Prof. Narinder K Mehra Prof. Narinder K Mehra Dept of Transplant Immunology & ImmunogeneticsDept of Transplant Immunology & Immunogenetics
AIIMS, New Delhi AIIMS, New Delhi [email protected]@hotmail.com
Hematopoietic Stem Cell Transplantation :Hematopoietic Stem Cell Transplantation :
Opportunities and challengesOpportunities and challenges
Hematopoietic Stem Cell TransplantationHematopoietic Stem Cell Transplantation1. 1. Hematologic Malignancies Hematologic Malignancies
Acute and Chronic Myeloid LeukemiaAcute and Chronic Myeloid Leukemia
Acute and Chronic lymphocytic leukemia Acute and Chronic lymphocytic leukemia Myeloddysplastic SyndromeMyeloddysplastic SyndromeLymphomas – Hadgkins and Non HodgkinsLymphomas – Hadgkins and Non HodgkinsMultiple MyelomasMultiple Myelomas
2. 2. Non Malignant DisordersNon Malignant Disorders Severe Aplastic Anemias (SAA)Severe Aplastic Anemias (SAA)Hurler’s SyndromeHurler’s SyndromeWiskott – Aldrich Syndrome Wiskott – Aldrich Syndrome Diamond – Blackfan AnemiaDiamond – Blackfan AnemiaOsteopetrosisOsteopetrosis
3. 3. Inherited Blood DisordersInherited Blood DisordersBeta Thalassaemia MajorBeta Thalassaemia Major
4. 4. Severe combined immunodeficiency (SCID)Severe combined immunodeficiency (SCID)
5. 5. Pathologic States including autoimmune diseasesPathologic States including autoimmune diseases
6. 6. Solid Tumors (Breast cancer, Teratomas, ovarian tumors etc.)Solid Tumors (Breast cancer, Teratomas, ovarian tumors etc.)
Bone Marrow
Peripheral Blood Stem Cells (PBSC) Cord Blood
Sources of Hematopoietic Stem CellsSources of Hematopoietic Stem Cells
NKM / AIIMS
Original disease of the patient
Prior therapy of the Pre-Tx conditioning regimen
Genetic disparity between donor and recipientGenetic disparity between donor and recipient
No. of hematopoietic stem cells transfused
Presence / absence of donor T cells
Post transplant immune suppression therapy
HSCT : FACTORS INFLUENCING SUCCESS
NKM / AIIMS
Chromosome 6 Chromosome 6
Gene map of the HLA regionGene map of the HLA region
Class IIClass II Class Class IIIIII
Class IClass I
1.8 Mb1.8 Mb
40 % of which have assumed immune functions 40 % of which have assumed immune functions
tel. Long arm cen. short arm tel. tel. Long arm cen. short arm tel.
6p 21.36p 21.3HLA regionHLA region
Bf Bf DP DP DM DM DQ DQ DR DR C4 C2Hsp70TNF C4 C2Hsp70TNF
B C E A G F B C E A G F
128 functional genes 128 functional genes Most polymorphic Most polymorphic
Ag presentation, crucial in organ and HSCTAg presentation, crucial in organ and HSCT
Compatible Donor Search -Compatible Donor Search -Matching HLAMatching HLA
Family- ¼ chanceFamily- ¼ chance
Unrelated – Unrelated –
1/500 - 0/10 million chance of 1/500 - 0/10 million chance of matchmatch
70% patients70% patientsdo not have family do not have family donordonor
Extraordinary polymorphism in HLAExtraordinary polymorphism in HLA
Num
ber
Class I AntigensClass I AllelesClass II AntigensClass II Alleles
Multiple DNA Based HLA TechnologiesMultiple DNA Based HLA Technologies
SSPSSP SSOPSSOP Reverse SSOP Reverse SSOP SBTSBT
Luminex (Flow)Luminex (Flow)
DNA extraction (optimum quality, quantity, contamination free) is critical DNA extraction (optimum quality, quantity, contamination free) is critical
DNA vs Serology: Example of a CML patientDNA vs Serology: Example of a CML patient
HLAHLA Pt (P)/Pt (P)/ SerologySerology PCR based DNA typingPCR based DNA typingCousinCousinDonor (CD)Donor (CD) Low ResolutionLow Resolution High ResolutionHigh Resolution
AA PP 34, 234, 2 3401, 023401, 02 3401, 02073401, 0207
CDCD 34, 234, 2 3402, 023402, 02 3402, 02113402, 0211
BB PP 8, 408, 40 0802, 40030802, 4003 0802, 40030802, 4003CDCD 8, 408, 40 0801, 40070801, 4007 0801, 40070801, 4007
CwCw PP 4, -4, - 07, 07, 0404 0704, 040704, 04
CDCD 4, 74, 7 07 ,07 ,05010501 07(01-03), 050107(01-03), 0501
DRDR PP 4, 24, 2 15, 0415, 04 1501, 04051501, 0405
CDCD 4, 24, 2 15, 0415, 04 1502, 04041502, 0404
DQB1DQB1 PP 3, 13, 1 03, 0103, 01 0301, 06010301, 0601
CDCD 3, 13, 1 03, 0103, 01 0302, 06020302, 0602Match Match FullFull PartialPartial MajorMajorGradeGrade MatchMatch MismatchMismatch MismatchesMismatches
NKM / AIIMS
Why Is It Difficult to Find an HLA Identical Why Is It Difficult to Find an HLA Identical Unrelated Donor ?Unrelated Donor ?
Extensive allele & haplotype diversity
Differences among human populations Allele & haplotype frequencies Novel & unique issues
MHC Diversity : MHC Diversity : Biological and clinical implications Biological and clinical implications
ExtremeExtreme MHCMHC
DiversityDiversity inin
India India
Novel Novel AllelesAlleles
- racial admixture- racial admixture
Founder effectFounder effect- Selective environmentalSelective environmental & microbial pressure& microbial pressure
Oriental Oriental influenceinfluence
Unique HLA Unique HLA haplotypeshaplotypes
-- disease associated disease associated
Unique Unique repertoire of repertoire of
peptide peptide presenting presenting moleculesmolecules
*02082%
*02032%
*02012%
*02067%
*02092%
*020734%
*020514%
*021137%
North Indians
Diversity of HLA-A2Diversity of HLA-A2
*020196%
*02111% *0213
1%*0205
1%
Caucasians
*020147%
*020636% *0207
17%
Japanese
A*020140%
A*020240%
A*020520%
Gambian Tissue Antigens 57, 502-507, 2001.
*021118%
*020182%
Hungarian Gypsies
Caucasoids
Japanese
B*2705
B*2702
Blacks
B*2705
B*2703
B*2705
B*2704
Thai
B*2705
B*2704
B*2706
B*2707
Asian Indians
B*2707 ( 11% )
B*2704 ( 29% )
B*2702 ( 1.6%) B*2714
( 0.01% )
B*2705 (57% )
AzoresB*2705
B*2702
B*2703 B*2708B*2707
AIIMS / NKM
** Multiple HLA-DR3 haplotypes Multiple HLA-DR3 haplotypes
CaucasiansCaucasians IndiansIndiansA1-B8-DR3A1-B8-DR3 (AH8.1) (AH8.1) A26-B8-DR3 (AH8.2*) A26-B8-DR3 (AH8.2*) UniqueUnique
A24-B8-DR3A24-B8-DR3A2-B8-DR3A2-B8-DR3
ChineseChinese Indians IndiansA33-B58-DR3A33-B58-DR3 (AH58.1) (AH58.1) A33-B58-DR3 A33-B58-DR3
IndiansIndians???????? A2-B50-DR3 A2-B50-DR3 UniqueUnique
*Witt , Mehra, Kaur,….et al Tissue Antigens *Witt , Mehra, Kaur,….et al Tissue Antigens 20022002
What if an HLA-A,-B,-DR compatible sibling is not available in the family ?
OPTIONSOPTIONS
Extended family searchExtended family search
Partially HLA compatible donorsPartially HLA compatible donors
Ante Natal HLA typingAnte Natal HLA typing
Unrelated Donors through registryUnrelated Donors through registryNKM / AIIMS
Antenatal HLA &
Predictive Genetic Testing
ETHICS ? ETHICS ? MORAL ISSUES ? MORAL ISSUES ? PSYCHOLOGY ?PSYCHOLOGY ?
ThalassemiasThalassemias Storage disordersStorage disorders
HLA MatchingHLA Matching SiblingsSiblings LeukemiasLeukemias Thal. M. Thal. M.
IdenticalIdentical 177 177 42 42
HaploidenticalHaploidentical 143 143 87 87
UnidenticalUnidentical 21 21 31 31
Donor Selection : AIIMS Experience Donor Selection : AIIMS Experience (1998 – Feb 2003)(1998 – Feb 2003)
Donor Selection : AIIMS Experience Donor Selection : AIIMS Experience (1998 – Feb 2003)(1998 – Feb 2003)
NKM / AIIMS
* CVS typing reconfirmed after birth in 3 cases * CVS typing reconfirmed after birth in 3 cases
* Out of these, two were transplanted* Out of these, two were transplanted
CVS*CVS*N+ = 33N+ = 33
14 (42.4%)14 (42.4%)
12 (36.4%)12 (36.4%)
7 (21.2%)7 (21.2%)
Unrelated Donor Tx : Unrelated Donor Tx : Thalassemia Major Thalassemia Major
Pt: S W
A26 A26B8 B8DR3 DR3
A26 A33B8 B35DR3 DRx
AH
A26 A11B8 B35DR3 DR15
A26 A11B8 B35DR3 DR15
AH
I
Pt: S Ch
A26 A11B8 B8DR3 DR3
AH AH
A1 A11B52 B8DR3 DR3
A26 A26B8 B8DR3 DR3
II
Ancestral Haplotypes Conserved in the race
How Can We Reduce Risk of Allorecognition ?How Can We Reduce Risk of Allorecognition ?
Select donor to resemble “self”
HLA compatible sib or 10/10 match
Remove mature immune cells from graft
T cell depletion
Umbilical cord blood as source HSC
Reduce “danger” signals
Reduced conditioning regimen
Inactivate alloreactive cells in transplanted recipient
Immune suppressive drugs- cyclosporine, methotrexate, corticosteroids, anti-thymocyte globulin
Balancing ActBalancing Act –Strategy to Reduce One Complication May
Increase Another
Reduce GvHDReduce GvHD
Decrease engraftmentDecrease engraftmentIncrease infectionIncrease infectionIncrease risk relapseIncrease risk relapse
T Cell DepletionT Cell Depletion
BMDWBMDW
France Greffe de Moelle 125,843 125,843 Austrian Bone
Marrow Donors
52,029 52,029
National Marrow Donor Program
3,927,577 3,927,577
Anthony Nolan Research Center
358,285 358,285
AustralianBone Marrow Donor
Registry
162,450 162,450
German Registry of Bone Marrow
Donors
2,299,322 2,299,322
Bone Marrow Donors WorldwideBone Marrow Donors Worldwide
13.8M Donors13.8M Donors
Jan, 2010B.M.Donors = 13.8 MCBUs = 176,779
75 Registries75 Registries
37 CB Banks37 CB Banks
Asian Indian Donor Marrow
Registry
3,630
19941994
History of Registries as Source of History of Registries as Source of Unrelated HLA Compatible DonorsUnrelated HLA Compatible Donors
Early on, unrelated donors through public drives by families & from HLA-typed platelet donor panels
1974 patient’s mother founds Anthony Nolan Bone Marrow Register
1988 World Marrow Donor Association
1988 Bone Marrow Donors Worldwide
Today, 58 registries in 43 countries; 38 cord blood banks in 21 countries with >10 million volunteer donors
>1/3 donors come from countries different than patient
Patient of Indian origin has 1/10th chance of finding a donor
0
50
100
150
200
250
300
Japan Taiwan Hongkong India Others
311,311311,311
277,081277,081
62,841
1205
Asians
Caucasians
BMDW- Asian ComponentTotal : 12.34M, July,2008
95%
5%
No
of
Do
no
rsN
o o
f D
on
ors
ASIANASIAN REGISTRIESREGISTRIES
Country Registry Year of Estb
No of Donors MUD Tx
JAPAN Japan Marrow Donor Program
1991 300,000 >8000
CHINA Chinese Marrow Donor Program
1992 950,000 >1117
SINGAPORE Bone Marrow Donor Program
1993 44,000 >215
TAIWAN Budhist Tzu Chi Marrow Donor Registry
1993 319,000 >1800
KOREA Korea Marrow Donor Program
1994 144,970 >1477
THAILAND Thai Stem Cell Donor Registry
Asian Indian Donor Marrow RegistryAsian Indian Donor Marrow Registry(AIDMR)(AIDMR)
• Needs and RequirementsNeeds and Requirements
• Genetic Diversity of HLAGenetic Diversity of HLA
• Novel Alleles, Unique Haplotypes Novel Alleles, Unique Haplotypes
Established in AIIMS in 1994 as a National level RegistryEstablished in AIIMS in 1994 as a National level Registry
Objectives Encourage donor drive through mass education
programs
Recruit donors with ‘novel alleles’ and ‘unique haplotypes’ from different ethnicities
Regional efforts
Develop and adopt good matching algorithms
Keep updating HLA typing technologies
Encourage and support research that will reduce the need of HLA matching requirements
Asian Indian Donor Marrow RegistryAsian Indian Donor Marrow Registry(AIDMR)(AIDMR)
AIDMR Targets
Atleast 50,000 donors representing various ethnicities
Special attention to donors with novel HLA alleles and unique haplotypes
Donor retention issues: involvement of volunteers/ family members/ social scientists
Technology development and updates including QC issues
In-house creation of HLA typing kits/ reagents
Financial issues: Test cost, BMT cost
BMDW participation: marrow exchange
International collaboration
Donor Centres
AIDMR-National Network
Transplant Centres
AIIMS, SGPGI, IIH, CMC, PGI
NBU, GSBTM
AIIMS, N Delhi
AHRR, N Delhi
CMC, Vellore
TMH, Mumbai
SGPGI, Lucknow
Apollo, Chennai
PGI, Chd
KEM, Mumbai
1. Patient Diagnosis
2. Family Screening for HLA Id sib- best option
3. If not-available Family in distress
4. Search for options :
a) extended family search: not always rewarding except in consanginous marriages
b) Prenatal genetic testing & HLA analysis- useful in disease like thalassemia major
unrelated HSCT
c) Search in national registry- donor pool too small
d) Search in international registry: BMDW, WMDA, ANT etc
STEPS INVOLVED IN MUD TRANSPLANTATION
Probability of Finding Matched Donor for Probability of Finding Matched Donor for Next PatientNext Patient
Antigen level ABDR, ZKRD German Registry More volunteers, more likely to find a match
Registry SizeRegistry Size
Mueller et al, Human Immunol 64:137, 2003Mueller et al, Human Immunol 64:137, 2003
Ottinger et al, Blood 2003
MFD, early diseaseMUD, early disease
ISD, early disease
ISD, advanced disease
MUD, advanced disease
MFD, advanced disease
Overall Survival after allo HSCT from ISDs, MFDs and MUDs
Years after Transplantation
Risk of acGvHD after HSCT from ISDs, MFDs and MUDs
MFD
MUD
ISD
Ottinger et al, Blood 2003
MFD, MM2
MFD, MM1
MUDISD
MFD, MMO
Impact of mismatched HLA Loci on the risk of Ac GvHD
Days after Transplantation
p
Ottinger et al, Blood 2003
Probability of survival after Allogeneic Stem Cell Transplant
Hows et al, BMT 2006
1. Umbilical Cord Blood (UCB)
Benefits and Drawbacks
Private and Public Cord Blood Banks
Affordability issues
Controversaries
2. Creation of HLA Compatible Stem Cell Banks
Human Embryonal Stem Cell lines (hESc)
150 lines covering most HLA
types
Homozygous HLA haplotype lines
Even 10 lines may be
sufficient for a given population, covering
common haplotypes
Alternate Sources Alternate Sources
HLA Compatible hES cell Lines
• Promising Source of Transplantation to replace diseased Promising Source of Transplantation to replace diseased or damaged tissue (Neurodegenerative , CVD, DM).or damaged tissue (Neurodegenerative , CVD, DM).
• Can be propagated indefinitely in an undifferentiated state Can be propagated indefinitely in an undifferentiated state while retaining pluripotencywhile retaining pluripotency
• Differentiated progeny express HLAs leading to graft Differentiated progeny express HLAs leading to graft rejection; matching strategies requiredrejection; matching strategies required
• Effective strategies need to be developed e.g. i) bank of Effective strategies need to be developed e.g. i) bank of HLA typed hES cells ii) Creation of HLA homozygous cell HLA typed hES cells ii) Creation of HLA homozygous cell lines (allelic, haplotypic)lines (allelic, haplotypic)
Important study that provides basis for Stem Cell Banking
A bank of 150 hESCs could provide a full match(A,B,DR) for ~ 20%, or a beneficial match (1MM) for a large majority
A panel of 10 highly selected homozygous donors could provide the max benefit for HLA matching
No line homozygous for all HLA loci has yet been derived
Population based HLA phenotype data necessary
Banking of Human embryonic stem cells: estimating the number of donor cell lines needed for HLA matching
Craig Taylor et al Lancet 366:2019-25, 2005
Creation of HLA Specific Somatic Cells by SCNT Technology: Indian effort• Skin Biopsies from 2 males homozygous for HLA haplotype AH 8.2,
which is ‘unique’ to the Indian population
• 3.03% of healthy Indians and >25% of those with Type 1 Diabetes and celiac disease possess this haplotype (Dr Mehra’s group, AIIMS)
• Banking of such known and common HLA haplotypes could benefit several patients requiring HSCT
• Making an ES cell line by SCNT is possible, but challenging. Recent success with iPS technology (Shinya Yamanaka’s group) provides further hope and ecouragement.
Source of Somatic CellsSource of Somatic CellsCollborative study between AIIMS and Collborative study between AIIMS and NIRRHNIRRH
Fetal fibroblastsFetal fibroblasts
Human fetal fibroblasts already being successfully grown at the Human fetal fibroblasts already being successfully grown at the NIRRH by Dr Deepa BhartiyaNIRRH by Dr Deepa BhartiyaAneuploid cases from a Cytogenetics lab (n= 10)Aneuploid cases from a Cytogenetics lab (n= 10)
Adult fibroblastsAdult fibroblasts
Selection of HLA Homozygous individuals positive for Ancestral Selection of HLA Homozygous individuals positive for Ancestral Haplotype , AH8.2 by the AIIMS Group of Prof N.K. MehraHaplotype , AH8.2 by the AIIMS Group of Prof N.K. Mehra
Adult skin biopsies, Cytogenetics lab (n=2)Adult skin biopsies, Cytogenetics lab (n=2)
cccc
cccc
SummarySummary
• Novel approaches to HCT are improving Novel approaches to HCT are improving outcomes and applicability of HCToutcomes and applicability of HCT
• HCT should be discussed early in the course of HCT should be discussed early in the course of the diseasethe disease
• A team approach involving the A team approach involving the Hematologist/Geneticist, Immunologist, Hematologist/Geneticist, Immunologist, Transplanter and Family/Patient advocate is Transplanter and Family/Patient advocate is essential essential
AIIMS – TII GROUP
Derivation and Characterization of two genetically Derivation and Characterization of two genetically unique huESC lines on in-house derived human feeders unique huESC lines on in-house derived human feeders (Stem cells and development 2008)(Stem cells and development 2008)
Project with a Strong Basic Research Component Project with a Strong Basic Research Component
as well as a Future Translational Goal in the Area of Stem Cell Researchas well as a Future Translational Goal in the Area of Stem Cell Research
Establishing the Technology Establishing the Technology to Derive Human Embryonic Stem Cell Lines by to Derive Human Embryonic Stem Cell Lines by
Somatic Cell Nuclear Transfer and ParthenogenesisSomatic Cell Nuclear Transfer and Parthenogenesis
NIRRH, Mumbai - Deepa Bhartiya and groupNIRRH, Mumbai - Deepa Bhartiya and groupAIIMS, New Delhi – Narinder Mehra, Gurvinder KaurAIIMS, New Delhi – Narinder Mehra, Gurvinder Kaur
Other collaboators…….Other collaboators…….
Creating HLA Homozygous ES Cell Lines
• If a particular HLA haplotype is prevalent, the derived cell line will have potential to treat several individuals
• Idea is to bank most common haplotype ES cell lines so that most of patients can benefit with relatively smaller cohort of cell lines
• Available literature suggests that such HLA homozygous cell lines could be derived by Parthenogenesis
• pES cell lines are pluripotent and have no greater safety risks compared to conventional hES cell lines.
• Such cell lines with uniparental disomy have unique advantage during transplantation since they are homozygous and can be transplanted into both homozygous and heterozygous recipients without rejection
Survival among HLA-A,B and DRB1 allele matched pairs by number of mismatched class I loci
Years after Transplant
1 Mismatch (n=317)
0 Mismatches (n=791)
2 Mismatches (n=117)
Flomenberg et al, Blood 2004
Molecular matching is critical for unrelated HSCT program
Allele level matching is associated with lower risks of graft failure, GVHD and transplant related mortality (TRM)
Clinical effects of multilocus mismatching are additive : Quantitative effects
Sequence matching to define ‘residues’ that are well tolerated without GVHD and TRM: Qualitative effects
Unrelated HSCT: lessonsUnrelated HSCT: lessonsHLA Matching StrategiesHLA Matching Strategies
NKM / AIIMS