Abstract
Donor-directed human leukocyte antigen (HLA)–specific allo-antibodies (DSAs) cause graft failure in animal models of hematopoietic stem cell transplantation (HCT). Archived pretransplantation sera from graft failure patients (n = 37) and a matched case-control cohort (n = 78) were tested to evaluate the role of DSAs in unrelated donor HCT. Controls were matched for disease, disease status, graft type, patient age, and transplantation year. Patients had acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, or myelodysplastic syndrome; 98% received myeloablative conditioning regimens 100% received T-replete grafts, 97% received marrow, 95% HLA-mismatched, and 97% received calcineurin-based graft-versus-host disease prophylaxis. Among the 37 failed transplantations, 9 (24%) recipients possessed DSAs against HLA-A, B, and/or DP, compared with only 1 (1%) of 78 controls. Therefore, the presence of DSAs was significantly associated with graft failure (odds ratio = 22.84; 95% confidence interval, 3.57-∞; P < .001). These results indicate that the presence of pretransplantation DSAs in recipients of unrelated donor HCT is associated with failed engraftment and should be considered in HCT donor selection.
Introduction
Hematopoietic stem cell transplantation (HCT) recipients may become alloimmunized to foreign human leukocyte antigens (HLAs) through pregnancy or blood transfusions. The resulting sensitization may include antibodies directed against mismatched HLA antigens of a potential stem cell donor. Recent National Marrow Donor Program (NMDP) analyses suggest that greater than 50% of unrelated donor HCTs are mismatched for at least one classic HLA-A, B, C, or DRB1 locus.1,2 In addition, mismatching at HLA-DP is observed in approximately 88% of all unrelated donor HSCT.1,2 Engraftment failure is observed at a rate of approximately 5% in unrelated donor HCT, and donor-directed HLA alloantibodies may increase the risk.3 In a murine model of allo-sensitization, rapid graft failure was shown to result from alloimmune rejection mediated by antibody-dependent cell-mediated killing.4 Prescreening of patient serum and the identification of specific HLA antibodies could be used as part of a donor selection strategy designed to avoid a potential deleterious incompatibility.
Only a few studies have demonstrated that recipient sensitization to mismatched donor HLA antigens affects engraftment. In a study of marrow transplantations from HLA-mismatched relatives, graft failure occurred in 13 of 21 patients (62%) with a positive pretransplantation cross-match (patient serum vs donor T or B lymphocytes), compared with 31 of 501 patients (7%) with a negative cross-match (P < .001).5,6 Ottinger et al also found that a positive lymphocyte cross-match was a predictor for graft failure and poor survival after HCT from HLA-mismatched donors.7
Although a lymphocyte cross-match is an effective tool to evaluate alloimmunization and potential donor/recipient incompatibility, the procedure is labor intensive, may detect non-HLA antibodies, and is logistically difficult for remotely located unrelated donors because of the requirement for live cells. Non-HLA antibodies may be important in HCT; however, studies have not been done that support this point unequivocally. New solid-phase antibody detection technologies can better identify HLA-specific alloantibodies and are more sensitive than cytotoxicity testing and flow cytometry.8–11 Using these methods, it may be possible to predict alloreactivity against HLA mismatches for unrelated donor recipient pairs before transplantation. Takanashi et al have reported that virtual cross-match–detected DSAs predict graft failure of unrelated umbilical cord blood transplantation.12
Methods
We designed a case-control study to retrospectively evaluate the effect of preexisting DSAs on engraftment in unrelated donor HSCT. Thirty-seven cases with available samples were selected based on the failure to achieve engraftment after transplantation and 78 controls that engrafted were selected for comparison. Cases and controls were matched for disease, disease status, patient age, year of transplantation, conditioning regimen, and graft type. Graft failure was defined as never achieving an absolute neutrophil count more than 500 with survival beyond 28 days. Patients had acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, or myelodysplastic syndrome; 98% received myeloablative conditioning regimens, 100% received T-replete grafts, 97% received marrow, and 97% received calcineurin-based graft-versus-host disease prophylaxis (Table 1). Cases and controls were preferentially selected for the presence of at least one HLA mismatch at HLA-A, -B, -C, DRB1, DQB1, or DPB1 to serve as a potential allogeneic target. All HLA typing was verified using high-resolution DNA-based methods as described previously.13
Table 1.
Characteristics of patients included in the study using cases (graft failure) versus controls (engraftment)
| Variable | Graft failure |
Engraftment |
||
|---|---|---|---|---|
| No. evaluated | No. (%) | No. evaluated | No. (%) | |
| No. of patients | 37 | 78 | ||
| No. of centers | 22 | 33 | ||
| Median age, y (range) | 37 | 35 (7-53) | 78 | 36 (5-54) |
| Age at transplantation, y | 37 | 78 | ||
| Less than 10 | 1 (3) | 1 (1) | ||
| 11-20 | 4 (11) | 9 (12) | ||
| 21-30 | 9 (24) | 18 (23) | ||
| 31-40 | 11 (30) | 22 (28) | ||
| 41-50 | 10 (27) | 24 (31) | ||
| More than 50 | 2 (5) | 4 (5) | ||
| Male sex | 37 | 17 (46) | 78 | 45 (58) |
| Karnofsky before treatment > 90% | 36 | 26 (72) | 76 | 51 (67) |
| Overall HLA matching | 37 | 78 | ||
| 12/12 | 2 (5) | 4 (5) | ||
| 11/12 | 4 (11) | 11 (14) | ||
| 10/12 | 6 (16) | 26 (33) | ||
| 9/12 | 11 (30) | 18 (23) | ||
| 8/12 | 7 (19) | 8 (10) | ||
| 7/12 | 5 (14) | 6 (8) | ||
| 6/12 | 2 (5) | 5 (7) | ||
| HLA mismatch distribution | ||||
| HLA-A | 13 (35) | 25 (32) | ||
| HLA-B | 13 (35) | 25 (32) | ||
| HLA-C | 22 (59) | 38 (49) | ||
| HLA-DRB1 | 3 (8) | 8 (10) | ||
| HLA-DQB1 | 10 (27) | 13 (17) | ||
| HLA-DPB1 | 33 (89) | 63 (81) | ||
| Disease at transplantation | 37 | 78 | ||
| AML | 12 (32) | 26 (33) | ||
| ALL | 2 (5) | 5 (6) | ||
| CML | 20 (54) | 41 (53) | ||
| MDS | 3 (8) | 6 (8) | ||
| Disease status at transplantation | 37 | 78 | ||
| Early | 13 (35) | 28 (36) | ||
| Intermediate | 14 (38) | 30 (38) | ||
| Advanced | 9 (24) | 19 (24) | ||
| Other | 1 (3) | 1 (1) | ||
| Stem cell source | 37 | 78 | ||
| Bone marrow | 36 (97) | 76 (97) | ||
| PBSC | 1 (3) | 2 (3) | ||
| Conditioning regimen | 37 | 78 | ||
| Myeloablative | 36 (97) | 76 (97) | ||
| Reduced intensity | 1 (3) | 2 (3) | ||
| GVHD prophylaxis | 37 | 78 | ||
| CsA or FK506 + MTX ± other | 36 (97) | 73 (93) | ||
| CsA or FK506 ± other (no MTX) | 0 | 2 (3) | ||
| MTX ± other | 1 (3) | 2 (3) | ||
| Other | 0 | 1 (1) | ||
| Donor/recipient sex match | 37 | 78 | ||
| Male/male | 5 (14) | 31 (40) | ||
| Male/female | 12 (32) | 20 (26) | ||
| Female/male | 12 (32) | 14 (18) | ||
| Female/female | 8 (22) | 13 (17) | ||
| Donor/recipient CMV match | 37 | 78 | ||
| Negative/negative | 4 (11) | 29 (37) | ||
| Negative/positive | 15 (41) | 24 (31) | ||
| Positive/negative | 10 (27) | 7 (9) | ||
| Positive/positive | 5 (14) | 17 (22) | ||
| Unknown | 3 (8) | 1 (1) | ||
| Donor median age, y (range) | 37 | 36 (21-52) | 78 | 35 (18-54) |
| Donor age, y | 37 | 78 | ||
| 18-29 | 11 (30) | 21 (27) | ||
| 30-39 | 12 (32) | 35 (45) | ||
| 40-49 | 13 (35) | 14 (18) | ||
| 50 and older | 1 (3) | 8 (10) | ||
| Year of transplantation | 37 | 78 | ||
| 1990 | 0 | 1 (1) | ||
| 1991 | 0 | 0 | ||
| 1992 | 1 (3) | 6 (8) | ||
| 1993 | 3 (8) | 7 (9) | ||
| 1994 | 3 (8) | 7 (9) | ||
| 1995 | 6 (16) | 5 (6) | ||
| 1996 | 5 (14) | 10 (13) | ||
| 1997 | 4 (11) | 6 (8) | ||
| 1998 | 2 (5) | 8 (10) | ||
| 1999 | 5 (14) | 8 (10) | ||
| 2000 | 3 (8) | 11 (14) | ||
| 2001 | 5 (14) | 7 (9) | ||
| 2002 | 0 | 2 (3) | ||
| Median follow-up of survivors, mo | 3 | 72 (70-74) | 21 | 65 (38-158) |
AML indicates acute myeloid leukemia; ALL, acute lymphoblastic leukemia; CML, chronic myelogenous leukemia; MDS, myelodysplastic syndrome; and MTX, methotrexate.
Pretransplantation recipient serum samples were obtained from the NMDP Research Repository. All surviving recipients included in this analysis were retrospectively contacted and provided informed consent for participation in the NMDP research program. Research was approved and conducted under supervision of the NMDP Institutional Review Board. A modeling process was used as previously described to adjust for any bias introduced by exclusion of nonconsenting survivors.1
The sera were tested in 2 different laboratories using solid-phase microparticle methods with 10% tested in duplicate for quality control purposes. HLA antibody screening was initially performed on all samples by flow cytometry using FlowPRA (One Lambda, Inc). All samples with a positive panel reactive antibody (PRA) were further evaluated using the Luminex-based LABScreen single antigen assay or single antigen flow beads (One Lambda Inc) to determine HLA specificities. Samples were considered positive for specific HLA antigens based on a background-adjusted mean fluorescence intensity of more than 2000 for Luminex-tested samples or a mean fluorescence channel shift of more than 40 for single antigen flow bead assays. DSA assessments were performed by comparing the HLA antibody profile with the mismatched HLA antigens in the graft-rejection direction.
Conditional logistic regression analysis was performed to compare the PRA and DSA positivity rates between the graft failure cases and engrafted controls. Cox regression models were developed to test the impact of HLA-C mismatches, cytomegalovirus (CMV), and cell dose on graft failure rates, as these were not included in the original matching schema.
Results and discussion
The PRA testing found that 37% of all patients carried preexisting HLA class I or II antibodies before transplantation. Forty-three percent of graft failure cases and 32% of engraftment controls possessed a positive PRA, and the distribution by class I and II is noted in Table 2. There was no significant difference in PRA positivity rates between the 2 groups (χ2, P = .221) or an association found between PRA and graft failure using conditional logistic regression (data not shown).
Table 2.
Results of univariate analysis comparing the rates of panel-reactive antibody positivity and donor-specific HLA antibody positivity between graft failure cases and engrafted controls
| Class I positive, no. (%) | Class II positive, no. (%) | Class I and/or II positive, no. (%) | P | |
|---|---|---|---|---|
| Panel-reactive antibody results | > .221 | |||
| Cases (n = 37) | 16 (43) | 10 (27) | 16 (43) | |
| Controls (n = 78) | 21 (27) | 13 (17) | 25 (32) | |
| Donor-specific HLA antibodies | < .001 | |||
| Cases (n = 37) | ||||
| A: 1 (3) | DP: 6 (16) | 9 (24) | ||
| B: 4 (11) | DR: 0 (0) | |||
| C: 0 (0) | DQ: 0 (0) | |||
| Controls (n = 78) | ||||
| A: 1 (1) | DP: 1 (1) | 1 (1) | ||
| B: 0 (0) | DR: 0 (0) | |||
| C: 0 (0) | DQ: 0 (0) |
Screening for the presence of DSAs in the PRA-positive graft failure cases and controls found that 8.7% were positive. Among the cases, 24% (9 of 37) carried DSAs, 3 with class I only, 4 with class II only, and 2 with both class I and II specific antibodies (Table 2). In the control group, only one case was positive for DSA (class I and II). The DSA positivity rates were significantly different between the groups (χ2, P < .001). The conditional logistic regression analysis found that the presence of class I (odds ratio [OR] = 11.34; 95% confidence interval [CI], 1.49-∞; P = .017), class II (OR = 12.00; 95% CI, 1.46-551.97; P = .014), or either class I or II (OR = 22.84; 95% CI, 3.57-∞; P < .001) DSAs in the recipients before transplantation was significantly associated with graft failure (Table 3).
Table 3.
Results of conditional logistic regression analysis evaluating the association of DSA directed against HLA class I and/or II and graft failure
| Odds ratio | 95% confidence interval | P | |
|---|---|---|---|
| Class I DSA | 11.34 | 1.49-∞ | .017 |
| Class II DSA | 12.00 | 1.46-551.97 | .014 |
| Class I and/or II DSA | 22.84 | 3.57-∞ | < .001 |
Further analyses were conducted using Cox regression models to evaluate a limited set of covariates not accounted for in the case-control matching, including patient CMV status, cell dose, and HLA-C match, because these variables had been associated with graft failure in a previous study of unrelated donor transplantations.14 Cell dose (< 2 × 109/kg) and CMV status (recipient positive) were independently predictive of engraftment (P = .01 and .03, respectively). No effect was observed for HLA-C match (P = .84). The presence of antidonor HLA class I or II antibodies was still predictive of engraftment failure when adjustment was made for either cell dose (OR = 15.49; 95% CI, 2.06-697.83; P = .002) or CMV status (OR = 7.94; 95% CI, 0.97-367.84; P = .05).
Interestingly, antibodies against HLA-DP were quite prominent and were present in 60% of antibody-positive failures (Table 2). HLA-DP mismatching has previously been implicated as affecting graft-versus-host disease and lower relapse rates in the T-cell replete myeloablative bone marrow transplantation setting, suggesting its ability to act as a transplantation antigen.1,15 This suggests that, even though matching for HLA-DP may not be important for overall survival, if a patient has HLA antibodies directed against the mismatched DP type of the donor, there may be an increased risk for graft failure. Although it is possible that a higher cell dose, such as provided by a peripheral blood stem cell graft, could help overcome the engraftment barrier posed by DSAs, there are no clinical data available to define a cell dose threshold. Thus, HLA antibody screening is indicated for potential HCT recipients. Moreover, if HLA antibody is present, a thorough assessment of antibody specificity and donor mismatches is warranted before transplantation.
Acknowledgments
The Center for International Blood and Marrow Transplant Research is supported by the National Cancer Institute, the National Heart, Lung and Blood Institute, and the National Institute of Allergy and Infectious Diseases (Public Health Service Grant/Cooperative Agreement U24-CA76518); the National Heart, Lung and Blood Institute and National Cancer Institute (Grant/Cooperative Agreement 5U01HL069294); the Health Resources and Services Administration (Department of Health and Human Services; contract HHSH234200637015C); the Office of Naval Research (grants N00014-06-1-0704 and N00014-08-1-0058); and grants from AABB; Aetna; American Society for Blood and Marrow Transplantation; Amgen Inc; anonymous donation to the Medical College of Wisconsin; Astellas Pharma US Inc; Baxter International Inc; Bayer HealthCare Pharmaceuticals; Be the Match Foundation; Biogen IDEC; BioMarin Pharmaceutical Inc; Biovitrum AB; BloodCenter of Wisconsin; Blue Cross and Blue Shield Association; Bone Marrow Foundation; Canadian Blood and Marrow Transplant Group; CaridianBCT; Celgene Corporation; CellGenix GmbH; Centers for Disease Control and Prevention; Children's Leukemia Research Association; ClinImmune Labs; CTI Clinical Trial and Consulting Services; Cubist Pharmaceuticals; Cylex Inc; CytoTherm; DOR = BioPharma Inc; Dynal Biotech, an Invitrogen Company; Eisai Inc; Enzon Pharmaceuticals Inc; European Group for Blood and Marrow Transplantation; Gamida Cell Ltd; GE Healthcare; Genentech Inc; Genzyme Corporation; Histogenetics Inc; HKS Medical Information Systems; Hospira Inc; Infectious Diseases Society of America; Kiadis Pharma; Kirin Brewery Co Ltd; Leukemia & Lymphoma Society; Merck & Company; Medical College of Wisconsin; MGI Pharma Inc; Michigan Community Blood Centers; Millennium Pharmaceuticals Inc; Miller Pharmacal Group; Milliman USA Inc; Miltenyi Biotec Inc; National Marrow Donor Program; Nature Publishing Group; New York Blood Center; Novartis Oncology; Oncology Nursing Society; Osiris Therapeutics Inc; Otsuka America Pharmaceutical Inc; Pall Life Sciences; PDL BioPharma Inc; Pfizer Inc; Pharmion Corporation; Saladax Biomedical Inc; Schering Corporation; Society for Healthcare Epidemiology of America; StemCyte Inc; StemSoft Software Inc; Sysmex America Inc; Teva Pharmaceutical Industries; THERAKOS Inc; Thermogenesis Corporation; Vidacare Corporation; Vion Pharmaceuticals Inc; ViraCor Laboratories; ViroPharma Inc; and Wellpoint Inc.
The views expressed in this article do not reflect the official policy or position of the National Institutes of Health, the Department of the Navy, the Department of Defense, or any other agency of the US government.
Footnotes
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.
Authorship
Contribution: S.S., R.B., and C.A. participated in the conception and design of this study and wrote the paper; S.S., S.F., M.H., and J.K. selected and defined the study population; R.B. and S.R.-B. performed laboratory testing; M.H. and C.V.-G. prepared the dataset for analysis and performed univariate assessments; J.K. performed the multivariate analysis; and S.F., M.H., S.R.-B., C.V.-G., and J.K. reviewed the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Stephen Spellman, Scientific Services, National Marrow Donor Program, 3001 Broadway St NE, Ste 100, Minneapolis, MN 55413; e-mail: sspellma@nmdp.org.
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