Single- or double-unit UCBT following RIC in adults with AL: a report from Eurocord, the ALWP and the CTIWP of the EBMT

Background The feasibility of cord blood transplantation (CBT) in adults is limited by the relatively low number of hematopoietic stem/progenitor cells contained in one single CB unit. The infusion of two CB units from different partially HLA-matched donors (double CBT) is frequently performed in patients who lack a sufficiently rich single CB unit. Methods We compared CBT outcomes in patients given single or double CBT following reduced-intensity conditioning (RIC) in a retrospective multicenter registry-based study. Inclusion criteria included adult (≥18 years) patients, acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL), complete remission (CR) at the time of transplantation, first single (with a cryopreserved TNC ≥ 2.5 × 107/kg) or double CBT between 2004 and 2014, and RIC conditioning. Results Data from 534 patients with AML (n = 408) or ALL (n = 126) receiving a first single (n = 172) or double (n = 362) CBT were included in the analyses. In univariate analysis, in comparison to patients transplanted with a single CB, double CB recipients had a similar incidence of neutrophil engraftment but a suggestion for a higher incidence of grade II–IV acute GVHD (36 versus 28%, P = 0.08). In multivariate analyses, in comparison to single CBT recipients, double CBT patients had a comparable incidence of relapse (HR = 0.9, P = 0.5) and of nonrelapse mortality (HR = 0.8, P = 0.3), as well as comparable overall (HR = 0.8, P = 0.17), leukemia-free (HR = 0.8, P = 0.2) and GVHD-free, relapse-free (HR = 1.0, P = 0.3) survival. Conclusions These data failed to demonstrate better transplantation outcomes in adult patients receiving double CBT in comparison to those receiving single CBT with adequate TNC after RIC. Electronic supplementary material The online version of this article (doi:10.1186/s13045-017-0497-9) contains supplementary material, which is available to authorized users.


Background
Allogeneic umbilical cord blood transplantation (CBT) is a treatment option for many patients with acute myeloid (AML) or acute lymphoblastic (ALL) leukemia who lack an HLA-matched donor [1][2][3][4]. In the last two decades, the development of reduced-intensity conditioning (RIC) regimens for CBT has allowed extending its use to patients who were deemed ineligible for myeloablative (MAC) conditioning because of older age or medical comorbidities [5][6][7][8][9][10][11]. We recently compared outcomes of AML or ALL patients given CBT after RIC (n = 415) versus MAC (n = 479) regimens. We observed that, in comparison to MAC patients, RIC recipients had a higher incidence of disease relapse and a lower nonrelapse mortality (NRM), translating to comparable leukemia-free (LFS), GVHD-free, relapse-free survival (GRFS), and overall (OS) survival [11].
Previous studies have demonstrated poor outcomes in patients receiving CB graft containing <2.5 × 10 7 total nucleated cells (TNC) per kilogram at cryopreservation, particularly in the presence of human leukocyte antigen (HLA)-mismatches [12]. Unfortunately, many adult patients lack a sufficiently rich CB unit to allow safe CBT. Based on these observations, the Minnesota group pioneered the infusion of two CB units from different partially HLA-matched donors (dCBT) for patients who lack a sufficiently rich single CB unit [13]. Based on preliminary encouraging results, this approach has been extended to patients who had a single CB unit containing >2.5 × 10 7 total nucleated cells (TNC) per kilogram at cryopreservation [14]. This has been particularly the case in the setting of RIC-CBT since it was hypothesized that in comparison with single CBT, double CBT might promote engraftment and increase graft-versus-leukemia effects [15]. The later might be due at least in part via graft-versus-graft alloreactivity as recently demonstrated [16].
In a previous study, we compared transplantation outcomes of adult AML or ALL patients transplanted with one single CB or two CB units after myeloablative conditioning regimen (n = 239) [17]. Among patients transplanted with one single CB unit (sCBT), those receiving a thiotepa, busulfan, and fludarabine (TBF) regimen had better LFS than those transplanted with busulfan-or total body irradiation (TBI)-based regimens. When the sCBT group was restricted to patients given TBF-based conditioning, transplantation outcomes were comparable between patients receiving sCBT or dCBT, with the exception for a higher incidence of grade II-IV acute GVHD in dCBT recipients. Similarly, two recent prospective randomized studies demonstrated that dCBT following myeloablative conditioning failed to improve transplantation outcomes in comparison to sCBT in children and/or young adult patients who had a sufficiently rich single CB unit [18,19].
In the current registry study, we investigated whether these observations remained true in the setting of adults after RIC CBT, which depends primarily on engraftment of donor immune cells and on graft-versus-leukemia effects for disease eradication.

Data collection
This survey is a retrospective, multicenter registrybased study performed by the Acute Leukemia Working Party (ALWP) of the European Society for Blood and Marrow Transplantation (EBMT) and by Eurocord. EBMT registry is a voluntary working group of more than 500 transplant centers, participants of which are required once a year to report all consecutive stem cell transplantations and follow-up. Audits are routinely performed to determine the accuracy of the data. Eurocord collects data on CBT performed in >50 countries worldwide and >500 transplant centers, mainly EBMT centers. Inclusion criteria were adult (≥18 years) patients, AML or ALL, complete remission (CR) at the time of transplantation, first single (with a cryopreserved TNC ≥2.5 × 10 7 /kg) or double CBT between 2004 and 2014, and RIC conditioning. RIC was defined as use of fludarabine associated with <6 Gy TBI, or busulfan ≤8 mg/kg, melphalan ≤140 mg/m 2 or other nonmyeloablative drugs, as previously reported [11,20,21]. HLA-compatibility requirements followed the current practice of antigen level typing for HLA-A and -B and allele level typing of HLA-DRB1. CB units were 4-6/6 HLA-A, -B, and -DRB1 matched to the recipient and to the other unit in case of dCBT in most patients. However, more recently, some centers are no longer matching the CB units between them with regard to HLA based on the study by Avery et al. [22]. HLA disparities between each unit and the recipient and between the two units were not necessarily at the same loci. Grading of acute and chronic GVHD was performed using established criteria [23].
For the purpose of this study, all necessary data were collected according to EBMT and Eurocord guidelines.

Statistical analyses
Data from all patients meeting the inclusion/exclusion criteria were included in the analyses. Start time was date of transplant for all endpoints. Neutrophil engraftment was defined as first of three consecutive days with a neutrophil count of at least 0.5 × 10 9 /L. Platelet engraftment was defined as the first of seven consecutive days of an unsupported platelet count of at least 20 × 10 9 /L [2].
To evaluate the relapse incidence, patients dying either from direct toxicity of the procedure or from any other cause not related to leukemia were censored. NRM was defined as death without experiencing disease recurrence. Patients were censored at the time of relapse or of the last follow-up. Cumulative incidence functions were used for relapse incidence and NRM in a competing risk setting since death and relapse were competing together.
For estimating the cumulative incidence of chronic GVHD, death was considered as a competing event. OS and LFS were estimated using the Kaplan-Meier estimates. GRFS was defined as being alive with neither grade III-IV acute GVHD, severe chronic GVHD nor disease relapse [24]. Univariate analyses were done using Gray's test for cumulative incidence function and log rank test for OS and LFS. Associations between single or double CBT and transplantation outcomes (chronic GVHD, relapse, NRM, LFS, and OS) were evaluated in
After adjusting for potential confounding factors in multivariate analyses, dCBT and sCBT recipients had a similar risk of relapse (HR = 0.9; 95% CI 0.6-1.3, P = 0.    Table 3). The only factor associated with lower OS in multivariate analysis was the use of ATG (HR = 1.8; 95% CI 1.2-2.8, P = 0.01). This was due to a significantly higher NRM in ATG in comparison with non-ATG recipients (HR = 2.4; 95% CI 1.3-4.3, P < 0.001) while relapse incidence was not affected by ATG (HR = 1.0, 95% CI 0.6-1.9, P = 1.0). As shown in the Table 4 and in the Additional file 1: Table S2, causes of death were not statistically different between sCBT and dCBT recipients. However, there was a suggestion for more deaths from GVHD (13/362 (3.5%) versus 3/172 (1.7%)) in dCBT than in sCBT recipients in the first 100 days after transplantation, while the incidence of death from infection in the first 100 days after CBT was comparable between the 2 groups (22/362 (6.1%) in dCBT versus 13/172 (7.6%) in sCBT recipients, respectively).

Subgroup analyses
To further dissect the impact of sCBT versus dCBT, we performed additional (univariate) Cox analyses separately for various pre-transplant/transplant variables. The results of these analyses are presented graphically using Forest plots in Figs. 2 and 3. There were no interactions between patient age at transplantation, patient gender, number of cells infused, disease type, disease status, HLA-matching and conditioning type (TCF versus other), and the association between sCBT versus dCBT and GRFS or OS. Further multivariate Cox models assessing possible interactions between ATG and sCBT versus dCBT demonstrated the

Impact of cell dose
We finally assessed what was the combined impact of cell dose and sCBT versus dCBT. In order to address this issue, we performed multivariate Cox models including four graft type groups: sCBT and Fig. 3 Forest plot analysis of GVHD-free relapse-free survival a and overall survival b. HR and 95% confidence intervals were computed using univariate Cox analyses TNC above median, dCBT and TNC above median, sCBT and TNC below median, and dCBT and TNC below median. As observed in the Table 5, in comparison to the reference group (sCBT and TNC above median), patients given sCBT with TNC below the median had a higher risk of relapse (HR = 2.0, 95% CI 1.0-3.9, P = 0.04) and a suggestion for a worse LFS (HR = 1.5, 95% CI 0.9-2.3, P = 0.11), while outcomes were comparable between patients receiving sCBT and TNC above median and those given dCBT (irrespective of the cell dose received).

Discussion
Umbilical CB units contain a limited number of hematopoietic cells. This is unfortunate given that cell dose is one of the main predictive factors for CBT outcomes [26][27][28]. Transplantation of two CB units has been introduced by investigators from the university of Minnesota to increase the cell dose infused [13,29]. Preliminary studies have demonstrated that this strategy allowed safe CBT in adult patients who lacked a sufficiently rich CB unit [30]. Further studies observed that dCBT induced graft-versusgraft reactions that could increase alloreactivity and perhaps graft-versus-leukemia effects [15]. This prompted us to compare post-transplantation outcomes in patients with acute leukemia receiving sCBT or dCBT after RIC, a transplantation approach that depends mainly on graft-versusleukemia effects for tumor eradication [31,32]. Several observations were made. A first observation was that indeed, dCBT allowed safe CBT in adult patients who lacked a CB unit containing at least 2.5 × 10 7 TNC/kg since OS and LFS were at least as good in these patients than in those transplanted with a single CB unit containing ≥2.5 × 10 7 TNC/kg. This is in concordance with the observations reported by the University of Minnesota [30].
A second observation was that patients who received dCBT had a similar incidence of relapse than those given sCBT. This was also true when comparing the relapse incidence in patients receiving sCBT with TNC > median to those receiving dCBT with TNC > median. These observations suggest that graft-versus-leukemia effects are comparable after sCBT or dCBT. A comparable incidence of relapse in patients receiving sCBT or dCBT has also been observed in recent registry [17,30,33] or prospective randomized [18,19] studies including patients given CBT after myeloablative conditioning. Other approaches to decrease relapse incidence after CBT might include post-transplant administration of disease-targeted medications [34][35][36] or of chimeric antigen receptor T cells [37].
In multivariate analyses, sCBT and dCBT patients had comparable NRM, LFS, GRFS, and OS. These observations are also in accordance with those made in patients receiving CBT after myeloablative conditioning [17-19, 30, 33, 38]. Subgroup analyses revealed no interaction between patient age at transplantation, patient gender, number of cells infused, disease type, disease status, HLA-matching, use of ATG and conditioning type (TCF versus other), and the associations between sCBT versus dCBT and GRFS or OS.
The current study also confirmed a detrimental impact of ATG on NRM (leading to a significantly inferior OS) as recently reported in a study including data from patents given CBT after myeloablative conditioning [39] or RIC dCBT [40]. Further, despite ATG not only induces in vivo T cell depletion of the graft but also promotes the generation of regulatory T cells [41,42], ATG failed to prevent chronic GVHD in the current study, in contrast to what has been observed in peripheral blood stem cell recipients [43][44][45]. These results are also in accordance with those reported by Admiraal et al. who demonstrated that reducing the exposure of ATG after CBT (allowing early CD4+ T cell recovery) improved outcomes in pediatric CBT [46]. There are some limitations in our study including its design (retrospective registry survey) and the relative imbalance in the two groups such as more frequent use of the TCF conditioning regimen but less frequent use of ATG in dCBT patients. These differences were carefully adjusted for in multivariate analyses. Another potential limitation of the study is a potential lack of statistical power to detect small advantages of one group to another. However, the number of patients included in the current study (n = 534) is higher than the number of patients included in prior registry studies in adults (n = 409 in the CIBMTR study [30] and n = 239 in the Eurocord/EBMT study [17]) or in recent prospective randomized studies in children (n = 224 in the study reported by Wagner et al. [18] and n = 151 in the study reported by Michel et al. [19]). Nevertheless, further prospective randomized studies in the RIC setting are needed to draw definitive conclusions. Finally, further studies should compare outcomes after CBT or HLA-haploidentical transplantation following RIC regimens [47][48][49].

Conclusions
In summary, we observed comparable outcomes in patients given dCBT or sufficiently rich sCBT with a TNC dose at cryopreservation >2.5 × 10e7/Kg. Recent advances in the field of CBT expansion are likely to improve outcomes of RIC sCBT [50].