Page 1807 - Hematology_ Basic Principles and Practice ( PDFDrive )

Chapter 105 Unrelated Donor Hematopoietic Cell Transplantation 1611

serologic to DNA-based methods, development of dictionaries of TABLE
HLA alleles and antigen equivalents has become a necessity because 105.5 Vector of Mismatch
many donors in the registries have been typed only by serologic
assays. Interpretation and use of molecular typing data for donor Examples
search and selection has required the development of informatics Vector Definition Donor Recipient
programs. a
Low-resolution DNA-based typing methods can define groups of HVG Presence of DRB1*01:01,04:01 b DRB1*01:01,04:10
alleles that are serologic equivalent (e.g., HLA-A*02 is DNA-defined donor alleles DRB1*01:01,04:01 DRB1*01:01,01:01
and is equivalent to HLA-A2 that is serologically-defined). not present in
Intermediate-resolution DNA typing methods provide additional the recipient
information but not to the level of the complete DNA sequence that GVH Presence of DRB1*01:01,04:01 a DRB1*01:01,04:10
distinguishes one allele from another (e.g., the information is suffi- recipient DRB1*01:01,01:01 b DRB1*01:01,04:10
cient to delineate one group of alleles that include HLA-A*02:01 and alleles not
another that include HLA*02:05 but cannot definitely assign the present in the
allele). High-resolution typing defines the unique DNA sequence of donor
an allele (e.g., HLA-A*02:01). The term “6/6” matched refers to a These combinations contain bidirectional (both HVG and GVHD) mismatch
recipients and donors who share the same low-resolution–defined vectors.
HLA-A, HLA-B, and HLA-DR genes. The term “8/8” refers to b Unidirectional mismatches.
high-resolution matching at the four loci HLA-A, HLA-B, HLA-C, GVH, Graft-versus-host; HVG, host-versus-graft.
and HLA-DRB1. When HLA-DQB1 is added, “10/10” refers to
high-resolution matching at the five loci. When HLA-DPB1 is
added, “12/12” refers to donor−recipient pairs that are allele matched higher rates of graft failure than heterozygous patients. These clinical
at all six genetic loci. observations led to the establishment of the “vector of HLA incom-
Several PCR-based HLA typing approaches are widely used by patibility” in allogeneic transplantation: whereas the presence of
clinical tissue typing laboratories in support of unrelated HCT pro- donor alleles not shared by the recipient determines HVG allorecog-
grams. The sequence-specific primer method uses a panel of primers nition, the presence of recipient alleles not shared by the donor
to amplify the HLA locus or alleles. The PCR products are electro- provides the immunologic basis for GVH allorecognition (Table
phoresed on a gel, and assignment of an HLA type is made by 105.5). “Bidirectional” mismatching refers to the situation in which
examining the composite pattern of positive and negative PCR reac- both HVG and GVH vectors are present at a given HLA locus.
tion methods. “Unidirectional” mismatching describes the situation in which either
The sequence-specific oligonucleotide probe hybridization the donor or the recipient is homozygous for the same allele at the
(SSOPH) method uses a solid phase support to immobilize PCR- mismatched locus. A unidirectional GVH vector mismatch occurs
amplified products. Nonradioactive-labeled oligonucleotide probes when the donor is homozygous and the recipient is heterozygous and
are allowed to hybridize to the support. Whereas probes with shares one allele with the donor (e.g., patient DRB1*01:01, *04:10
sequences complementary to the target DNA will hybridize, probes versus donor DRB1*01:01, *01:01). A unidirectional HVG vector
with as few as one nucleotide difference will fail to hybridize. Alter- mismatch occurs when the patient is homozygous and the donor is
natively, SSOPH methods can use probes that are immobilized to the heterozygous and shares one allele with the patient (e.g., patient
solid phase support and allow PCR-amplified target DNA to hybrid- DRB1*01:01, *01:01 versus donor DRB1*01:01, *04:01).
ize to the support. Clinical outcomes analyses that evaluate the association between
A variation of the SSOPH method is oligonucleotide array tech- HLA disparity and risk of graft failure or GVHD should specify the
nology. Arrays can simultaneously query multiple regions of polymor- vector of incompatibility that is used to define the comparison
phisms in many HLA genes. Oligonucleotide probes can be designed groups. In a recent evaluation of unrelated donor transplants per-
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to all four potential nucleotides, thereby enabling detection of new formed through the NMDP and CIBMTR, unidirectional GVH
sequence polymorphisms with the same sensitivity and specificity as vector mismatching among patients and HLA 7/8–matched donors
sequencing-based typing. Redundancy of probe sequences allows was associated with similar risk as bidirectional HLA mismatches
combinations of alleles to be distinguished in heterozygous individu- among HLA 7/8 transplant pairs. Patients who were homozygous at
als. Commercial platforms are now available and provide quality- an HLA locus, receiving a transplant from a donor heterozygous at
controlled reagents for high throughput genotyping. 17,18 that locus (HLA 7/8 HVG vector mismatch) had lower risk of
In addition to probe-based assays, Sanger sequencing of HLA GVHD than patients receiving an HLA 7/8 transplant involving a
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genes has been an established method for high-resolution typing. GVH vector mismatch. These observations are consistent with the
Newer sequencing approaches include “next generation sequencing” early haploidentical transplant experience and demonstrate the
(NGS) platforms that provide not only high resolution of HLA alleles importance of the HLA vector of incompatibility in defining risks of
but also have the advantage of short-range phasing of exons 2, 3, and GVHD and graft failure.
4 of class I genes, and of exons 2 and 3 of class II genes. 20,21 The
capability of linking sequences across exons substantially reduces the
number of theoretical ambiguities of allele combinations. NGS ASSESSMENT OF HUMAN LEUKOCYTE ANTIGEN
approaches for HLA typing are supported by software for automated HAPLOTYPES
22
assignment of HLA alleles. Because many samples may be tested
simultaneously, NGS is a cost-effective typing method for typing Patients who are candidates for allogeneic transplantation undergo a
donors recruited into registries. 23,24 pedigree analysis to determine the availability of potential HLA
genotypically identical siblings who could serve as a donor (Box
105.2). The family study, which includes typing of the propositus’
ASSESSMENT OF THE VECTOR OF MISMATCHING mother, father, and all full siblings, provides an internal verification
of the patient’s HLA haplotypes. Because HLA genes segregate in
The “vector” or “direction” of HLA compatibility between a donor classic Mendelian fashion, the probability that a sibling inherits the
and a recipient has biologic relevance in defining the risks of graft same parental haplotypes is 25% (genotypically identical). The prob-
failure and GVHD. The concept of the vector was first demonstrated ability that a sibling inherits one identical paternal or maternal
in cases of haploidentical related mismatched transplantation and haplotype plus one nonshared haplotype is 50% (haploidentical).
25
defines HVG and GVH alloreactivity. Patients homozygous for the The probability of inheriting neither of the same haplotypes is 25%
mismatched HLA locus had lower rates of severe acute GVHD but (complete mismatch).