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.

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).

Table 1.

Correlation of HA-1 Disparity With Acute GVHD

Skin Stage HA-1 Disparity (P = .02)
Absent Present
0  124 (62) 14 (41)  
1  5 (2)  3 (9)  
2  26 (13)  2 (6) 
3  42 (21)  15 (44)  
4  4 (2)  
Gut Stage  HA-1 Disparity (P = .0007)  
Absent  Present  
0  154 (77) 16 (47)  
1  29 (14)  13 (38)  
2  8 (4) 1 (3)  
3  5 (2)  3 (9)  
4  5 (2)  1 (3) 
Liver Stage  HA-1 Disparity (P = .92)  
Absent  Present  
0  152 (76) 25 (74)  
1  18 (9)  5 (15)  
2  16 (8)  2 (6) 
3  10 (5)  2 (6)  
4  5 (2)  
Overall Grade  HA-1 Disparity (P = .04)  
Absent  Present  
115 (57)  12 (35)  
II  53 (26)  15 (44)  
III 29 (14)  7 (21)  
IV  4 (2)  
Skin Stage HA-1 Disparity (P = .02)
Absent Present
0  124 (62) 14 (41)  
1  5 (2)  3 (9)  
2  26 (13)  2 (6) 
3  42 (21)  15 (44)  
4  4 (2)  
Gut Stage  HA-1 Disparity (P = .0007)  
Absent  Present  
0  154 (77) 16 (47)  
1  29 (14)  13 (38)  
2  8 (4) 1 (3)  
3  5 (2)  3 (9)  
4  5 (2)  1 (3) 
Liver Stage  HA-1 Disparity (P = .92)  
Absent  Present  
0  152 (76) 25 (74)  
1  18 (9)  5 (15)  
2  16 (8)  2 (6) 
3  10 (5)  2 (6)  
4  5 (2)  
Overall Grade  HA-1 Disparity (P = .04)  
Absent  Present  
115 (57)  12 (35)  
II  53 (26)  15 (44)  
III 29 (14)  7 (21)  
IV  4 (2)  

Data indicate the n and (%) in each category. P values for differences in the distribution of skin, liver, and gut stages and overall grade were calculated from Wilcoxon rank sum tests.

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).

Table 2.

Distribution of Risk Factors for GVHD According to HA-1 Disparity

Risk Factor Recipient HA-1 Disparity P Value
Absent (n = 201)Present (n = 34)
Median age in years (range) 31 (1-65)  36 (2-58)  .56  
Recipient age (n; %)   .71  
 ≤16 yrs  22 (11)  3 (9)  
 >16 yrs 179 (89)  31 (91)  
Transplant year (n; %)    .48 
 Before 1991  148 (74)  27 (79)  
 1991 or later 53 (26)  7 (21)  
Donor/recipient gender (n; %)   .49  
 Male/male  59 (29)  10 (29) 
 Male/female  50 (25)  4 (12)  
 Parous female/female 24 (12)  7 (21)  
 Nulliparous female/female  20 (10) 4 (12)  
 Parous female/male  22 (11)  6 (18) 
 Nulliparous female/male  17 (8)  2 (6)  
 Female, unknown parity* 9 (4)  1 (3)  
Advanced malignancy (n; %)   .03  
 No  153 (76)  20 (59)  
 Yes 48 (24)  14 (41)  
TBI (n; %)    .17  
 ≤12 Gy 81 (40)  13 (38)  
 >12 Gy  54 (27)  14 (41) 
 None  66 (33)  7 (21) 
Risk Factor Recipient HA-1 Disparity P Value
Absent (n = 201)Present (n = 34)
Median age in years (range) 31 (1-65)  36 (2-58)  .56  
Recipient age (n; %)   .71  
 ≤16 yrs  22 (11)  3 (9)  
 >16 yrs 179 (89)  31 (91)  
Transplant year (n; %)    .48 
 Before 1991  148 (74)  27 (79)  
 1991 or later 53 (26)  7 (21)  
Donor/recipient gender (n; %)   .49  
 Male/male  59 (29)  10 (29) 
 Male/female  50 (25)  4 (12)  
 Parous female/female 24 (12)  7 (21)  
 Nulliparous female/female  20 (10) 4 (12)  
 Parous female/male  22 (11)  6 (18) 
 Nulliparous female/male  17 (8)  2 (6)  
 Female, unknown parity* 9 (4)  1 (3)  
Advanced malignancy (n; %)   .03  
 No  153 (76)  20 (59)  
 Yes 48 (24)  14 (41)  
TBI (n; %)    .17  
 ≤12 Gy 81 (40)  13 (38)  
 >12 Gy  54 (27)  14 (41) 
 None  66 (33)  7 (21) 
*

This group included 7 male and 3 female patients.

Patients with hematologic malignancy in relapse, accelerated phase, or blast phase at the time of transplantation.

Table 3.

Multivariable Logistic Regression Model of Risk Factors for Grades II-IV Acute GVHD

Risk Factor Odds Ratio 95% CI P Value
Patients age (per decade)3-150 1.47  1.16-1.86  .002 
Transplant year  
 ≥1991  1.0  
 <1991  0.84 0.39-1.81  .65  
Donor/recipient gender  
 Male/male 1.0  
 Male/female  1.69  0.77-3.71  .19  
 Parous female/female  0.94  0.37-2.38  .89  
 Nulliparous female/female  0.92  0.31-2.70  .88  
 Parous female/male 1.90  0.70-5.18  .21  
 Nulliparous female/male  1.34 0.44-4.09  .61  
 Missing parity data  0.96  0.23-4.02 .95  
Advanced malignancy3-151 
 No  1.0  
 Yes 1.86  0.96-3.59  .06  
TBI  
 ≤12 Gy  1.0 
 >12 Gy  2.08  1.03-4.20  .04  
 None  0.52 0.24-1.11  .09  
HA-1 disparity  
 Absent  1.0 
 Present  2.1  0.91-4.68  .08 
Risk Factor Odds Ratio 95% CI P Value
Patients age (per decade)3-150 1.47  1.16-1.86  .002 
Transplant year  
 ≥1991  1.0  
 <1991  0.84 0.39-1.81  .65  
Donor/recipient gender  
 Male/male 1.0  
 Male/female  1.69  0.77-3.71  .19  
 Parous female/female  0.94  0.37-2.38  .89  
 Nulliparous female/female  0.92  0.31-2.70  .88  
 Parous female/male 1.90  0.70-5.18  .21  
 Nulliparous female/male  1.34 0.44-4.09  .61  
 Missing parity data  0.96  0.23-4.02 .95  
Advanced malignancy3-151 
 No  1.0  
 Yes 1.86  0.96-3.59  .06  
TBI  
 ≤12 Gy  1.0 
 >12 Gy  2.08  1.03-4.20  .04  
 None  0.52 0.24-1.11  .09  
HA-1 disparity  
 Absent  1.0 
 Present  2.1  0.91-4.68  .08 
F3-150

Modeled as a continuous variable.

F3-151

Patients with hematologic malignancy in relapse, accelerated phase, or blast phase at the time of transplantation.

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.15,16Further 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%.18On 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.

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.

REFERENCES

1
Ferrara
JL
Joachim Deeg
H
Graft-versus-host disease.
N Engl J Med
324
1991
667
2
Sullivan
KM
Graft-versus-host disease
Bone Marrow Transplantation.
Forman
SJ
Blume
KG
Thomas
ED
1994
339
Blackwell Scientific
Boston, MA
3
Beatty
PG
Clift
RA
Mickelson
EM
Nisperos
BB
Flournoy
N
Martin
PJ
Sanders
JE
Steward
P
Buckner
CD
Storb
R
Thomas
ED
Hansen
JA
Marrow transplantation from related donors other than HLA-identical sibling.
N Engl J Med
313
1985
765
4
Hansen
JA
Gooley
TA
Martin
PJ
Appelbaum
F
Chauncey
TR
Clift
RA
Petersdorf
EW
Radich
J
Sanders
JE
Storb
RF
Sullivan
KM
Anasetti
C
Bone marrow transplants from unrelated donors for patients with chronic myeloid leukemia.
N Engl J Med
338
1998
962
5
van Els
CA
D’Amaro
J
Pool
J
Blokland
E
Bakker
A
van Elsen
PJ
van Rood
JJ
Goulmy
E
Immunogenetics of human minor histocompatibility antigens: Their polymorphism and immunodominance.
Immunogenetics
35
1992
161
6
Wang
W
Meadows
LR
den Haan
JM
Sherman
NE
Chen
Y
Blokland
E
Shabanowitz
J
Agulnik
AI
Hendrickson
RC
Bishop
CE
Hunt
DF
Goulmy
E
Engelhard
VH
Human H-Y: A male-specific histocompatibility antigen derived from the SMCY protein.
Science
269
1995
1588
7
Goulmy
E
Schipper
R
Pool
J
Blokland
E
Falkenburg
F
Vossen
J
Gratwohl
A
Vogelsang
GB
van Houwelingen
HC
van Rood
JJ
Mismatches of minor histocompatibility antigens between HLA-identical donors and recipients and the development of graft-versus-host disease after bone marrow transplantation.
N Engl J Med
334
1996
281
8
Perreault
C
Roy
DC
Fortin
C
Immunodominant minor histocompatibility antigens: The major ones.
Immunol Today
19
1998
69
9
Goulmy
E
Minor histocompatibility antigens in man and their role in transplantation
Transplantation Reviews 2.
Morris
PJ
Tilney
NL
1988
29
Saunders
Philadelphia, PA
10
den Haan
JM
Meadows
LM
Wang
W
Pool
J
Blokland
E
Bishop
TL
Reinhardus
C
Shabanowitz
J
Offringa
R
Hunt
DF
Engelhard
VH
Goulmy
E
The minor histocompatibility antigen HA-1: A diallelic gene with a single amino acid polymorphism.
Science
279
1998
1054
11
Tseng
LH
Lin
MT
Martin
PJ
Pei
J
Smith
AG
Hansen
JA
Definition of the gene encoding the minor histocompatibility antigen HA-1 and typing for HA-1 from genomic DNA.
Tissue Antigens
52
1998
305
12
Thomas
ED
Storb
R
Clift
RA
Fefer
A
Johnson
FL
Neiman
PE
Lerner
KG
Glucksberg
H
Buckner
CD
Bone marrow transplantation.
N Engl J Med
292
1975
832
13
Martin
PJ
Nash
R
Sanders
J
Leisenring
W
Anasetti
C
Deeg
HJ
Storb
R
Appelbaum
FR
Reproducibility in retrospective grading of acute graft-versus-host disease after allogeneic marrow transplantation.
Bone Marrow Transplant
21
1998
273
14
Nash
RA
Pepe
MS
Storb
R
Longton
G
Pettinger
M
Anasetti
C
Appelbaum
FR
Bowden
RA
Joachim Deeg
H
Doney
K
Martin
PJ
Sullivan
KM
Sanders
J
Witherspoon
RP
Acute graft-versus-host disease: Analysis of risk factors after allogeneic marrow transplantation and prophylaxis with cyclosporine and methotrexate.
Blood
80
1992
1838
15
de Bueger
M
Bakker
A
Van Rood
JJ
Van der Woude
F
Goulmy
E
Tissue distribution of human minor histocompatibility antigens. Ubiquitous versus restricted tissue distribution indicates heterogeneity among human cytotoxic T lymphocyte-defined non-MHC antigens.
J Immunol
149
1992
1788
16
van Lochem
E
van der Keur
M
Mommaas
AM
deGast
GC
Goulmy
E
Functional expression of minor histocompatibility antigens on human peripheral blood dendritic cells and epidermal Langerhans cells.
Transplant Immunol
4
1996
151
17
Wilke
M
Pool
J
den Haan
JM
Goulmy
E
Genomic identification of the minor histocompatibility antigen HA-1 locus by allele-specific PCR.
Tissue Antigens
52
1998
312
18
Martin
PJ
Increased disparity for minor histocompatibility antigens as a potential cause of increased GVHD risk in marrow transplantation from unrelated donors compared with related donors.
Bone Marrow Transplant
8
1991
217
19
Martin PJ: Applicability of matching for minor histocompatibility antigens in human bone marrow transplantation, in Roopenian DC, Simpson E, Goulmy E (eds): Minor Histocompatibility Antigens. Austin, TX, Landis Bioscience (in press)

Author notes

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@fhcrc.org.