.et al., The clinical significance of human leukocyte antigen (HLA) allele compatibility in patients receiving a marrow transplant from serologically HLA-A, HLA-B, and HLA-DR matched unrelated donors. Blood. 2002;99;
Petersdorf EW. HLA matching in allogeneic stem cell transplantation. Curr Opin Hematol. 2004;11:
Petersdorf EW, Anasetti C, Martin PJ et al. Limits of HLA mismatching in unrelated hematopoietic cell transplantation. Blood. 2004;104:
Flomenberg N, Baxter-Lowe LA, Confer D et al. Impact of HLA class I and class II high-resolution matching on outcomes of unrelated donor bone marrow transplantation: HLA-C mismatching is associated with a strong adverse effect on transplantation outcome. Blood. 2004;104:
Lee SJ, Klein J, Haagenson M et al. High-resolution donor-recipient HLA matching contributes to the success of unrelated donor marrow transplantation. Blood. 2007;110:
Petersdorf EW, Gooley T, Malkki M, Horowitz M. Clinical significance of donor-recipient HLA matching on survival after myeloablative hematopoietic cell transplantation from unrelated donors. Tissue Antigens. 2007;69 Suppl 1:25-30.
Petersdorf E, Bardy P, Cambon-Thomsen A et al. 14thInternational HLA and Immunogenetics Workshop: report on hematopoietic cell transplantation. Tissue Antigens. 2007;69 Suppl 1:17-24.
Shaw, B. E., Gooley, T. A., Malkki, M., et al. 2007, "The importance of HLA-DPB1 in unrelated donor hematopoietic cell transplantation", Blood, vol. 110, no. 13, pp
Petersdorf EW. Optimal HLA matching in hematopoietic cell transplantation. Curr Opin Immunol. 2008;20:
Macmillan ML, Davies SM, Nelson GO et al. Twenty years of unrelated donor bone marrow transplantation for pediatric acute leukemia facilitated by the National Marrow Donor Program. Biol Blood Marrow Transplant. 2008;14:16-22.
Bray RA, Hurley CK, Kamani NR et al. National marrow donor program HLA matching guidelines for unrelated adult donor hematopoietic cell transplants. Biol Blood Marrow Transplant. 2008;14:45-53.
Shaw P.J. et al Outcome of paediatric bone marrow transplantation for leukaemia and myelodysplasia using matched sibling, mismatched related or matched unrelated donors. Blood 2010
The purpose of this slide is to show that there are many publications on HLA typing in stem cell transplantation but all of these studies involve retrospective typing, multicentre (up to 450 centres) multi disease and differing age groups and not direct observations from their transplant programs.
What we shall show is prospective high resolution matching for a single centre, single disease category and single age group in a what you see is what you get basis.
NHSBT - H & I Dept & Bristol Royal Hospital for Children

7. Evidence for the influence of high resolution HLA matching NMDP 2001
HLA - DRB1 single high resolution mismatches negatively affects transplant outcome.
HLA - A,B & C single antigen serological defined mismatch transplants negatively affects transplant outcome.
HLA - A,B & C single high resolution DNA defined mismatch did not affect transplant outcome.
If we now look back at some of the literature on the impact of High resolution typing we see that early on the picture was very confused. In these two retrospective papers by Petersdorf - DRB1 allele mismatches were reported to have a negative impact upon survival, while at class one only antigen (low resolution) matches impacted survival but allelic mismatches did not.
Petersdorf EW et al. Blood 2001; 98:2922–2929
Petersdorf EW et al. New England Journal of Medicine, 2001; 345:
NHSBT - H & I Dept & Bristol Royal Hospital for Children 7

8. Evidence for the influence of high resolution HLA matching JMDP 2002
HLA - A & B high resolution mismatch reduced overall survival
HLA – C & DRB1 or DQB1 high resolution mismatch did not reduce overall survival
The following year this larger retrospective HLA typing Japanese study by Morishima showed the opposite with class I being important and Class II and C reported not to affect survival.
Six months before this publication at NHSBT Bristol, we started prospectively matching for HLA A,B,C,DRB1 and DQB1 at high resolution for the Bristol Royal Hospital for Children transplant program in full agreement with our transplant consultants in this considering the confusion in the literature we were very much pathfinders showing our field of endeavour.
Morishima Y et al.
Blood. 2002;99:
NHSBT - H & I Dept & Bristol Royal Hospital for Children 8

9. Evidence for the influence of high resolution HLA matching
NMDP 2004 and 2010
HLA – A,B,C & DR single high resolution mismatches associate with a significant reduction in overall survival.
Antigenic mismatches had a stronger effect than allelic mismatches.
HLA – DQ and DP mismatches not associated with decreased overall survival.
HLA – C should be included in matching algorithims
60% of mismatches were only detected by high resolution typing.
Low resolution matching gives only a 56% chance that the transplant pair will be matched at high resolution
Two years later Flomenberg published what was to be a seminal article on the value of high resolution typing. (this was another retrospective HLA typing study).
In this paper single allele mismatches at HLA A,B,C and DR were associated with a significant reduction in overall survival.
HLA DQ and DP were found to not influence overall survival
60% of mismatches could only be detected by high resolution typing
And low resolution typing gave only a 56% chance that the transplant pair will be matched at high resolution.
Flomenberg N et al. Blood. 2004;104:
Woolfrey A.E. et al. Blood 2010;112:563
NHSBT - H & I Dept & Bristol Royal Hospital for Children 9

10. Evidence for the influence of high resolution HLA matching NMDP 2007
Confirmed the importance of high resolution HLA – A, B,C, and DRB1 matching on improved patient survival and stated that both antigenic and allelic mismatches had equal weighting.
Single mismatches at HLA DQ and DP was not associated with changes in overall survival.
Three years later Lee et al confirmed the principle findings of Flomenberg in a larger study but stated that HLA DQ allele mismatches when present with an allelic mismatch for HLA A,B,C,or DRB1 appeared to have a weak additive effect (however this possible additive effect remains controversial)
Lee SJ et al.
Blood. 2007;110:
NHSBT - H & I Dept & Bristol Royal Hospital for Children 10

11. NMDP HLA Matching Guidelines for UD Adult SCT
Took the literature and distilled HLA donor matching guidelines for optimal stem cell transplant outcome in adults
Recommend high resolution HLA – A,B,C & DRB1 match as minimum requirement for optimal patient survival (8/8 match)
They also state that where the patients condition may rapidly deteriorate then it is reasonable to accept a single high resolution (or antigen) mismatch and progress rapidly to transplant
The following year Bray et al issued NMDP guidelines on optimum donor selection in which high resolution matching for HLA ABC&DRB1 was put forward as the minimum requirement to optimise patient survival with HLA matching.
They also state that where the patients condition may rapidly deteriorate then it is reasonable to accept a single allele mismatch and progress rapidly to transplant
Bray RA, Hurley CK, Kamani NR et al.
Biol Blood Marrow Transplant. 2008;14:45-53
NHSBT - H & I Dept & Bristol Royal Hospital for Children 11

12. NHSBT - H & I Dept. & Bristol Royal Hospital for Children
Current UK Practice
Cross sectional study in 2009 of the interaction between the HLA laboratory and the transplant units they served from the laboratory perspective (100% return of survey).
Very diverse arrangements throughout the UK in every step of donor selection.
60% of matched unrelated transplants in the UK are low to intermediate matched at class I and high resolution matched at class II.
This is not put forward a major study but it is of relevance in terms of practice in the UK towards the end of 2009.
In this cross sectional study I approached every H & I laboratory in the United Kingdom and posed questions concerning the interaction of the laboratory with the transplant unit they served and the levels of HLA resolution required for final donor selection plus questions on their typing practice and when and if high resolution for HLA typing was introduced
We found that the arrangements throughout the UK were very diverse and that 60% of the transplants performed at that time selected donors using intermediate level typing for class I and high resolution typing for class II.
Only 40% of transplants conformed with the recommendations on donor selection issued by the NMDP.
Harvey J, Green A.
Survey of human leukocyte antigen matching criteria used in donor selection for haematopoietic stem cell transplantation in the United Kingdom and Ireland.
Hum Immunol. 2009;70 supplement 1:S97
NHSBT - H & I Dept & Bristol Royal Hospital for Children 12

13. Cost of transplant between £80k & £100k
How does this relate to the costs of Transplantation?
Assuming a high res. HLA class II and an intermediate res. Class I would be performed
Additional cost of HR typing patient and 4 donors = £700
Cost of transplant between £80k & £100k
Additional cost is less than 1% of transplant
Nothing is free so what are the costs associated with high resolution donor selection.
Assuming that the all transplants will be paying the cost of Class I intermediate and class II high resolution typing the additional costs using NHSBT prices for the average transplant is £693.82
And this represents less than 1% of the overall cost of the transplant.
CUT THE COSTS BIT HERE – WILL PUT IT INTO CONCLUSION
NHSBT - H & I Dept & Bristol Royal Hospital for Children
Bristol Royal Hospital for Children 13

14. Bone Marrow Transplant in Bristol
1st transplant September 1987
872 paediatric allogeneic BMT :-
ALL 406
AML 155
Other leuks 69
MDS 60
SAA 76
Inborn errors 73
Other 30
Thank you John
The transplant unit in Bristol has been open since 1987 and has performed almost 1500 allogeneic transplants in that time. As you can see our single largest experience is in allogeneic transplant for paediatric acute lymphoblastic leukaemia.
602 adult allogeneic BMT
NHSBT - H & I Dept & Bristol Royal Hospital for Children

15. Unrelated donor BMT for Paediatric ALL Bristol 1988–99
MUD
MMUD
In 1999 we published the outcome of unrelated donor transplant in Bristol for paediatric ALL.
All donor recipient pairs had been low resolution typed.
It can be seen that at that time matched and mismatched unrelated donor paediatric ALL transplants had identical survival.
Green,A et al
Blood. 1999;94:
NHSBT - H & I Dept & Bristol Royal Hospital for Children
Bristol Royal Hospital for Children

16. Unrelated donor BMT for Paediatric ALL Bristol 1988–99
40% mismatch when HR typed
MUD
MMUD
However, as expected from the retrospective NMDP data, re-typing of a sample of these ‘matched’ patients revealed a 40% mismatch rate when high resolution typed.
Green,A et al
Blood. 1999;94:
NHSBT - H & I Dept & Bristol Royal Hospital for Children
Bristol Royal Hospital for Children

17. NHSBT - H & I Dept. & Bristol Royal Hospital for Children
Does the introduction of high resolution matching at HLA class I and II improve Survival in Matched Unrelated Donor Transplant for Childhood ALL?
We therefore wanted to look at what benefit, if any, there was when we changed to a high resolution matching strategy at HLA class I and II.
Examining a series of transplants in a single disease from a single centre, though it reduces the number of cases and therefore statistical power, has the advantage of providing a uniform cohort where the influence of disease specific factors and conditioning regimen is reduced.
NHSBT - H & I Dept & Bristol Royal Hospital for Children
Bristol Royal Hospital for Children

18. NHSBT - H & I Dept. & Bristol Royal Hospital for Children
Improved Survival in Matched Unrelated Donor Transplant for Childhood ALL since the introduction of high resolution matching at HLA class I and II?
SCT for paediatric ALL
SCT after the introduction of HR typing
We analysed 356 allogeneic transplants for ALL from of these were performed after 2002 when we introduced high resolution typing at both class I and II.
All the unrelated donor transplants were T-cell depleted – a notable difference from patients published in the large NMDP retrospective analyses mentioned earlier.
Harvey et al. BMT in press
NHSBT - H & I Dept & Bristol Royal Hospital for Children
Bristol Royal Hospital for Children

19. Bristol MUD Transplants for ALL 1988 – 2001
Outcome according to HLA typing epoch
Pre 2002
(OS 50%)
We divided the transplants into epochs according to typing methodology as shown by John earlier.
Looking just at matched unrelated donor BMT, we see that outcomes were stable with a survival of approximately 50% from 1988 – This is despite the introduction of intermediate resolution typing at class I and high resolution typing at class II in 2000.
Harvey et al. BMT in press
NHSBT - H & I Dept & Bristol Royal Hospital for Children
Bristol Royal Hospital for Children

20. Bristol MUD Transplants for ALL 1988 – 2007 by HLA typing epoch
HR typed
(OS 78.8%)
Pre 2002
(OS 50%)
However, there was an approximately 25% improvement in survival for matched unrelated transplants performed after 2002 following the introduction of high resolution typing at class I and II.
Harvey et al. BMT in press
NHSBT - H & I Dept & Bristol Royal Hospital for Children
Bristol Royal Hospital for Children 20

21. Bristol Transplants for ALL 2002 – 2007
MSD
MUD
MMUD
Secondly, if we look at outcomes for all donor types since 2002, we see that in the era of high resolution typing, outcomes for matched unrelated donor and sibling transplants are identical.
For simplicity this is the data for CR2 transplants, but the same picture is seen for the whole cohort.
Harvey et al. BMT in press
NHSBT - H & I Dept & Bristol Royal Hospital for Children
Bristol Royal Hospital for Children

22. Bristol Transplants for ALL 1988 – 2007
Notably, unlike the improvement seen in matched unrelated donor BMT, the outcome for the mismatched unrelated donors has not improved with the introduction of high resolution typing, despite changes in the degree of mismatch seen.
Harvey et al. BMT in press
NHSBT - H & I Dept & Bristol Royal Hospital for Children
Bristol Royal Hospital for Children 22

23. Bristol Transplants for ALL 1988 – 2007
MMUD
50% 1 Ag MM
50% >1Ag MM
50% 1 All MM
50% 1Ag MM
Prior to 2002 just over half of the mismatch group had one antigen mismatches whilst the rest had 2 or more antigen mismatches.
From 2002 onwards half of the mismatched unrelated donor group had a single allele mismatch only (i.e. detectable only by high resolution typing). There was no difference in outcome between those with allele and antigen level mismatch in this group.
This adds further support to the NMDP data published by Lee et al that allele mismatch is as significant as antigen mismatch in terms of overall survival.
Harvey et al. BMT in press
NHSBT - H & I Dept & Bristol Royal Hospital for Children
Bristol Royal Hospital for Children 23

24. Bristol Transplants for ALL 1988 – 2007
MSD
The astute amongst you will notice that outcomes for matched sibling donor transplants have also improved since 2002 – and this clearly cannot be due to improved typing.
Harvey et al. BMT in press
NHSBT - H & I Dept & Bristol Royal Hospital for Children
Bristol Royal Hospital for Children 24

25. NMDP 2 yr OS in paediatric ALL and AML UD transplants
2003 -
We are not the 1st group to notice this improved outcome in more recent times.
MacMillan showed that for allogeneic transplants in paediatric acute leukaemia from the NMDP, there is a clear improvement in survival from 2003 onwards.
This was largely attributable to a significant reduction in TRM from 28% overall to 15% and the authors also ascribe this improvement to better tissue typing strategies.
MacMillan et al BBMT 2008
NHSBT - H & I Dept & Bristol Royal Hospital for Children

26. Incidence of relapse categorised by epoch and HLA match group.
MSD
17/54 (31%)
4/26 (15%)
MUD
47/134 (35%)
5/33 (15%)
MMUD
22/75 (29%)
3/16 (20%)
Overall
86/263 (32%)
12/75 (16%)
However, disease related factors also play an important part in transplant outcome, particularly the risk of post-transplant relapse.
High risk leukaemias have a much higher relapse rate post transplant.
However, despite no change in the percentage of high risk cases, there has been some reduction in relapse rate across the whole cohort, with a relapse rate of 16% since 2002 compared to 32% prior to that.
This is the explanation for the improved survival in the matched sibling donor transplants. It also partly explains the improvement in matched unrelated transplants.
This reduction in relapse rate is due to better pre-transplant chemotherapy.
The relapse rate of 16% that we see is very similar to that found in those patients who reach transplant from the recently published UK relapse study R3 and is not unique to our institution.
Harvey et al. BMT in press
NHSBT - H & I Dept & Bristol Royal Hospital for Children
Bristol Royal Hospital for Children

27. NHSBT - H & I Dept. & Bristol Royal Hospital for Children
Non Relapse Mortality
p=n.s.
p=0.026
If we look at non-relapse mortality, we see a different story.
The improvement in survival seen in the matched unrelated donor group was in large part due to a reduction in transplant related mortality from an historical level of near 20% to 6%.
Harvey et al. BMT in press
NHSBT - H & I Dept & Bristol Royal Hospital for Children

28. Acute Graft versus Host Disease
aGvHD (gr II–IV) 53/356 = 15%
associated with increased TRM (p=0.002)
Incidence reduced since = 9% (p= n.s.)
Death with GvHD 64% pre 2002,14% post 2002 (p=0.001)
Epoch
No. patients developed > grade II aGvHD in each epoch (aGvHD and died)
53 (34)
12 (7)
9 (8)
15 (10)
10 (8)
7 (1)
p=0.001*
*analysis of OS and aGvHD using chi square test for trends
GvHD rates are very low in T-cell depleted transplants, but GvHD is associated with an increased transplant related mortality.
At 9% the aGvHD rate since 2002 and the introduction of high resolution typing is slightly, but not significantly, lower than previous epochs.
What is notable, however, is that survival with a diagnosis of GvHD is much better since 2002.
Whilst this might be due to better supportive care, this did not translate into lower TRM in the mismatched group, implying that better matching may influence survivability more than incidence of GvHD, at least in the T-cell depleted setting.
Harvey et al. BMT in press
NHSBT - H & I Dept & Bristol Royal Hospital for Children
Bristol Royal Hospital for Children

29. NHSBT - H & I Dept. & Bristol Royal Hospital for Children
Conclusion
HR typing improves the outcome of MUD BMT by reducing NRM
This improvement is not seen in less than fully matched transplants
HR-MUD have equivalent outcomes to MSD transplants
HR typing at class I and II should be standard for UD BMT and is cost effective.
Thus, in conclusion, we believe that our clinical experience of using high resolution class I and II typing since 2002 concurs with the NMDP retrospective data and does result in significantly improved survival for high resolution fully matched unrelated donor transplants.
This is because of reduced transplant related mortality
No improvement is seen in less than fully matched transplants.
The outcome and clinical course of HR matched unrelated donor transplants is identical to matched sibling transplants. This intriguingly suggests that high resolution matched unrelated donors are biologically and immunologically similar to matched sibling donors.
Class I and II high resolution typing is a cost-effective intervention and should be routine for unrelated donor transplants in the 21st century.
NHSBT - H & I Dept & Bristol Royal Hospital for Children
Bristol Royal Hospital for Children

30. NHSBT - H & I Dept. & Bristol Royal Hospital for Children
Acknowledgements
Scientific
Dr Ann Green
Dr Leigh Keen
Dr Steve Culliford
Mrs Elizabeth Wroe
Clinical
Dr Jackie Cornish
Dr Colin Steward
Dr Michelle Cummins
Statistical
Dr Linda Hunt
Dr Yi Li
I would like to thank our scientific and clinical collaborators who helped with the preparation of this data
NHSBT - H & I Dept & Bristol Royal Hospital for Children
Bristol Royal Hospital for Children

31. Additional Data

32. Retrospective Retyping analysis
15 Consecutive Matched Unrelated Transplant pairs from 1996 & 1997 retyped at high resolution
Six of the fifteen pairs had previously unidentified HLA mismatches (40%).
The allelic mismatches detected were:
Two examples of HLA-DRB1*04:01 v DRB1*04:04;
Single examples of DRB1*14:01 v DRB1*14:04,
B*44:02 v B*44:03 and
C*07:01 v C*07:02.
Two further HLA Cw antigen mismatches: HLA Cw*1203 and
Cw*1601( previously HLA-Cw blanc specificities).
NHSBT - H & I Dept & Bristol Royal Hospital for Children
Bristol Royal Hospital for Children

34. NHSBT - H & I Dept. & Bristol Royal Hospital for Children
The following parameters were analysed using Cox Prop. Hazard and Binary logistic regression modelling for both univariate and multivariate analysis
-Disease status at time of transplant
-CMV match status
-Stem Cell Source
-Gender match status
-HLA match grade
-Age of donor and recipient
-Days to engraftment
-Time in days from diagnosis to transplant
-Incidence of relapse
NHSBT - H & I Dept & Bristol Royal Hospital for Children
Bristol Royal Hospital for Children