Prof. Narinder K Mehra Prof. Narinder K Mehra Dept of Transplant Immunology & ImmunogeneticsDept of Transplant Immunology & Immunogenetics

AIIMS, New Delhi AIIMS, New Delhi narin98@hotmail.comnarin98@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

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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