Histocompatibility & Immunogenetics Blood and Transplant
PRINCIPLES OF HLA TYPING; HLA MATCHING IN HSCT
David SmillieH & I, NHSBT, Sheffield
Histocompatibility & Immunogenetics Blood and Transplant
• successful HSCT depends on many factors
(disease, stage, age, treatment regime etc)
• not least is HLA compatibility between patient and donor!
Histocompatibility & Immunogenetics Blood and Transplant
HLA TYPING REPORT – a collection of letters and numbers, how do we arrive at this and what use is it?
Histocompatibility & Immunogenetics Blood and Transplant
DEFINITIONS• HLA = Human Leucocyte Antigen• membrane glycoproteins on all nucleated cells• 6 ‘classical’ HLA loci, Class I (A,B,C) & Class II (DR,DQ,DP)
each encoded by separate genes• recognised by the immune system as ‘self’ or ‘non self’• this determines histocompatibility = acceptance/rejection of
foreign tissue (e.g. transplant) - host vs graft, graft vs host, graft vs leukaemia)
• cellular immunity• antibody response
• most polymorphic system in human genome - challenges for HLA typing and donor selection!
Histocompatibility & Immunogenetics Blood and Transplant
AMINO ACID POLYMORPHISM(this is what the immune system recognises)
HLA molecule
e.g HLA-A1 e.g HLA-A2
Histocompatibility & Immunogenetics Blood and Transplant
DNA POLYMORPHISM(resolved by DNA typing)
• SNP = Single Nucleotide Polymorphism• alleles differ by 1 or more SNP
Histocompatibility & Immunogenetics Blood and Transplant
AB
CDR
DQ
DP
AB
CDR
DQ
DPmaternal haplotype
paternal haplotype
HLA ANTIGENS ON NUCLEATED CELLS
Histocompatibility & Immunogenetics Blood and Transplant
INHERITANCE OF HLA HAPLOTYPES
01 08 07 03 02
A* B* C* DRB1* DQB1*
03 07 07 15 06
02 44 05 04 03
30 13 06 10 05
02 44 05
(a)
(b)
(c)
(d)
(r) 10 05
PARENTS
CHILDREN
a/c a/d b/c b/d b/r
Father + Mother = 4 haplotypes (25% chance of identical sib)
a b c d
Histocompatibility & Immunogenetics Blood and Transplant
ORGANISATION OF HLA GENESCHROMOSOME 6
Histocompatibility & Immunogenetics Blood and Transplant
1950’s discovery of HLA system
1960’s serological typing
1980’s first HLA genes cloned, sequenced
1990’s DNA/PCR based HLA typing
1999 sequence entire MHC (HGP)
2000 database of all HLA alleles
2000’s SBT, Luminex SSO
HLA TYPING METHODS
Histocompatibility & Immunogenetics Blood and Transplant
• alloantisera
• patient/donor lymphocytes
(e.g. A2)
• add complement
anti HLA-A1
anti HLA-A2
anti HLA-A3
anti HLA-A24
HLA TYPING BY SEROLOGY(Complement Dependent Cytotoxicity - using HLA-A as an example)
Histocompatibility & Immunogenetics Blood and Transplant
ADVANTAGES OF DNA BASED TECHNIQUES
• not dependent on cell viability or cell surface expression of antigens
• standardisation of reagents (synthetic c.f. alloantisera, complement)
• more accurate and more precise
Histocompatibility & Immunogenetics Blood and Transplant
3 LEVELS OF RESOLUTION
1. low resolution (2 digit) - identifies broad families of alleles belonging to the same serotypic group (e.g. A*02)
2. intermediate resolution (allele string) - identifies alleles that have common sequence determinants and thus share hybridisation pattern (e.g. A*02:05/08/22)
3. high resolution (minimum 4 digit) - identifies single allele
Histocompatibility & Immunogenetics Blood and Transplant
LEVELS OF RESOLUTION FOR HSCT
• European Federation for Immunogenetics (EFI) Standards v5.6 (stipulated by JACIE)
• related donor - ‘adequate testing to definitively establish HLA identity by descent’
• unrelated donor - ‘low resolution HLA-A/B/C (2 digit) and high resolution DRB1 typing (4 digit)’
• confirmatory typing
Histocompatibility & Immunogenetics Blood and Transplant
HLA TYPING BY DNA TECHNOLOGY – ACRONYMS!
gene polymorphism detected by:
• primer specificity (PCR-SSP)• probe specificity (PCR-SSOP) e.g. Luminex(primers/probes are short lengths of syntheticDNA which hybridise only to their exactcomplementary sequence and this hybridisationcan be detected)
• sequencing based typing (SBT)
Histocompatibility & Immunogenetics Blood and Transplant
A*01
A*02
A*03
A*24
allele-specific sequences (primer/probe)
conserved sequence
PRINCIPLE OF DNA TYPING(using HLA-A gene as an example)
Histocompatibility & Immunogenetics Blood and Transplant
HIGH RESOLUTION HLA TYPING WHY SEQUENCING BASED TYPING ?
• complete view of HLA gene sequence (cf PCR-SSP, SSOP etc); detects new alleles
• ‘gold standard’ for HSCT
Histocompatibility & Immunogenetics Blood and Transplant
DONOR SELECTION
Histocompatibility & Immunogenetics Blood and Transplant
GUIDELINES FOR HLA MATCHING IN HSCT
Histocompatibility & Immunogenetics Blood and Transplant
MATCHED DONOR OF CHOICE1. HLA identical sibling
– confirmed by family studies– identical for other genes in MHC region
2. HLA identical family member– differences at other gene loci possible
3. HLA identical unrelated donor– differences at other gene loci probable
4. HLA mismatched unrelated donor
5. cord blood unit(s)
Histocompatibility & Immunogenetics Blood and Transplant
HSCT – TYPICAL HLA TYPING PROTOCOL
PATIENT & FAMILYLOW RESR HLA-A, B, C, DRB1, DQB1
MATCHHAPLOTYPE ASSIGNMENT
CONFIRMATORY TESTING
DONOR & RECIPIENT
SBT DRB1 (& TO ESTABLISH HAPLOTYPES)
NO MATCHSBT RECIPIENT HLA-A, B, C,
DRB1, DQB1
MUD SEARCH
BBMR/AN/WBMR/BMDW
SELECT LOW RES MATCHED DONORS
MUD’s: CONFIRMATORY LOW RES & SBT HLA-
A,B,C,DRB1,DQB1, CMV, BLOOD GROUP etc
TRANSPLANT
Histocompatibility & Immunogenetics Blood and Transplant
HLA MATCHING IN RELATED HSCT
Histocompatibility & Immunogenetics Blood and Transplant
HLA: A* B* C* DRB1* DQB1*
Patient * 02 29 44 51 15 16 07 - 02 -
Sib 1 * 02 29 44 51 15 16 07 - 02 -
Sib 2 24 29 07 44 07 16 07 15 02 06
Sib 3 02 - 13 51 06 15 07 - 02 -
Sib 4 24 29 07 44 07 16 07 15 02 06
Sib 5 02 - 13 51 06 15 07 - 02 -
Sib 6 02 - 13 51 06 15 07 - 02 -
FAMILY WITH 4 HAPLOTYPES(1 HLA identical sibling)
Histocompatibility & Immunogenetics Blood and Transplant
FAMILY WITH 5 HAPLOTYPES(0 HLA matches!)
HLA: A* B* C* DRB1* DQB1*
Patient 02 11 35 52 04 12 01 15 05 06
Sib 1 01 03 07 08 07 - 03 13 02 06
Sib 2 01 11 08 52 07 12 03 15 02 06
Sib 3 01 - 08 - 07 - 03 - 02 -
Sib 4 02 03 07 35 04 07 01 13 05 06
Sib 5 01 03 07 08 07 - 03 13 02 06
Sib 6 01 11 08 52 07 12 03 15 02 06
Histocompatibility & Immunogenetics Blood and Transplant
HLA MATCHING IN UNRELATED HSCT
• donor identification via national/international registries
• best results - allele match at 5 loci (A,B,C,DRB1,DQB1 =10/10)
• Caucasian patients have a 40-50% chance of having a high
resolution matched donor at HLA-A, -B, -C, -DRB1 and -DQB1
(10/10 match)
• the chance of a 10/10 match in other ethnic groupings is lower
• comparable disease free survival in good risk patients
• increased frequency of post-transplant complications
Histocompatibility & Immunogenetics Blood and Transplant
UNRELATED DONOR MATCHING - TYPICAL STRATEGY
• HLA-A, B, C, DRB1 & DQB1 (5 loci = 10 alleles) at low resolution
• if matched at low resolution, proceed to SBT (minimum A, B, DRB1)
• if matched at high resolution, select on:• gender• CMV• blood group
• if not matched at high resolution• widen search (BMDW ~20 million)• single allele mismatch • single or double CBU, 6/6 > 5/6 > 4/6 and cell dose
Histocompatibility & Immunogenetics Blood and Transplant
UK REGISTRIES UK Stem Cell Strategic Forum 2010
Recommendations – Transplantation:
• streamline registry activities in the UK
• data collection and outcome monitoring at every stage
• alternative donor clinical trials network
• cord blood transplantation concentrated into designated Centres of Excellence
Histocompatibility & Immunogenetics Blood and Transplant
UK REGISTRIES UK Stem Cell Strategic Forum 2010
Recommendations – Cord Blood:
• increase from ~8000 to 50,000 high dose units in 5 years
• 30 to 50% of donations from black and ethnic minority women
• newly banked units to have > 90 x 107 TNC (ethnic minority donors) or 120 x 107 TNC (Caucasian donors)
Histocompatibility & Immunogenetics Blood and Transplant
UK STEM CELL STRATEGIC FORUM 2011 (£4 million)
• align provision of stem cell donations - AN to become the single contact point for all searches (access >700,000 adult donors)
• select 20,000 young adult donors with common phenotypes for high resolution HLA typing
• increase collection at 8 cord blood collection sites, additional 2,000 CBU’s per year
• genotype prediction algorithm to speed up searches (? 2012) -probability estimates for finding a 10/10 donor based on HLA haplotype and allele frequencies in relevant population is highly predictable
Histocompatibility & Immunogenetics Blood and Transplant
TOTAL STEM CELL PROVISION WORLDWIDE
WMDA ANNUAL REPORT
Histocompatibility & Immunogenetics Blood and Transplant
PROBLEMS ASSOCIATED WITH UNRELATED DONOR SEARCHING
problems are:
1. incomplete registry data (e.g. no HLA-C or DQB1)
2. HLA polymorphism (only 40-50% Caucasians have 10/10 HLA match, other groups less)
• rare alleles/allelic variants
• ethnicity
• linkage disequilibrium
3. donor drop out
Histocompatibility & Immunogenetics Blood and Transplant
PROBLEMS ASSOCIATED WITH UNRELATED DONOR SEARCHING (1)
incomplete registry data• HLA
• not all donors typed by DNA techniques• not all donors typed for DRB1• not all donors typed for C &/or DQB1• very few donors high resolution typing
• gender, blood group, ethnicity, CMV not always available
• costs
Histocompatibility & Immunogenetics Blood and Transplant
PROBLEMS ASSOCIATED WITH UNRELATED DONOR SEARCHING (2)
HLA polymorphism
• rare alleles/allelic variants
(5,880 Class I, 1647 Class II alleles)
• linkage disequilibrium
Histocompatibility & Immunogenetics Blood and Transplant
HLA: A* B* C* DRB1* DQB1*
JM 02:05 03:01 07:02 40:02 02:02 07:02 13:01 14:01/54 05:03 06:03
DEDKM 2127057 02:01 03:01 07:02 40:02 02:02 07:02 13:01 14:01/54 05:03 06
GB 1300733 02#1 - 07 40:02 02 07 13:01 14 05 06
DEDKM 620547 02#1 03 07 40:01 03 07 13:01 14 05 06
#1 Not HLA A*02:05
NO 10/10 DONORBECAUSE OF RARE ALLELES
Histocompatibility & Immunogenetics Blood and Transplant
HLA: A* B* C* DRB1* DQB1*
SH 03 24 15:18 18 05 07 04:07 13:01 03:01 06
TO03 3992 01 24 15:10 18 03 12:03/06 04:02 11 03:02 05
AKB-142342 24 25 15:18 18 07 12:03/06 04:01 13:01 03:02 06
HLA: Panel A* B* C* DRB1* DQB1* Vol(ml)
TNC(107)
SH N/A 03:01 24:02 15:18 18:01 05:01 07:04 04:07 13:01 03:01 06 N/A N/A
6504125 ACCB 03 24:02 38:01 35:23 07:02 12:03 04:07 13:01 03:02 06:03 121 318
999293104 UICB 03 24 35 - 04 04:07 13:01 N/T 30 208
290055467 AUCB 03 24 27 35 N/T 04:07 13:01 N/T 60 91.9
CBU’s (matching for HLA-A, B & DRB1 only)
MUD’s
NO 10/10 DONORBECAUSE OF RARE ALLELES
Histocompatibility & Immunogenetics Blood and Transplant
HLA: A* B* C* DRB1* DQB1*
DM 02:11 11:01 35:03 40:06 04:01 15:02 10:01 15:01 05:01 06:01
DEDKM 3571314 02:11 11:01 35:03 40:06 04:01 15:02 10:01 15:01 05:01 06:01
0564-3760-1 02:11 11:01 35:03 40:06 12:03 15:02 10:01 15:01 05:01 06:01
SUITABLE DONORDESPITE RARE ALLELES
Histocompatibility & Immunogenetics Blood and Transplant
LINKAGE DISEQUILIBRIUM• some alleles occur more frequently together than expected by random
association
• extended (ancestral) haplotypes e.g.A*01, B*08, C*07, DRB1*03,
DQB1*02
• commonly found HLA-B & C, HLA-DRB1 & DQB1
• patients with common HLA-B and -C or HLA-DRB1 and -DQB1
associations have a positive impact on the likelihood of finding a donor
• patients with uncommon HLA-B and -C or HLA-DRB1 and -DQB1
associations have a negative impact on the likelihood of finding a donor
Histocompatibility & Immunogenetics Blood and Transplant
LINKAGE DISEQUILIBRIUM HLA-B & C
B*27 C*01
B*27 C*02
B*55 C*03:03(Cw9)
B*60 C*03:04(Cw10)
B*35 C*04
B*44:02 C*05
B*57 C*06:02
B*07 C*07
B*08 C*07
B*14 C*08
B*15:02 C*08
B*52 C*12:02
B*38 C*12:03
B*51 C*15
B*44:03 C*16:01
B*41 C*17
Histocompatibility & Immunogenetics Blood and Transplant
LINKAGE DISEQUILIBRIUM HLA-DRB1 & DQB1
DRB1*01 DQB1*05
DRB1*15 DQB1*06:02
DRB1*16 DQB1*05
DRB1*03:01 (DR17) DQB1*02:01
DRB1*03:02 (DR18) DQB1*04
DRB1*04 DQB1*03:01/03:02
DRB1*11 DQB1*03:01
DRB1*12 DQB1*03:01
DRB1*13 DQB1*06:03/06:04
DRB1*14 DQB1*05
DRB1*07 DQB1*02:02/03:03
DRB1*08 DQB1*04/03:01
DRB1*09 DQB1*03:03
DRB1*10 DQB1*05
Histocompatibility & Immunogenetics Blood and Transplant
COMPOUNDING EFFECT OF LINKAGE DISEQUILIBRIUM
HLA: A* B* C* DRB1* DQB1*
KaM 02:01 68:01 27:05 44:02 02 07 08:03 13:02 03 06:04
GB 1545503 (AN) 02:01 68:01 27:05 44:02 02 07 11:01 13:02 03 06:04
1/026701 (BBMR) 02 68:02 27 44 02 05 08 13:01 04 06:03
DE-BBB 12355 02 68:01 27 44 05 - 08 13:01 03 06:03
DE-DKM 336800 02 68 27 44 01 07 08 13:02 04 06:04
DE-DKM 513225 02 68:01 27 44 01 05 08 13:01 04 06:03
DE-DKM 2473710 02 68:01 27 44 05 07 08 13:01 04 06:03
0213-6872-5 02 68:01 27 44 03 07 08 13:02 04 06:04
0491-2420-9 02 68 27 44 01 07 08 13:02 04 06:04
Histocompatibility & Immunogenetics Blood and Transplant
WHAT IS THE RISK OF HLA MISMATCHING ?
• graft failure (rejection)
• GVHD (but GVL; ?HLA-DPB1)
• selecting a mismatch at 1 locus may affect
other loci due to linkage disequilibrium
Histocompatibility & Immunogenetics Blood and Transplant
16th IHW PROJECT INTO HLA MISMATCHING
Histocompatibility & Immunogenetics Blood and Transplant
16th IHW PROJECT - RESULTS
Grades III-IV aGVHD
Relapse Survival
HLA mismatch OR, P value HLA 10/10 (10,930) 1 1 1 Single HLA-A (1,430) 1.64, <0.001 1.10, 0.11 1.36, <0.001 Single HLA-B (651) 1.88, <0.001 0.93, 0.47 1.38, <0.001 Single HLA-C (2,600) 1.55, <0.001 1.00, 1 1.29, <0.001 Single HLA-DRB1 (383) 1.47, <0.003 1.04, 0.74 1.14, 0.07 Single HLA-DQB1(1,139) 1.00, 0.99 1.07, 0.29 1.05, 0.27
Single DQB1 mismatch is tolerated the best OR, P value HLA 10/10 (10,930) 1 1 1 Single Class I (4,681) 1.62, <0.001 1.02, 0.58 1.32, <0.001 Single Class II (1,522) 1.11, 0.15 1.06, 0.29 1.07, 0.07
Class I mismatches are more detrimental than Class II
Histocompatibility & Immunogenetics Blood and Transplant
HLA MISMATCHING – NO CONSENSUS IN UK!
Histocompatibility & Immunogenetics Blood and Transplant
HLA NOMENCLATURE