PRINCIPLES OF HLA TYPING HLA MATCHING IN HSCT

PRINCIPLES OF HLA TYPING; HLA MATCHING IN HSCT David Smillie H & I, NHSBT, Sheffield Histocompatibility & Immunogenetics Blood and Transplant

• successful HSCT depends on many factors (disease, stage, 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-A 1 Histocompatibility & Immunogenetics e. g HLA-A 2 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

HLA ANTIGENS ON NUCLEATED CELLS DR DQ C DP B A DP A B DQ DR Histocompatibility & Immunogenetics paternal haplotype maternal haplotype C Blood and Transplant

INHERITANCE OF HLA HAPLOTYPES Father + Mother = 4 haplotypes (25% chance of identical sib) PARENTS A* a b c d CHILDREN a/c a/d b/c b/d Histocompatibility & Immunogenetics b/r B* C* DRB 1* DQB 1* (a) 01 08 07 03 02 (b) 03 07 07 15 06 (c) 02 44 05 04 03 (d) 30 13 06 10 05 (r) 02 44 05 10 05 Blood and Transplant

ORGANISATION OF HLA GENES CHROMOSOME 6 Histocompatibility & Immunogenetics Blood and Transplant

HLA TYPING METHODS 1950’s 1960’s 1980’s 1999 2000’s discovery of HLA system serological typing first HLA genes cloned, sequenced DNA/PCR based HLA typing sequence entire MHC (HGP) database of all HLA alleles SBT, Luminex SSO Histocompatibility & Immunogenetics Blood and Transplant

HLA TYPING BY SEROLOGY (Complement Dependent Cytotoxicity - using HLA-A as an example) anti HLA-A 1 • alloantisera anti HLA-A 2 anti HLA-A 3 anti HLA-A 24 • patient/donor lymphocytes (e. g. A 2) • add complement 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 v 5. 6 (stipulated by JACIE) • related donor - ‘adequate testing to definitively establish HLA identity by descent’ • unrelated donor - ‘low resolution HLA-A/B/C digit) and high resolution DRB 1 typing (4 digit)’ • confirmatory typing Histocompatibility & Immunogenetics (2 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 synthetic DNA which hybridise only to their exact complementary sequence and this hybridisation can be detected) • sequencing based typing (SBT) Histocompatibility & Immunogenetics Blood and Transplant

PRINCIPLE OF DNA TYPING (using HLA-A gene as an example) A*01 A*02 A*03 allele-specific sequences (primer/probe) A*24 conserved sequence 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 CHOICE 1. 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 & FAMILY LOW RESR HLA-A, B, C, DRB 1, DQB 1 MATCH HAPLOTYPE ASSIGNMENT CONFIRMATORY TESTING DONOR & RECIPIENT SBT DRB 1 (& TO ESTABLISH HAPLOTYPES) TRANSPLANT Histocompatibility & Immunogenetics NO MATCH SBT RECIPIENT HLA-A, B, C, DRB 1, DQB 1 MUD SEARCH BBMR/AN/WBMR/BMDW SELECT LOW RES MATCHED DONORS MUD’s: CONFIRMATORY LOW RES & SBT HLAA, B, C, DRB 1, DQB 1, CMV, BLOOD GROUP etc Blood and Transplant

HLA MATCHING IN RELATED HSCT Histocompatibility & Immunogenetics Blood and Transplant

FAMILY WITH 4 HAPLOTYPES (1 HLA identical sibling) HLA: A* B* C* DRB 1* DQB 1* 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 - Histocompatibility & Immunogenetics Blood and Transplant

FAMILY WITH 5 HAPLOTYPES (0 HLA matches!) HLA: A* B* C* DRB 1* DQB 1* 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, DRB 1, DQB 1 =10/10) • Caucasian patients have a 40 -50% chance of having a high resolution matched donor at HLA-A, -B, -C, -DRB 1 and -DQB 1 (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, DRB 1 & DQB 1 (5 loci = 10 alleles) at low resolution • if matched at low resolution, proceed to SBT (minimum A, B, DRB 1) • 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 DQB 1) 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 DRB 1 • not all donors typed for C &/or DQB 1 • 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

NO 10/10 DONOR BECAUSE OF RARE ALLELES HLA: A* B* C* DRB 1* DQB 1* 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 Histocompatibility & Immunogenetics Blood and Transplant

NO 10/10 DONOR BECAUSE OF RARE ALLELES MUD’s HLA: A* B* C* DRB 1* DQB 1* SH 03 24 15: 18 18 05 07 04: 07 13: 01 06 TO 03 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 CBU’s (matching for HLA-A, B & DRB 1 only) HLA: Panel SH N/A 6504125 A* B* C* DRB 1* DQB 1* Vol (ml) TNC (107) 03: 01 24: 02 15: 18 18: 01 05: 01 07: 04 04: 07 13: 01 06 N/A 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 04: 07 13: 01 N/T 60 91. 9 Histocompatibility & Immunogenetics N/T Blood and Transplant

SUITABLE DONOR DESPITE RARE ALLELES HLA: A* B* C* DRB 1* DQB 1* DM 02: 11 11: 01 35: 03 40: 06 04: 01 15: 02 10: 01 15: 01 06: 01 DEDKM 3571314 02: 11 11: 01 35: 03 40: 06 04: 01 15: 02 10: 01 15: 01 06: 01 0564 -3760 -1 02: 11 11: 01 35: 03 40: 06 12: 03 15: 02 10: 01 15: 01 06: 01 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, DRB 1*03, DQB 1*02 • commonly found HLA-B & C, HLA-DRB 1 & DQB 1 • patients with common HLA-B and -C or HLA-DRB 1 and -DQB 1 associations have a positive impact on the likelihood of finding a donor • patients with uncommon HLA-B and -C or HLA-DRB 1 and -DQB 1 associations have a negative impact on the likelihood of finding a donor Histocompatibility & Immunogenetics Blood and Transplant

LINKAGE DISEQUILIBRIUM HLA-B & C Histocompatibility & Immunogenetics Blood and Transplant

LINKAGE DISEQUILIBRIUM HLA-DRB 1 & DQB 1 Histocompatibility & Immunogenetics Blood and Transplant

COMPOUNDING EFFECT OF LINKAGE DISEQUILIBRIUM HLA: A* B* C* DRB 1* DQB 1* Ka. M 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-DPB 1) • selecting a mismatch at 1 locus may affect other loci due to linkage disequilibrium Histocompatibility & Immunogenetics Blood and Transplant

16 th IHW PROJECT INTO HLA MISMATCHING Histocompatibility & Immunogenetics Blood and Transplant

16 th IHW PROJECT - RESULTS Histocompatibility & Immunogenetics Blood and Transplant

HLA MISMATCHING – NO CONSENSUS IN UK! Histocompatibility & Immunogenetics Blood and Transplant

HLA NOMENCLATURE Histocompatibility & Immunogenetics Blood and Transplant