Graft Source

A prospective investigation of cell dose in single-unit umbilical cord blood transplantation for adults with high-risk hematologic malignancies

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Abstract

Umbilical cord blood (UCB) as an allogeneic transplant source is generally limited to units with pre-cryopreservation total nucleated cell (TNC) doses 2.5 × 107 NC/kg. We prospectively investigated single UCB transplantation, with cord units as low as 1 × 107 NC/kg, all processed with post-thaw albumin–dextran dilution. We transplanted 104 adult patients with 84% having relapsed/refractory disease. The median TNC dose was 2.1 × 107 NC/kg (range: 1.0–4.4 × 107) and median CD34+ cell dose was 1.0 × 105/kg (range: 0.0–3.7 × 105/kg). Post-manipulation cell recovery and viability were 96% and 99%, respectively. Median times to neutrophil and platelet engraftment were 16 and 43 days, respectively. Univariate factors predicting neutrophil engraftment included TNC (P=0.03) and CD34+ cell dose (P=0.01). CD34+ dose predicted platelet engraftment (P<0.001). In multivariate analysis, CD34+ dose remained significant for neutrophil and platelet engraftment (P<0.0001 and P<0.0001, respectively). The 100-day and 1-year overall survival were 70% and 46%, respectively (95% confidence interval: 36%–56% at 1 year). The subset transplanted with 1–1.5 × 107 NC/kg had similar 100-day and 1-year survivals of 73% and 45%, respectively. Single-unit UCB transplantation using small units, processed as described, leads to favorable engraftment and acceptable outcomes in poor prognosis patients. CD34+ cell dose (1.5 × 105/kg) helps predict faster engraftment and can aid in graft selection.

Introduction

Umbilical cord blood (UCB) is an alternative source of hematopoietic stem cells for patients with hematologic malignancies. It is often the only source of potential stem cells for patients without a matched (or mismatched) sibling or unrelated donor. Limitations to UCB transplantation (UCBT) for adult patients have been identified, including delayed engraftment due to the low dose of infused stem cells.1 Unnecessary cell loss during processing, estimated to be around 20% using washing techniques,2 can further reduce the infused stem cell dose. Larger umbilical cord grafts have been shown to improve time to engraftment and decrease transplant-related mortality.3 The recommended total nucleated cell (TNC) dose for an adult patient is 2.5 × 107 NC/kg,4, 5 with a higher recommended dose for HLA-mismatched grafts.5, 6

A benefit of UCBT is the reduced risk of severe GvHD, allowing for less stringent HLA matching.1 UCBT may be as effective as unrelated bone marrow or peripheral blood stem cell transplantation, when a full (6/6) HLA-matched cord is used.7 Preliminary studies suggest HLA-C antigen matching in UCBT may be beneficial in terms of engraftment8 and transplant-related mortality.9 Furthermore, allele-level matching has also been described as a potential factor influencing outcome10 but is not yet a standard practice. Extensive research has been devoted to the use of double cord blood transplantation; however, with higher costs and increased risk of infusion reactions a survival advantage has not yet been demonstrated.

In addition to TNC and HLA match, CD34+ cell dose appears important in UCB unit selection.4, 5 One study showed a significant impact of CD34+ cell dose on event-free survival,11 another showed an impact on engraftment.12 A reported challenge is the lack of standardized CD34+ cell measurements among different cord blood banks.13, 14 There are also data using flow cytometry as a measure of CD34+ cell viability, with higher viability being predictive of engraftment in double cord transplantation.15

We present the outcome of 104 patients enrolled on a single institution prospective trial of UCBT for relapsed/refractory hematologic malignancies. The primary purpose was to investigate the effect of TNC and CD34+ cell dose on engraftment and outcome. The secondary aim was to establish a standard laboratory processing procedure for low cell count UCB units, with a hypothesis that a technique for post-thaw processing developed in the 1970s16 would minimize cell loss and lead to faster engraftment.

Patients and methods

Patients

Eligible patients had high risk or relapsed hematologic malignancies necessitating allogeneic transplantation. Patients were required to have no compatible related or unrelated HLA-matched living donor or could not have received a prior allogeneic transplant. Consecutive eligible patients were prospectively enrolled on this trial at time of UCBT and signed institutionally approved informed consent. They had to have a compatible (4–6/6 HLA match; antigen matched at HLA-A and B, and allele matched at HLA-DR) umbilical cord graft consisting of at least 1 × 107 TNC/kg. Exclusion criteria included ejection fraction <45%, FEV1 <60%, serum bilirubin 3 or AST/ALT>three times the upper limit of normal, and a creatinine clearance <60 mL/min. Patients could not have evidence of acute, uncontrolled infection or active central nervous system leukemia.

UCB preparation

RBC-deplete cord blood units were transported/stored in the vapor phase of liquid nitrogen and thawed at the time of transplant in a 37 °C decontaminated water bath. Detailed dilution procedure is listed in Supplementary Material. Briefly, thawed cells were transferred aseptically into a 600-mL plasma transfer bag via a 60-mL syringe, after which a diluent medium was added, consisting of 8.5% albumin combined with 6.7% dextran. The volume of diluent medium prepared was four times the volume of cord blood. The cord blood was diluted at progressively faster rates designed to deliver 10%, 30% and 60% of the cell volume over 2, 4 and 6 min, respectively, giving a final ratio of thawed cells to diluent of 1:4. The cells were not washed or manipulated further. When dilution was complete, a sample was removed to determine the TNC count, sterility and viability testing by Trypan Blue dye exclusion. Subsequently, unfiltered cells were infused immediately over 15 min.

Conditioning regimen, GvHD prophylaxis and supportive care

Patients were conditioned with either a myeloablative or reduced-conditioning regimen (defined in Table 1). Tacrolimus (FK506) or cyclosporine A, was used for all patients for GvHD prophylaxis. Tacrolimus (0.03 mg/kg per day) infusion started on day −2 and was titrated to therapeutic serum levels (10–15 ng/mL). Cyclosporine A infusion was administered at 5 mg/kg starting on day −2 and adjusted for a goal level of 200–400 ng/mL. Antithymocyte globulin (Thymoglobulin, Sanofi US, Bridgewater, NJ, USA, 1.5 mg/kg per day on days −6 and −5) was administered to patients who were at an increased risk of acute rejection, that is, those who had never received immune suppressive chemotherapy or had not received it in the 3 months before transplant.

Table 1 Patient and cord unit characteristics

Prophylactic antimicrobials for bacteria, superficial fungal infections and HSV-1 were started on day +1 or earlier if clinically indicated. G-CSF (Filgrastim 5 mcg/kg per day) was administered post stem cell infusion on day 0 and continued until an ANC reached 1500/μL for 3 days or 5000/μL on one occasion. Donor chimerism testing was performed using PCR amplification of micro-satellite short tandem repeat loci at approximately day +60 and +90.

Statistical analysis and definitions

Standard descriptive statistics including medians (range) and frequencies (percentage) were used to describe characteristics of the study population. Primary endpoints included neutrophil and platelet engraftment, overall survival (OS), PFS and non-relapse mortality (NRM). Survival estimates were calculated via standard Kaplan–Meier methods17 using SPSS version 18 (SPSS Inc., Chicago, IL, USA). Differences between Kaplan–Meier curves were assessed by the log-rank test and differences between cumulative incidence curves were assessed using Gray’s test of equality. SAS version 9.3 (SAS Institute Inc., Cary, NC, USA) was used to calculate the 95% confidence intervals and to conduct multivariate analyses. Multivariate analyses were performed using Cox proportional hazards models; final models were chosen based on Akaike Information Criterion using backward selection approach. Significance was assessed at α=0.05.

Neutrophil engraftment was defined as the first of three consecutive days for ANC500/μL. Platelet engraftment was defined as the first of seven consecutive days of platelet count20 000/μL without transfusion support. Graft failure was defined as failure to engraft neutrophils to >500/μL by day 45 or ANC500/μL with no donor engraftment as shown by chimerism studies.

Patients were categorized into high/intermediate- or low-risk disease as defined by the American Society for Blood and Marrow Transplantation.18 High/intermediate-risk disease was defined as AML and ALL in CR2, CR3+, primary induction failure, or relapsed disease; myelodysplastic syndrome was categorized as refractory anemia with excess blasts-1, -2 and -T, and chronic myelomonocytic leukemia; Hodgkin lymphoma in CR2, PR1, PR2, primary refractory or relapse chemoresistant; Non-Hodgkin lymphoma in CR2, PR, primary refractory or relapse chemoresistant. All others were defined as low risk.

OS was evaluated from time of UCBT to death from any cause. PFS was measured from UCBT until disease progression, relapse or death from any cause, whichever occurred first. NRM was defined as all deaths other than disease progression or relapse occurring anytime after transplantation. Acute and chronic GvHD (aGvHD and cGvHD) was classified according to standard criteria.19, 20 Hematopoietic cell transplantation-specific comorbidity index was calculated for each patient as previously described.21

Results

Patient characteristics

One hundred and four patients underwent a single UCBT in this study between January 2002 and July 2013 (Table 1). Their median age was 51 years (range: 18–72 years), median weight was 79 kg (range: 49–127 kg) and 55 patients (53%) were male. A majority, 84%, of patients had high-risk disease. Twenty six percent of patients had undergone prior autologous stem cell transplantation. A majority of patients, 77%, had a good performance status (Karnofsky performance status 90%–100%). Median hematopoietic cell transplantation-specific comorbidity index for all patients was 2 (range: 0–11).

Transplant characteristics

The median TNC dose for the 104 patients was 2.1 × 107 NC/kg (range: 1.0–4.4 × 107) and the median CD34+ cell dose was 1.0 × 105/kg (range: 0.0–3.7 × 105/kg). TNC was measured at the time of unit collection at the cord blood bank and was repeated after thawing and dilution locally. CD34+ cell dose was measured at the time of unit collection; CD34+ viability by flow cytometry was not performed in this trial. The median TNC dose for patients receiving myeloablative and reduced-conditioning regimens was 2.0 × 107 and 2.1 × 107/kg, respectively. The median post-thaw cell recovery for all 104 units was 96% (range: 78%–100%) and the post-thaw cell viability was 99% (range: 85%–100%).

Nine percent of patients were HLA-matched at 6/6 loci, while 36% were matched at 5/6 loci and 55% at 4/6 loci (Table 1). HLA-C antigen match was known for 94 patients, only 10% were C locus matched. Sixty percent of patients were conditioned with a reduced-intensity conditioning regimen. Infusion reactions were mild, affecting 17% (n=18) of patients. Hypertension was the most common reaction (n=11), followed by nausea (n=3), fever (n=3) and pruritis (n=1). There were no grade 4 or 5 reactions.

Hematopoietic recovery

The cumulative incidence of neutrophil engraftment by day 45 was 87% (95% confidence interval (CI): 79%–92%), with a median time to neutrophil engraftment for all patients of 16 days (range: 6–38; Table 2). In univariate analysis, patients receiving a high TNC dose ( 2.5 × 107 NC/kg) had a better probability of engraftment (P=0.03; Figure 1) as did patients receiving a higher CD34+ celldose (1.5 × 105/kg, P=0.01; Figure 2). The median neutrophil engraftment for patients receiving a TNC dose of 1.5 × 107 NC/kg was 24 days (range:15–50), whereas for patients receiving an intermediate dose (1.6–2.4 × 107 NC/kg) it was 17 days (range: 9–43), and for patients receiving a dose 2.5 × 107 NC/kg it was 13 days (range: 6–45). The median neutrophil engraftment for patients receiving a CD34+ cell dose of <1.5 × 105/kg was 21 days (range: 9–43), whereas for patients receiving a CD34+ cell dose of 1.5 × 105/kg, it was 13 days (range: 8–45). HLA match and conditioning regimen did not have a significant impact on neutrophil engraftment. The median time to neutrophil engraftment for patients receiving a myeloablative or reduced-conditioning regimen was 17 and 16 days, respectively (P=0.48) (Table 3).

Table 2 Results and outcome
Figure 1
figure1

Cumulative incidence of neutrophil engraftment based on TNC dose.

Figure 2
figure2

Cumulative incidence of neutrophil engraftment based on CD34+ cell dose.

Table 3 Cell dose and engraftment based on conditioning regimen

In multivariate analysis, CD34+ cell dose remained significant in predicting neutrophil engraftment (P<0.0001; Table 4). Other variables significant for neutrophil engraftment included female sex, no history of prior transplantation and a low hematopoietic cell transplantation-specific comorbidity index score (Table 4). TNC dose and HLA match had no significant impact on neutrophil engraftment after adjusting for other variables included in the model.

Table 4 Results of multivariate analysis

The cumulative incidence of platelet engraftment by day 95 was 78% (95% CI: 68%–85%), with a median platelet engraftment for all patients of 43 days (range: 19–95). Higher CD34+ cell dose predicted faster platelet engraftment, in univariate analysis (P<0.001; Figure 3). The median platelet engraftment for patients receiving a CD34+ cell dose of <1.5 × 105/kg was 60 days (range: 30–90) and for patients receiving a CD34+ cell dose of 1.5 × 105/kg it was 39 days (range: 19–89). In univariate analysis, higher TNC dose showed a trend in faster platelet engraftment but was not statistically significant (P=0.19). In this analysis, HLA match and conditioning regimen did not have a significant impact on platelet engraftment.

Figure 3
figure3

Cumulative incidence of platelet engraftment based on CD34+ cell dose.

In multivariate analysis, CD34+ cell dose remained highly significant for platelet engraftment (P<0.0001; Table 4). Other significant predictors included 6/6 HLA match, female sex and low-risk disease (Table 4).

Eighty-three patients had chimerism results; of these, 70 were full donor chimeras. Thirteen patients (13%) had evidence of graft failure confirmed by chimerism studies. Three had primary graft failure and 10 had autologous reconstitution at the time of relapse. The majority of patients with graft failure received a reduced-conditioning regimen (n=9), had 4/6 HLA match (n=9) and/or received low CD34+ cell doses (n=12). Neither TNC dose, disease risk, HLA match nor regimen type was significant for graft failure in this study.

aGvHD and cGvHD

The cumulative incidence of aGvHD and cGvHD for all patients was 40% (95% CI: 30%–49%) and 39% (95% CI: 29%–48%), respectively. Grade 3/4 aGvHD occurred in 5% of the group. aGvHD consisted predominantly of skin and gastrointestinal pathology. Per NIH20 scoring system, 29 patients had evidence of mild cGvHD, 4 patients had moderate cGvHD involving the skin and 3 patients had severe cGvHD involving the skin and the gastrointestinal tract.

Patient outcomes and prognostic factors

The median follow-up time for this study is 5.8 years. The 100-day, 1-year and 2-year OS for all patients is 70%, 46% and 36%, respectively, with a median survival of 8.4 months (95% CI: 36%–56% at 1 year; Figure 4a). The PFS is 63% at 100 days, 38% at 1 year and 30% at 2 years, with a median PFS of 5 months (95% CI: 29%–48% at 1 year; Figure 4b). The NRM for all patients was 22% at 100 days and 36% at 1 year (95% CI: 27%–47% at 1 year).

Figure 4
figure4

Kaplan–Meier estimates of (a) overall survival and (b) PFS.

Notably, patients receiving the lowest TNC dose (1–1.5 × 107 NC/kg) had similar survival as the entire cohort, 73% at 100 days and 46% at 1 year. In univariate analysis, no identifiable graft factor had a significant impact on OS. In multivariate analysis, only age and disease-risk group had a significant impact on survival (P=0.03 and P=0.05, respectively; Table 4). For PFS, only HLA-match and disease-risk group had a significant impact on univariate analysis (P=0.03 and P=0.04, respectively). In multivariate analysis, age and disease-risk group were the only significant predictors of PFS (P=0.04 and P= 0.04, respectively; Table 4).

In univariate analysis, history of prior transplantation significantly had an impact on NRM. The 100-day NRM for patients with prior transplantation was 46% vs 13% without prior transplant (P=0.01). In multivariate analysis, the following variables had a significant impact on NRM: age, low TNC dose, myeloablative conditioning regimen and prior transplantation (Table 4).

Relapse and causes of death

A total of 44% have relapsed (n=39), with a median time to relapse of 4 months (range: 0.3–79). The major cause of death was relapsed disease, accounting for 50% of deaths. The most common cause of NRM was infection: ARDS (n=13; 18%), sepsis (n=10; 14%) and pneumonia (n=7; 10%). Other causes included veno-occlusive disease (n=2; 3%), post-transplant lymphoproliferative disease (n=3; 4%) and solid tumor malignancy (n=2; 3%). Of note, 81% of patients (n=22/27) failing a previous autologous transplant have died, 8 of relapsed disease, 12 of infection and 1 each of veno-occlusive disease and solid malignancy.

Discussion

This prospective, single-center trial investigated the effect of TNC dose and CD34+ cell dose on engraftment and patient outcome with single UCBT in high-risk patients. We explored the lower limits of TNC dose and showed acceptable engraftment of 24 days in patients receiving a very low TNC of 1–1.5 × 107 NC/kg (n=23). This subset also had relatively favorable outcomes given their high-risk disease with 100-day and 1-year OS of 73% and 45%, respectively, which is similar to the OS for the entire cohort. As this was a very high-risk group, this lack of a difference however may not have been seen had we transplanted patients with predominately low-risk disease. Not surprisingly and in line with previously published data,3, 6, 22 we show that higher TNC count predicts faster engraftment. Furthermore, the analysis of CD34+ cell dose appears promising and predictive of both neutrophil and platelet engraftment. CD34+ cell dose 1.5 × 105/kg was highly statistically significant for faster engraftment in both univariate and multivariate analysis, suggesting CD34+ cell dose should have a role in single cord unit selection, even though CD34+ measurement has not be standardized. Standardized methods of CD34+ cell count across cord banks and further investigation of its value should be undertaken.

We also present our 11-year experience with a unique post-thaw progressive dilution process based on optimal in vitro stem cell recovery data from the 1970s16 with excellent post-thaw cell recovery and viability. Rather than being washed, which can result in ~20% cell loss,2 cells were diluted with albumin–dextran, producing a safe dimethyl sulfoxide concentration that is non-toxic to stem cells and carries no additional risk of infusion reactions. A similar approach was recently explored and published in children and adults receiving double cord blood units.23, 24 Infusion reactions were mild and actually lower in our trial than previously reported with this technique.23, 24 We believe that this technique permits centers to choose units as low as 1 × 107 NC/kg when larger units are not available and still achieve favorable outcomes.

In our study, HLA match (6/6 vs 4/6) may have had an impact on platelet engraftment, as seen in multivariate analysis. However, in contrast to previously published data7, 8, 10 HLA match (including HLA-C) did not appear to have a significant impact on neutrophil engraftment, OS, PFS or NRM. This may be due to the difference in the size of the graft, patient population treated and a very limited number of patients with 6/6 HLA match. Studies showing a significant impact of HLA match on mortality and/or outcome typically include pediatric patients7 who receive large units (median of 5 × 107 NC/kg in one study).10 In line with our data, studies restricted to the adolescent and adult patients do not show an association between HLA match and outcome.25, 26, 27 Recent data suggests a benefit to HLA allele-level matching,10 however, analysis of this variable was not possible in our patients, owing to limited allele information of the donor units.

It is important to note that neither TNC nor CD34+ cell dose had a significant impact on OS or PFS, despite their significant effect on engraftment. In multivariate analysis, it was patient-specific factors that had a significant impact: age and disease risk, both of which are beyond the control of a transplant physician but may impact patient selection. In multivariate analysis, NRM was significantly worse in patients with a TNC <2.5 × 107 NC/kg, receiving a myeloablative conditioning regimen and having a personal history of prior transplantation. The high rate of fatal complications in patients failing a previous transplant (81%), with statistical significance in our multivariate analysis, has led to a more careful consideration of this subpopulation at our institution. Clinical trials exploring other treatment options may be preferred for these patients.

Double cord blood transplantation has been extensively investigated but, to date, a survival advantage has not been shown over single UCB units or unrelated donor grafts7, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 and additional efforts are being investigated in clinical trials.4, 38, 39 As these attempts are explored and potentially practice-changing data emerges, improvements and expertise in our current transplant procedures are of great importance.

Single cord blood units continue to be an option in high-risk patients who decline or are not eligible for enrollment on a clinical trial and can increase the number of patients potentially benefiting from this procedure. Expertise in UCBT, exceptional supportive care, optimal graft characteristics and conditioning regimen may allow for transplantation with smaller cord units and overcome some of the risks with such transplantation, namely delayed neutrophil engraftment. As demonstrated in our trial, a dilution step, rather than washing, can help minimize unnecessary cell loss and may contribute to favorable outcomes. We show positive outcomes in patients receiving low-dose units (median of 2.1 × 107 NC/kg), including those receiving a very low unit (1–1.5 × 107 NC/kg). In addition, CD34+ cell dose (1.5 × 105/kg) can favorably impact and predict engraftment, and should be considered in graft selection.

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Acknowledgements

We thank our transplant center’s nursing staff, advance practice nurses, research assistants, social workers and case managers for their invaluable ongoing contributions to patient care.

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Correspondence to P Stiff.

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The authors declare no conflict of interest.

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Supplementary Information accompanies this paper on Bone Marrow Transplantation website

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