CD34 Cell Dose Research Articles

Characteristics of CliniMACS® System CD34-Enriched T Cell-Depleted Grafts in a Multicenter Trial for Acute Myeloid Leukemia-Blood and Marrow Transplant Clinical Trials Network (BMT CTN) Protocol 0303

Eight centers participated in the Blood and Marrow Transplant Clinical Trials Network (BMT CTN) protocol 0303 to determine the effect of extensive T cell depletion (TCD) on the outcome of HLA matched sibling donor transplantation for acute myeloid leukemia. One goal of the study was to determine if TCD could be performed uniformly among study sites. TCD was achieved using the CliniMACS(®) CD34 Reagent System for CD34 enrichment. Processed grafts needed to contain ≥ 2.0 × 10(6) CD34(+)cells/kg with a target of 5.0 × 10(6) CD34(+) cells/kg and <10(5) CD3(+) T cells/kg. Up to 3 collections were allowed to achieve the minimum CD34(+) cell dose. In total, 86 products were processed for 44 patients. Differences in the starting cell products between centers were seen in regard to total nucleated cells, CD34(+) cells, and CD3(+) T cells, which could in part be ascribed to a higher dose of granulocyte-colony stimulating factor used for mobilization early in the trial. Differences between centers in processing outcomes were minimal and could be ascribed to starting cell parameters or to differences in graft analysis methods. Multivariate analysis showed that CD34(+) cell recovery (66.1% ± 20.3%) was inversely associated with the starting number of CD34(+) cells (P = .02). Median purity of the CD34 enriched fraction was 96.7% (61.5%-99.8%) with monocytes and B cells the most common impurity. All patients received the minimum CD34(+) cell dose, and 39 patients (89%) came within 10% or exceeded the target CD34(+) cell dose without exceeding the maximum T cell dose. All patients proceeded to transplantation and all achieved initial engraftment. Products processed at multiple centers using the CliniMACS System for CD34 enrichment were comparably and uniformly highly enriched for CD34(+) cells, with good CD34(+) cell recovery and very low CD3(+) T cell content.

Cryopreserved mobilized autologous blood progenitors stored for more than 2 years successfully support blood count recovery after high-dose chemotherapy

The ability of hematopoietic progenitor cells-apheresis (HPC-A) that have been stored for many years after cryopreservation to reconstitute hematopoiesis following high-dose chemo/radiotherapy has not been well-documented. In this retrospective study, eight Canadian centers contributed data from 53 autologous stem cell transplants (ASCT) performed using HPC-A that had undergone long-term storage (>2 years, range 2-7 years) and 120 ASCT using HPC-A stored for <6 months (short-term storage). The doses of nucleated and CD34(+) cells per kilogram recipient weight were similar between the short- (mean ± SD, 4.7 ± 4.9 × 10(8) and 6.8 ± 4.3 × 10(6), respectively) and long- (4.0 ± 4.9 × 10(8) and 6.1 ± 3.4 × 10(6), respectively) term storage groups. The median days to neutrophils (absolute neutrophil count; ANC) >0.5 × 10(9)/L (median 11 days for both short- and long-term storage) and platelets >20 × 10(9)/L (median 12 and 11 for short- and long-term storage, respectively) post-ASCT were not significantly different between the two groups. When ASCT performed with <5 × 10(6)/kg CD34(+) cells was compared there was also no difference in ANC or platelet recovery (median 12 days for both after short-term storage, and 12 and 11 days, respectively, after long-term storage). Fourteen HPC-A products stored for >5 years also showed similar count recoveries as the entire long-term storage group (median 11 days for both ANC and platelets). Cryopreserved HPC-A can be stored for at least 5 years with no apparent loss in their ability to support hematopoietic reconstitution after high-dose chemotherapy.

A risk-based approach to optimize autologous hematopoietic stem cell (HSC) collection with the use of plerixafor

Autologous hematopoietic stem cell (HSC) transplant is an effective treatment for patients with hematological malignancies. Unfortunately, 15-30% of patients fail to mobilize a sufficient number of HSCs for the transplant. Plerixafor is now used as a salvage mobilization regimen, with good success. We describe here a risk-based approach for the use of plerixafor, based on the circulating CD34(+) cell count and the CD34(+) cell dose collected after 4 days of G-CSF, that identifies potential poor HSC mobilizers upfront. A total of 159 patients underwent HSC collections using this approach. Of these, 55 (35%) were identified as high risk owing to low CD34(+) cell number or low yield on day 1 of collection, and received plerixafor on the subsequent days of collection. Of the 159 patients, 151 (95%) were able to provide adequate collections with the first mobilization attempt in a median of 1.7 days using this approach. Of the eight who failed initial mobilization, 5 successfully underwent re-mobilization with plerixafor and G-CSF and 3 (1.9%) were mobilization failures. This approach helped to control the overall cost of HSC collections for our BMT program by decreasing the need for remobilization, reducing the number of collection days and avoiding the use of plerixafor in all patients.

Higher CD3+ and CD34+ cell doses in the graft increase the incidence of acute GVHD in children receiving BMT for thalassemia

We evaluated the incidence of GVHD, risk factors and the impact of graft composition on acute GVHD (aGVHD) in 92 children who underwent BMT for thalassemia following busulfan/cyclophosphamide (BUCY)-based conditioning regimens and GVHD prophylaxis with CSA/short-MTX and methylprednisolone. The incidence of grade 2-4 and 3-4 aGVHD was 35% (95% confidence interval (CI) 25-44) and 9% (95% CI 4-16), respectively. We found that CD3(+) and CD34(+) cell doses above the median were associated with high incidence of grade 2-4 aGVHD (49 vs 20%, P=0.005 and 46 vs 23%, P=0.021, respectively). In multivariate analysis, high CD3(+) (hazard ratio (HR) 4.6; 95% CI 1.4-14.7; P=0.010) and CD34(+) (HR 4.3; 95% CI 1.4-12.7; P=0.011) cell doses were associated with grade 2-4 aGVHD. We further examined the effect of CD3(+) and CD34(+) cell doses on aGVHD using quartile cutoff points and found a minimum threshold for CD3(+) (38 × 10(6)/kg) and CD34(+) (4 × 10(6)/kg) cells above which the incidence of grade 2-4 aGVHD is significantly increased. This study shows for the first time a positive correlation between the number of CD3(+) and CD34(+) cells and aGVHD in children receiving sibling BMT, and indicates that using tailored and more intensive post transplant immunosuppression may permit to better control aGVHD.

Impact of CD34 Cell Dose on Platelet Engraftment and Platelet Transfusions Following Autologous Hematopoietic Stem Cell Transplantation

Maximizing the CD34 cell content of the graft has been reported to lead to faster platelet engraftment and lower requirement for platelet transfusions in patients undergoing autologous stem cell transplantation (A-HSCT). We evaluated the impact of CD34 cell dose on platelet recovery and transfusion requirements in 114 consecutive adults undergoing A-HSCT with a CD34 cell dose of > 2 × 106/kg recipient body weight (BW) infused.

Evaluation of mobilized peripheral stem cells according to CD34 and aldehyde dehydrogenase expression and effect of SSClo ALDHbr cells on hematopoietic recovery

We evaluated hematopoietic stem cells according to CD34 expression and aldehyde dehydrogenase (ALDH) activity in peripheral blood and apheresis product samples from patients after mobilization with granulocyte-colony-stimulating factor (G-CSF) alone or G-CSF after high-dose cyclophosphamide (4 g/m²) once daily, intravenously on day 1). We also investigated the relationship between the number of SSC(lo) CD45(dim) CD34(hi) cells, SSC(lo) ALDH(br) cells and engraftment. Thirty patients (20 males and 10 females), who were candidates for autologous peripheral blood stem cell transplantation, were included in the study. Cyclophosphamide + G-CSF was used for 17 and G-CSF alone for 24 mobilizations. Primary diagnoses were multiple myeloma (n = 14), Hodgkin's lymphoma (n = 7), non-Hodgkin's lymphoma (n = 2), acute myloid leukemia (n = 2), chronic lymphocytic leukemia (n = 1) and germ cell testis tumor (n = 1). Numbers of SSC(lo) CD45(dim) CD34(hi) cells and SSC(lo) ALDH(br) cells were highly correlated in both peripheral blood and apheresis products (P < 0.001). We could not find a relationship between the transplanted SSC(lo) CD45(dim) CD34(hi) cell dose or SSC(lo) ALDH(br) cell dose and platelet or neutrophil recovery. The optimal thresholds for SSC(lo) CD45(dim) CD34(hi) cells were 5.40 × 10⁶/kg for neutrophil recovery and 7.22 x 10⁶/kg for platelet recovery. The optimal thresholds for SSC(lo) ALDH(br) cells were 6.53 x 10⁶/kg for neutrophil recovery and 8.72 x 10⁶/kg platelet recovery. According to our data, numbers of SSC(lo) ALDH(br) cells are in very good agreement with numbers of SSC(lo) CD45(dim) CD34(hi) cells and can be a predictor of stem cell mobilization.

Transplanted CD34 + Cell Dose Is Associated with Long-Term Platelet Count Recovery following Autologous Peripheral Blood Stem Cell Transplant in Patients with Non-Hodgkin Lymphoma or Multiple Myeloma

Autologous hematopoietic stem cell transplantation (ASCT) is an established treatment for patients with hematologic malignancies, yet the impact of transplanted CD34(+) cell dose on clinical outcomes is unresolved. We conducted post hoc analyses of transplanted CD34(+) cell dose and hematopoietic recovery following ASCT in 438 patients with non-Hodgkin lymphoma (NHL) or multiple myeloma (MM), using data from 2 multicenter phase 3 clinical studies that compared plerixafor plus granulocyte-colony stimulating factor (G-CSF) versus placebo plus G-CSF as stem cell mobilization regimens. Days to engraftment and the proportion of patients who reached predetermined blood count thresholds were compared across 3 CD34(+) cell dose levels: 2-4 × 10(6) cells/kg, 4-6 × 10(6) cells/kg, and >6 × 10(6) cells/kg, regardless of mobilization treatment. Short-term neutrophil and platelet engraftment times were similar regardless of cell dose. A significant linear trend was observed between transplanted CD34(+) cell dose and the proportion of patients with platelet count >150 × 10(9)/L at 100 days (P < .001), 6 months (P = .026), and 12 months (P = .020) in patients with NHL, and at 100 days in patients with MM (P = .004). A linear trend was also observed between transplanted cell dose and the proportion of patients with platelet count >100 × 10(9)/L at 100 days (P < .001) and 6 months (P = .023) in patients with NHL. A higher cell dose was associated with a lower percentage of NHL patients requiring red blood cell transfusions (P = .006). Our analyses confirm previous findings that transplanted CD34(+) cell dose may be associated with better long-term platelet recovery after ASCT.

Interactions Between Total Nucleated and CD34 Cell Dose and Their Impact on Outcomes After Single and Double Unrelated Cord Blood Transplantation for Patients with Hematological Malignancies. An Analysis on Behalf of Societe' Française De Greffe De Moelle Et Therapie Cellulaire (SFGM-TC) and Eurocord

AbstractAbstract 361Pre-freezing (pf) and post-thawing (pt) total nucleated cells (TNC) is one of the most important factor for outcomes after UCBT. Its impact has been demonstrated after single UCBT (sUCBT); a minimum cell dose has been established as pfTNC of 2.5 ×107/kg, but its impact on outcomes after double UCBT (dUCBT) has not been shown. Also number of pfCD34 cells/kg is associated with outcomes after sUCBT, but only small series of patients have been analyzed. In order to evaluate the interactions between pf and pt TNC and CD34 and their impact on outcomes, we have studied separately 600 patients with hematological malignancies receiving a first sUCBT and 397 a first dUCBT in France (Table1). Methods:for all prognostic analysis pf and pt TNC and CD34 were divided into 4 categories at 25th, 50th and 75th percentiles. Results:Single UCBT: there was a highly statistical significant correlation between pf and pt TNC and CD34 (p<0.001, respectively). Median time to ANC recovery was 26 days (6-84). Cumulative incidence (CI) of ANC recovery at day 60 was 83%. In univariate analysis, the best cutoff point (associated with greater ANC recovery) of pfTNC and pfCD34 were ≥3.6 ×107/kg and '1.6 ×105/kg and ptTNC and ptCD34 were ≥3.3 ×107/kg and ≥1.3 ×105/kg. In multivariate analysis pf or pt TNC or CD34 were independently associated with ANC recovery (pfTNC HR=1.4, p=0.005; pfCD34 HR=1.3, p=0.01; ptTNC HR=1.4, p=0.01, ptCD34 HR=1.4, p<0.0001). We have not found any association of number of HLA disparities and ANC recovery. When analyzing only adults, patients receiving <2.0 ×107/kg, CI of ANC recovery was only 70% compared to 81% for ≥2.0 ×107/kg (p=0.02). CI of aGVHD at day 100 was 36% and was not associated with TNC or CD34. Estimated 2 year (y) OS was 47%, 2 y DFS was 40%. Interestingly, in a multivariate analysis, pfTNC, pfCD34 or ptCD34 were not associated with OS or DFS, but only higher ptTNC (≥3.3 ×107/kg) was associated with OS (51%vs43%, HR: 0.78, p=0.05) but not with DFS. Double UCBT: there was a highly correlation between pf and pt TNC and CD34 (p<0.001, respectively). CI of ANC recovery at day 60 was 81% in a median time of 23 days (5-64). In univariate analysis, pfTNC, pfCD34 and ptTNC were not associated with ANC recovery. However, there was an association of ptCD34 cell dose (Figure1) and ANC recovery. Chimerism data was available for 75% of the patients (n=298) during the first 100 days: 76% were full donor, 14% were mixed and 11% of the patients had autologous recovery. Autologous recovery was also associated with lower CD34 cell dose, it was 49% in patients receiving (<0.9×105/kg), and 25% for remainders (p<0.001). In multivariate analysis, ptCD34 >0.9x ×105/kg was the only independent factor associated with ANC recovery (HR=1.6, p=0.001). CI of acute GVHD at day 100 was 42% and was not associated with TNC or CD34 cell dose. At 1 y NRM was 22% and relapse incidence 26%. Estimate 1 y DFS was 50±3%. TNC or CD34 cell dose were not associated with any of the above outcomes. Only disease status at transplant impacts outcomes (64% early phase, 49% intermediate phase and 37% for more advanced disease). In conclusion, our study confirms the impact of cell dose measured by pf and ptTNC and CD34 on neutrophil recovery after sUCBT and the minimum cell dose recommended should be pfTNC≥2.5 ×107/kg and ptTNC≥2×107/kg, however only ptTNC is associated with survival in sUCBT. This is the first time that an impact of ptCD34 cell dose on neutrophil recovery after dUCBT is demonstrated and may be used to choose the best CB units. The different associations of cell dose in sUCBT and dUCBT can be explained by biological and immunological properties of other CB cells in the graft. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Validation of the Cord Blood (CB) APGAR Score System to Predict Neutrophil Engraftment of Plasma Depleted CB Products

AbstractAbstract 3549Cord blood potency is important for transplantation product selection, and various tests have been proposed to measure potency and predict engraftment potential, such as total nucleated cell dose (TNC), CD34+ cell dose (CD34), and colony forming unit dose (CFU). Though TNC has been the most widely used measure for CB selection to date, its predictive value is not as robust as the progenitor cell measurements. Unfortunately, most progenitor cell tests suffer from high inter-bank/laboratory coefficient of variance (CV) - decreasing the clinical utility of these tests as potency measures. Recently, the Duke Group proposed a CB APGAR scoring system composed of (1) a Pre-Cryopreserved Score (PCS) reflecting pre-freeze TNC, CD34, CFU and CB collected volume, as well as a (2) Composite Score (CS) which combines the PCS score with post-thaw TNC, CD34, CFU and mononuclear cell dose (MNC). Based on single, myeloablative and first transplants of mostly pediatric patients performed at a single institution (Duke) and using largely red cell reduced CB, the PCS and CS scores have been shown to be predictive of neutrophil and platelet engraftment probability. With CIBMTR-audited outcome data of transplanted CB products from a large multi-national CB bank, we sought to validate the CB APGAR system (1) on a patient population made up of mostly adults, (2) with heavy representation of minority and international patients, (3) on both single myeloablative and first transplants, as well as (4) other types of transplants, and lastly (5) using plasma depleted (red cell replete) CB products. The table below shows the day 42 neutrophil engraftment probability (ANC500) comparison of the original Duke data with StemCyte transplants using PCS and CS scores for both single, myeloablative and first transplants (SMF) and all transplants (All). For each of the PCS and CS strata that were compared, neutrophil engraftment probability appeared to be similar among the Duke SMF, StemCyte SMF and StemCyte All cohorts. We conclude that the Duke CB APGAR score, especially the PCS score, may serve as an easy-to-use and reproducible potency measurement that is highly predictive of neutrophil engraftment probability for (1) red cell reduced as well as plasma depleted CB, (2) for pediatric patient population as well as for mixed populations of adults and children, and (3) for minority and international patients. Lastly, the CB APGAR appears to be applicable to cord blood units with or without RBC depletion providing a tool that can be used for units processed and stored by various methods at different cord blood banks. Disclosures:Chow:StemCyte: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Wang:StemCyte: Consultancy, Equity Ownership. Lin:StemCyte: Employment. Kang:StemCyte: Employment. Petz:StemCyte: Employment, Equity Ownership. Kurtzberg:StemCyte: Consultancy.

Effect of Peripheral Blood Stem Cell (PBSC) Graft Composition on Graft Versus Host Disease (GVHD) and Mortality After Allogeneic Transplantation

AbstractAbstract 676We investigated the relationship of PBSC graft composition on GVHD and survival in 2,350 allogeneic transplant recipients from a single institution. Characteristics of the cohort included: median age: 50 (0.5-79); disease risk: low 12%, intermediate 41%, high 35%; HLA-matched sibling donor 53% and reduced-intensity conditioning 36%. Graft composition cell subsets included stem/progenitors (CD34+), monocytes (CD14+), B (CD20+), NK (CD3-CD56+), NK/T (CD3+CD56+), dendritic cell subsets (DC1 and DC2), and T-cell subsets (CD3+CD4+, CD3+CD8+, Memory, Naïve, Regulatory), measured by multicolor FACS analyses and expressed as infused cell subset/kg of actual recipient weight. Cell doses were modeled as continuous linear variables, by quartiles, or by cubic splines (allowing for non-linear models). Results:In contrast with previous findings, increasing CD34+ cell dose was not statistically associated with acute GVHD (p=0.49), chronic GVHD (p=0.91) or survival (p=0.74). However, several other cell subsets showed important associations. Shown in Figure 1 is a cubic spline graph of the hazard ratio (HR) of chronic GVHD as a function of CD3+CD4+ T-cell dose relative to a relatively low dose of 0.5 × 107/kg. [Display omitted] A highly significant correlation was found between larger doses of CD14+ monocytes and less chronic GVHD (p<0.001). Figure 2 displays the HR results compared to a dose of 0.5 × 108 CD14 cells/kg, indicating a strong association in the alternative donor setting (p<0.001: by quartile analysis HR=0.59 for Q4 vs. Q1; p=.0004), and remained a suggestive trend in the matched sibling group. For the overall mortality analysis, several cell subsets showed significant associations. Patients receiving higher doses of NK cells had lower survival (p=0.007). Higher doses of memory CD4 was also found to be associated with lower survival, but the effect appeared to be restricted to the matched-sibling donor group (p=0.007). Increasing doses of DC1 were also associated with worse survival (p=0.02) for both reduced intensity and ablative conditioning groups, although the dose response was stronger in the ablative group (HR=1.17 Q4 vs. Q1). In contrast, higher doses of with NK/T cells correlated with lower mortality (p=0.01) but only in the matched-sibling group (Q4 vs. Q1, HR=0.78, p=0.05). [Display omitted] Conclusions:In this large, single center study, no associations of CD34 cell dose with GVHD or overall mortality were found. Further, our results suggest that previously reported associations between T cell dose and chronic GVHD are primarily driven by CD4 content in PBSC, and restricted to the matched-sibling donor (Figure 1). The lack of correlation with alternative donors may reflect differences in T-cell responses to the greater minor histocompatibility divergence with alternative donors, or changes in donor T-cell functionality associated with the higher dose of G-CSF (16 μg/kg/day) used for sibling donor mobilization at our center. To our knowledge, the correlation of higher CD14 dose with a reduced incidence of chronic GVHD is a novel finding, perhaps reflecting the known shift to a more immunosuppressive function of monocytes after G-CSF exposure. Similarly, reduced survival associated with higher doses of NK, memory CD4, and DC1 cells, and improved survival with greater numbers of NK/T cells represent new findings that will require additional analyses. Altogether, these correlations of GVHD and mortality with PBSC cell subsets support the concept that engineering of grafts could result in improved outcomes with allogeneic transplantation. Disclosures:No relevant conflicts of interest to declare.

CXCR4 expression on transplanted peripheral blood CD34+ cells: relationship to engraftment after autologous transplantation in a cohort of multiple myeloma patients

Expression of the chemokine receptor CXCR4 by haematopoietic stem cells (HSCs) is believed to influence the process of these cells 'homing' back to the bone marrow post-transplantation, in response to the stromal cell-derived factor-1 gradient, followed by engraftment. The primary aim of this retrospective study was to compare reinfused CD34(+) cell dose, assessed from the fresh collection, with the post-thaw viable (v) CD34(+) and vCD34/CXCR4(+) dual positive cell dose as predictors of haematopoietic recovery in multiple myeloma patients undergoing autologous stem cell transplantation. Cryopreserved samples from stem cell collections of 27 myeloma patients were analysed for CD34 and CXCR4 expression and times to haematological engraftment measured. Dosage of transplanted vCD34(+) cells was on average 79% of the original calculation from the fresh collection bag (range 29-98%). The median percentage of vCD34+ cells co-expressing CXCR4 was 37% (3.7-97%). Surface expression of CXCR4 by thawed vCD34(+) cells was closely correlated to complementary DNA levels. The median dose of CD34/CXCR4(+) cells in the autografts was 1.2 × 10(6)/kg (0.2-3.0 × 10(6)/kg) compared with 3.3 × 10(6)/kg for transplanted vCD34(+) cells (1.2-5.5 × 10(6)/kg). Both CD34 and vCD34 doses correlated with neutrophil engraftment (p < 0.005) although vCD34/CXCR4(+) dose did not. However, patients given a higher dose of CD34/CXCR4(+) cells (≥1.75 × 10(6)/kg) showed a faster time to platelet recovery (p < 0.05) than those given a lower dose (≤0.42 × 10(6)/kg). These results warrant further study of CD34/CXCR4 expression by mobilised HSCs and the relationship to platelet recovery post-transplantation on a larger cohort of patients.

Adult Dual Umbilical Cord Blood Transplantation Using Myeloablative Total Body Irradiation (1350 cGy) and Fludarabine Conditioning

High treatment-related mortality (TRM) and high graft failure rate are serious concerns in HLA-mismatched umbilical cord blood (UCB) transplantation with myeloablative conditioning. We conducted a prospective trial of dual UCB transplantation using modified myeloablation consisting of total-body irradiation (TBI; 1350 cGy) and fludarabine (Flu) (160 mg/m(2)). Twenty-seven patients (median age, 33 years; range: 20-58 years) with hematologic malignancies were enrolled. The median combined cryopreserved total nucleated cell (TNC) dose was 4.3 × 10(7)/kg (range: 3.2-7.7 × 10(7)/kg). The cumulative incidences of neutrophil (≥500/μL) and platelet (≥50,000/μL) engraftment were 80% (95% confidence interval [CI], 58%-91%) and 68% (95% CI, 46%-83%), respectively. Among engrafted patients, a single cord blood unit was predominant by 100 days posttransplantation. A higher cryopreserved and infused TNC dose and infused CD3(+) cell dose were significant factors associated with the predominant UCB unit (P = .032, .020, and .042, respectively). TRM and relapse rates at 2 years were 28% (95% CI, 12%-47%) and 20% (95% CI, 7%-37%), respectively. Cumulative incidences of grades II-IV and grades III-IV acute graft-versus-host disease (aGVHD) were 37% (95% CI, 20%-55%) and 11% (95% CI, 3%-26%), respectively, and that of chronic GVHD was 31% (95% CI, 15%-49%). With a median follow-up of 23 months, overall survival and disease-free survival ratesat2years were 58% (95% CI, 34%-75%) and 52% (95% CI, 29%-70%), respectively. This study supports the use of TBI 1350 cGy/Flu as an alternative to conventional myeloablative conditioning for dual UCB transplantation.

Reduced-intensity conditioning with Fludarabin, oral Busulfan, and thymoglobulin allows long-term disease control and low transplant-related mortality in patients with hematological malignancies

The development of reduced-intensity conditioning regimens rather than myeloablative regimens for allogeneic stem cell transplantation has led to decreased treatment-related mortality and increased use of this treatment modality, especially in older patients with hematological malignancies. No randomized controlled trials have been performed resulting in determining effectiveness on phase II studies, which rarely report on long-term survival. In an attempt to address this limitation, we analyzed a single-center cohort of 100 consecutive patients with hematological malignancies undergoing allogeneic stem cell transplantation from a human leukocyte antigen-matched related donor with median follow-up of 60 months. The reduced-intensity conditioning regimen consisted of oral Busulfan, rabbit anti-thymocyte globulin, and Fludarabin. Median age was 50 years (range, 18-64 years). The incidences of acute and chronic graft-vs.-host disease were 43% and 81%, respectively. The probability of nonrelapse mortality at 1 and 5 years was 15% and 25%, respectively. Nonrelapse mortality was adversely associated with acute graft-vs.-host disease (hazard ratio = 6; p = 0.0002). Of the 52 patients with measurable disease, 37 (71%) achieved a response. Relapse/progression occurred at a median of 11 months (range 1-52 months) in 21 patients, for a cumulative incidence of 22%. The probability of overall survival and progression-free survival at 5 years were 60% and 54%, respectively. Overall survival and progression-free survival were favorably influenced by having had previous autologous stem cell transplantation and a low CD34(+) cell dose. Overall survival, progression-free survival, and nonrelapse mortality improved over time in this cohort of patients. These results are encouraging for populations different in term of age, diagnosis, and disease status.

GVHD after unrelated cord blood transplant in children: characteristics, severity, risk factors and influence on outcome

We reviewed our experience in 79 children who had unrelated cord blood transplant (UCBT) between 1996 and 2007 with a major focus on GVHD, comparing both traditional and National Institute of Health (NIH) criteria. The cumulative incidence (CI) of acute GVHD (aGVHD, by day +100) was 0.42 for grade II-IV and 0.22 for grade III-IV. The CI of all aGVHD (NIH, that is, no time limit) at 1 year was 0.45 for grade II-IV and 0.32 for grade III-IV. Infused CD34 cell dose (>1 × 10(5)/kg), pretransplant bacterial infection and nonmalignant disorders were risk factors for grade II-IV aGVHD on univariate analysis. Infused CD34 cell dose remained significant on multivariate analysis. At 1 year, the CI of chronic GVHD (cGVHD) using the Seattle criteria was 0.27, whereas that for cGVHD (NIH) was 0.08. By NIH criteria, the classic form of cGVHD was uncommon (5%) after UCBT. Instead, the acute (71%) and overlap (24%) GVHD variants predominated. Grade II-IV aGVHD was a significant risk factor for cGVHD by both Seattle and NIH criteria. We conclude that GVHD after day +100 after UCBT typically carries features of aGVHD. Moreover, and in marked contrast to adult unrelated donor hematopoietic stem cell transplantation, the GVHD observed in this series did not adversely affect survival.

Strategy for a multicenter phase I clinical trial to evaluate globin gene transfer in β‐thalassemia

Globin gene transfer in autologous hematopoietic stem cells offers a potentially curative treatment option for patients suffering from beta-thalassemia major who lack an HLA-matched hematopoietic stem cell donor. Based on extensive preclinical investigation, we are initiating a phase I clinical trial using G-CSF mobilized, autologous CD34(+) cells transduced with a vector similar to the original TNS9 vector. Our first mobilizations in adult beta-thalassemic subjects have been well tolerated and yielded the required CD34(+) cell dose. To minimize toxicity to enrolled subjects, and in the absence of a demonstrated requirement for myeloablative conditioning, our trial will use a reduced intensity conditioning regimen. Because low vector titers may adversely affect efficacy and safety, we have focused on vector manufacturing processes. We are now in a position to transfer our globin lentiviral vectors in a clinically relevant dosage (averaging 0.8 vector copy per cell in bulk CD34(+) cells) and to supply clinical grade vector to collaborating centers in the U.S.A. and in Europe. We anticipate that the first U.S. trial of globin gene transfer will start in 2010.

Single-Unit Umbilical Cord Blood Transplantation from Unrelated Donors in Adult Patients with Chronic Myelogenous Leukemia

Clinical studies focused on outcomes of umbilical cord blood transplantation (UCBT) for patients with chronic myelogenous leukemia (CML) in need of allogeneic stem cell transplantation and lacking an HLA-matched adult donor are limited. We analyzed the outcome of 26 adults with CML receiving single-unit UCBT from unrelated donors after myeloablative conditioning at a single institution. Conditioning regimens were based on combinations of thiotepa, busulfan, cyclophospamide or fludarabine, and antithymocyte globulin. At the time of transplantation, 7 patients (27%) were in first chronic phase (CP), 11 (42%) were in second CP, 2 (8%) were in accelerated phase (AP), and 6 (23%) were in blast crisis (BC). The cumulative incidence (CI) of myeloid engraftment was 88% at a median time of 22 days and was significantly better for patients receiving higher doses of CD34(+) cells. The CI of acute graft-versus-host disease (GVHD) grade II-IV was 61%, that of acute GVHD grade III-IV was 39%, and that of chronic extensive GVHD was 60%. Treatment-related mortality (TRM) was 41% for patients undergoing UCBT while in first or second CP and 100% for patients in AP or BC (P < .01). After a median follow-up of 8 years, none of the patients relapsed, giving an overall disease-free survival (DFS) at 8 years of 41%. The DFS for patients undergoing UCBT while in any CP was 59%. These results demonstrate that UCBT from unrelated donors can be a curative treatment for a substantial number of patients with CML. Advances in supportive care and better selection of cord blood units and patients are needed to improve TRM.

Higher CD34 + and CD3 + Cell Doses in the Graft Promote Long-Term Survival, and Have No Impact on the Incidence of Severe Acute or Chronic Graft-versus-Host Disease after In Vivo T Cell-Depleted Unrelated Donor Hematopoietic Stem Cell Transplantation in Children

The aim of our study was to compare the results of unrelated donor (UD) peripheral blood stem cell transplantation versus UD bone marrow transplantation and to analyze the impact of infused CD34(+) and CD3(+) cell doses on survival and incidence of severe graft-versus-host disease (GVHD) in 187 children who underwent UD hematopoietic cell transplantation with the use of in vivo T cell depletion (antithymocyte globulin or CAMPATH-1H). HLA typing was performed at the "high-resolution" level. Patients receiving > or =10 x 10(6) CD34(+) cells/kg and > or =4 x 10(8) CD3(+) cells/kg had better overall and disease-free survival. Multivariate analysis has shown that both infused CD34(+) cell dose <10 x 10(6)/kg and CD3(+) cell dose <4 x 10(8)/kg were independent risk factors for mortality (relative risk [RR] 1.8 and 1.71, P = .009 and .016, respectively). Regarding disease-free survival, multivariate analysis has revealed another independent risk factor for poor outcome apart from the 2 earlier-mentioned cell doses, which was the use of donors mismatched at 2 HLA antigens or 3 HLA allele/antigens (RR 2.5, P = .004). In age groups 0-10 years and 10-20 years, CD34(+) cell doses higher than the age-adjusted median dose clearly favored survival. Higher infused doses of CD34(+) and CD3(+) cells did not result in an increased rate of severe GVHD. The use of mismatched donors was the only independent risk factor for the incidence of severe acute GVHD (RR 2.2, P = .046). The report demonstrates for the first time in a pediatric cohort, that higher doses of transplanted CD34(+) and CD3(+) cells lead to an improved survival without an increased risk of severe GVHD. The study findings may be limited to the population of patients receiving in vivo T cell depletion, which is now broadly used in unrelated donor setting in Europe.

Cutpoint selection for discretizing a continuous covariate for generalized estimating equations

We consider the problem of dichotomizing a continuous covariate when performing a regression analysis based on a generalized estimation approach. The problem involves estimation of the cutpoint for the covariate and testing the hypothesis that the binary covariate constructed from the continuous covariate has a significant impact on the outcome. Due to the multiple testing used to find the optimal cutpoint, we need to make an adjustment to the usual significance test to preserve the type-I error rates. We illustrate the techniques on one data set of patients given unrelated hematopoietic stem cell transplantation. Here the question is whether the CD34 cell dose given to patient affects the outcome of the transplant and what is the smallest cell dose which is needed for good outcomes.

Donor KIR Genes 2DL5A, 2DS1 and 3DS1 Are Associated with a Reduced Rate of Leukemia Relapse After HLA-Identical Sibling Stem Cell Transplantation for Acute Myeloid Leukemia but Not Other Hematologic Malignancies

Stem cell transplantation (SCT) from a healthy donor can be curative for patients with hematologic malignancies resistant to other treatments. Elimination of malignant cells through a graft-versus-leukemia (GVL) effect involves donor T and natural killer (NK) cells, but their relative contribution to this process is poorly defined. NK cell alloreactivity and GVL effects are controlled by the nature of the interaction of NK activation receptors and killer-immunoglobulin-like-receptors (KIR) with major histocompatibility locus class I antigens on the target cell. We performed KIR-genotyping of HLA-identical sibling donors in 246 T cell-depleted SCTs to identify genetic factors affecting transplant outcome (treatment-related mortality [TRM], leukemic relapse, and survival). Univariate and multivariate analysis of transplant-related risk factors and KIR genotyping was performed to identify independent variables predictive of outcome for different forms of leukemia. Further to confirming known predictive factors for TRM and survival (CD34 cell dose, patient age, disease stage), statistical analysis revealed that 3 donor B haplotype KIR genes, 2DL5A, 2DS1, and 3DS1, were associated with significantly less relapse in patients with acute myelogenous leukemia (AML) (13% versus 57%) but not in patients with other myelogenous or lymphoid malignancies. AML patients receiving SCT from donors with these KIR genes relapsed 4 times less frequently than patients transplanted from donors with other KIR genotypes. These findings suggest specific, genetically determined, interactions between NK cells and AML cells that facilitate the GVL effect, and have implications for donor selection for AML patients.

Donor-Derived Second Hematologic Malignancies after Cord Blood Transplantation

Double umbilical cord blood transplantation (UCBT) with a reduced-intensity conditioning regimen is an effective strategy for adult patients without a matched donor. The risk of second malignancies in these patients has not yet been established, however. In the present study, 98 adults with a hematologic malignancy underwent double UCBT. Seventy patients received a reduced-intensity conditioning regimen of fludarabine 30 mg/m(2)/day for 6 days, melphalan 100 mg/m(2)/day for 1 day, and rabbit antithymocyte globulin 1.5 mg/kg/day for 4 days, and 28 patients received a myeloablative total body radiation-containing conditioning regimen. Sixty-three patients received sirolimus-based graft-versus-host disease (GVHD) prophylaxis, and 35 patients received non-sirolimus-based GVHD prophylaxis. The median patient age was 48 years (range, 19-67 years). Eighteen patients developed a second malignancy at a median of 134 days after transplantation. Sixteen patients had lymphoma, and 2 patients had myelodysplasic syndrome/myeloproliferative disorder (MDS/MPD). Sixteen of these second malignancies (both cases of MDS/MPD and 14 of the lymphomas) were donor-derived; the origins of the others were not determined. GVHD prophylaxis, HLA matching, primary disease, age, total nucleated cell dose, and CD34(+) cell dose were not associated with a higher rate of second malignancy. Second myelogenous malignancies of donor origin occur after double UCBT, suggesting that a search for donor origin should be performed in all patients with suspected relapse.