PRINCIPLES OF HLA TYPING; HLA MATCHING IN HSCT David Smillie H & I, NHSBT, Sheffield

successful HSCT depends on many factors (disease, stage, age, treatment regime etc) not least is HLA compatibility between patient and donor!

HLA TYPING REPORT – a collection of letters and numbers, how do we arrive at this and what use is it?

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!

AMINO ACID POLYMORPHISM (this is what the immune system recognises) HLA molecule e.g HLA-A1 e.g HLA-A2

DNA POLYMORPHISM (resolved by DNA typing) SNP = Single Nucleotide Polymorphism alleles differ by 1 or more SNP

HLA ANTIGENS ON NUCLEATED CELLS DR C DQ B DP paternal haplotype A A maternal haplotype DP B DQ C DR

INHERITANCE OF HLA HAPLOTYPES Father + Mother = 4 haplotypes (25% chance of identical sib) PARENTS A* B* C* DRB1* DQB1* (a) 01 08 07 03 02 a b c d CHILDREN (b) 03 07 07 15 06 (c) 02 44 05 04 03 (d) 30 13 06 10 05 (r) 02 44 05 10 05 a/c a/d b/c b/d b/r

ORGANISATION OF HLA GENES CHROMOSOME 6

HLA TYPING METHODS 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 BY SEROLOGY (Complement Dependent Cytotoxicity - using HLA-A as an example) anti HLA-A1 anti HLA-A2 anti HLA-A3 anti HLA-A24 alloantisera patient/donor lymphocytes (e.g. A2) add complement

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

3 LEVELS OF RESOLUTION low resolution (2 digit) - identifies broad families of alleles belonging to the same serotypic group (e.g. A*02) intermediate resolution (allele string) - identifies alleles that have common sequence determinants and thus share hybridisation pattern (e.g. A*02:05/08/22) high resolution (minimum 4 digit) - identifies single allele

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

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 synthetic DNA which hybridise only to their exact complementary sequence and this hybridisation can be detected) sequencing based typing (SBT)

PRINCIPLE OF DNA TYPING (using HLA-A gene as an example) allele-specific sequences (primer/probe) A*03 A*24 conserved sequence

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

DONOR SELECTION

GUIDELINES FOR HLA MATCHING IN HSCT

MATCHED DONOR OF CHOICE HLA identical sibling confirmed by family studies identical for other genes in MHC region HLA identical family member differences at other gene loci possible HLA identical unrelated donor differences at other gene loci probable HLA mismatched unrelated donor cord blood unit(s)

HSCT – TYPICAL HLA TYPING PROTOCOL PATIENT & FAMILY LOW RESR HLA-A, B, C, DRB1, DQB1 NO MATCH SBT RECIPIENT HLA-A, B, C, DRB1, DQB1 MATCH HAPLOTYPE ASSIGNMENT MUD SEARCH BBMR/AN/WBMR/BMDW CONFIRMATORY TESTING DONOR & RECIPIENT SBT DRB1 (& TO ESTABLISH HAPLOTYPES) SELECT LOW RES MATCHED DONORS MUD’s: CONFIRMATORY LOW RES & SBT HLA-A,B,C,DRB1,DQB1, CMV, BLOOD GROUP etc TRANSPLANT

HLA MATCHING IN RELATED HSCT

FAMILY WITH 4 HAPLOTYPES (1 HLA identical sibling) DRB1* DQB1* Patient * 02 29 44 51 15 16 07 - Sib 1 * Sib 2 24 06 Sib 3 13 Sib 4 Sib 5 Sib 6

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 03 07 08 - 13 Sib 2 Sib 3 Sib 4 Sib 5 Sib 6

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

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

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

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)

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

TOTAL STEM CELL PROVISION WORLDWIDE WMDA ANNUAL REPORT

PROBLEMS ASSOCIATED WITH UNRELATED DONOR SEARCHING problems are: incomplete registry data (e.g. no HLA-C or DQB1) HLA polymorphism (only 40-50% Caucasians have 10/10 HLA match, other groups less) rare alleles/allelic variants ethnicity linkage disequilibrium donor drop out

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

PROBLEMS ASSOCIATED WITH UNRELATED DONOR SEARCHING (2) HLA polymorphism rare alleles/allelic variants (5,880 Class I, 1647 Class II alleles) linkage disequilibrium

BECAUSE OF RARE ALLELES NO 10/10 DONOR BECAUSE OF RARE ALLELES HLA: A* B* C* DRB1* DQB1* JM 02:05 03:01 07:02 40:02 02:02 13:01 14:01/54 05:03 06:03 DEDKM 2127057 02:01 06 GB 1300733 02#1 - 07 02 14 05 DEDKM 620547 03 40:01 #1 Not HLA A*02:05

BECAUSE OF RARE ALLELES NO 10/10 DONOR BECAUSE OF RARE ALLELES MUD’s HLA: A* B* C* DRB1* DQB1* SH 03 24 15:18 18 05 07 04:07 13:01 03:01 06 TO03 3992 01 15:10 12:03/06 04:02 11 03:02 AKB-142342 25 04:01 CBU’s (matching for HLA-A, B & DRB1 only) 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 06 6504125 ACCB 03 38:01 35:23 07:02 12:03 03:02 06:03 121 318 999293104 UICB 24 35 - 04 N/T 30 208 290055467 AUCB 27 60 91.9

SUITABLE DONOR DESPITE RARE ALLELES 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 0564-3760-1 12:03

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

LINKAGE DISEQUILIBRIUM HLA-B & C

LINKAGE DISEQUILIBRIUM HLA-DRB1 & DQB1

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) 11:01 1/026701 (BBMR) 68:02 27 44 05 08 13:01 04 06:03 DE-BBB 12355 - DE-DKM 336800 68 01 DE-DKM 513225 DE-DKM 2473710 0213-6872-5 0491-2420-9

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 41

16th IHW PROJECT INTO HLA MISMATCHING

16th IHW PROJECT - RESULTS

HLA MISMATCHING – NO CONSENSUS IN UK!

HLA NOMENCLATURE