Volume 13, Issue 10 , Pages 1233-1243, October 2007
Similar Outcomes of Cryopreserved Allogeneic Peripheral Stem Cell Transplants (PBSCT) Compared to Fresh Allografts
Article Outline
- Abstract
- Introduction
- Methods and Materials
- Granulocyte-colony stimulating factor (G-CSF) Mobilized PBSC Collections from Healthy Donors
- Cryopreservation, Thawing, and Infusion of Allogeneic PBSCs
- Clinical Transplant Procedures
- Quantification of CD34+ Cells by Flow Cytometry and Clonogenic Hematopoietic Progenitor Cells in Culture
- Definitions
- Statistics
- Results
- Peripheral Blood Stem Cell Collection, Cryopreservation, and Infusion after Thawing
- Comparison of the Transplant Outcomes between Cryopreserved and Fresh PBSCT
- Stem Cell Loss after Cryopreservation
- Correlation of Clonogenic Progenitors with CD34+ Cells and Total Nucleated Cells
- Factors Affecting Engraftment after Allogeneic PBSCT
- Evaluation of Cryopreservation as a Risk Factor for OS, NRM, and Disease Recurrence
- Discussion
- Acknowledgments
- References
- Copyright
Abstract
The BMT program at Princess Margaret Hospital performed 105 transplants using cryopreserved peripheral blood stem cells (PBSC) from related allogeneic donors. The outcomes were compared with those of a historic control of 106 patients transplanted with freshly procured PBSC. The infusions were tolerated with limited toxicity related to nausea/vomiting or bradycardia, correlated with the total amount of DMSO infused. The average viability of the total nucleated cell (TNC) population after thawing was 71%. The survival of clonogenic progenitors amounted to 75% for colony-forming unit-granulocyte-macrophage (CFU-GM), 69% for burst-forming units erythroid (BFU-E), and 78% for colony-forming units granulocyte-erythrocyte-monocyte-megakaryocyte (CFU-GEMM). In contrast, colony-forming units megakaryocyte (CFU-MEG) was significantly more cryosensitive with recovery rates of 39%. The number of viable CD34+ cells transplanted was correlated with the number of transplanted viable CFU-GM (P < .001), BFU-E (P < .001), CFU-MEG (P < .001), and CFU-GEMM (P = .049), but not with the TNC dose. The number of transplanted CD34+ cells was correlated with engraftment of neutrophils (P = .012) and platelets (P = .013). The outcomes of cryopreseved or fresh PBSC transplants (PBSCT) with respect to engraftment of neutrophils (P = .178) and platelets (P = .785), lymphocyte recovery (P = .926), acute (P = .113), and chronic graft-versus-host disease (P = .673), recurrence (P = .295), nonrelapse mortality (P = .340), and overall survival (P = .668) were not significantly different. It is therefore reasonable to consider the option of cryopreserved allografts.
Key Words: Allogeneic peripheral blood stem cell transplantation, Cryopreservation
Introduction
The efficacy of cryopreserved hematopoietic progenitor cells is well established for blood and marrow-derived autografts [1, 2, 3, 4]. Despite some toxicities related to dimethylsulfoxide (DMSO) [5, 6, 7] and high cell numbers [8], the procedure has an excellent safety record [9, 10, 11, 12]. Although the experience with cryopreserved allografts is limited [13, 14], the technology is used predominantly to store umbilical cord blood [15]. In addition, there is a recent trend to use cryopreserved grafts obtained from unrelated donors [13, 16]. Theoretical concerns over cryopreserving allografts include the possibility of damaging selective cell populations, particularly T cells, that may reduce the antitumor effect of cryopreserved grafts, contamination of grafts secondary to the more extensive manipulation, and toxicity related to the use of DMSO as cryoprotectant. On the other hand, cryopreserved allografts may enhance the safety by guaranteeing the availability of a quality controlled graft prior to initiating myeloablative therapies, and may increase the flexibility of scheduling transplants. These issues were discussed in a recent review [16] suggesting that there are insufficient data available to recommend the use of fresh allografts over cryopreserved preparations.
It was the objective of the current study to gain the necessary information and develop recommendations by comparing the safety and clinical outcomes of a series of cryopreserved peripheral blood derived (PBSC) consecutive allografts with that of a historic control series of freshly procured PBSC. Secondary objectives included impact assessment of cryopreservation on the survival of clonogenic hematopoietic progenitors and their contribution to engraftment as well as lymphocyte recovery after transplantation.
Methods and Materials
Granulocyte-colony stimulating factor (G-CSF) Mobilized PBSC Collections from Healthy Donors
A total of 105 consecutive recipients of cryopreserved allogeneic PBSC transplants (PBSCT) collected from related donors between September 2003 and December 2005 were included in a prospective study. These represented all patients with hematopoietic malignancies who received transplants with PBSC from a related donor during the indicated time interval. One hundred six consecutive patients with hematopoietic malignancies transplanted with freshly collected PBSC between June 2001 and August 2003 served as a historic control. The characteristic features of recipients and donors of both cohorts are summarized in Table 1. All donors were screened for risk factors and medically examined before commencement of G-CSF injections. The evaluation included a bone marrow study. The marrow samples of all donors in this study were morphologically normal. Written consents were obtained from both recipients and donors.
Table 1. Properties of Patients, Donors, Grafts, and Transplant Procedures for Both Cohorts Receiving Either Cryopreserved or Freshly Procured PBSCT
| Cryopreserved PBSCT (n = 105) | Fresh PBSCT (n = 106) | P-Value | |
|---|---|---|---|
| Recipients | |||
 Sex (F/M) | 48/57(46%/54%) | 46/69(43%/57%) | NS |
| Age (years) | 51(17-71) | 49(21-69) | NS |
| Weight (kg, mean ± SE) | 73.5±1.4 | 71.2±1.9 | |
| Diseases | NS | ||
| AML/ALL | 42/7(40%/7%) | 44/14(42%/13%) | |
| MDS/MF | 7/9(7%/8%) | 9/1(8%/1%) | |
| CML/CLL | 9/15(8%/14%) | 15/7(14%/7%) | |
| NHL/HD | 13/1(12%/1%) | 12/1(11%/1%) | |
| MM/solid | 2/0(2%/0%) | 1/2(1%/2%) | |
| Donors | |||
| Sex (F/M) | 50/55(48%/52%) | 65/41(61%/39%) | NS |
| Age (years) | 47(12-75) | 48.5(13-73) | NS |
| Weight (kg, mean±SE) | 74.5±1.8 | 73.9±2.0 | NS |
| CMV status (R/D, n = 193) | NS | ||
| (pos/pos)/(pos/neg) | 44/11(44%11%) | 41/10(44%/11%) | |
| (neg/pos)/(neg/neg) | 20/25(20%/25%) | 19/23(20%/25%) | |
| PBSC collection | |||
| 1-/2-/≥3 times | 90/12/3(86%/11%/3%) | 79/24/3(75%/23%/3%) | NS |
| Transplant CD34+ cell dose | |||
| CD34+ cells (×106/kg) | 4.8(2.3-14.8) | 5.2(1.7-13.4) | |
| CD34+ cells, ≥ 5 × 106/kg | 41(45%) | 43(54%) | NS |
| CFUs, infused (×104/kg) | |||
| CFU-GM | |||
| (mean ± SE) | 134.87±8.44 | 132.86±16.12 | NS |
| (median, range) | 118.67(8.95-451.75) | 108.76(5.12-1111.81) | |
| BFU-E | |||
| (mean ± SE) | 141.19±7.73 | 126.38±12.53 | NS |
| (median, range) | 134.02(4.41-350.70) | 112.56(1.40-942.18) | |
| CFU-MEG | |||
| (mean ± SE) | 24.11±2.27 | 42.28±6.37 | .009 |
| (median, range) | 19.68(0-86.16) | 32.75(0.47-475.70) | |
| CFU-GEMM | |||
| (mean ± SE) | 8.77±0.46 | 4.63±0.35 | <.001 |
| (median, range) | 8.39(0-21.04) | 2.65(0.47-20.28) | |
| Conditioning regimen | NS | ||
| Myeloablative | 74(70%) | 79(74%) | |
| TBI based/non-TBI based | 47/27(45%/26%) | 65/14(61%/13%) | |
| Reduced intensity | 31(30%) | 27(26%) | |
| GVHD prophylaxis | NS | ||
| CSA/MTX | 62(59%) | 74(70%) | |
| CSA/MMF | 31(29%) | 26(24%) | |
| Others⁎ | 12(12%) | 5(5%) | |
| Type of PBSCT | NS | ||
| Syngeneic | 3(3%) | 1(1%) | |
| Allogeneic sibling | 98(93%) | 94(89%) | |
| Allogeneic family | 4(4%) | 11(10%) | |
| HLA-disparity | NS | ||
| HLA-identical | 95(91%) | 93(88%) | |
| 1-antigen mismatched | 10(9%) | 13(12%) |
⁎Others included cyclosporine alone (n = 4 in cryopreserved PBSCT and n = 5 in fresh PBSCT group), FK506 with mycophenolate mofetil or methotrexate (n = 4 in cryopreserved PBSCT and n = 1 in fresh PBSCT group), and others (experimental ATG prophylaxis protocol, n = 4 in cryopreserved PBSCT group). |
All donors were mobilized with subcutaneous injections of G-CSF (Neupogen®, Amgen Canada, Mississauga, Canada) at a dose of 10 μg/kg/day. G-CSF was routinely administered for 4 consecutive days prior to the PBSC collection on day 5. The minimum target of CD34+ cells was defined as 4 × 106/kg actual weight of the recipient. Provisions were made for additional injections of G-CSF and collections if the target number was not obtained on the first day of collection. The apheresis procedure was performed with a COBE Spectra cell separator (Gambro BCT, Lakewood, CO) using acid-citrate-dextrose formula A (ACD-A) as anticoagulant as well as 2000 units of heparin. The median blood volume processed during 1 collection was 18 L (range: 7.7-53.3 L).
Cryopreservation, Thawing, and Infusion of Allogeneic PBSCs
PBSC products were prepared in a cell-processing laboratory under current good manufacturing standards. Cells were cryopreserved at total nucleated cell (TNC) concentrations of <3 × 108/mL using 10% DMSO as the cryoprotectant, and where necessary, using autologous plasma and/or plasmalyte as a diluent [17, 18, 19, 20]. The products were placed in −86°C freezer for a minimum of 3 hours and a maximum of 5 days before being moved into the vapor phase of a liquid nitrogen freezer for long-term storage.
Grafts were thawed at bedside by immersing the cryobags into a 40°C water bath. Viability was assessed via trypan blue exclusion.The thawed PBSC products were infused within 10-15 minutes through a central venous catheter without filtration. All patients received 20 mg of dexamethasone, 25 mg of meperidine, 50 mg of diphenhydramine, and 1 mg of granisetron intravenously, and 2 mg of lorazepam sublingually 30 minutes prior to infusion. Heavy meals were avoided for 3 to 4 hours prior to infusion.
Clinical Transplant Procedures
The transplant procedure and posttransplant management followed previously described institutional policies [21, 22, 23, 24]. They are summarized in Table 1. The clinical data were updated as of July 2006, and included the following events: engraftment of neutrophils, platelets, and lymphocytes, acute and chronic graft-versus-host disease (aGVHD, cGVHD), recurrence of disease, nonrelapse mortality (NRM), and overall survival (OS). Absolute lymphocyte counts (ALCs) were recorded at 1, 2, 3, 6, 9, 12, 18, 24, and 36 months after PBSCT.
Quantification of CD34+ Cells by Flow Cytometry and Clonogenic Hematopoietic Progenitor Cells in Culture
Each PBSC product was evaluated before cryopreservation to determine the number of TNCs and CD34+ cells, as well as the presence and frequency of multicolony-forming units granulocyte-erythrocyte-monocyte-megakaryocyte (CFU-GEMM) and single [CFU-GM, Burst-forming units erythroid (BFU-E), and colony-forming units megakaryocyte (CFU-MEG)] lineage clonogenic hematopoietic progenitors. After thawing, each PBSC product was assessed by trypan blue exclusion for viability of TNCs. The data were used to calculate the number of infused viable CD34+ cells. The content of viable clonogenic progenitors was quantified in culture as well as calculated using the trypan blue data. As described previously, clonogenic progenitor cells were assessed by growing 5 × 104 TNCs for 14 days in semisolid methylcellulose cultures [8, 9]. CD34+ cell counts were obtained by flow cytometry on fresh cells before cryopreservation [25, 26].
Definitions
The day of the PBSC infusion was defined as day 0. Engraftment was determined as a peripheral absolute neutrophil count (ANC) of >0.5 × 109/L for 2 consecutive days, and a peripheral platelet count of >20 × 109/L for at least 3 consecutive days without requiring transfusion. aGVHD and cGVHD were diagnosed and graded using established criteria [27, 28]. OS was defined as the time from transplantation until death from any cause. NRM was defined as death not related to disease recurrence or progression. The incidence of recurrence was defined as the time from transplantation until disease progression. The disease risk of the recipients was defined as low, intermediate, and high, as previously described [29].
Statistics
Clinical characteristics of patients transplanted with cryopreserved or fresh PBSC were compared by Fisher’s exact or Mann-Whitney’s U-test. The OS, incidence of engraftment, GVHD, NRM, and recurrence were assessed by log rank test. A logistic regression analysis was performed to evaluate the effect of various parameters on the occurrence of infusion-related toxicity using a forward conditional procedure [5, 8]. These parameters included sex and body weight of recipients, TNCs infused, volume of DMSO, and the interval between PBSC collection and infusion.
The relationship of TNCs, CD34+ cells, CFU-GM, BFU-E, CFU-MEG, and CFU-GEMM within each graft was evaluated by applying the Pearson’s correlation test. The influence of each cell subset on neutrophil and platelet engraftment was assessed by treating each subset as categoric variable defined as either above or below the median. A Cox’s proportional hazard regression model was used to test parameters for their independence. Lymphocyte counts were determined serially and compared for both cohorts at 1, 2, 3, 6, 9, 12, 18, 24, and 36 months after PBSCT using a general linear model based on serial data.
The contribution of potential prognostic factors to OS, NRM, and recurrence were examined by uni- and multivariate analyses of parameters identified by Cox’s proportional hazard regression. They included the following variables: cryopreservation (fresh versus cryopreserved PBSCT), conditioning regimen (nonmyeloablative vs. myeloablative), disease status (low or intermediate versus high risk), age of recipients (≤50 years versus >50 years), sex mismatch between recipient and donor (female donors/male recipients versus others), aGVHD (no or grade one versus grades II-IV), cGVHD (no versus yes). P-values of <.05 were considered significant. The statistical analyses were performed using the SPSS/PC+ software, version 13.0 (Chicago, IL).
Results
Peripheral Blood Stem Cell Collection, Cryopreservation, and Infusion after Thawing
PBSC collections to harvest CD34+ cells over 4.0 × 106/kg were completed on 1 (n = 90, 86%), 2 (n = 12, 11%), or 3 consecutive days (n = 3, 3%). A median of 2.99 × 1010 TNCs was stored per bag in a median volume of 117 mL. The median volumes of PBSCs infused amounted to 357 mL, ranging from 201 to 709 mL in a median number of 3 bags (range 2-8 bags) per patient. The median length of cryopreservation between collection and thawing was 15 days (range: 5-238 days). The median viability of cells after thawing was 71.2% ± 1.0% (range: 50% to 98%). The median respective number of viable TNC and CD34+ cells transplanted was 8.9 × 108/kg and 4.8 × 106/kg. As determined after thawing, the grafts contained CFU-GM, BFU-E, CFU-MEG, and CFU-GEMM at a frequency of 118, 134, 19, and 8.4 × 104/kg, respectively. All infused PBSC products were microbiologically negative before cryopreservation and after thawing.
The most common side effects encountered during the infusion included nausea and vomiting [5], followed by abdominal pain, bradycardia, hypotension, and chest tightness (Table 2). In general, the symptoms were rated at a toxicity grade of 1 or 2. Only 1 patient experienced grade 3 toxicity with nausea and vomiting. The following factors were found to be associated with infusion related toxicities: (1) nausea of any grade was more frequently observed in female patients (53.5%, 23 of 43 patients) compared to male recipients (30.9%, 17 of 55 patients; P = .025, odds ratio [OR] 2.571 [1.123-5.885]). (2) Hypotension (P = .049, OR 1.219 [1.001-1.483]), and bradycardia (P = .048, OR 1.077 [1.001-1.159]), were asssociated with the total volume of infused DMSO per patient.
Table 2. Infusion-Related Toxicities of Thawed PBSC
| Type of Toxicity | Grade 1 N (%) | Grade 2 N (%) | Grade 3 N (%) |
|---|---|---|---|
| Nausea/vomiting | 24(24.5) | 15(15.3) | 1(1.0) |
| Abdominal pain | 18(18.4) | 3(3.1) | 0(0) |
| Hypotension | 1(1.0) | 2(2.0) | 0(0) |
| Bradycardia | 4(4.1) | 2(2.0) | 0(0) |
| Chest tightness | 2(2.0) | 0(0) | 0(0) |
| Chills/Fever | 0(0) | 0(0) | 0(0) |
| Neurologic signs | 0(0) | 0(0) | 0(0) |
Comparison of the Transplant Outcomes between Cryopreserved and Fresh PBSCT
The median follow-up of survivors after cryopreserved and fresh PBSCT was 411 days (range: 56-1346 days) and 1192 days (range: 800-1809 days), respectively. Both groups showed similar engraftment kinetics. The median time to reach neutrophil counts of ≥0.5 × 109/L was 17 and 18 days for recipients of cryopreserved and fresh PBSCT (P = .446; Figure 1A) and 21 days for neutrophils ≥1.0 × 109/L. Both groups achieved a platelet count of ≥20 × 109/L after 13.0 days (P = .785; Figure 1B) and required similar time intervals to increase their platelet counts to over 50 × 109/L (15 days versus 16 days, P = .858) or 100 × 109/L (22 days versus 22 days, P = .563).
Figure 1.
Engraftment kinetics of neutrophils (A), platelets (B), and lymphocytes (C) after cryopreserved and fresh PBSCT. D, Shows similar serial recoveries of lymphocyte counts after cryopreserved and fresh PBSCT.
Overall, 6 patients did not achieve engraftment until the latest observation time point, including 1 case receiving cryopreserved PBSCT and 5 cases receiving fresh PBSCT. As shown in Table 3, 3 cases out of 6 cases (50%) were 1 antigen mismatched transplants and 4 cases (67%) developed veno-occlussive disease (VOD) of liver, which might be of significant contribution to the development of engraftment failure. However, their transplant doses of CD34+ cells or PBSC collection times were not different from those of the overall group.
Table 3. Summary of Cases Failing Engraftment
| Source of PBSC | Sex/Age | Disease | Conditioning | CD34+ Cells⁎ | Time(s) of Collection | Donor | HLA Match | Events | Status | |
|---|---|---|---|---|---|---|---|---|---|---|
| 1 | Cryo | F/19 | CML, 2nd BMT | BUCY | 5.3 | 1 | Sibling | 6/6 | VOD+HUS/TTP | A+60 |
| 2 | Fresh | M/45 | Ph+ ALL | CY/TBI | 5.0 | 1 | Daughter | 9/10 | VOD | D+26 |
| 3 | Fresh | F/52 | CLL | CY/TBI | 4.7 | 3 | Sibling | 9/10 | VOD | D+34 |
| 4 | Fresh | M/58 | AML | CY/TBI | 5.2 | 1 | Nephew | 9/10 | GVHD+Infection | D+39 |
| 5 | Fresh | F/56 | AML | CAT | 2.7 | 1 | Sibling | 6/6 | Recurrence | D+290 |
| 6 | Fresh | F/57 | NHL | BUCY | 3.9 | 1 | Sibling | 6/6 | VOD | D+27 |
⁎×106/kg of recipients body weight. |
The median number of days to achieve lymphocyte counts above 0.5 × 109/L was 22 days for each group (P = .703), that above 1.0 × 109/L was 33 days for each group (P = .425), and that above 2.0 × 109/L was 257 and 279 days (P = .687), respectively (Figure 1C). Serial measurements of lymphocytes after allogeneic PBSCT revealed a gradual increase in their frequency from 1 month to 9 months, followed by a plateau. The recovery kinetics after transplantation of cryopreserved or fresh PBSC were not different as determined by a general linear model based on serial data (P = .926; Figure 1D).
The cumulative incidence of aGVHD grades II-IV was 78.2% ± 4.3% after cryopreserved and 81.2% ± 4.5% after fresh PBSCT (P = .113; Figure 2A). The respective cumulative incidence of cGVHD at 1 year amounted to 83.8% ± 5.1% and 90.6% ± 3.4% (P = .673; Figure 2B). The OS rates at 1 and 2 years for recipients of cryopreserved PBSCT were 64.3% ± 5.1% and 52.7% ± 6.5% compared to 65.1% ± 4.6% and 59.4% ± 4.8% for the group of patients that had received a fresh PBSCT (P = .668, Figure 3A). The NRM and relapse rates were not significantly different in both groups. The respective 1-year NRM rates were 24.6% ± 4.6% and 20.4% ± 4.2% (P = .340; Figure 3B), with a 2-year relapse rate of 26.6% ± 5.8% and 19.4% ± 4.3% (P = .295; Figure 3C).
Figure 2.
Incidence of acute (A) and cGVHD (B) after cryopreserved and fresh PBSCT.
Figure 3.
Overall survival, nonrelapse mortality, and recurrence rates of patients receiving cryopreserved or freshly procured allografts.
Stem Cell Loss after Cryopreservation
The viability assessment of TNCs by trypan blue exclusion yielded a median recovery of 71.2% ± 1.0% after thawing. The recovery rates ranged from 53% to 98%. Clonogenic hematopoietic progenitors were evaluated in cell culture before and after cryopreservation to determine the relevance of trypan blue data for this subset of cells (Figure 4). The loss of CFU-GM (24.6% ± 2.5%), BFU-E (30.5% ± 2.5%), and CFU-GEMM (21.3% ± 2.1%) was similar to calculated values based on trypan blue exclusion. In contrast, the recovery of CFU-MEG as determined by culture studies was significantly lower than calculated values (61.4% ± 3.1% versus 28.8% ± 1.0%; P < .001 by paired t-test).
Figure 4.
Frequency of clonogenic hematopoietic progenitors in PBSC preparation before cryopreservation and after thawing. Colony counts after 14 days’ incubation of 5 × 104 TNCs. Abbreviations: CFU-GM, colony-forming units granulocyte-macrophage; BFU-E, Burst-forming units erythroid; CFU-MEG, colony-forming units megakaryocyte; CFU-GEMM, colony-forming units granulocyte-erythrocyte-monocyte-megakaryocyte.
Correlation of Clonogenic Progenitors with CD34+ Cells and Total Nucleated Cells
The number of transplanted CD34+ cells was generally well correlated with the frequency of transplanted clonogenic progenitors including CFU-GM (r = 0.666, P < .001), BFU-E (r = 0.670, P < .001), CFU-MEG (r = 0.478, P < .001), and CFU-GEMM (r = 0.052, P .052) (Table 4). In contrast, the number of transplanted TNCs was neither correlated with CD34+ cells (P = .511) nor with clonogenic pregenitors except for CFU-GEMM (r = 0.438, P < .001).
Table 4. Correlation of Transplanted Doses of TNCs, CD34+ Cells, and Clonogenic Hematopoietic Precursors
| Correlation Coefficient (R) | ||||||
|---|---|---|---|---|---|---|
| Cell Type | TNCs | CD34+ Cells | CFU-GM | BFU-E | CFU-MEG | CFU-GEMM |
Factors Affecting Engraftment after Allogeneic PBSCT
Four of the 105 recipients of cryopreserved PBSCT failed to show engraftment of neutrophils. All 4 suffered from severe infections: pneumonia (n = 1), hepatosplenic candidiasis (n = 1), necrotizing fasciitis (n = 1), and Escherichia coli sepsis (n = 1). The remaining 101 patients reached neutrophil levels of ≥1.0 × 109/L with a median of 21 days. Patients receiving a higher number of either cryopreserved or fresh CD34+ cells engrafted their neutrophils faster than patients with lower cell doses when treating the number of CD34+ cells as a continuous (P = .003) or categoric variable below and above the median (16 days versus 18 days, P = .015). For both measures of neutrophil engraftment (≥0.5 or ≥1.0 × 109/L), there was no association with the transplanted dose of TNCs (P = .880 and .685), CFU-GM (P = .985 and .560), BFU-E (P = .539 and .663), CFU-MEG (P = .297 and .087), or CFU-GEMM (P = .805 and .619).
One (1%), 6 (6%), and 16 patients (15%) did not reach a platelet count over 20, 50, and 100 × 109/L, respectively. The median number of days to achieve these counts was 13.0, 15.0, and 21.0 days. Patients receiving a higher CD34+ cell dose increased their platelet count more rapidly. Patients receiving a higher number of CD34+ cells engrafted their platelets above ≥100 × 109/L faster than patients with lower cell doses when treating the number of CD34+ cells as a categoric variable below and above the median (18 days versus 28 days, P = .005).
To analyze the influence of the transplanted dose of CFU-MEG on the platelet engraftment, patients were grouped in quartiles based on the transplanted dose of CFU-MEG. The time to achieve a platelet count of over 20 or 50 × 109/L was similar in all 4 groups independent of using cryopreserved or fresh PBSCs. The time to reach a platelet count of over 100 × 109/L was not different in all 4 groups when analyzed in overall patients or in the group receiving fresh PBSCT. However, the time to reach platelet counts over 100 × 109/L after transplantation of cryopreserved PBSC was slow for patients receiving a dose of CFU-MEG in the lowest quartile compared to the patients with the other 3 quartiles of CFU-MEG doses (median day 29 versus 18 days, P = .046). In addition, lower proportion of patients in this subgroup achieved a platelet count of over 100 × 109/L (67% versus 89%).
Evaluation of Cryopreservation as a Risk Factor for OS, NRM, and Disease Recurrence
A uni- and multivariate analysis using a Cox’s proportional hazard model was performed to evaluate the contribution of cryopreservation on OS, NRM, and recurrence in the context of a number of clinically relevant parameters. The data are shown in Table 5. The development of cGvHD influenced positively all 3 clinical outcomes. In addition, older age was associated with poorer OS and increased NRM. Patients with high-risk disease experienced reduced OS and increased relapse rates. None of the outcome measures was influenced by cryopreservation.
Table 5. Uni- and Multivariate Analyses to Evaluate the Contribution of Cryopreservation in the Context of Clinical Risk Ractors on OS, NRM, and Disease Recurrence
| Prognostic Factor | Univariate | Multivariate | ||
|---|---|---|---|---|
| P-Value | HR (95% CI) | P-Value | HR (95% CI) | |
| Overall Survival | ||||
| Cryopreservation | .768 | 1.100(0.601-1.503) | NS | — |
| Myeloablative conditioning | .103 | 1.455(0.926-2.283) | NS | — |
| High risk disease | .030 | 2.247(1.082-4.663) | .027 | 4.095(1.170-14.330) |
| Older age (≥50 years) | .015 | 1.711(1.110-2.638) | .021 | 1.801(1.094-2.967) |
| Female donor/male recipient | .824 | 0.949(0.597-1.509) | NS | — |
| Acute GVHD, grade 2-4 | .833 | 0.944(0.553-1.612) | NS | — |
| Chronic GVHD | <.001 | 0.216(0.128-0.365) | <.001 | 0.197(0.114-0.339) |
| Non-relapse mortality | ||||
| Cryopreservation | .342 | 1.330(0.739-2.395) | NS | — |
| Myeloablative conditioning | .841 | 1.070(0.551-2.080) | NS | — |
| High-risk disease | .439 | 0.457(0.063-3.321) | NS | — |
| Older age (≥50 years) | .020 | 2.113(1.124-3.974) | .041 | 2.299(1.036-5.102) |
| Female donor/male recipient | .745 | 1.110(0.591-2.087) | NS | — |
| Acute GVHD, grade 2-4 | .091 | 2.439(0.866-6.896) | NS | — |
| Chronic GVHD | .021 | 0.372(0.161-0.858) | .006 | 0.302(0.129-0.709) |
| Recurrence | ||||
| Cryopreservation | .297 | 1.432(0.729-2.811) | NS | — |
| Myeloablative conditioning | .008 | 0.411(0.214-0.790) | NS | — |
| High risk disease | <.001 | 8.431(3.816-18.625) | <.001 | 4.854(2.041-11.628) |
| Older age (≥50 years) | .111 | 1.719(0.883-3.348) | NS | — |
| Female donor/male recipient | .146 | 0.543(0.239-1.238) | NS | — |
| Acute GVHD, grade 2-4 | .094 | 0.888(0.419-1.883) | NS | — |
| Chronic GVHD | <.001 | 0.121(0.058-0.249) | <.001 | 0.135(0.064-0.285) |
Discussion
The marrow repopulating ability of hematopoietic progenitor cells is sustained after cryopreservation. Graft cryopreservation technology is the cornerstone of autologous transplantation and has facilitated the development of umbilical cord blood banking. The experience with cryopreserved allografts outside of cord blood transplants is limited and restricted to marrow as a source of repopulating cells [13, 16]. A series using cryopreserved allogeneic PBSC has not yet been published. In part, the reluctance to use cryopreserved allografts is based on concerns that the procedure may alter viability and function of various cellular subsets that might negatively influence graft function. Specifically, the question is raised as to whether or not potential alterations of lymphoid subsets may reduce the antimalignancy effect of the allograft. In addition, the use of DMSO as cryoprotectant may result in toxicities that are not encountered with the use of fresh allografts [1, 2, 3, 4].
These issues were addressed in our current study comparing the outcome of 2 consecutive cohorts of patients transplanted with either cryopreserved or freshly collected PBSCs. The cohorts did not differ in engraftment kinetics including lymphocyte recovery, OS, NRM, recurrence, aGVHD, and cGVHD. We were able to demonstrate that (1) there was no delay in engraftment of neutrophils and platelets, especially in terms of long-term platelet engraftment beyond 100 × 109/L. (2) The infusion-related toxicity was acceptable: 99%, ≤grade 2. Based on our data, the DMSO volume infused per patient appears to be associated with cardiovascular adverse events such as hypotension or bradycardia [5, 8]. (3) All 105 cryopreserved allografts were free from bacterial contamination [16, 30, 31]. (4) The frequency of PBSC collections was acceptable, because 97% of all donors required only 1 or 2 procedures [16].
The use of cryopreserved allografts is associated with benefits. As discussed in a review article by Frey et al. [16], it provides the following opportunities: (1) greater flexibility to schedule collections and transplants, (2) evaluation of the quality of grafts particularly prior to initiating myeloablative preparation of the respective recipient, (3) rescheduling of collections in the event of poor mobilization and potential use of different mobilization strategies such as administration of AMD 3100 [32, 33] or the use of different donors, and (4) collection and cryopreservation of sufficient cells for multiple infusion of DLIs through a single procedure.
Our data on PBSCT are largely consistent with previous studies comparing cryopreserved and fresh bone marrow [14]. Similar engraftment kinetics of neutrophils and platelets, as well as the day 100 survival were reported [14, 34]. There was some discrepancy with respect to published data for aGVHD. Stockschlader et al. [14] reported a similar incidence of grade II-IV aGVHD for both recipients of fresh and cryopreserved marrow compared to a lower incidence observed for recipients of cryopreserved marrow by Eckardt et al. [34]. As mentioned above, similar rates of aGVHD (P = .113) and cGVHD (P = .673), as well as recurrence, were observed for both cohorts receiving PBSCT, suggesting that cryopreservation does not selectively alter graft function including the graft-versus-malignancy effect. This view is supported by a similar lymphocyte count recovery in both cohorts. Future studies should include the determination of more detailed functional profiles of the emerging lymphocyte populations such as flow cytometric profiling, T cell receptor rearrangement excision circles (TRECs), CD3R spectratyping, or ELISA/ELISPOT assays [35].
The rates of aGVHD and cGVHD were relatively high in both patient cohorts. One may speculate that this observation results from the relatively old patient population (median age, 50 years) and the use of PBSC as a source of stem cells. In addition, 10% of donors were 1 antigen mismatched donors, which might also contribute to a higher incidence of GVHD.
The possibility that cryopreservation may influence cell populations present at low concentrations, however, should at least be considered. In our study, attention was drawn to this possibility by differences in the survival of CFU-MEGs, indicating their increased sensitivity to cold damage compared to other clonogenic progenitors [36].
In summary, although not randomized, the presented study on 105 prospectively studied recipients of cryopreserved allogeneic PBSC and 106 historic controls that had received fresh allogeneic PBSC, suggests that cryopreserved allografts are not inferior to freshly collected preparations. Side effects related to the collection and infusion are generally of limited severity and clinical outcomes including engraftment kinetics, OS, NRM, aGVHD, cGvHD, and recurrence rates are similar.
Acknowledgments
D.K. and H.M. contributed to the design of the study, the supervision of data collection, and of data interpretation, data analysis, and writing of the manuscript, equally. N.J. and R.S. were involved in the laboratory investigations, the data collection, and critical revision of the manuscript. D.L. and J.W. were responsible for the clinical data collection, and critical revision of the manuscript. V.G., J.K., J.L., and M.M. were involved in the supervision of the data collection, interpretation of the data, and critical revision of the manuscript.
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PII: S1083-8791(07)00348-5
doi:10.1016/j.bbmt.2007.07.003
© 2007 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.
Volume 13, Issue 10 , Pages 1233-1243, October 2007
