Introduction

The use of umbilical cord blood (UCB) for transplantation of patients with hematological malignancies or hereditary diseases increases progressively. In October 2006, the International NETCORD Foundation maintained the inventory of more than 124 000 UCB units and documented more than 4900 unrelated UCB transplants.¹ Better outcome after hematopoietic stem cell transplant may be attributable to close matching of human leukocyte antigens (HLAs) of the donor–recipient pair. Both retrospective and prospective studies on unrelated bone marrow or peripheral blood stem cell transplants have demonstrated that donor–recipient matching for HLA class I and class II at the high-resolution level would result in an improved outcome.^{2, 3, 4, 5, 6, 7} Despite pertinent data derived from similar analyses of UCB transplant being scanty, studies on unrelated UCB transplant suggested that the HLA matching may not have to be as close as that needed for bone marrow or peripheral blood stem cell transplants. Recently, Kogler et al.⁸ reported that the numbers of HLA mismatches on high-resolution level were not associated with acute graft-versus-host disease (GvHD) and overall survival after UCB transplant. We performed high-resolution HLA typing for 65 UCB-recipient pairs who had been typed previously at low-resolution level and evaluated the correlation of HLA disparity with engraftment, graft failure, acute GvHD, transplant-related mortality and long-term overall survival.

Materials and methods

Patients and grafts

From March 1999 to September 2005, a total of 65 patients (58 children, seven adults) transplanted with unrelated UCB from Guangzhou Cord Blood Bank at 24 transplant centers in China (see Acknowledgments for details of transplant centers and numbers of patients contributed) were studied on the basis of available pretransplanted DNA samples from UCB-recipient pairs for high-resolution HLA typing. None of the patients received multiple units of UCB, nor had they undergone previous hematopoietic stem cell transplantation. UCB units were unmanipulated; there was no ex vivo expansion or T-cell depletion. The selection of an appropriate UCB unit was at the discretion of the transplanters according to their institutional protocols.

Firstly, HLA-identical UCB units with high cellularity were chosen. Additionally, due weight was given to the least HLA-disparate UCB units with a nucleated cell dose of not less than 1.5 10⁷/kg recipient body weight. UCB units were collected and processed in Guangzhou Cord Blood Bank using good manufacturing practices and standard operating procedures for donor eligibility, collection, characterization, processing, storage and release from quarantine.⁹

Data for clinical outcome were collected from transplant centers using a standard template and analyzed. The median duration of follow-up was 18 months (range: 3.5–72). Table 1 shows the demography, conditioning regimen, GvHD prophylaxis, immunotherapy and cell dose infused of the cohort of patients in the study.

Table 1 - Demography of patients having undergone UCB transplant.

Full table

HLA typing

Blood samples from UCB units and patients were typed before transplant by low-resolution polymerase chain reactions using sequence-specific oligonucleotide probes and sequence-specific primers for HLA-A, -B and -DRB1 as described previously.⁹ DNA samples from UCB-recipient pairs were retyped by high-resolution sequence-based typing in this study.

Clinical outcome

Hematopoietic recovery was defined as an absolute neutrophil count rebounding from a nadir to >0.5 10⁹/l for three consecutive days and independency from platelet transfusion with a count >20 10⁹/l. Failure of engraftment was defined by the absence of neutrophil recovery at day 60 post transplant, or the necessity for a second transplant, or for hematopoietic reconstitution with autologous hematopoietic stem cells. In the analysis of immunoreactivity, the GvH vector was labeled when the recipient's alleles were not shared by UCB donor, whereas host-versus-graft (HvG) vector was attributed to alleles of the UCB donor not shared by the recipient. All patients were considered at risk for the occurrence of GvHD post-transplant. Acute and chronic GvHD were graded at each transplant center according to previously described criteria.¹⁰ Transplant-related mortality was identified as all causes of nonrelapse deaths occurring within 100 days post-transplant. Overall survival was the time between transplant and death due to any cause.

Statistical analysis

The following parameters and end points were analyzed for potential prognostic values of UCB transplant: sex, age, ABO-compatibility, disease (malignancy vs nonmalignancy), pretransplant disease status in patients with leukemia (progression vs remission), infused nucleated cells and CD34⁺ cells per kg body weight of patient, conditioning regimen and use of antithymocyte globulin. Correlation of low- and high-resolution HLA disparity with clinical outcome was analyzed. Probabilities of neutrophil engraftment, acute GvHD, transplant-related mortality and overall survival were evaluated by Kaplan–Meier analysis. Univariate analyses were conducted because of the relatively small cohort of patients. All statistics were performed using SPSS version 13.0 statistical software package (SPSS Inc., Chicago, IL, USA).

Results

Types of mismatches at HLA-A, -B and -DRB1

High-resolution sequence-based typing delineated HLA alleles that were hardly demonstrated by low-resolution typing (high-resolution vs low-resolution: 6.2% (4) vs 12.3% (8) full matches, 27.7% (18) vs 63.1% (41) one-antigen mismatches, 35.4% (23) vs 24.6% (16) two-antigen mismatches, 20% (13) vs 0% (0) three-antigen mismatches, 10.8% (7) vs 0% (0) four-antigen mismatches). HLA disparities in the GvH direction (recipient's alleles not shared by UCB donor) and HvG direction (UCB donor's alleles not shared by the recipient) were noted in 91.4% (53/58) and 94.8% (55/58) UCB-recipient pairs, respectively (Table 2).

Table 2 - Low- and high-resolution HLA typing of 65 UCB-recipient pairs.

Full table

Engraftment

In the cohort of 65 patients, three died within 28 days and were not evaluable for engraftment. There were 15 episodes of non-engraftment accounting to 24.2%. A median number of days of neutrophil engraftment of 47 patients was 18 (9–42), inferring a probability of 86% at day 60 (Figure 1a). A median time for platelet engraftment was day 38 (12–116). Pertaining to the pathologic disease, the engraftment failure rate was 15.4% (2/13) in acute myeloblastic leukemia, 9.1% (2/22) in acute lymphoblastic leukemia, 10% (1/10) in chronic myelocytic leukemia, 75% (3/4) in severe aplastic anemia, 66.7% (6/9) in -thalassemia and 100% (1/1) in metabolic disease. Graft rejection was evident in a patient transplanted for -thalassemia and another for acute myeloblastic leukemia, accounting for 4% (2/50) of engrafted cases.

Figure 1.

Kaplan–Meier analyses of patients who underwent umbilical cord blood transplant. (a) Neutrophil recovery in 62 patients, (b) acute GvHD grade II–IV in 63 patients, (c) acute GvHD grade III–IV, (d) transplant-related mortality and (e) long-term 6-year overall survival in 64 patients.

Full figure and legend (48K)

Univariate analysis of the patient-, disease- and graft-related characteristics depicted three contributing factors of engraftment failure: disease (malignancy vs nonmalignancy), status before transplant in leukemia (progression vs remission) and conditioning (total body irradiation (TBI) vs non-TBI, Table 3). On the basis of low-resolution typing, one to two HLA-A disparities in the HvG direction were associated with an increasing risk of non-engraftment (Table 4). However, this scenario was not noted in transplants with other HLA-locus mismatches at either low- or high-resolution level of HLA typing (Table 4).

Table 3 - Univariate analyses of transplant-related parameters in the cohort of 65 patients.

Full table

Table 4 - Univariate analyses of engraftment failure, acute GvHD, mortality and survival.

Full table

GvHD

Among 63 evaluable patients, acute GvHD was not evident in 19 (30.2%), whereas 44 (69.8%) patients developed grade I–IV acute GvHD (grade I: 17 (27.0%), grade II: 16 (25.4%), grade III: 6 (9.5%), and grade IV: 5 (7.9%)). The probabilities of acute GvHD grades II–IV and III–IV at day 100 were 57 and 33%, respectively (Figure 1b–c). It was noteworthy that patients transplanted for malignancy were prone to develop acute GvHD as compared to patients transplanted for nonmalignant disease or disorder (Table 3). On the contrary, there was no correlation of acute GvHD with recipient sex, age, ABO compatibility, disease status, conditioning and the use of antithymocyte globulin (Table 3). Forty-four patients who developed acute GvHD were stratified into three arms of HLA disparity (HLA-A/-B, DRB1 and HLA-A/B+DRB1). Statistical analyses showed no association of the extent of HLA disparity, on both low- and high-resolution level, to the development of grade II–IV acute GvHD (Table 4). Five patients developed chronic GvHD (four limited and one extensive). The association of HLA disparity with chronic GvHD was not analyzed in this small cohort of patients.

Transplant-related mortality and overall survival

There were 14 episodes of transplant-related mortality accounting for 23% at day 100 (Figure 1d). The long-term 6-year overall survival was 59% (Figure 1e). Univariate analyses demonstrated that the long-term overall survival correlated to the numbers of infused nucleated cells and CD34⁺ cells (Table 3). TBI in the conditioning of patients for UCB transplant was also noted to exert a significant impact on transplant-related mortality and overall survival, despite not being able to rule out readout bias attributable to the small number of patients undergoing TBI (Table 3). There was no association of HLA disparity with transplant-related mortality (Table 4). A significantly lower rate of overall survival was noted in patients transplanted with HLA-disparate UCB at locus A or B and DRB1 on low-resolution level. Nonetheless, the trend failed to reach a statistical significance when the HLA results of UCB-recipient pairs were reanalyzed by high-resolution sequence-based typing (Table 4).

Discussion

Data from the retrospective study in a limited cohort of 65 ethnic Chinese patients transplanted with unrelated UCB from Guangzhou Cord Blood Bank in China suggest that high-resolution typing for HLA-A, -B and -DRB1 helped little in predicting improved clinical outcome. There was no significant difference between non-engraftment, development of acute GvHD, transplant-related mortality and overall survival with the extent of HLA disparity. The results revealed for the first time that graft failure might be attributed to low-resolution HLA-A disparity in the HvG direction between Chinese UCB-recipient pairs, and that the disparities of HLA-A/B and DRB1 might compromise the long-term overall survival of UCB transplant patients.

HLA-C and HLA DQB1 typing were not taken into account in this study. However, our findings were in accordance with the pioneer work of Kogler et al.,⁸ who reported that the probabilities of acute GvHD grade II–IV and 2-year overall survival had no correlation with the numbers of high-resolution mismatches for HLA-A, -B and -DRB1 and HLA-A, -B, -C, -DRB1 and -DQB1 in a heterogeneous group of 122 patients who underwent UCB transplant. In both studies, only a few UCB-recipient pairs were identical for HLA-A, -B and -DRB1 on a high-resolution level (HLA-matched UCB-recipient pairs: the present series vs the French series, 6.2% (4/65) from 12.3% (8/65) vs 5.7% (7/122) from 13.1% (16/122)). The HLA disparity between UCB-recipient pairs was higher than expected. If the clinical relevance of high-resolution HLA alleles is unclear in UCB transplant,^{11, 12} this may raise unnecessary barriers in deciding the best available UCB donor for a recipient.

Transplantation outcome is correlated with the infused cell dose.¹³ The cellularity of a UCB unit must be taken into account when considering the best available UCB graft for a particular recipient. As shown in this study, the infused cell dose is an important prognostic factor for survival (Table 3). More rapid hematopoietic recovery in UCB recipients of higher cell doses was reported.^{14, 15, 16} Wagner et al.¹³ noted a high survival rate in recipients of no more than two HLA-mismatched UCB grafts that contained at least 1.7 10⁵ CD34⁺ cells/kg of recipient body weight. Gluckman et al.¹⁷ observed no correlations of the number of HLA disparities to the treatment-related mortality and the overall survival of UCB recipients, despite the fact that the higher the number of HLA disparities, the higher the probability of acute GvHD grades III and IV and the lower the risk of relapse.

In routine hematopoietic stem cell transplantation, UCB will only be chosen should there be no HLA-identical sibling or HLA-matched unrelated donor available for the respective patients for bone marrow or peripheral blood stem cell transplant. Hematopoietic stem cells derived from bone marrow, or peripheral blood after cytokine mobilization, are biologically different to those derived from UCB in terms of the dynamics of hematopoietic and immune reconstitution of recipients post-transplant. In unrelated bone marrow or peripheral blood hematopoietic stem cell transplantation, high-resolution HLA typing was reported to improve clinical outcome.^{7, 18} However, the prerequisite for UCB transplant is entirely different. The probability and severity of acute GvHD observed in pediatric and adult recipients of HLA class I- and class II-disparate UCB grafts are lower compared to recipients of unrelated adult donor grafts.^{13, 19, 20, 21} The immaturity and low number of immunocompetent donor T cells may underline the reduced allo-reactivity after UCB transplant.^{22, 23} The probabilities of acute GvHD grades II–IV and III andIV were 57 and 33%, respectively, being higher to those reported by others in the literature.^{8, 24, 25} These scenarios might be attributable to the infrequent administration of antithymocyte globulin in conditioning patients for UCB transplant (9.2% (6/65)), which had been proven to effectively reduce the incidence of both acute and chronic GvHD in a dose-dependent manner.²⁶

HLA is well known as a highly polymorphic genetic system. To date, more than 1900 alleles for HLA gene loci have been identified. Despite the ethnic Chinese population being the largest in the world, gene frequencies of many HLA alleles wait to be unveiled. There is little literature describing the ethnic Chinese HLA diversity, hence a relatively homogeneous to a more heterogeneous HLA allele distribution cannot be generalized and compared to other ethnic populations. Apart from the benefit of the HLA homogeneity of the donor population, the likelihood of finding an HLA-matched unrelated donor for patients is related to the number of donors and the racial origin of the patient and of the majority of the donors on the registry. The difficulty in establishing consensual guidelines for donor selection based on HLA incompatibilities could also be related to the heterogeneity of the patient population. Nonetheless, ethnic donor registries are important to cater for populations of specific ethnic origin. Hawkins et al.²⁷ documented the genetic heterogeneity of the Chinese population in the analysis of the Hong Kong Marrow Match Foundation data, where >95% of the registered donors were ethnic Chinese. The lack of influence of high-resolution matching of UCB on several clinical outcome measures, despite the heterogeneous study population, is significant and should be validated. The choice of the best available UCB unit of a high cellularity might partially negate the passive impact of HLA disparity in UCB transplant.

In China, there is a great demand for hematopoietic stem cell transplant for patients with high-risk malignancies and hereditary disorders who are hardly manageable by conventional therapies. However, the maneuver of hematopoietic stem cell transplant is hampered by the lack of HLA-matched donors and the constraint of resources. Data from the study suggest that the use of high-resolution sequence-based typing did not improve substantially the clinical outcome of UCB transplant, whereas low-resolution HLA typing appears to be a feasible approach in matching UCB-recipient pairs for transplant in developing countries with limited resources. Should our observation be confirmed in a larger study, the practice of evidence-based strategy in choosing the best UCB donation can benefit more patients in need of hematopoietic stem cell transplant.

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Acknowledgements

We thank the clinicians of 24 transplant centers in China for performing UCB transplants and providing clinical data of 65 patients in this study, namely; The Second Affiliated Hospital of Sun Yat Sen University, Guangzhou, Guangdong Province (n=18); Anhui Provincial Hospital, Hefei, Anhui Province (n=11); Shanghai Children's Medical Center, Shanghai (n=4); The First Affiliated Hospital of Jinan University, Guangzhou, Guangdong Province (n=4); Guangzhou Maternal and Neonatal Hospital, Guangzhou, Guangdong Province (n=3); Lu Dao Pei Ward, Affiliated Hospital, Peking University, Beijing (n=3); Peking Children's Hospital, Beijing (n=2); Naval General Hospital, Beijing (n=2); Guangdong Provincial People Hospital, Guangzhou, Guangdong Province (n=2); Guangzhou First Municipal People's Hospital, Guangzhou, Guangdong Province (n=2); Affiliated Xijing hospital, The Fourth Military Medical University, Xian, Shanxi Province (1); Affiliated Hospital of Guiyang Medical College, Guiyang, Guizhou Province (n=1); The 12th Municipal People's Hospital, Guangzhou, Guangdong Province (n=1); Haikou People Hospital, Haikou, Hainan Province (n=1); Henan Cancer Hospital, Zhengzhou, Henan Province (n=1); Institute of Hematology, Huazhong University of Science and Technology, Wuhan, Hubei Province (n=1); Military General Hospital, Guangzhou, Guangdong Province (n=1); Nanfang Hospital, Guangzhou, Guangdong Province (n=1); Soochow University Affiliated Children's hospital, Suzhou, Jiangsu Province (n=1); The First People's Hospital of Foshan, Foshan, Guangdong Province (n=1); The Second Guangdong Provincial People's Hospital, Guangzhou, Guangdong Province (n=1); The Third Affiliated Hospital of Sun Yat Sen University, Guangzhou, Guangdong Province (n=1); Tongji Hospital of Tongji Medical College, Wuhan, Hubei Province (n=1) and West China Hospital, Sichuan University, Chengdu, Sichuan Province (n=1).

This work was supported in part by funding of projects #96-Z-64-1 and #2003C34006 of Guangdong Provincial Scientific Technological Committee, the project #99-Z-008-01 of Guangzhou Scientific Technological Committee, Five Ones Project #1996-23 of Guangdong Provincial Health Bureau and the scientific technological project of Guangzhou Health Bureau in 1996, China.