The Significance of the Direction of the HLA Mismatch in Cord Blood Matching and the Implication of...

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The Significance of the Direction of the HLA Mismatch in Cord Blood Matching and the Implication of Graft-

Specific Anti-HLA Antibodies

Marcelo A. Fernández Viña, Ph.D. Department of Pathology

Medical School Stanford University

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Goals of Allogeneic HSCT

• Achieve Engraftment • Absence of Graft versus Host Disease • Prevent Relapse • Effective Immune reconstitution

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Search for Allogeneic HSC

Nuclear family Extended family

CBU and Unrelated Donors

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Factors Influencing Decision to Transplant

Quality of HLA match Recipient factors

Diagnosis and stage of disease Age Major organ function CMV serology

Donor/CBU factors Age – Period of collection Body size - Cell Dose CMV serology – Infectious Disease Markers Pregnancy and transfusion history

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Patient and Donor/CBU are Heterozygous in all HLA loci Difference (mismatch) in One allele of HLA-A

• The HLA-A mismatched antigen in the Donor/CBU can be recognized as foreign by the Patient’s Immune System (Host versus Graft; rejection)

• The HLA-A mismatched antigen in the Patient can be recognized as foreign by

the Donor/CBU’s Immune System (Graft versus Host; GvHD and GvL)

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Molecules encoded in the HLA systemMolecules encoded in the HLA system

Maternal origin

PATIENT

Encoded in HLA-DRB1 locus

(Class II)

Encoded in HLA-A locus

(Class I)

Paternal origin

Encoded in HLA-B locus

(Class I)

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Patient is Homozygous in HLA-A and Donor/CBU is Heterozygous in HLA-A

Mismatch Only in the HvG direction

• The HLA-A mismatched antigen in the Donor/CBU can be recognized as foreign by the Patient’s Immune System (Host versus Graft; rejection)

• No mismatch in the Graft versus host direction (No Graft versus Host; No

GvHD/ No GvL)

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Maternal origin

DONOR/CBU


(Class II)


(Class I)

Paternal origin


(Class I)

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Patient is Heterozygous in HLA-A, Donor/CBU is Homozygous in HLA-A for one allele present in the Patient

Mismatch Only in the HvG direction

• The HLA-A mismatched antigen in the Patient can be recognized as foreign by the Donor/CBU ’s Immune System (Graft versus Host; GvHD and GvL)

• No mismatch int Host versus Graft direction

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Maternal origin

PATIENT


(Class II)


(Class I)

Paternal origin


(Class I)

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“HLA mismatch direction in cord blood transplantation: impact on outcome and implications for cord blood unit selection”

Stevens CE, Carrier C, Carpenter C, Sung D, Scaradavou A

Blood. 2011 Oct 6;118(14):3969-78. • 1202 single CBU TX • 1993-2006 (57% before 2000) • Engraftment at day 77 • 75 % <16 y/o • 67% Malignancies • Early (23%), Intermediate

(46%), Advanced 30%) • 6/6 – 6% • 5/6 – 38% • 364 1 bidirectional MM • 58 GvH-only MM • 40 HvG-Only MM

• 76% Neutrophil engraftment • 51 % Platelet engraftment • Match grade (p<0.001) and cell

dose (p<0.006) changed over time

• Engraftment Faster in the GvH-only (HR = 1.6, P = .003)

• GvH-only had Less TRM, Mortality, Treatment Failure in patients with Maligancy

• Comparable to Matched transplants

Cumulative probability of achieving ANC 500 during the first 77 days after transplantation.

Stevens C E et al. Blood 2011;118:3969-3978

©2011 by American Society of Hematology

Cumulative probability of TRM during the first 3 years after transplantation.

Stevens C E et al. Blood 2011;118:3969-3978


Patients with malignancies

HLA-matched or GVH-only mismatched CB grafts

Patients with other diseases

1 bidirectional mismatch

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• “Evaluation of HLA matching in unrelated hematopoietic stem cell transplantation for nonmalignant disorders”

• John Horan, Tao Wang, Michael Haagenson, Stephen R. Spellman, Jason Dehn, Mary Eapen, Haydar Frangoul, Vikas Gupta, Gregory A. Hale, Carolyn K. Hurley, Susana Marino, Machteld Oudshoorn, Vijay Reddy, Peter Shaw, Stephanie J. Lee and Ann Woolfrey

• Blood. 2012 Oct 4;120(14):2918-24.

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BMT in Non-Malignant Diseases CBU

• Similar to HSCT in non-Malignant Diseases, absence of Engraftment may have played a major role in causing morbidity and mortality

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Goals of Allogeneic BMT

Achieve Engraftment: • Patient’s immune system may cause

rejection • Donor’s immune system may enhance the

engraftment through the destruction of patient’s immune cells that cause rejection

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Histocompatibility Factors Affecting Engraftment

• HLA-mismatches in the Host versus graft direction (HvG mismatch)

• Patient’s Homozygosity (HvG mismatch) • Donor-specific anti-HLA Antibodies • ABO Major Mismatch

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Homozygosity at a given locus vs Heterozygosity

Major-Histocompatibility-Complex class I alleles and antigens in hematopoietic-cell transplantation.

Petersdorf EW, Hansen JA, Martin PJ, Woolfrey A, Malkki M, Gooley T, Storer B, Mickelson E, Smith A, Anasetti C.

N Engl J Med. 2001 Dec 20;345(25):1794-800

• CML, No ATG

• RESULTS: A single HLA allele mismatch did not increase the risk of graft failure, whereas a single antigen mismatch significantly increased the risk

• The risk was also increased if the recipient was HLA homozygous at the mismatched class I locus or if the donor had two or more class I mismatches

• CONCLUSIONS: HLA class I antigen mismatches that are serologically detectable confer an enhanced risk of graft failure after hematopoietic-cell transplantation. Transplants from donors with a single class I allele mismatch that is not serologically detectable may be used without an increased risk of graft failure

Impact of High Resolution Mismatches (Allele Level) & Broad Mismatches (Serologically Detectable) in Alleles of HLA Loci

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The impact of HLA unidirectional mismatches on the outcome of myeloablative hematopoietic stem

cell transplantation with unrelated donors

• Hurley CK, Woolfrey A, Wang T, Haagenson M, Umejiego J, Aljurf M, Askar M, Battiwalla M, Dehn J, Horan J, Oudshoorn M, Pidala J, Saber W, Turner V, Lee SJ, Spellman S

• Blood. 2013 May 1. [Epub ahead of print] PubMed PMID: 23637130

• 2,687 myeloablative MUD; malignant disease • 7/8 bidirectional MM transplants, n=1393 • 7/8 host vs. graft (HVG) vector MM , n=112 • 7/8 graft vs. host (GVH) vector MM, n=119 • 8/8 matches, n=1063

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HvG and GvH mismatches (Conclusions)

• The 7/8 groups differed only for grades 3-4 acute GVHD where HVG MM had less GVHD than the 7/8 bidirectional MM (HR 0.52, p=0.0016) and GVH MM (HR 0.43, p=0.0009) but not the 8/8 group (HR 0.83, p=0.39)

• There were no differences between the 7/8 groups for relapse, chronic GVHD, neutrophil engraftment or graft failure

• Unidirectional GVH vector mismatches have the same risk as 7/8 bidirectional mismatches

• Recipients with a 7/8 HVG MM have a reduced risk of acute GVHD without an increased risk of disease relapse or graft failure compared to a 7/8 bi-directional MM at a heterozygous locus

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Causes of Death

• The 7/8 bi-directional and GVH MM groups had slightly higher deaths attributed to GVHD, compared to the 8/8 and 7/8 HVG MM groups, 18.2% and 18.0% vs. 14.9% and 14.3%, respectively

• The 7/8 HVG MM group had more deaths attributed to graft rejection than the 8/8, 7/8 GVH MM and 7/8 bidirectional groups, 9.5% vs. 0.6%, 1.1% and 1.6%, respectively

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Differences in the number of Mismatched Epitopes when the Patient is Homozygous or Heterozygous at the Mismatched locus

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• Patients homozygous at a given locus when presenting humoral sensitization tend to make anti-HLA antibodies reactive with many (in some instances all but self antigens) HLA antigens of the same locus

• Novel anti-HLA antibody screening techniques allow to precisely identify anti-HLA antibodies defining unacceptable or high risk mismatches. Therefore, donors can be excluded or given lower priority on the basis of their HLA mismatch and the patient’s antibody reactivity.

• Current conditioning/immunosupression (ATG) this risk for primary graft loss mediated by T-cells may be greatly reduced

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• Current conditioning/immunosupression (ATG) this risk for primary graft loss mediated by T-cells may be greatly reduced

DSA in TCD Haploidentical Stem Cell Transplantation Ciurea SO, de Lima M, Cano P, Korbling M, Giralt S, Shpall EJ,

Wang X, Thall PF, Champlin RE, Fernandez-Vina M

DSA were detected in 5 of 24 consecutive patients (21%)

4/24 patients had primary graft failure (PGF) 3/4 (75%) patients with DSA failed to engraft as

compared with 1/20 (5%) without DSA (P=0.008)

DSA\Engraftment YES NO

YES 1 3

NO 19 1

“High risk of graft failure in patients with anti-HLA antibodies undergoing haploidentical stem-cell transplantation”

Transplantation. 2009 Oct 27;88(8):1019-24

Recently published work: Anti-HLA Antibodies and Virtual Cross Match in MUDs

Retrospective study - DSA found in 9/37 MUDT patients with

graft failure 1/78 had DSA in the matched control group of patients who

engrafted (TCR grafts, 85% mismatched at DP locus)

Spellman S, Bray R, Rosen-Bronson S, Haagenson M, Klein J, Flesch S, Vierra-Green C, Anasetti C. The detection of donor-directed, HLA-specific alloantibodies in recipients of unrelated hematopoietic cell transplantation is predictive of graft failure. Blood. 2010 Apr 1;115(13):2704-8

PGF Control

DSA 9/37 (24%) 1/78 (1%)

Class I +/- Anti-DP 5 1

Anti-DP alone 4 0

DSA and PGF

RR = 24.8 – 28.1

Ciurea SO, Thall PF, Wang X, Wang SA, Hu Y, Cano P, Aung F, Rondon G, Molldrem JJ, Korbling M, Shpall EJ, de Lima M, Champlin RE, Fernandez-Vina M. Donor-specific anti-HLA Abs and graft failure in matched unrelated donor hematopoietic stem cell transplantation. Blood. 2011 Nov 24;118(22):5957-64

592 MUD TX 75 % of the 8/8 transplants present at least one mismatch in DP, DQ, DRB3/4/5 in the HvG vector 20 % of HSC patients are immunized against HLA HLA Immunization in FEMALE HSCT patients: 30% HLA Immunization in MALE HSCT patients: 10% 3.3 % present antibodies against HLA-DP (1/2 MUD TX have one or two DP mismatches and DSA anti HLA-DP)

Other Associations: Gender, Number of Pregnancies and anti-HLA antibodies

Variable N Coefficie

nt SE P-value OR 95% CI Intercept -2.71 0.60 <0.0001 - - Male vs. Females 0

pregnancies 356 0.73 0.62 0.24 2.1 0.62 - 6.94

Female: Number of pregnancies = 1 (vs. 0)

37 1.85 0.70 0.008 6.3 1.62 - 24.85

Female: Number of pregnancies ≥ 2 (vs. 0)

152 2.25 0.62 0.0003 9.5 2.83 - 32.02

Significant association between gender and the presence of AHA: 30.8% females vs 12.1% males had anti-HLA antibodies (p<0.0001) 7/8 pts with DSA were females

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Lower Expression of HLA in CBU-CD34

Bone Marrow CD34

Cord Blood CD34

Anti-HLA antibodies in CBU transplantation DSA in Single Unit CBU TX Double CBU and DSA: • DSA against both Units • DSA against one Unit • No DSA

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Evaluation of DSA by a Solid Phase Assay with Single Antigen Beads

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“The impact of anti-HLA antibodies on unrelated cord blood transplantations” Takanashi M, Atsuta Y, Fujiwara K, Kodo H, Kai S, Sato H, Kohsaki M, Azuma H, Tanaka H, Ogawa A, Nakajima K, Kato S

Blood. 2010 Oct 14;116(15):2839-46 • 386 Single CBT; myeloablative, Median age 33 • HLA Immunization: 89 patients (23%); 20 cases with

DSA • Neutrophil recovery @ day +60 : • 83% for the ab-negative • 73% for ab-positive/No-DSA • 32% ab-positive/Yes-DSA • Anti-HLA antibodies should be tested and considered

pre-transplantation in the selection of cord blood

Cumulative incidence of neutrophil/platelet recovery and survival for 386 CBT cases. ab-negative indicates patient does not have anti-HLA antibody; ab-positive, patient has anti-HLA

antibody but the CB does not have the corresponding antigen for the antibod...

Takanashi M et al. Blood 2010;116:2839-2846


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“Donor-specific anti-HLA antibodies predict outcome in double umbilical cord blood transplantation” Cutler C, Kim HT, Sun L, Sese D, Glotzbecker B, Armand P, Koreth J, Ho V, Alyea E, Ballen K, Ritz J, Soiffer RJ, Milford E, Antin JH

Blood. 2011 Dec 15;118(25):6691-7 • 73 Double CBT; myeloablative (27%)or

RIC/ATG (73%); Median age 48 • 18 patients with DSA (11 Single Unit DSA,

7 DSA against both CBU)

Cumulative incidence of graft failure.

Cutler C et al. Blood 2011;118:6691-6697


no DSA

DSA against one unit

DSAs against both units

Cumulative incidence of neutrophil engraftment.



no DSA

DSA against one or both units

Cumulative incidence of early death or relapse.



no DSA

DSA against one unit

DSA against both units

DSA in the Study by Brunstein et al

• Lower cut-off for calling positve anti-HLA antibodies • DSA against 1 Unit: n = 12; 2 graft failures • DSA> 3000 MFI: n = 3; 1/3 had graft failure

• DSA against both Units: n= 6; 1 graft failure • One had DSA> 3000 MFI : n = 4 ; 1/3 had graft failure

Impact of donor specific anti-HLA antibodies on graft failure and survival after reduced intensity conditioning-unrelated cord blood transplantation. A Eurocord, Societe Francophone d'Histocompatibilite et d'Immunogenetique (SFHI) and Societe Francaise de Greffe de Moelle et de Therapie Cellulaire (SFGM-TC) analysis

Boudifa A, Coeffic B, Devys A, De Matteis M, Dubois V, Hanau D, Hau F, Jollet I, Masson D, Pédron B, Perrier P,

Picard C, Ramouneau-Pigot A, Volt F, Charron D, Gluckman E, Loiseau P

Haematologica. 2012 Dec 14. [Epub ahead of print] PubMed

PMID: 23242594

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Anti-HLA antibodies in CBU transplantation

• HvG vector, Serologic Level mismatches (class I) • Sensitivity, specificity and quantitation of antibody detection are greatly

enhanced by novel solid phase assays • Variations in end points and cut-off values • Cross-match testing is not be practical in HSCT

• High risk of graft rejection in patients with D.S. Antibodies • Higher mortality and morbidity associated to DSA • Allo-antibodies directed against both class I and II HLA antigens • Less mismatches in MUD than in CBU • Anti-DP antibodies in MUD (mostly matched at other HLA loci)

• Anti-HLA antibody testing is warranted to all HSCT with known or presumed

HLA mismatches • Further analysis for different graft sources and diseases is warranted

“Complement (C1q) fixing solid-phase screening for HLA antibodies increases the availability of compatible platelet components for refractory patients” Fontaine MJ, Kuo J, Chen G, Galel SA, Miller E, Sequeira F, Viele M, Goodnough LT, Tyan DB. Transfusion. 2011 Dec;51(12):2611-8

• 13 highly sensitized refractory patients received 177 PLT units incompatible by the IgG-SAB method

• The mean CPRA value was significantly lower by C1q-SAB (60%) than by IgG-SAB (94%; p < 0.05)

• Patients showed significantly better corrected count increment with C1q-compatible than with C1q-incompatible PLTs

• Results show that 75% of PLT units previously considered incompatible were actually compatible

• For highly refractory patients to PLT transfusion, the C1q-based SAB binding assay may be a better method for identifying clinically relevant HLA antibodies and selecting PLT units that will result in acceptable CCI

0

5000

10000

15000

20000

25000A

*23:

01A

*80:

01A

*01:

01A

*24:

02A

*36:

01A

*29:

02A

*24:

03A

*66:

02A

*33:

01A

*33:

03A

*29:

01A

*34:

01A

*32:

01A

*11:

02A

*43:

01A

*11:

01A

*03:

01A

*74:

01A

*26:

01A

*34:

02A

*69:

01A

*68:

02A

*25:

01A

*02:

06A

*66:

01A

*02:

01A

*68:

01A

*02:

03A

*30:

01A

*30:

02A

*31:

01

IgG C1Q

Fluo

resc

ence

Inte

nsity

Self HLA-A A*02, A*24, A*25, A*26,A*34, A*68, A*69, A*43

may be acceptable by the C1q Assay

Antibody Tests by

Assays

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Anti-HLA antibodies in CBU transplantation

• Only 16-24 % PGF have DSA • T-cell immunity • Conditioning, Immunosuppressive regimens

(ATG, post TX Cyclophosphamide) • Preservation of patient’s Immune System (Primary

Disease and Stage) • Graft type and variations in HLA expression • Graft Manipulation

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Goals of Allogeneic BMT • Absence of Graft versus Host Disease: GvHD is mediated by donor’s T-cells • Effective Immune reconstitution: T-cells of donor origin may interact with patient’s tissues

and in some instances patient’s APCs • Prevent Relapse: GvL may be mediated by both, T-cells and NK cells of

donor origin; donor’s T cells causing GvHD may also prevent relapse

• GvH: Major factor in Mortality and Morbidity: Once engraftment is achieved; the mismatches in the GvH vector play a major role in transplant outcome




• However, in light of current conditioning/immunosupression (ATG) this risk for primary graft loss mediated by T-cells may be greatly reduced

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Differences in the number of Mismatched Epitopes when the Patient is Homozygous or Heterozygous at the Mismatched locus

Neutrophil Engraftment (absence @ day+28)

Mismatch n RR L-R HR p

8/8 4779 1.00 - - 0.0009

C*03 PMM 134 0.84 0.33 2.16 0.72

C-allele 61 0.41 0.04 4.69 0.47

C-antigen 700 1.66 1.27 2.18 0.0002

A, B or DRB1 959 1.46 1.13 1.89 0.0034

Histocompatibility Factors Affecting Engraftment

• HLA-mismatches in the Host versus graft direction (HvG mismatch)

• Serologic level HLA-class I mismatches • Patient’s Homozygosity (HvG mismatch) • Donor-specific anti-HLA Antibodies • ABO Major Mismatch

Univariate Logistic Regression Model for Graft Failure in MUDT

Variable Parameter Estimate

Parameter Standard Error

Univariate P-value Odds Ratio

Male (vs. female) -0.53 0.47 0.26 0.59

Black (vs. white) 1.17 0.78 0.14 3.22

Others (vs. white) -12.15 316.51 0.97 0.00

AHA= yes (vs. no) 0.91 0.49 0.06 2.48

DSA = yes (vs. no) 3.06 0.77 0.0001 21.33

HEL HvG = 8 (vs. 7) -0.31 0.64 0.62 0.73

LEL HvG = 6 (vs. <6) -0.53 0.64 0.41 0.59

CD34 cell numbers infused

-0.51 0.38 0.18 0.60

CMV mismatch = yes (vs. no)

0.27 0.47 0.56 1.31

Sex mismatch = yes (vs. no)

0.60 0.47 0.21 1.82

ABO mismatch = yes (vs. no)

1.35 0.63 0.03 3.86

AHA – anti HLA antibodies; DSA – donor-specific AHA; HEL – high expression loci; LEL – low expression loci; HvG – host versus graft direction

MVA: DSA (p=0.0001) and ABO mm (p=0.04) remained significantly associated with GF

Cumulative incidence of neutrophil and platelet recovery in subgroups of CD34 cell dose. ab-negative indicates patient does not have anti-HLA antibody; ab-positive, patient has anti-HLA

antibody but the CB does not have the corresponding antigen for the ant...

Takanashi M et al. Blood 2010;116:2839-2846


Progression-free (A) and overall survival (B)



no DSA

no DSA



Anti-HLA antibodies in CBU transplantation DSA in Single Unit CBU TX Double CBU and DSA DSA against both Units DSA against one Unit

Even though the authors do not agree:

Higher mortality and morbidity associated to DSA. Variations in end points and cut-off values

The Significance of the Direction of the HLA Mismatch in Cord Blood Matching and the Implication of...

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