Cord Blood Stem Cells

Double umbilical cord blood transplantation with reduced intensity conditioning and sirolimus-based GVHD prophylaxis

Abstract

The main limitations to umbilical cord blood (UCB) transplantation (UCBT) in adults are delayed engraftment, poor immunological reconstitution and high rates of non-relapse mortality (NRM). Double UCBT (DUCBT) has been used to circumvent the issue of low cell dose, but acute GVHD remains a significant problem. We describe our experience in 32 subjects, who underwent DUCBT after reduced-intensity conditioning with fludarabine/melphalan/antithymocyte globulin and who received sirolimus and tacrolimus to prevent acute GVHD. Engraftment of neutrophils occurred in all patients at a median of 21 days, and platelet engraftment occurred at a median of 42 days. Three subjects had grade II–IV acute GVHD (9.4%) and chronic GVHD occurred in four subjects (cumulative incidence 12.5%). No deaths were caused by GVHD and NRM at 100 days was 12.5%. At 2 years, NRM, PFS and OS were 34.4, 31.2 and 53.1%, respectively. As expected, immunologic reconstitution was slow, but PFS and OS were associated with reconstitution of CD4+ and CD8+ lymphocyte subsets, suggesting that recovery of adaptive immunity is required for the prevention of infection and relapse after transplantation. In summary, sirolimus and tacrolimus provide excellent GVHD prophylaxis in DUCBT, and this regimen is associated with low NRM after DUCBT.

Introduction

Umbilical cord blood (UCB) transplantation (UCBT) is a viable option for patients requiring allogeneic transplantation when a suitable adult donor is not available. The main limitation to the broader use of UCBT is the small number of hematopoietic progenitors found in an UCB unit. As a result of this low number, engraftment is often delayed, immunologic reconstitution is less complete, and treatment-related mortality often exceeds that seen with traditional adult donors.1, 2 Several strategies have been used to ameliorate outcomes after UCBT, including the use of reduced-intensity conditioning (RIC), and the simultaneous or sequential transplantation of two UCB units (double UCBT, DUCBT). The engraftment characteristics of DUCBT appear to be similar to that of traditional BMT, with a median time to neutrophil engraftment of approximately 12–23 days.3, 4

Although UCBT is often associated with a reduction in the incidence and severity of acute GVHD,2 our previous experience and the experience of the Minnesota group has demonstrated that GVHD remains a challenging problem after DUCBT.3, 4 In adults, the use of cyclosporine and mycophenolate mofetil is associated with a grade II–IV acute GVHD rate of 40–58%.3, 5 We hypothesized that the use of sirolimus and tacrolimus in the DUCBT setting would lead to a reduction in the rate of grade II–IV acute GVHD.

Sirolimus is a potent immunosuppressant that prevents T cell-mediated alloimmunity through a variety of mechanisms.6 In addition, it may also be immunosuppressive through its effects on APCs.7 We have previously demonstrated that sirolimus is efficacious in preventing acute GVHD after related and unrelated adult donor transplantation.8 The use of sirolimus in UCBT would be particularly attractive, as CMV reactivation after UCBT is common, and sirolimus may have independent suppressive effects on CMV.9

This report details our phase II experience using the immunosuppressive regimen of sirolimus and tacrolimus after RIC and DUCBT.

Methods

This research protocol was reviewed and approved by the Institutional Review Board of the Dana Farber/Harvard Cancer Center. Written informed consent was obtained from all patients before the enrollment and participation. The trial was prospectively registered at http://www.clinicaltrials.gov (NCT00133367).

Patients

Patients were eligible to participate in this research study if they had no 6/6 or 5/6 HLA-matched related donor or 10/10 matched unrelated donor, or if an unrelated donor was not available within the time frame necessary to perform a potentially curative SCT. Patients were between the ages of 18–65 years, had an Eastern Cooperative Oncology Group performance status of 0–2, had adequate measures of hepatic and renal function and met standard transplant eligibility criteria including cardiac ejection fraction >40% and a diffusing capacity of the lung for carbon monoxide >50% of predicted.

Malignant disease criteria for the entry included acute leukemia in second or subsequent remission or in first remission with adverse cytogenetics or an antecedent hematologic disorder. Patients with myelodysplastic syndrome were eligible with any World Health Organization subtype. Patients with CML were eligible if they had accelerated or second stable phase disease, or were intolerant to tyrosine kinase inhibitors. Patients with lymphoma were eligible in second or subsequent CR or with chemotherapy-sensitive PR. Patients with CLL had Rai stage III or IV disease, a lymphocyte doubling time of <6 months, or with earlier stage disease after disease progression with 2 chemotherapy regimens, while in PR.

HLA typing and umbilical cord unit selection

UCB units were obtained from a variety of national and international registries. UCB units had to meet a minimum combined pre-cryopreservation cell dose of 3.7 × 107 total nucleated cells (TNC)/kg and each individual unit was required to have a minimum of 1.5 × 107 TNC/kg prior to cryopreservation. Confirmatory HLA typing was performed on all UCB units before the transplantation using PCR and sequence-specific primer technology (One Lambda, Canoga Park, CA, USA). UCB units were required to be a 4/6 match or better at the allele level for HLA-A, -B and -DRβ1 with each other and with the recipient. Typing at HLA-C and -DQ was performed but was not used in the search strategy. The choice of UCB units, when multiple units were available, was hierarchically based on a higher cell dose, greater HLA compatibility and a younger age of the cord blood unit. Other factors considered in selection were, when available, CD34+ cell dose, colony-forming unit assays and cellular viability testing. All units were screened for hemoglobinopathies.

Treatment plan

Patients received pretransplant conditioning therapy with fludarabine (30 mg/m2/day) on 6 consecutive days (days −8 through −3; total dose 180 mg/m2), melphalan (100 mg/m2) on day −2, and rabbit antithymocyte globulin (thymoglobulin, 1.5 mg/kg/day) on 4 alternating days (days −7, −5, −3, −1; total dose 6.0 mg/kg). UCB stem cells were infused on day 0. UCB units were thawed according to the methods of Rubinstein et al.,10 and administered sequentially between 1 and 6 h apart. The larger of the two units, based on precryopreservation TNC dose, was administered first.

GVHD prophylaxis began on day −3 and consisted of continuous intravenous infusion tacrolimus (target serum level 5–10 ng/mL) and a 12 mg oral loading dose of sirolimus. Sirolimus was then dosed orally once daily to maintain a serum trough level of 3–12 ng/mL, as previously described.8 Tacrolimus was given orally before the discharge. In the absence of GVHD, both GVHD prophylaxis agents were tapered from day 100 through 180, or earlier at the discretion of the treating physician. GVHD was graded according to the consensus criteria.11

After transplantation, patients received supportive care with transfusion support. Bacterial prophylaxis consisted of levofloxacin until neutrophil engraftment. Antifungal prophylaxis consisted of fluconazole until day +100. All patients received acyclovir as antiviral prophylaxis. Prophylaxis against Pneumocystis jirovecii was started upon hospital discharge. Monitoring for reactivation of CMV was performed weekly, whereas monitoring for EBV and human herpesvirus-6 reactivation were performed every other week. Pre-emptive therapy against CMV was administered upon viral reactivation.

Filgrastim (5 mcg/kg/day) was administered from day +5 until the absolute neutrophil count was greater than 2.0 × 109 cells/L for 2 consecutive days. Donor chimerism was monitored according to previously described methodology.3 Briefly, unfractionated donor chimerism was determined from PBMCs by analyses of informative short tandem repeat loci using the ABI Profiler-Plus Kit (Applied Biosystems Inc, Bedford, MA, USA) and the ABI 310 Genetic Analyzer. Chimerism was measured routinely at 30 and 100 days, and then regularly through the first 2 years after transplantation. Results are expressed as a median of two simultaneous assays, with a 5% margin of error.

Immunologic reconstitution monitoring

Whole blood was collected in EDTA containing lavender top tubes (Kendall Vacutainer, Mansfield, MA, USA). The blood samples were obtained from patients immediately before the transplantation and at 4, 8, 12, 26 and 52 weeks after transplantation. Leukocyte populations and specific lymphocyte subpopulations were analyzed by flow cytometry using fluorescence-conjugated monoclonal antibodies directed against lineage-specific cell surface markers. Flow cytometry was performed using either a FC500 (Beckman Coulter, Miami, FL, USA) or a BD FACSCanto (BD Biosciences, San Jose, CA, USA) flow cytometer and data was analyzed using either Beckman Coulter CXP software or BD FACSDiva software.

Thymopoiesis was measured via analysis of T cell receptor excision circles (TRECs). DNA was isolated from PBMCs using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA), and the DNA concentration determined by standard spectrophotometry. The quantitation of signal-joint TREC DNA was performed using Taqman real-time PCR following a previously described method using a Rotor-Gene 6000 thermal cycler (Corbett Life Science, Sydney, Austrialia).12 Quantitation of the signal-joint TREC copy number for each patient sample was performed using a standard curve prepared with 10-fold dilutions of a plasmid containing the signal-joint TREC sequence (kindly provided by Dr D Douek, NIH, Bethesda, MD, USA).

Statistical design and analysis

The study was designed as a single stage phase II trial. A sample size of 32 patients was planned with the hypothesis that the rate of GVHD would be less than 50%. With this design, the probability of concluding the rate is acceptable is 0.84 if the true underlying rate of developing grade II–IV acute GVHD was 40% and 0.09 if the true rate was 60%, using an exact binomial distribution.

Patients were enrolled between September 2005 and November 2007. The dataset for analysis was closed on March 1, 2009 and analyzed thereafter. Survival data were updated to 1 March 2010. Patient baseline characteristics were reported descriptively. Neutrophil engraftment was defined as the first of three consecutive days with neutrophil recovery to at least 0.5 × 109 cells/L. Platelet engraftment was defined as the first day of a platelet count of at least 20 × 109 cells/L, without supporting transfusion in the previous 3 days.

Cumulative incidence (CI) curves for acute GVHD and chronic GVHD were constructed reflecting early death and death or relapse as competing risks, respectively. CI curves for non-relapse mortality (NRM) and relapse with or without death were constructed reflecting time to relapse and time to NRM as competing risks. Time to relapse and time to NRM were measured from the date of stem cell infusion. OS was defined as the time from transplant to death from any cause, whereas PFS was defined as the time from transplant to progression or death from any cause. Surviving patients were censored at their date of last known follow-up. OS and PFS estimates were calculated using the method of Kaplan and Meier.13 Univariable and multivariable Cox regression analyses were performed for OS and PFS. Cellular phenotype data were included as log10-transformed time-varying covariates in the Cox model. All P-values are based on two-sided tests, and were computed using SAS v9.2 software (SAS Institute, Cary, NC, USA).

Results

In all, 32 sequential eligible subjects underwent DUCBT and are all included in this analysis. Patient and UCB characteristics are shown in Table 1. The median age was 53 years, whereas the median weight was 75.9 kg. The median time from diagnosis to DUCBT was 24.9 months (range 1.9–176.9 months), and the majority of patients had advanced lymphoid malignancies. The majority of patients had high-risk malignancies per standard criteria (63%).

Table 1 Subject and graft characteristics

UCB units and engraftment

Full cellular characteristics of the UCB units are available for 30 of the 32 recipients. The median single UCB size was 2.43 × 107 TNC/kg before the cryopreservation (range 1.51–3.94). The first infused unit had a median cell count of 2.67 × 107 TNC/kg, whereas the smaller second infused unit had a median cell count of 2.33 × 107 TNC/kg before the cryopreservation. When combined, the median total cell dose administered to recipients was 5.16 × 107 TNC/kg before the cryopreservation (range 3.66–7.58). The median number of CD34+ progenitors before the cryopreservation in the individual UCB units was 0.9 × 105 CD34+/kg (n=29 and 25 for the two units, range 0.2–3.4). When combined, the median dose of CD34+ progenitors was 1.9 × 105 CD34+/kg (range 0.5–4.1).

The CI of neutrophil engraftment was 100%. Recipients attained an absolute neutrophil count of 0.5 × 109/L at a median of 21 days from transplantation (range 13–70 days) (Figure 1). There were no cases of primary graft failure, however, three subjects had late graft loss. All three of these patients eventually had a recurrence of their malignancy, although graft rejection occurred in the absence of obvious malignant relapse. Three subjects did not attain platelet engraftment and transfusion independence. Overall, the median time to platelet engraftment was 42 days (range 25–162 days) (Figure 1). A total of 23 subjects attained a platelet count of 100 × 109/L and the median time to attain this platelet count was 89 days (range 26–209 days).

Figure 1
figure1

Cumulative incidence of neutrophil and platelet engraftment.

When stratified into two groups, based on the combined TNC dose above or below a cutoff of 5.0 × 107 TNC/kg, selected based on the median, there was no relationship between TNC and the time to neutrophil or platelet engraftment (P=0.32 and P=0.25 respectively). Similarly, there was no relationship with the combined CD34+ cell dose (cutoff of 2.0 × 105 CD34+/kg) administered and neutrophil or platelet engraftment (P=0.55 and P=0.91, respectively).

GVHD

Three subjects experienced grade II–IV acute GVHD at a median of 21 days from transplantation. Two subjects had isolated skin stage 3 GVHD (overall grade II GVHD), whereas the third subject had both skin and hepatic stage 3 disease (overall grade III GVHD). The CI of acute GVHD at 100 days was 9.4% (s.e.=5.2%). There were no deaths attributable to acute GVHD.

Four subjects were diagnosed with de novo chronic GVHD at a median of 212.5 days from transplantation. The CI of chronic GVHD at 1 year was 12.5% (s.e. 6.0%). All four subjects had cutaneous involvement, and one subject had additional oral involvement and bronchiolitis obliterans. One patient died of sepsis, while on immunosuppression for chronic GVHD. The other three patients died of relapse, secondary malignancy and trauma, respectively.

Treatment-related toxicity and mortality

The conditioning regimen was well tolerated. There were no cases of idiopathic pneumonia syndrome, thrombotic microangiopathy or veno-occlusive disease of the liver. One subject developed massive proteinuria requiring discontinuation of sirolimus, and two others developed an acneiform rash related to sirolimus not requiring discontinuation. CMV disease occurred in one subject despite a DNA-based monitoring and pre-emptive treatment strategy. Four patients developed meningoencephalitis, with the causative agent being human herpesvirus-6 and EBV in two subjects each. EBV reactivation led to the occurrence of post-transplantation lymphoproliferative disease (PTLD) in five individuals; all of these cases eventually were fatal.

NRM at 100 days from transplantation was 12.5% (s.e. 5.9%). At 2 years, NRM was 34.4% (s.e. 8.6%) (Figure 2).

Figure 2
figure2

Cumulative incidence of treatment-related mortality (TRM) and relapse.

Chimerism

Chimerism measurements at day 100 (±30 days) were assessed to determine the relative contribution to hematopoiesis in 29 surviving patients. At day 100, single umbilical cord dominance (defined as >80% contribution to hematopoiesis) was noted in 18 of 29 patients (62%), with both cords contributing to hematopoiesis in eight subjects. Three subjects had no evidence of cord engraftment at day 100 despite having evidence of donor chimerism at earlier time points. Of the 18 subjects with single cord predominance at day 100, 12 subjects had single cord hematopoiesis only, with no evidence of contribution by the other unit. Five of these 12 subjects had evidence of single unit early graft rejection or loss, as there was never any evidence of dual chimerism. Of the 18 subjects with single unit predominance at day 100, the first infused cord was responsible for the majority of the hematopoiesis in 11 (61%), whereas the second infused cord blood unit was responsible in the remaining 7 (39%) subjects. Thus, there was no statistical evidence that the initial cord infused was more likely to contribute to hematopoiesis in this limited sample (90% CI for the likelihood of equal contribution of two cord units 29–71%).

PFS and OS

The median follow-up of survivors is 48 months (range 30–62). To date, 12 subjects have relapsed. Seven of these patients had advanced lymphoid malignancies at the time of transplantation; however, five of these subjects remain alive. The median time to relapse after transplantation was 12.6 months (range 2.7–30.0), and the CI of relapse was 34.4% (s.e. 8.6%) at two years (Figure 2). PFS at 2 years is 31.2% (s.e. 8.2%), and OS at 2 years is 53.1% (s.e. 8.8%) (Figure 3). Causes of death are summarized in Table 2.

Figure 3
figure3

Kaplan–Meier estimates of PFS and OS.

Table 2 Causes of death

In a univariate analysis of factors related to outcome, age 50 years at the time of transplantation was associated with inferior OS, but not PFS (hazard ratio (HR) 3.37, 95% CI 1.14–9.88, P=0.03 for OS, HR 1.76, 95% CI 0.75–4.16, P=0.19 for PFS). In a multivariable model adjusted for age, sex, risk group and HLA matching, age retained significance for OS (HR 7.42, 95% CI 2.13–25.83, P=0.001) and now attained statistical significance for PFS (HR 2.87, 95% CI 1.11–7.40, P=0.03). The combination of two 4/6 HLA matches was associated with inferior OS (HR 4.05, 95% CI 1.37–12.05, P=0.01), but was not significant in the adjusted model for PFS when compared with other HLA combinations (HR 1.94, 95% CI 0.79–4.74, P=0.15) (Table 3). Disease risk was unassociated with overall and PFS outcomes in univariate analysis. Adding TNC dose (dichotomized by the cutoff of 5.0 × 107 TNC/kg) or CD34+ cell dose (dichotomized by the cutoff of 2.0 × 105 CD34+/kg) did not meaningfully change the HRs for OS, and themselves were not associated with outcome (N=30, HR 1.03, 95% CI 0.31–3.46 P=0.96 for TNC; N=24, 1.74, 95% CI 0.56–5.43 P=0.34 for CD34+ cell dose).

Table 3 Multivariable analysis of clinical factors related to OS and PFS

Immunologic reconstitution

The reconstitution of lymphocyte and monocyte populations was measured by flow cytometry in 27 subjects (Figure 4). The recovering CD4+ T cell population had a predominantly memory phenotype (CD45RO+) and the naive CD4+ population (CD45RA+) did not begin to recover until 6 months after transplantation (Figure 4b). Restoration of normal median values for both CD4+CD45RA+ and CD4+CD45RO+ T cells was not observed until we examined six patients who survived 2 years from transplantation (data not shown). Similarly, CD4+ regulatory T cells (CD4+CD25+) recovered very slowly and remained at low levels in peripheral blood throughout the first year after transplantation. CD8+ T cells also recovered gradually and were also predominately of memory phenotype (CD45RO+) in the first 3 months after transplantation (Figure 4c). CD8+ T cells with a naive phenotype (CD45RA+) began to recover 3–6 months after transplantation. In contrast to CD4+ T cells, the median values of CD8+CD45RA+ and CD8+CD45RO+ T cells reached the lower limits of normal values by 1 year after transplantation.

Figure 4
figure4

(ae) Immunologic reconstitution after transplantation. (a) Lymphocyte subsets (b) CD4+ lymphocyte subsets (c) CD8+ lymphocyte subsets (d) B lymphocyte and natural killer subsets (e) monocyte subsets. Median values are plotted on a log 10 scale and zero values are set equal to one. Error bars extend to the twenty-fifth and seventy-fifth percentiles.

Commencing at 6 months and continuing through 1 year after transplantation, there was a dramatic increase in CD20+ B cells resulting in the restoration of normal cell numbers. CD56+16+ natural killer cells maintained their pretransplantation level throughout the course of this study, at values slightly below the normal range, until 1 year after transplantation when they reached normal levels (Figure 4d). The median value for CD14+ monocytes doubled their pretransplantation level at 1 month after transplantation before falling to near pretransplantation values at 8 weeks and remained steady up to 12 months after transplantation. The CD14+ monocyte population remained at or above the upper limit of the normal control range throughout our study (Figure 4e).

In accordance with our analysis of CD4+ and CD8+ T cell numbers, median TREC values were below the limit of detection until 12 weeks after transplantation. We saw a substantial increase in the TREC copy number at 6 months (median number 117 copies/μg DNA), 1 year (median number 2136 copies/μg DNA) and 2 years after transplantation (median number 6749 copies/μg DNA). This increase in TREC copy number paralleled the increase in CD4+CD45RA+ naive T cell numbers from 6 months to 12 months after transplantation and the concordant plateau in CD4+CD45RO+ memory cells.

In multivariable models adjusted for age, HLA matching and risk group examining reconstitution of time-varying cellular immune parameters and outcome, several parameters were associated with both PFS and OS. Total lymphocyte count and lymphocyte subsets (CD3+, CD4+, CD4+CD45RA+, CD4+CD45RO+, CD8+ and CD8+CD45RA+) were all associated with improved OS. The maximal TREC value at 2 years (2000 vs <2000 copies/μg DNA) achieved per patient (N=22), was also associated with improved OS, in a non-time varying covariate model. Only recovery of CD56+CD16+ natural killer cells and maximal TREC copy number at 2 years were associated with improved PFS in univariate time-varying and non-time varying univariate models, respectively (Table 4).

Table 4 Multivariable modeling of outcomes with a time varying covariate adjusted for age, HLA match, and disease risk group

Discussion

In this study, we report favorable GVHD outcomes after reduced-intensity double UCB SCT when sirolimus and tacrolimus are used as GVHD prophylaxis. GVHD rates were very low, and in addition, we demonstrate early engraftment and low 100 day NRM. At 2 years, OS was over 50% in a cohort of patients with high-risk malignancies, including a high proportion of patients with resistant lymphoid malignancies.

The most striking difference in this experience in comparison with previous DUCBT studies is the rate of acute GVHD. Using an identical RIC regimen and UCB selection algorithm, we previously reported a 40% incidence of grade II–IV acute GVHD when cyclosporine and mycophenolate mofetil were used as GVHD prophylaxis.3 When comparing cohorts, this reduction was statistically significant (P=0.035) and not because of an excess in the former series, as the rate of 40% was similar to the larger Minnesota series, in which the rate of grade II–IV acute GVHD was reported to be as high as 58% in a sample size of 185 indivduals,5 although HLA matching for class I loci was only at the Ag level. In that analysis, when compared with single UCB transplantation, the use of two UCB units, omission of ATG in the preparative regimen and RIC were risk factors for the development of acute GVHD. Similar risk factors for acute GVHD were found in a retrospective database review by Eurocord-Netcord focusing on patients with lymphoid malignancies, where univariate analysis demonstrated an association between recipient age, total body irradiation use, lack of ATG use and RIC as risks for acute GVHD.14 Although the risk of acute GVHD was increased in patients receiving double in comparison with single UCB transplantation (32 vs 22%), this result was not statistically significant.

The reduction in acute GVHD is likely related to the introduction of sirolimus and tacrolimus as GVHD prophylaxis, and not the use of ATG, which was used in a similar fashion in our previous experience. Used extensively at our center since 2000, our previous results in adult PBSC transplantation have demonstrated impressive reductions in grade II–IV and grade III–IV acute GVHD in the related and unrelated donor settings.8 Although associated with an increase in the relative risk of treatment-related morbidity associated with thrombotic microangiopathy15 and veno-occlusive disease,16 these apparent increases were not noted in this experience, as much of this increased risk is thought to be related to the interplay between myeloablative conditioning regimens and endothelial injury associated with sirolimus.

In all, 11 subjects relapsed, 7 of whom had lymphoid malignancies, leading to a CI of relapse at 2 years of 34.4%. This is similar to a recently described series of 65 patients with low or intermediate grade lymphoid malignancies, who received RIC followed by single (14%) or double (86%) UCB transplants, in which the CI of relapse at 3 years was 42%, with longer follow-up than in our series.17 We have previously described an effect in relapse prevention when sirolimus is used following RIC and transplantation of adult stem cells in patients with lymphoid malignancies.18 It is well known that the mammalian target of rapamycin inhibitors possess independent antitumor activity in lymphoid malignancies,19 and therefore, the postulate that maintenance immunotherapy with these agents after transplantation can both prevent GVHD and relapse is attractive, as other attempts to prevent GVHD (such as T cell depletion) generally increase the risk of relapse.20 The utility of sirolimus to prevent relapse in the cord blood setting remains unknown as small samples sizes and differences in tumor characteristics preclude direct comparison of relapse rates between cohorts.

In this experience, we noted a higher than expected occurrence of reactivation of EBV with subsequent EBV-related PTLD (crude incidence 15.6%). This rate of EBV-related disease is in line with other large retrospective reviews of the literature, wherein the incidence of EBV-PTLD was noted to be the highest in patients who underwent non-myeloablative conditioning with ATG as part of the preparative regimen.21 As sirolimus use is associated with a reduced incidence of reactivation of CMV in adult stem cell9 and less clearly in renal transplantation,22, 23 we hypothesized that the administration of sirolimus as primary GVHD prophylaxis would result in a reduced incidence of EBV reactivation and PTLD. Some of the mechanisms behind sirolimus’ inhibitory function in EBV disease have been elucidated, and include the inhibition of IL-10 secretion by transformed tumor cells, a necessary autocrine signal for tumor growth.24, 25 Presumably, in the absence of a memory T cell response against EBV Ags, and in the presence of other immunosuppressants, a permissive environment for EBV-transformed lymphocytes was established and PTLD ensued. This underscores the need for the development of novel therapeutics for the treatment of established PTLD, as our high incidence of transformation to aggressive PTLD occurred despite alternate weekly DNA-based EBV monitoring, in contrast to other reports.26 In addition, others have reported favorable outcomes with the early administration of rituximab,26 whereas all patients treated with this agent in our series had progressive disease, and some individuals had a disease course that was too fulminant to permit the administration of this drug. Moving forward, we plan on reducing the dose of ATG in the preparative regimen in an attempt to reduce the rate of PTLD. An alternative strategy would be to administer rituximab to all transplant recipients early after transplantation.

In this analysis, we were unable to demonstrate a correlation between pre-transplantation graft characteristics and overall and PFS outcomes. However, several parameters associated with post-transplantation graft recovery were significantly associated with improved outcome. For example, improved CD3+ lymphocyte recovery (of both CD4+ and CD8+ subsets) was associated with an improvement in OS, whereas measures of T cell neogenesis and thymopoiesis such as TREC measurements were also correlated with an improvement in PFS. Small sample sizes precluded correlations between dominant cord blood units and transplant outcome.

Recovery of thymopoiesis as determined by TREC analysis was delayed and incomplete compared with recipients of adult stem cell grafts, who reach normal TREC numbers within 3–12 months after transplantation.27, 28 Quantitative TREC recovery was also inferior to that observed in pediatric recipients of cord blood allografts.29, 30 The differences in the kinetics and the quantitative recovery of thymopoiesis in these patient groups may be related to the regenerative properties of UCB, residual thymic activity in pediatric patients, the use of two UCB units or the immunomodulatory effects of our GVHD prophylaxis regimen. There is reason to suspect that either or both of the latter two explanations may be responsible, as our patient group displayed an improved thymic regeneration profile compared with a previously reported group of adult recipients of single UCB grafts, who had a nearly universal lack of thymic recovery for 12 months after transplantation31. Regardless of the reasons contributing to this difference, our data provides evidence that functional thymic regeneration can successfully recover after cord blood transplantation in adults and represents a critical parameter that governs PFS. The recovery of innate immunity after transplant may also have been improved, as the recovering natural killer cell population remained relatively constant after transplantation, and these cells may have antitumor activity after UCBT.32

In summary, we demonstrate that DUCBT in adult recipients using sirolimus and tacrolimus as GVHD prophylaxis is effective therapy in advanced hematologic malignancies. The rates of acute and chronic GVHD using this GVHD prophylaxis regimen are low, and the treatment-related morbidity and mortality is also acceptably low. Immunologic reconstitution after DUCBT is related to overall outcome, related to either fewer opportunistic infections, or potentially even to enhanced graft-vs-tumor effects.

References

  1. 1

    Laughlin MJ, Eapen M, Rubinstein P, Wagner JE, Zhang MJ, Champlin RE et al. Outcomes after transplantation of cord blood or bone marrow from unrelated donors in adults with leukemia. N Engl J Med 2004; 351: 2265–2275.

  2. 2

    Rocha V, Labopin M, Sanz G, Arcese W, Schwerdtfeger R, Bosi A et al. Transplants of umbilical-cord blood or bone marrow from unrelated donors in adults with acute leukemia. N Engl J Med 2004; 351: 2276–2285.

  3. 3

    Ballen KK, Spitzer TR, Yeap BY, McAfee S, Dey BR, Attar E et al. Double unrelated reduced-intensity umbilical cord blood transplantation in adults. Biol Blood Marrow Transplant 2007; 13: 82–89.

  4. 4

    Brunstein CG, Barker JN, Weisdorf DJ, DeFor TE, Miller JS, Blazar BR et al. Umbilical cord blood transplantation after nonmyeloablative conditioning: impact on transplantation outcomes in 110 adults with hematologic disease. Blood 2007; 110: 3064–3070.

  5. 5

    MacMillan ML, Weisdorf DJ, Brunstein CG, Cao Q, DeFor TE, Verneris MR et al. Acute graft-versus-host disease after unrelated donor umbilical cord blood transplantation: analysis of risk factors. Blood 2009; 113: 2410–2415.

  6. 6

    Sehgal SN . Sirolimus: its discovery, biological properties, and mechanism of action. Transplant Proc 2003; 35 (3 Suppl): S7–S14.

  7. 7

    Wang GY, Chen GH, Li H, Huang Y, Wang GS, Jiang N et al. Rapamycin-treated mature dendritic cells have a unique cytokine secretion profile and impaired allostimulatory capacity. Transpl Int 2009; 22: 1005–1016.

  8. 8

    Cutler C, Li S, Ho VT, Koreth J, Alyea E, Soiffer RJ et al. Extended follow-up of methotrexate-free immunosuppression using sirolimus and tacrolimus in related and unrelated donor peripheral blood stem cell transplantation. Blood 2007; 109: 3108–3114.

  9. 9

    Marty FM, Bryar J, Browne SK, Schwarzberg T, Ho VT, Bassett IV et al. Sirolimus-based graft-versus-host disease prophylaxis protects against cytomegalovirus reactivation after allogeneic hematopoietic stem cell transplantation: a cohort analysis. Blood 2007; 110: 490–500.

  10. 10

    Rubinstein P, Dobrila L, Rosenfield RE, Adamson JW, Migliaccio G, Migliaccio AR et al. Processing and cryopreservation of placental/umbilical cord blood for unrelated bone marrow reconstitution. Proc Natl Acad Sci USA 1995; 92: 10119–10122.

  11. 11

    Przepiorka D, Weisdorf D, Martin P, Klingemann HG, Beatty P, Hows J et al. 1994 Consensus Conference on Acute GVHD Grading. Bone Marrow Transplant 1995; 15: 825–828.

  12. 12

    Douek DC, McFarland RD, Keiser PH, Gage EA, Massey JM, Haynes BF et al. Changes in thymic function with age and during the treatment of HIV infection. Nature 1998; 396: 690–695.

  13. 13

    Kaplan E, Meier P . Nonparametric estimation from incomplete observations. J Am Stat Assoc 1958; 53: 457–481.

  14. 14

    Rodrigues CA, Sanz G, Brunstein CG, Sanz J, Wagner JE, Renaud M et al. Analysis of risk factors for outcomes after unrelated cord blood transplantation in adults with lymphoid malignancies: A Study by the Eurocord-Netcord and Lymphoma Working Party of the European Group for Blood and Marrow Transplantation. J Clin Oncol 2009; 27: 256–263.

  15. 15

    Cutler C, Henry NL, Magee C, Li S, Kim HT, Alyea E et al. Sirolimus and thrombotic microangiopathy after allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2005; 11: 551–557.

  16. 16

    Cutler C, Stevenson K, Kim HT, Richardson P, Ho VT, Linden E et al. Sirolimus is associated with veno-occlusive disease of the liver after myeloablative allogeneic stem cell transplantation. Blood 2008; 112: 4425–4431.

  17. 17

    Brunstein CG, Cantero S, Cao Q, Majhail N, McClune B, Burns LJ et al. Promising progression-free survival for patients low and intermediate grade lymphoid malignancies after nonmyeloablative umbilical cord blood transplantation. Biol Blood Marrow Transplant 2009; 15: 214–222.

  18. 18

    Armand P, Gannamaneni S, Kim HT, Cutler CS, Ho VT, Koreth J et al. Improved survival in lymphoma patients receiving sirolimus for graft-versus-host disease prophylaxis after allogeneic hematopoietic stem-cell transplantation with reduced-intensity conditioning. J Clin Oncol 2008; 26: 5767–5774.

  19. 19

    Costa LJ . Aspects of mTOR biology and the use of mTOR inhibitors in non-Hodgkin's lymphoma. Cancer Treat Rev 2007; 33: 78–84.

  20. 20

    Ho VT, Soiffer RJ . The history and future of T-cell depletion as graft-versus-host disease prophylaxis for allogeneic hematopoietic stem cell transplantation. Blood 2001; 98: 3192–3204.

  21. 21

    Brunstein CG, Weisdorf DJ, DeFor T, Barker JN, Tolar J, van Burik JA et al. Marked increased risk of Epstein-Barr virus-related complications with the addition of antithymocyte globulin to a nonmyeloablative conditioning prior to unrelated umbilical cord blood transplantation. Blood 2006; 108: 2874–2880.

  22. 22

    Mathew T, Kreis H, Friend P . Two-year incidence of malignancy in sirolimus-treated renal transplant recipients: results from five multicenter studies. Clin Transplant 2004; 18: 446–449.

  23. 23

    Kirk AD, Cherikh WS, Ring M, Burke G, Kaufman D, Knechtle SJ et al. Dissociation of depletional induction and posttransplant lymphoproliferative disease in kidney recipients treated with alemtuzumab. Am J Transplant 2007; 7: 2619–2625.

  24. 24

    Krams SM, Martinez OM . Epstein-Barr virus, rapamycin, and host immune responses. Curr Opin Organ Transplant 2008; 13: 563–568.

  25. 25

    Nepomuceno RR, Balatoni CE, Natkunam Y, Snow AL, Krams SM, Martinez OM . Rapamycin inhibits the interleukin 10 signal transduction pathway and the growth of Epstein Barr virus B-cell lymphomas. Cancer Res 2003; 63: 4472–4480.

  26. 26

    Kinch A, Öberg G, Arvidson J, Falk KI, Linde A, Pauksens K . Post-transplant lymphoproliferative disease and other Epstein-Barr virus diseases in allogeneic haematopoietic stem cell transplantation after introduction of monitoring of viral load by polymerase chain reaction. Scand J Infect Dis 2007; 39: 235–244.

  27. 27

    Hochberg EP, Chillemi AC, Wu CJ, Neuberg D, Canning C, Hartman K et al. Quantitation of T-cell neogenesis in vivo after allogeneic bone marrow transplantation in adults. Blood 2001; 98: 1116–1121.

  28. 28

    Lewin SR, Heller G, Zhang L, Rodrigues E, Skulsky E, van den Brink MR et al. Direct evidence for new T-cell generation by patients after either T-cell-depleted or unmodified allogeneic hematopoietic stem cell transplantations. Blood 2002; 100: 2235–2242.

  29. 29

    Talvensaari K, Clave E, Douay C, Rabian C, Garderet L, Busson M et al. A broad T-cell repertoire diversity and an efficient thymic function indicate a favorable long-term immune reconstitution after cord blood stem cell transplantation. Blood 2002; 99: 1458–1464.

  30. 30

    Weinberg K, Blazar BR, Wagner JE, Agura E, Hill BJ, Smogorzewska M et al. Factors affecting thymic function after allogeneic hematopoietic stem cell transplantation. Blood 2001; 97: 1458–1466.

  31. 31

    Komanduri KV, St John LS, de Lima M, McMannis J, Rosinski S, McNiece I et al. Delayed immune reconstitution after cord blood transplantation is characterized by impaired thymopoiesis and late memory T-cell skewing. Blood 2007; 110: 4543–4551.

  32. 32

    Beziat V, Nguyen S, Lapusan S, Hervier B, Dhedin N, Bories D et al. Fully functional NK cells after unrelated cord blood transplantation. Leukemia 2009; 23: 721–728.

Download references

Acknowledgements

Supported by NCI P01 CA142106, NCI R01 CA123855, the Jock and Bunny Adams Research and Education Fund, and the Ted and Eileen Pasquarello Research Fund. Funded in part by unrestricted research grants from Astellas, Inc and Genzyme, Inc. CC is supported by the Stem Cell Cyclists of the Pan Mass Challenge.

Author information

Correspondence to C Cutler.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Cutler, C., Stevenson, K., Kim, H. et al. Double umbilical cord blood transplantation with reduced intensity conditioning and sirolimus-based GVHD prophylaxis. Bone Marrow Transplant 46, 659–667 (2011). https://doi.org/10.1038/bmt.2010.192

Download citation

Keywords

  • umbilical cord blood
  • graft-vs-host disease
  • sirolimus

Further reading