The role of HLA in AllogeneicHematopoietic Stem Cell … · The role of HLA in AllogeneicHematopoietic Stem Cell Trans plantation and Platelet ... Polymorphism of the Major Histocompatibility

The role of HLA in Allogeneic Hematopoietic Stem Cell Transplantation and Platelet p

Refractoriness.

Robert Liwski, MD, PhD, FRCPCMedical Director

HLA Typing Laboratory

Department of Pathology

Dalhousie University

DALHOUSIE UNIVERSITYInspiring minds

IntroductionIntroduction

• Hematopoietic stem cell transplant (HSCT) is aHematopoietic stem cell transplant (HSCT) is a commonly used treatment for hematologic malignancies or bone marrow failuremalignancies or bone marrow failure syndromes/immunodeficiency

Indications for Allogeneic HSCTIndications for Allogeneic HSCT

– Chronic myeloid leukemia (CML)Chronic myeloid leukemia (CML)– Acute myeloid leukemia (AML)– Acute lymphoblastic leukemia (ALL)Plasma cell myeloma– Plasma cell myeloma

– Aplastic anemia– Congenital bone marrow failure syndromes– Congenital immunodeficiency (SCID)– Sickle cell anemia– ThalassemiaThalassemia– Selected solid tumors

Allogeneic HSCTAllogeneic HSCT

• Conditioning with high dose chemotherapy and g g pyradiation therapy – lethal

• Transplant with stem cell from healthy allogeneic donor.

PBSC mobili ed with G CSF– PBSC mobilized with G‐CSF – BM– Umbilical cord

HematopoiesisHematopoiesis

Allogeneic HSCTAllogeneic HSCT

• Transplant of the immune system.

• Immunosuppression necessary to prevent/minimize Host vs Graft (HVG) and Graft vs Host (GVH) Disease.

• May get cure due to Graft vs Leukemia (GVL) effect.

HVG, GVH,GVLHVG, GVH,GVL

• T cell mediated reactions against the graft (stemT cell mediated reactions against the graft (stem cells), host tissue, or leukemia (malignancy).

• Due to T cell mediated recognition of either major or minor histocompatibility differencesmajor or minor histocompatibility differences (MHC, and mHC) between the donor an recipient.

• In humans MHC molecules are named Human• In humans MHC molecules are named Human Leukocyte Antigen (HLA).

HLA class I and class II antigens

• Monomer with non‐ • Heterodimercovalently associated subunit (β2m)

• Presents antigenic

• Presents antigenic peptides to CD4+ T cellsese ts a t ge c

peptides to CD8+ T cells

E d b ll

ce s

• Restricted expression on antigen presenting ll (d d iti ll B• Expressed by all

nucleated cells including endothelium

cells (dendritic cells, B cells, macrophages)

• Inducible on other cells (endothelium and epithelium)

Human Leukocyte Antigen (HLA)

Menu FB


ACBDRDQDP

Class IClass II

αβ1α1β1α1β1 β3,4,5maternal

ACBDRDQDP paternal

HLA inheritanceACB

DRDQ

Mother Father

Sib 1 Sib 2 Sib 3 Sib 4

HLA inheritanceACB 4 haplotypes

DRDQ

Mother Father


HLA inheritanceACB 4 haplotypes

DRDQ

Mother Father


Approximately 25‐30% chance of having an HLA matched sibling

Polymorphism of the Major Histocompatibility Complex in humans HLAin humans ‐ HLA

89356914313 81435 10628 136 89356933 835 068 36

68143111652 63726 7716 118

Polymorphism of the Major Histocompatibility Complex in humans HLAin humans ‐ HLA

89356914313 81435 10628 136 89356933 835 068 36

68143111652 63726 7716 118

2139181 2612 136 22 Effective(caucasians)

Polymorphic residues on Class I HLA molecules (polymorphisms are concentrated around peptide binding groove)

Top view Side views

HLA-A

β2 i l b liHLA-B β2 microglobulin

HLA-C


Menu FB


Relevance of HLARelevance of HLA

• Necessary to mount immune responses against hpathogens

– Polygenic ‐ survival advantage to individual– Polymorphic ‐ survival advantage to speciesy p g p

• Transplantation– Difficulty finding compatible donorsDifficulty finding compatible donors.– Causes sensitization (T cell response and antibody response)

– Can lead to graft rejection (failure to engraft) orCan lead to graft rejection (failure to engraft) or GVHD

Major mismatch/direct allorecognition

Self T cell Donor T cell

Normal situation HSCT situation

Vi l nti n

TCR

Self HLA

Viral antigen(peptide)

Allo HLA

Foreign viral antigenand self HLA

Self antigenand allo HLA and self HLA and allo HLA

Self body cell Recipient cell

5‐10% T cells able to respond to allo‐MHCPotent reaction

HLA antibody development

Your (“self”) HLA


Your (“self”) HLA “allo” HLA





Sensitizing events:TransfusionPregnancy Transplantation

Studies investigating the impact of HLA mismatch in HSCTmismatch in HSCT

Lee et. al. Blood 2007

Lee et. al. Blood 2007

Donor selection for allo HSCTDonor selection for allo HSCT

• HLA inheritance pattern

• Extreme HLA polymorphism

Probability of finding HLA‐Identical DonorProbability of finding HLA Identical Donor

• Sibling 1:4 (30% on average)Sibling 1:4 (30% on average)

• Parent 1:100

C i l 0 000• Cousin, uncle, etc 1:10,000

• Unrelated 1:1,000,000

• Worldwide Registry of Bone Marrow DonorsWorldwide Registry of Bone Marrow Donors

• >10,000,000 donors

Patient workupPatient workup

New patientHLA TypingA, B, C, DR, DQ, , , , Q

Siblings and/or related donors?

Yes NoHLA TypingA, B, C, DR, DQ

70%

Match No match

70%

Unrelated donor searchHLA TypingA, B, C, DR, DQ

30%

Matched donor Mismatched donor

50% 20%

CasesCases

Family study 1Family study 1

Patient

Family study 1A 02 24B 07 62C 07 9C 07 9

DRB1 13 04

DQB1 06 8

Patient brother 1 brother 2 brother 3 sister

Family study 1A 02 24 02 24 01 02 02 01 02 24B 07 62 50 62 08 50 07 08 07 62C 07 9 06 9 07 06 07 07 07 9C 07 9 06 9 07 06 07 07 07 9

DRB1 13 04 17 04 17 17 13 17 13 04

DQB1 06 8 02 8 02 02 06 02 06 8



DRB1 13 04 17 04 17 17 13 17 13 04

DQB1 06 8 02 8 02 02 06 02 06 8

Brother 1 shares 1 haplotype



DRB1 13 04 17 04 17 17 13 17 13 04

DQB1 06 8 02 8 02 02 06 02 06 8

Brother 1 h 1 h l tshares 1 haplotype



DRB1 13 04 17 04 17 17 13 17 13 04

DQB1 06 8 02 8 02 02 06 02 06 8

Brother 1 h 1 h l t

Brother 2 I t t hshares 1 haplotype Is not a match



DRB1 13 04 17 04 17 17 13 17 13 04

DQB1 06 8 02 8 02 02 06 02 06 8

Brother 1 h 1 h l t

Brother 2 I t t h

Brother 3 h 1 h l tshares 1 haplotype Is not a match shares 1 haplotype



DRB1 13 04 17 04 17 17 13 17 13 04

DQB1 06 8 02 8 02 02 06 02 06 8

Brother 1 h 1 h l t

Brother 2 I t t h

Brother 3 h 1 h l t

Sisteri 10/10 t hshares 1 haplotype Is not a match shares 1 haplotype is a 10/10 match

patient brother 1 brother 2 brother 3 sister

Family study 1p

A 02 24 02 24 01 02 02 01 02 24B 07 62 50 62 08 50 07 08 07 62C 07 9 06 9 07 06 07 07 07 9C 07 9 06 9 07 06 07 07 07 9

DRB1 13 04 17 04 17 17 13 17 13 04

DQB1 06 8 02 8 02 02 06 02 06 8

Parent 1 Parent 2

A

BB

C

DRB1DRB1

DQB1


Family study 1p

A 02 24 02 24 01 02 02 01 02 24B 07 62 50 62 08 50 07 08 07 62C 07 9 06 9 07 06 07 07 07 9C 07 9 06 9 07 06 07 07 07 9

DRB1 13 04 17 04 17 17 13 17 13 04

DQB1 06 8 02 8 02 02 06 02 06 8

Parent 1 Parent 2

A 02 24B 07 62B 07 62C 07 9

DRB1 13 04DRB1 13 04

DQB1 06 8


Family study 1p

A 02 24 02 24 01 02 02 01 02 24B 07 62 50 62 08 50 07 08 07 62C 07 9 06 9 07 06 07 07 07 9C 07 9 06 9 07 06 07 07 07 9

DRB1 13 04 17 04 17 17 13 17 13 04

DQB1 06 8 02 8 02 02 06 02 06 8

Parent 1 Parent 2

A 02 02 24B 07 50 62B 07 50 62C 07 06 9

DRB1 13 17 04DRB1 13 17 04

DQB1 06 02 8


Family study 1p

A 02 24 02 24 01 02 02 01 02 24B 07 62 50 62 08 50 07 08 07 62C 07 9 06 9 07 06 07 07 07 9C 07 9 06 9 07 06 07 07 07 9

DRB1 13 04 17 04 17 17 13 17 13 04

DQB1 06 8 02 8 02 02 06 02 06 8

Parent 1 Parent 2

A 02 02 24 01B 07 50 62 08B 07 50 62 08C 07 06 9 07

DRB1 13 17 04 17DRB1 13 17 04 17

DQB1 06 02 8 02

Family Study 2Family Study 2

Family study 2

PatientA 02 01B 27 08C 02 07DRB1 04 03(17)DQB1 03(8) 02DQB1 03(8) 02

Family study 2

Brother Patient02 02 A 02 0127 44 B 27 0802 05 C 02 0704 04 DRB1 04 03(17)03(8) 03(7) DQB1 03(8) 0203(8) 03(7) DQB1 03(8) 02

Family study 2

Father MotherA 02 01 01 02B 27 08 08 44C 02 07 07 05DRB1 04 03(17) 03(17) 04DQB1 03(8) 02 02 03(7)


Family study 2

Father MotherA 02 01 01 02B 27 08 08 44C 02 07 07 05DRB1 04 03(17) 03(17) 04DQB1 03(8) 02 02 03(7)

01


Family study 2

Father MotherA 02 01 01 02Identical haplotypes

B 27 08 08 44C 02 07 07 05DRB1 04 03(17) 03(17) 04DQB1 03(8) 02 02 03(7)

01


Family study 2

Father MotherA 02 01 01 02Identical haplotypes

B 27 08 08 44C 02 07 07 05DRB1 04 03(17) 03(17) 04DQB1 03(8) 02 02 03(7)

01


Father and son are a 10/10 HLA match!

Family study 3Family study 3

Family study 3Patient sister 1 sister 2 sister 3

A 02 68 01 11 01 68 02 11B 44 14(65) 08 35 08 14(65) 44 35C 05 08 07 04 07 08 05 04C 05 08 07 04 07 08 05 04DRB1 04 13 03(17) 14 03(17) 13 04 14DQB1 03(7) 03(7) 02 05 02 03(7) 03(7) 05

Predicted parental typing

P t 1 P t 2Parent 1 Parent 2A 02 01 68 11B 44 08 14(65) 35C 05 07 08 04DRB1 04 03(17) 13 14DQB1 03(7) 02 03(7) 05


A 02 68 01 11 01 68 02 11B 44 14(65) 08 35 08 14(65) 44 35C 05 08 07 04 07 08 05 04C 05 08 07 04 07 08 05 04DRB1 04 13 03(17) 14 03(17) 13 04 14DQB1 03(7) 03(7) 02 05 02 03(7) 03(7) 05

Predicted parental typing

P t 1 P t 2Parent 1 Parent 2A 02 01 68 11B 44 08 14(65) 35C 05 07 08 04DRB1 04 03(17) 13 14DQB1 03(7) 02 03(7) 05

No MatchDouble cousins in the family!

GP 1 GP 2 GP 3 GP 4

Sister Parent 1 Parent 2 Brother¼ chance HLA ID

¼ chance HLA ID

Patient Sister 1 Sister 2 Sister 3

HLA ID HLA ID

Patient Sister 1 Sister 2 Sister 3

¼ x ¼ = 1/16 (6.25%) chance of presence of all 4 haplotypes

Cousin 1 Cousin 2Double cousins

¼ X 1/16 = 1/64 (1.5%) chance of inheriting the same haplotypes as the patient


A 02 68 01 11 01 68 02 11B 44 14(65) 08 35 08 14(65) 44 35C 05 08 07 04 07 08 05 04C 05 08 07 04 07 08 05 04DRB1 04 13 03(17) 14 03(17) 13 04 14DQB1 03(7) 03(7) 02 05 02 03(7) 03(7) 05

Double cousins

Cousin 1 Cousin 2A 02 68 02 68B 44 14(65) 44 14(65)C 05 08 05 08C 05 08 05 08DRB1 04 13 04 13DQB1 03(7) 03(7) 03(7) 03(7)

Both double cousins were matches with the patient!

Platelet Transfusion fRefractoriness

• Definition“a post-transfusion platelet count that is less than expected” 1h corrected count increment (CCI)

9< 5 – 10 x 109/Lpercent platelet recovery (PPR) < 20%1h CCI < 5 x 109/L x2 (twice) using ABO-identical platelets

56


• Incidence7-34% in hematology/oncology patients depending on a study and definition of refractoriness (Hod and Schwartz, 2008).

57

Platelet Transfusion fRefractoriness - Etiology

• Non-immune causesfeversepsissplenomegalyDICDICbleedingVODGVHDGVHD

58


• Immune causesImmune causes

Anti-ABO antibodies

Anti-HPA antibodies (2-11%, may be auto and/or transient)and/or transient)

Anti-HLA antibodies (Class I HLA only, A and B)Anti HLA antibodies (Class I HLA only, A and B) account for the majority of cases of immune causes.


causes of alloimmunization: transfusion (contaminating leukocytes)- transfusion (contaminating leukocytes)

- pregnancy - transplantation- transplantation

60

TRAP Study, Schlichter et. al. 1997TRAP Study, Schlichter et. al. 1997

Leukocyte reduction and ultraviolet B irradiation of platelets to prevent alloimmunization and refractoriness to platelet transfusionsprevent alloimmunization and refractoriness to platelet transfusions. N Engl J Med 1997; 337:1861-9


Proportion of cases due to immune vs non immune etiologies is not clearnon-immune etiologies is not clear.

Incidence varies depending on a study and HLAIncidence varies depending on a study and HLA antibody detection methods.

Most studies performed in the 1980s and 1990s utilizingMost studies performed in the 1980s and 1990s utilizing older and less reliable HLA antibody detection techniques.

62

HLA antibody identification by Luminex (solid phase) Assay(solid phase) Assay

HLA antigen coated beads

Tells the instrument which bead is being examined

2 lasers

Tells the instrument how much antibody is bound to the bead

HLA antibody detection by Luminex assay

1 2 3 4 8 109765


1 2 3 4 8 109765

A1 A2 A3 A11 A23 A24 A25 A26 A29 A30


9

A29

78

A26

1810

A29

A30

4

A11 5

6

A24

A25

910

A301

3 65

A1A26A30

2

A2

1

A1

5

A23

A24A29

A30

24

7

6

A3

A11

A23 A24

A25

A2

3

A3A2

A11A3


9

A29

78

A26

1810

A29

A30

4

A11 5

6

A25A26

910

A301

3 65

A1A26A30

2

A2

1

A1

5

A23

A24A29

A30

24

7

6

A3

A11

A23 A24

A25

A2

3

A3A2

A11A3


9

A29

78

A26

1810

A29

A30

4

A11 5

6

A25A26

910

1

3 65

A1A26A30

2

A2

1

A1

5

A23

A24A29

A30

2

3

47

65

A3

A11

A23 A24

A25

A2

3

A3A2

A11A3


9

A29

78

A26

1810

A29

A30

4

A11 5

6

A24

A25A26

910

A301

3 65

A1A26A30

2

A2

1

A1

5

A23

A24A29

A30

24

7

6

A3

A11

A23 A24

A25

A2

3

A3A2

A11A3


PE-α-IgG

9

A29

78

A26

1810

A29

A30

4

A11 5

6

A24

A25A26

910

A301

3 65

A1A26A30

2

A2

1

A1

5

A23

A24A29

A30

24

7

6

A3

A11

A23 A24

A25

A2

3

A3A2

A11A3


9

A29

78

A26

1810

A29

A30

4

A11 5

6

A24

A25

910

A301

3 65

A1A26A30

2

A2

1

A1

5

A23

A24A29

A30

24

7

6

A3

A11

A23 A24

A25

A2

3

A3A2

A11A3

HLA Class I antibody analysis

Patient A3 31 B7 60 DR1 14 (52) DQB5 6Patient A3,31 B7,60 DR1,14 (52) DQB5,6


Patient A3 31 B7 60 DR1 14 (52) DQB5 6Patient A3,31 B7,60 DR1,14 (52) DQB5,6Donor A1, B8 DR7,17 (53,52) DQB2



HLA Class II antibody analysis


HLA Class II antibody analysis


CPRA Calculator

http://optn.transplant.hrsa.gov/ Resources, professional resources, choose cPRA calculator from options

CDHA PolicyCDHA Policy• platelet count < 10 at 24 hours on two occasions following buffy coat platelet pool ‐ give single donorfollowing buffy coat platelet pool ‐ give single donor apheresis platelets (ABO‐matched)

• platelet count < 10 at 24 hours on two occasions following single donor apheresis platelets (ABO‐o o g s g e do o ap e es s p ate ets ( Omatched) ‐ give HLA‐matched platelets

• platelet count < 10 at 24 hours on two occasions following single donor apheresis platelets (HLA‐

79

matched) ‐ give HPA‐matched platelets

HLA Alloimmunization Rate In The Setting OfHLA Alloimmunization Rate In The Setting Of Refractoriness To Platelet Transfusion: A

Single Center StudySingle Center Study

Sharpe C, Liwski R, Couban S, Sadek I, Kahwash E

Patients/MethodsPatients/Methods• 33 platelet refractory patients identified at the QEII hospital

between 01/06 and 06/11between 01/06 and 06/11.

– 18 male

– 15 female

– 28 diagnosed with a hematological malignancy

• platelet refractoriness: platelet count rise of <10X109/L within 24 hours post‐tx of ABO‐matched single‐donor platelets on at least 2 occasions.

• HLA antibody analysis was performed by single antigen• HLA antibody analysis was performed by single antigen luminex assay at the CBS laboratory in Winnipeg.

• PRA was calculated using OPTN cPRA calculator

Results• HLA antibodies were found in 42% of patients

– 4/18 males (22%)4/18 males (22%)

– 10/15 females (67%)

• Degree of sensitization– males were mildly sensitized with an average PRA of 33% y g(range 11‐62%)

– females were highly sensitized (PRA values 95‐100%)

• No association between the presence of HLA tib di d ith ti t di i hi tantibodies and either patient diagnosis or a history

of allogeneic stem cell transplantation.

Conclusions/Implications• We found that in most patients (60%) platelet refractoriness

was due to non‐immunological causes.

• Approximately 2/3 of sensitized patients (all females) were hi hl iti d (PRA 95 100%) d ld i HLAhighly sensitized (PRA = 95‐100%) and would require HLA matched platelets.

• Approximately 1/3 of the sensitized patients (all males) had low PRA values and could probably be managed with using ti ti l t l tantigen negative platelets.

Suspect Alloimmune refractoriness

ABO identical platelets

Measure increment x 2Not refractory, supportWith standard platelets

Define antibodyMeasure PRA Crossmatchrandom platelets unitp

HLA ab present HLA absent< 20% > 20%

HLA matched or antigen negative

Consider and treat non immune causesManage bleeding as needed

Modified from Hod and Schwartz, 2008

Suspect alloimmune platelet refractoriness

ABO identical platelets

Measure increment x 2Not refractory, supportWith standard platelets

Define antibodyMeasure PRA Crossmatchrandom platelets unitp

HLA ab present HLA absent< 20% > 20%

HLA matched or antigen negative

Consider and treat non immune causesManage bleeding as needed

Modified from Hod and Schwartz, 2008

Thank youThank you

The role of HLA in AllogeneicHematopoietic Stem Cell … · The role of HLA in AllogeneicHematopoietic Stem Cell Trans plantation and Platelet ... Polymorphism of the Major Histocompatibility

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