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Blood, Vol. 94 No. 8 (October 15), 1999:
pp. 2911-2914
Correlation Between Disparity for the Minor Histocompatibility Antigen
HA-1 and the Development of Acute Graft-Versus-Host Disease After
Allogeneic Marrow Transplantation
By
Li-Hui Tseng,
Ming-Tseh Lin,
John A. Hansen,
Ted Gooley,
Ji Pei,
Anajane G. Smith,
Emily G. Martin,
Effie W. Petersdorf, and
Paul J. Martin
From the Division of Clinical Research, Fred Hutchinson Cancer
Research Center, Seattle, WA; the Department of Medical Genetics and
Oncology, National Taiwan University Hospital, Taipei, Taiwan; and the
Department of Internal Medicine, University of Washington,
Seattle, WA.
 |
ABSTRACT |
Results of a previous study suggested that recipient mismatching for
the minor histocompatibility antigen HA-1 is associated with acute
graft-versus-host disease (GVHD) after allogeneic marrow transplantation. In that study, most patients received either cyclosporine or methotrexate for GVHD prophylaxis, and a cytotoxic T-cell clone was used to test for HA-1 disparity. To facilitate large-scale testing, we developed a method that uses genomic DNA to
identify HA-1 alleles. A retrospective study was conducted to correlate
HA-1 disparity and the occurrence of acute GVHD in 237 HLA-A2-positive
white patients who had received a marrow or peripheral blood stem cell
transplant from an HLA-identical sibling. All patients received both
methotrexate and cyclosporine for GVHD prophylaxis. The presence of
HLA-A*0201 was confirmed in 34 of the 36 HA-1 disparate pairs by
sequencing the HLA-A locus. Grades II-IV GVHD occurred in 22 (64.7%)
of these 34 patients, compared with 86 (42.8%) of the 201 patients
without HA-1 disparity (odds ratio, 2.45; 95% confidence interval
[CI], 1.15 to 5.23; P = .02). Recipient HA-1 disparity
showed a trend for association with acute GVHD (odds ratio, 2.1; 95%
CI, 0.91 to 4.68; P = .08) when a multivariable logistic
regression model was used to include additional risk factors. These
data are consistent with results of the previous study, suggesting an
association between HA-1 disparity and risk of acute GVHD, but the
strength of this association may be lower in patients who received both
methotrexate and cyclosporine than in those who received methotrexate
or cyclosporine alone.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
ACUTE GRAFT-VERSUS-HOST disease (GVHD) is
a major cause of mortality and morbidity after hematopoietic cell
transplantation.1,2 Recipient disparity for major
histocompatibility antigens encoded on the short arm of chromosome 6 is
the most important risk factor for GVHD.3,4 Recipient
disparity for minor histocompatibility antigens (mHA) encoded by
sex-linked or autosomal loci also contributes to the risk of
GVHD.5-8 To date, only 1 autosomal mHA has been implicated
as a cause of GVHD in humans.7 This mHA, termed HA-1, was
first identified by a cytotoxic T-cell clone recovered from a marrow
transplant recipient who had GVHD.9
A polymorphism at position 504 of the HA-1 cDNA sequence (GenBank
accession no. D86976) encodes either histidine (HA-1H) or
arginine (HA-1R) at position 3 of a 9-mer peptide presented
by HLA-A*0201 molecules.10 HA-1-specific cytotoxic T cells
recognize the HA-1H peptide/HLA-A*0201 complex. Another
polymorphism at position 500 of HA-1 cDNA is silent. Based on these 2 polymorphisms, we developed a well-validated and easily applied method
for typing HA-1 alleles with genomic DNA.11 We have now
used this method to examine the correlation between HA-1 disparity and
GVHD in a large number of patients who received both methotrexate and cyclosporine after marrow transplantation from an HLA-identical sibling.
| |
MATERIALS AND METHODS |
Patient selection.
DNA samples were extracted from HLA-A2-positive white patients who
received a marrow (n = 229) or peripheral blood stem cell (n = 8)
transplant from an HLA-identical sibling at the Fred Hutchinson Cancer
Research Center (Seattle, WA) between 1981 and 1996. Methods for HLA
typing of donors and recipients have been described.12 All
patients received methotrexate and cyclosporine for GVHD prophylaxis and had either grade 0 or grades II-IV acute GVHD. Patients with grade
I GVHD, patients with renal failure requiring dialysis, and patients
without GVHD who died before day 80 after transplantation were
excluded. Because there were differences in GVHD grading according to
the reviewers who assigned the grades before and after
1991,13 sample selection was designed to preserve a balance in the numbers of patients with grades 0 or II-IV GVHD before and after
1991. With evaluation of 200 donor/recipient pairs, a 50% incidence of
grades II-IV GVHD, an 11.3% expected incidence of HA-1
disparity,7 and a 2-sided significance level of .05, the
study was estimated to have 80% power to detect an odds ratio of 4.5 for the association between HA-1 disparity and grades II-IV GVHD.
HA-1 genotyping.
Testing was performed by individuals who did not know whether the
sample had come from a patient who had GVHD. Samples containing 100 ng
DNA were subjected to 40 cycles of denaturation (94°C for 30 seconds), annealing (58°C for 30 seconds), and elongation (72°C for 60 seconds) using primers HA-1a and HA-1c, as described
elsewhere.11 Five microliters of the amplified product was
digested with 0.4 U Tsp45I or 0.5 U Fnu4HI (New England Biolabs,
Beverly, MA) for 2 hours and then analyzed by
electrophoresis in 2.2% agarose. When recipient HA-1 disparity was
detected by analysis of restriction fragments, results were confirmed
by allele-specific amplification. In this assay, samples containing 100 ng DNA were subjected to 40 cycles of denaturation (94°C for 30 seconds), annealing (60°C for 30 seconds), and elongation (72°C
for 60 seconds) using allele-specific 5' primer HA-1h for the
HA-1H allele or 5' primer HA-1r for the
HA-1R allele, together with 3' primer HA-1c as
described previously.11 Five microliters of the amplified
product was analyzed by electrophoresis in 2.2% agarose.
Sequencing of HLA-A2.
The presence of HLA-A*0201 in HA-1 disparate pairs was confirmed by
sequencing exon 2 and exon 3 of amplified HLA-A or HLA-A2 gene from the
donor. Samples containing 100 ng DNA were subjected to 40 cycles of
denaturation (94°C for 30 seconds), annealing (61°C for 30 seconds), and elongation (72°C for 90 seconds) using HLA-A2-specific 5' primer A2F2M13R
(5'-caggaaacagctatgaccTCTCAGCCACTCCTCGTCCCCAGGCTCT-3', positions 705-732, GenBank accession no. K02883; lower case letters
indicate M13 reverse primer) and HLA-A-specific 3' primer AR1M13F (5'-tgtaaaacgacggccagtCGGGAGATCTACAGGCGATCAG-3',
positions 1561-1540; lower case letters indicate M13 forward primer).
Alternatively, samples were subjected to 40 cycles of denaturation
(94°C for 30 seconds), annealing (61°C for 30 seconds), and
elongation (72°C for 90 seconds) using 5' primer IN1CONSM13R
(5'-caggaaacagctatgaccGTGAGTGCGGGGTCGGGA-3', positions
599-616) and HLA-A-specific 3' primer 18C182TM13F
(5'-tgtaaaacgacggccagtGTGGCCCCTGGTACCCGT-3', positions
1528-1511). The amplified product was electrophoresed in agarose,
eluted, and subjected to sequencing with the ABI Prism dye primer cycle
sequencing kit and an ABI 373A automated fluorescent sequencer system
(PE Applied Biosystems, Foster City, CA).
Statistical analysis.
GVHD scores were unblinded after all HA-1 typing was completed.
Recipient HA-1 disparity was defined as the presence of
HA-1H in the recipient but not in the donor. Univariate and
multivariable logistic regression models were used to analyze the
association between risk factors and the probability of acute GVHD.
Wilcoxon rank-sum tests were used to compare the distributions of organ stages and overall grades of GVHD for patients with HA-1 disparity and
those without HA-1 disparity. All P values are 2-sided, and no
adjustments were made for multiple comparisons.
| |
RESULTS |
Recipient HA-1 disparity was detected in 36 of the 237 donor/recipient
pairs (15.2%). HLA-A*0201 was present in 34 of the 36 donors. The
other 2 had HLA-A*0205 instead of HLA-A*0201 and were excluded from
further analysis. Twenty-two (64.7%) of the 34 patients with HA-1
disparity developed grades II-IV acute GVHD, compared with 86 (42.8%)
of the 201 patients without HA-1 disparity (Table 1). In univariate analyses,
recipient HA-1 disparity was significantly associated with an increased
probability of grades II-IV GVHD (odds ratio, 2.45; 95% CI, 1.15 to
5.23; P = .02). The distribution of organ stages showed more
severe skin and gut involvement among patients with HA-1 disparity
compared with those without HA-1 disparity, but the severity of liver
involvement was similar in the 2 groups (Table 1).
In a previous study of patients who received methotrexate and
cyclosporine for prevention of GVHD, donor/recipient gender, donor
parity, advanced malignancy, and total body irradiation (TBI) were
identified as risk factors for grade II-IV GVHD.14 In the
present study, patients with HA-1 disparity had more advanced malignancy than those without HA-1 disparity
(Table 2), but the 2 groups were otherwise
similar in risk factors for GVHD. In a multivariable logistic
regression analysis, increased patient age and greater than 12 Gy TBI
were significantly associated with higher risk of GVHD
(Table 3). Other risk factors were not
significantly associated with grades II-IV GVHD in this study
population. With all factors included in the model, recipient HA-1
disparity showed a trend toward an increased probability of grades
II-IV GVHD (odds ratio, 2.1; 95% CI, 0.91 to 4.68; P = .08).
| |
DISCUSSION |
Results of this study support the conclusion that recipient disparity
for the HA-1 antigen is associated with an increased risk of GVHD, but
the odds ratio in our study (2.1) is lower than the value reported by
Goulmy et al7 (5.4). These results could reflect
differences in methods used for sample selection, variability in GVHD
grading, or the use of more effective GVHD prophylaxis in the
population we selected for study. In the study by Goulmy et
al,7 only 15% of the patients received both methotrexate and cyclosporine for GVHD prophylaxis, and all 10 adult patients with
HA-1 disparity developed acute GVHD. In our study, all patients received both methotrexate and cyclosporine, and those who received reduced doses of cyclosporine because of acute renal failure were excluded. Twelve of the 34 patients (and 10 of the 31 more than 16 years of age) with HA-1 disparity did not develop GVHD. These data
suggest that the combination of methotrexate and cyclosporine may have
prevented the development of GVHD in some patients with HA-1 disparity.
Our finding that HA-1 disparity is associated with selectively
increased severity of GVHD in the skin and gut but not the liver
suggests that the tissue distribution of alloantigens in the recipient
might influence the clinical manifestations of GVHD. Results of
previous studies have suggested that the HA-1 antigen is expressed by
hematopoietic cells, dendritic cells, and Langerhans cells but not by
cultured fibroblasts, keratinocytes, or melanocytes.15,16 Further studies are needed to assess expression of the HA-1 antigen in
vivo and to determine how the tissue distribution of this antigen is regulated.
Although the current data are consistent with the conclusion that HA-1
disparity is associated with increased risk of GVHD, the implications
for hematopoietic cell transplantation remain to be determined. The
availability of a simple DNA-based assay would make it feasible to type
and match prospectively for HA-1 compatibility between the donor and
recipient,11,17 but the opportunity for selecting among
multiple HLA-identical related donors is low because of limited family
size. Moreover, the prevalence of HLA-A2 among white patients is less
than 50%, and the proportion of unselected recipients with HA-1
disparity is only 10% to 15%.7,11,17 With unrelated
transplantation, multiple donors are available for patients who have
common HLA haplotypes, and the proportion of unselected pairs with
recipient HA-1 disparity is approximately 20% to 25%.18
On the other hand, the association between recipient HA-1 disparity and
GVHD after unrelated marrow transplantation has yet to be demonstrated.
Results of previous studies have suggested that the reduction in risk
of GVHD from typing and matching for a single mHA is likely to be
small, but substantial benefit could come from typing and matching for
multiple mHA.19 Additional studies will be needed to assess
the effects of HA-1 disparity on other important endpoints such as
chronic GVHD, leukemia relapse, and survival.
| |
ACKNOWLEDGMENT |
The authors thank Alison Sell for help in preparing the manuscript.
| |
FOOTNOTES |
Submitted March 24, 1999; accepted June 2, 1999.
Supported by National Institutes of Health Grants No. AI33484, CA18029,
CA15704, and CA18211.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Paul J. Martin, MD, Fred Hutchinson Cancer
Research Center, 1100 Fairview Ave N, D2-100, PO Box 19024, Seattle,
WA, 98109-1024; e-mail: pmartin{at}fhcrc.org.
| |
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ABSTRACT
INTRODUCTION