16/01/2018 Pre-Transplant Immunogenetic and Immunological assessment for Hematopoietic Stem Cell Transplantation Dr UMA KANGA Dept. of Transplant Immunology & Immunogenetics umakanga@hotmail.com January 2018 1
16/01/2018 Bone Marrow Transplantation Bone Marrow Transplantation Hematopoietic Stem Cell Transplantation Hematopoietic Stem Cell Transplantation Recognized curative therapy Recognized curative therapy Indications Indications • Leukemia , lymphoma Leukemia , lymphoma • Metabolic disorders Metabolic disorders • Aplastic anemia Aplastic anemia • Immunodeficiencies Immunodeficiencies • Hemoglobinopathies Hemoglobinopathies • MS, Rheumatologic MS, Rheumatologic disorders disorders • MDS myeloproliferative MDS myeloproliferative • Thalassemia Thalassemia Rapid strides – Rapid strides – understanding understanding intricacies intricacies of HSCT of HSCT 2 Uma Kanga, AIIMS, NDelhi, Jan 2
16/01/2018 15 th March 1920 ‐ Oct 20 th 2012 1950 : First Transplant Between identical twins LEUKEMIA 3 Uma Kanga, AIIMS, NDelhi, Jan 2018 3
16/01/2018 Hematopoietic Stem Cell Transplantation Stem cells extracted from Donor HLA Matching Conditioning Despite good HLA match, Regimen Space created in milder conditioning, better bone marrow immunosupression Engraftment of Major cause of morbidity Donor Cells in and mortality is GVHD?? Recipient Marrow Patient Improved, now on GVHD prophylaxis 4 Uma Kanga, AIIMS, NDelhi, Jan 2018 4
16/01/2018 Centre for International Blood and Marrow Transplant Research 5 Uma Kanga, AIIMS, NDelhi, Jan 2018 5
16/01/2018 Sources of Stem Cells CORD BLOOD BONE MARROW HARVEST PERI PHERAL BLOOD STEM CELLS 6 Uma Kanga, AIIMS, NDelhi, Jan 2018 6
16/01/2018 Factors Influencing HSCT Outcome Original disease- malignant / non-malignant Pre-Transplant conditioning regimen Recipient – donor Genetic disparity Hematopoietic Stem Cell Dose Presence / absence of donor T cells Immunosuppression 7 Uma Kanga, AIIMS, NDelhi, Jan 2018 7
16/01/2018 Genomic Organization of the HLA Complex Chromosome 6 Highly polymorphic genomic region Immunologically relevant- Antigen presentation Polymorphism restricted to functionally crucial peptide binding regions Crucial in Organ and Stem Cell transplantation Many diseases associated with genes encoding HLA molecules 8 Uma Kanga, AIIMS, NDelhi, Jan 2018 8
16/01/2018 Polymorphism of Polymorphism of the HLA System the HLA System (2017- IMGT/HLA) Numbers of HLA Alleles Class I - 12544 + Class II - 4622 = 17166 9 Uma Kanga, AIIMS, NDelhi, Jan 2018 9
16/01/2018 WHO Nomenclature Committee HLA- Nomenclature System 2010 HLA-typing at the DNA level requires nomenclature for specific DNA sequences Allele family 15 Subregion Gene region Third allele HLA-DRB1*15:03 ‐ or ‐ chain polypeptide Gene locus 10 Uma Kanga, AIIMS, NDelhi, Jan 10
16/01/2018 AIIMS, New Delhi : HLA Typing for BMT/PBSCT Low resolution Intermediate High resolution methods methods resolution methods CDC (Serology) PCR-SSP PCR-SSP PCR-SSP PCR-RLS PCR-SSOP Sequenced Based Typing PCR-SSOP PCR-SSOP (SBT) Next Generation Sequencing (NGS) CDC-Complement Dependent Cytotoxicity SSP- Sequence Specific Primer SSOP-Sequence Specific Oligoneucleotide Probes RLS- Reverse Line Strips 11 Uma Kanga, AIIMS, NDelhi, Jan 2018 11
16/01/2018 Serological Typing: Microlymphocytotoxicity Complement antibody Positive reaction to antibody kills cells. Dead cells pick up dye. Buffy coat Negative reaction to antibody: from patient cells survive and exclude dye. Limitations Fresh blood, viable lymphocytes, in CML patients ‐ Blasts?? Cross reactivity Surface expression of antigens ‐ heterozygous/ homozygous Throughput ‐ Low 10 ‐ 15 samples/ day Resolution ‐ Low 12 Uma Kanga, AIIMS, NDelhi, Jan 12
16/01/2018 Molecular methods – DNA based HLA typing SSP ‐ PCR Sequence ‐ specific PCR ‐ allele ‐ specific primers Amplification internal controls Allele ‐ specific product SSP= Sequence ‐ specific primer Amplification SSP No amplification SSP SSP does not match allele Throughput ‐ Low 4 ‐ 8 samples/ day Resolution ‐ Low and High 13 Uma Kanga, AIIMS, NDelhi, Jan 2018 13
16/01/2018 PCR-SSO: Reverse Hybridization Throughput ‐ intermediate 16 A,B,DR / run Resolution ‐ Low /intermediate 14 Uma Kanga, AIIMS, NDelhi, Jan 14
16/01/2018 PCR-SSO on the Chip Throughput ‐ High 32 samples (A,B,DR)/ day Resolution ‐ Low/ intermediate/high 15 Uma Kanga, AIIMS, NDelhi, Jan 2018 15
16/01/2018 Luminex Based assay- PCR-SSO Genotyping and Antibody • Controls + 96 specificities (class I) or 76 specificities (class II)/ well • Performed in a 96 ‐ well format (8 well strips) • Assay time = 2 hours • Higher sensitivity than flow SA beads Tells the instrument how much antigen is bound to Laser 2 the bead Tells the instrument which bead is being examined Laser 1 Throughput ‐ High 32 samples/ day (A,B,DR) Resolution ‐ Low and High Class I beads: Class II beads: HLA-A, -B, -C loci HLA-DR, -DQ, -DP 16 Uma Kanga, AIIMS, NDelhi, Jan 2018 16
16/01/2018 Sequence Based Typing Reverse PCR primer Forward PCR primer E x o n 2 E x o n 3 HLA ‐ B Sequencing primers Applied Bio ‐ systems 3130xl Genetic Analyzer Exon 3 Throughput ‐ 12 samples/ day (A,B,DR) Resolution ‐ High 17 Uma Kanga, AIIMS, NDelhi, Jan 17
16/01/2018 HLA Allele assignment- Sequencing Technologies 18 Uma Kanga, AIIMS, NDelhi, Jan 18
16/01/2018 HLA- Next Generation Sequencing- NGS Roche • 454 PacBio • RS II Oxford • Sequel Nanopore Illumina • MinION Thermo Fisher • MiSeq • MiniSeq • Promethion • Ion Proton • HiSeq • NovaSeq • Ion PGM • NextSeq • Ion S5 Long reads (>10 kb) Short reads (<1 kb) 19 Uma Kanga, AIIMS, NDelhi, Jan 2018 19
16/01/2018 HLA- Next Generation Sequencing- NGS Applications in Clinical Histocompatibility Testing • Gene Segments Covered-detects all intragenic mutations • Accuracy and Reproducability- Ability to get correct types • Extremely high resolution. No secondary tests needed • Ability to find errors and novel alleles • TAT similar or better than Sanger SBT • Automatic Genotype assignment and Less analysis time • Loci Tested (in HSCT and in solid organ transplantation) 20 Uma Kanga, AIIMS, NDelhi, Jan 2018 20
16/01/2018 Donor Selection based on HLA match Par Parents : s : Both 50 % Both 50 % Match Match 50% Sibs: 50% Match 25% Sibs: 0% Match 25% Sibs: 100% Match 21 Uma Kanga, AIIMS, NDelhi, Jan 2018 21
16/01/2018 What if no HLA compatible donor in the family ? OPTIONS ?? 22 Uma Kanga, AIIMS, NDelhi, Jan 2018 22
16/01/2018 23 Uma Kanga, AIIMS, NDelhi, Jan 23
16/01/2018 Possibility of additional HLA compatible family donors 24 Uma Kanga, AIIMS, NDelhi, Jan 2018 24
16/01/2018 2013 ‐ Graft failure. required second transplant Donor? ‐ Sibling, the first donor? 25 Uma Kanga, AIIMS, NDelhi, Jan 2018 25
16/01/2018 26 Uma Kanga, AIIMS, NDelhi, Jan 2018 26
16/01/2018 27 Uma Kanga, AIIMS, NDelhi, Jan 2018 27
16/01/2018 Matched Unrelated Donor Parents : Both 50 % Match A26, B8, DR3 50% Sibs: 50% Match A2, B44, DR4 25% Sibs: 0% Match 25% Sibs: 100% Match 28 Uma Kanga, AIIMS, NDelhi, Jan 28
16/01/2018 29 Uma Kanga, AIIMS, NDelhi, Jan 2018 29
16/01/2018 75 Registries Bone Marrow Donors 53 CB Banks Worldwide- 53 countries (Aug 2017) 28.9 M Donors France Greffe Australia de Moelle 273,027 Bone Marrow Donor Austrian Bone Registry Marrow Donors 170,680 67,059 BM Donors + CBUs 28,935,264 German Registry of BMDW Be the Match Bone Marrow Donors NMDP 7,542,435 8,403,592 India IN1 ‐ 9580 IN2 ‐ 223,067 Anthony Nolan IN3 ‐ 6225 278,053 Trust IN4 ‐ 5200 659,486 IN5 ‐ 33,977 30 Uma Kanga, AIIMS, NDelhi, Jan 2018 30
16/01/2018 Centre for International Blood and Marrow Transplant Research 31 Uma Kanga, AIIMS, NDelhi, Jan 2018 31
16/01/2018 Transplant Outcomes By Year: Sibling vs Unrelated Hematology 2012 Improved overall 1 year survival rates: • Improvement in HLA ‐ matching techniques • better donor selection • better overall patient selection for transplantation • improvements in supportive care 32 Uma Kanga, AIIMS, NDelhi, Jan 2018 32
16/01/2018 Survival among HLA-A,B and DRB1 allele matched pairs by number of mismatched class I loci Flomenberg et al, Blood 2004 0 Mismatches (n=791) 1 Mismatch (n=317) 2 Mismatches (n=117) Years after Transplant 33 Uma Kanga, AIIMS, NDelhi, Jan 2018 33
16/01/2018 Overall Survival after allo HSCT from ISDs, MFDs and MUDs Ottinger et al, Blood 2003 ISD, early disease MUD, early disease MFD, early disease ISD, advanced disease MUD, advanced disease MFD, advanced disease 34 Uma Kanga, AIIMS, NDelhi, Jan 2018 34
16/01/2018 Impact of mismatched HLA Loci on the risk of Ac GvHD Ottinger et al, Blood 2003 MFD, MM2 MFD, MM1 p MUD MFD ISD, MMO Days after Transplantation 35 Uma Kanga, AIIMS, NDelhi, Jan 2018 35
16/01/2018 SCT Workshop Component: Survival among matched pairs 36 Uma Kanga, AIIMS, NDelhi, Jan 2018 36
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