Peripheral Blood Progenitor Cell Grafts Research Articles

Quantification of Acute Lymphoblastic Leukemia Clonotypes in Leukapheresed Peripheral Blood Progenitor Cells Predicts Relapse Risk Following Autologous Hematopoietic Cell Transplantation

BackgroundAlthough multiple studies have shown superiority of allogeneic hematopoietic cell transplantation (alloHCT) over autologous hematopoietic cell transplantation (autoHCT) for patients with "high-risk" acute lymphoblastic leukemia (ALL), these findings may be explained, in part, by contamination of the peripheral blood progenitor cell (PBPC) leukapheresis product by residual leukemic cells in patients undergoing autoHCT.MethodsWe retrospectively evaluated minimal residual disease (MRD) via next-generation sequencing (NGS) (Adaptive Biotechnologies, S. San Francisco, CA) in the PBPC leukapheresis products from 32 ALL patients who underwent autoHCT. All patients had "high-risk" ALL, as defined by B-lineage disease with WBC at diagnosis >30,000/uL; high-risk cytogenetics including t(9;22), t(4;11), other 11q23 abnormalities, or monosomy 7; or primary refractory disease.Peripheral hematopoietic cell mobilization consisted of cytarabine 2000mg/m2 IV every 12 hours (16,000mg/m2 cumulative) concurrent with etoposide 40mg/kg cumulative by continuous IV infusion over 4 days. The autoHCT conditioning consisted of 1320cGy total body irradiation (TBI) given in 11 fractions from day -8 to -5, etoposide 60mg/kg IV on day -4, and cyclophosphamide 100mg/kg IV on day -2. Tyrosine kinase inhibitors (TKI) were allowed for patients with Philadelphia chromosome-positive (Ph+) B-ALL. Seven patients (22%) participated in a multi-institutional trial in which the PBPC graft underwent ex vivo complement-mediatedpurging using monoclonal antibodies against CD9/CD10/CD19/CD20 for patients with B-lineage disease, or against CD2/CD3/CD4/CD5/CD8 for patients with T-lineage disease.Kaplan-Meier curves were generated using GraphPad Prism (GraphPad Software, La Jolla, CA) and statistical differences assessed via Log-Rank and Wilcoxon analyses, with a p-value of <0.05 considered significant.ResultsTwenty-eight patients (88%) had diagnostic bone marrow samples with quantifiable immunoglobulin or T cell receptor (Ig/TCR) gene rearrangements suitable for MRD quantification in the PBPC collections. Twelve (38%) patients had Ph+ B-ALL, 12 (38%) had Ph-neg B-ALL, and 4 (14%) had T-cell ALL. The majority of patients were male, in first complete remission, and autografted between 2000-2009, with a median age at autoHCT of 32 (range 19-55).With a median follow-up of 41 months (range 3-217), median relapse-free survival (RFS) and overall survival (OS) for the entire cohort are 3.2 and 4.2 years, respectively. At 5 years post-autoHCT, 42% of patients remain alive and relapse-free.Of the 28 diagnostic bone marrow specimens, 21 (75%) had rearrangements in more than 1 immunoreceptor locus (Figure 1A). A total of 73 rearrangements were identified across all patients. Clonal Ig heavy chain (IGH) sequences with complete VDJ rearrangement were the most commonly identified rearrangement, present in 17 of 28 patients (61%) and representing 30 of the 73 (41%) total rearrangements.When stratified by graft MRD burden, the median RFS for patients with MRD detectable at a level ≥10-6 (n=13) was 6.5 months, and has not been reached for patients without detectable MRD above this threshold (n=15; p=0.0005; Figure 1B). Ex vivo antibody-based purging failed to eradicate leukemia cells from the PBPC collections of 3 patients with detectable MRD who received a purged graft (Figure 1C), all of whom ultimately relapsed.Of the Ph+ cohort, 6 (50%) had detectable MRD in the PBPC graft. Two (33%) of these patients were not treated with a TKI; 1 relapsed at 4 months post-autoHCT and the other died from non-relapse mortality. Of the 4 Ph+ patients with detectable MRD who received a TKI, 2 (50%) remain long-term relapse-free survivors. Of the 6 patients without MRD who were treated with a post-autoHCT TKI, 5 (83%) remain relapse-free.ConclusionWe demonstrate that the NGS-based immunosequencing platform identifies ALL MRD in leukapheresed PBPC collections. The presence of MRD in the autograft strongly predicts relapse, with only 15% RFS in patients harboring ≥10-6 MRD at stem cell collection versus 73% in patients with undetectable MRD. The absence of MRD in PBPC may thus identify a subset of "high-risk" patients likely to achieve long-term remissions without alloHCT. TKI therapy for patients with Ph+ B-ALL may also, in some cases, abrogate the need for alloHCT, even with quantifiable MRD prior to high-dose therapy. [Display omitted] DisclosuresDamon:Atara: Consultancy; Sunesis: Research Funding; Sigma-Tau: Research Funding; McGraw-Hill: Patents & Royalties: Book Chapter. Andreadis:McGraw Hill: Honoraria; Novartis: Consultancy; Cellerant: Consultancy; Pharmacyclics: Honoraria. Olin:Daiichi-Sankyo: Research Funding. Kong:Adaptive Biotechnologies: Employment, Equity Ownership. Faham:Adaptive Biotechnologies: Employment, Equity Ownership.

Impact of clinical factors and allograft leukocyte content on post-transplant lymphopenia, monocytopenia, and survival in patients undergoing allogeneic peripheral blood haematopoietic cell transplant.

BackgroundWe have previously shown that lymphopenia and monocytopenia at 2–3 months post-allogeneic haematopoietic cell transplant (HCT) is associated with poor survival in recipients of both myeloablative and reduced intensity conditioning regimens. It is not known whether the graft leukocyte content has a role in early lymphocyte and monocyte recovery following allogeneic T-cell replete peripheral blood HCT.MethodsHaematologic recovery data, including absolute lymphocyte and monocyte counts (ALC and AMC, respectively) at day +15, +30, +60, and +100, and outcomes data were pooled from two prior independent cohorts, and parameters were correlated with leukocyte graft content in those individuals receiving peripheral blood progenitor cell grafts. 216 consecutive patients from 2001–2010 were included in the analysis.ResultsNeither infused allograft lymphocyte, monocyte, granulocyte, nor CD34+ cell number per kilogram recipient body weight correlated with haematologic recovery parameters or overall survival in this cohort. Prognostic factors for overall survival based on multivariate analysis were as expected from the results of the previous independent cohorts and included severity of chronic GVHD (p < 0.001), development of post-transplant relapse (p = 0.001), day +60 AMC > 0.3 x 109 cells/L (p = 0.0015), and day +100 ALC > 0.3 x 109 cells/L (p < 0.001). Low monocyte and lymphocyte counts at the day +60 and day +100 time points were significantly associated with acute GVHD and/or CMV viraemia.ConclusionsThis study suggests that graft cell count does not influence post-transplant monocyte and lymphocyte recovery following T-cell replete allogeneic peripheral blood HCT. Post-transplant complications such as acute GVHD and/or CMV viraemia negatively influenced monocyte and lymphocyte recovery, and hence the survival. Further studies aimed at understanding the mechanisms behind sustained lymphopenia and monocytopenia post-transplant are needed.

Low CD34 Cell Dose Is Associated With Higher Non-Relapse and Overall Mortality After Reduced Intensity Conditioning Hematopoietic Cell Transplantation For Acute Myeloid Leukemia and Myelodysplastic Syndrome

In allogeneic hematopoetic cell transplantation (HCT) using myeloablative conditioning, the nucleated and CD34 cell doses were found to be of importance for several outcome parameters including survival, relapse and graft- versus- host disease (GVHD). Reduced intensity conditioning (RIC) HCT is increasingly used for older patients and for younger patients with co-morbidities. Peripheral blood progenitor cells (PBPC) is the predominant graft for RIC HCT. There is no large study of the effect of CD34 cell dose in the setting of RIC transplantation. Therefore, we studied the effect of CD34 dose on 1057 patients aged 45 – 75 years with acute myeloid leukemia (AML) or myelodysplasic syndrome (MDS) who received RIC regimen HCT between 2000 and 2011. Patients received grafts from HLA-matched siblings (n = 370) or from volunteer adult unrelated donors matched at the allele-level at HLA-A, -B, -C and –DRB1 (8/8; n = 522) or mismatched at 1 HLA-locus (7/8; n = 178). All patients received peripheral blood progenitor cells (PBPC), low dose TBI regimens (200 cGy) or alkylating agent plus fludarabine containing regimens. Separate analyses were conducted for HLA-matched and unrelated donor transplants. For HLA-matched sibling transplants (AML n=301; MDS n=69), exploratory analysis identified differences in survival for CD34 dose less than 4 x 106/kg. Multivariate modeling, adjusting for performance score, disease status, cytogenetic risk and transplant period, showed transplantation of PBPC with CD34 dose < 4 x 106/kg was associated with higher risks of overall mortality (HR 1.48, p=0.008) and non-relapse mortality (HR 2.03, p=0.004) compared to transplantation of PBPC with CD34 dose ≥ 4 x 106/kg. The effect of CD34 dose was independent of the other factors associated with mortality. The likelihood of neutrophil (HR 0.76, p=0.03) and platelet recovery (HR 0.76, p=0.03) was also lower with CD34 dose < 4 x 106/kg containing PBPC grafts. CD34 dose was not associated with acute or chronic GVHD or disease recurrence. In contrast, after unrelated donor transplantation (AML n=557; MDS n=130), the effect of CD34 cell dose on mortality was marginal. Exploratory analysis identified differences in survival for CD34 dose less than 6 x 106/kg. After adjusting for age, performance score, disease status, interval from diagnosis to HCT, and HLA-match, transplantation of PBPC with CD34 dose < 6 x 106/kg was associated with marginally higher risks for overall mortality (HR 1.20, p=0.05) and non-relapse mortality (HR 1.38, p=0.02) compared to transplantation of PBPC with CD34 dose ≥ 6 x 106/kg. There were no significant differences in hematopoietic recovery, acute or chronic GVHD and disease recurrence by CD34 dose. Further, we did not identify a CD34 dose for either donor source above which acute or chronic GVHD risks were higher. In conclusion, in the setting of RIC transplants for AML and MDS, the data support optimizing PBPC collections such that the CD34 dose delivered is in excess of 4 x 106/kg for HLA-matched sibling and CD34 dose in excess of 6 x 106/kg for unrelated donor transplants. Disclosures:No relevant conflicts of interest to declare.

Definition of the Variables Affecting Efficacy of Immunodepletion Ex Vivo of Peripheral Blood Progenitor Cell Grafts by Alemtuzumab (Campath in the Bag)

The immunodepleting effects of alemtuzumab on peripheral blood progenitor cell (PBPC) grafts for stem cell transplantation need to be better defined. The optimal graft cell concentration, antibody dose, need for complement, and whether alemtuzumab is infused with the graft during transplantation remain unclear. PBPC from 6 normal allogeneic stem cell donors harvested by apheresis were first quantitated and the cellular content defined by flow cytometry. Mononuclear cells were then incubated with incremental concentrations of alemtuzumab (.00001, .0001, .001, and .01 mg/mL) for 30 minutes at 20°C or in cell dose responses with 1, 5, and 10 × 106 mononuclear cells/mL added to a fixed dose of .001 mg/mL of alemtuzumab with or without a source of complement. Cells were enumerated and analyzed by flow cytometry before and after exposure to alemtuzumab. To determine the presence of unbound anti-CD52, the supernatant of the cell dose responses were tested using the ELISA assay. Selected CD34+ lineage-negative cells were incubated with antibody at the same working concentrations and conditions and cultured in granulocyte-macrophage colony-forming unit assay. The colony numbers were compared with control cultures devoid of the antibody. Incremental concentrations of alemtuzumab led to a significant (2 log) reduction in CD3, CD4, and CD8 populations, which plateaued at .001 mg/mL. Addition of complement led to a further significant reduction in the CD4 and CD8 cells. The maximum CD4 (3 log) and CD8 (2 log) cell death was obtained at 10 × 106 cells/mL. Analysis of supernatants for soluble alemtuzumab by ELISA showed a significant reduction in the free antibody concentration when the cell number was increased from 1 to 10 × 106 cells/mL implying utilization/binding of the antibody by target cells. Incremental concentrations of alemtuzumab did not affect the number of granulocyte-macrophage colony-forming units. Alemtuzumab depletes all cells expressing the CD52 antigen and has higher activity on CD3, CD8, and particularly on CD4 cells, which are depleted in excess of 2 logs. From this study, we were able to derive that the optimal cell kill in the graft without detectable free alemtuzumab in the supernatant can be achieved with 1 mg of antibody per 100 mL containing 10 × 109 cells and active complement (AB serum).

Effect of Stem Cell Source From Unrelated Donors on Transplant Outcomes In Severe Aplastic Anemia (SAA): a Comparison of Unrelated Bone Marrow (BM) and Peripheral Blood Progenitor Cells (PBPC)

AbstractAbstract 531Unrelated donor HSCT has been considered a therapy of last choice in SAA, because of poor overall survival reported prior to 1998 (32% @ 5-years). This is due in large measure to low resolution donor-recipient HLA typing and the selection of patients. Survival after unrelated donor BM HSCT for SAA has improved in recent years with survival rates of approximately 75% @ 5-years when the donor and recipient are matched at HLA-A, -B, -C and –DRB1. Furthermore, improvements in conditioning regimen may also have reduced the rate of graft rejection. In recent years PBPC instead of BM are increasingly used and PBPC grafts now account for 25% of unrelated donor HSCT for SAA. An earlier report from the CIBMTR and the EBMT identified higher chronic graft-versus-host disease (GVHD) incidence and mortality after transplantation of PBPC from HLA-matched siblings compared to BM in patients with SAA aged ≤20 years. GVHD rates are higher after unrelated adult donor HSCT regardless of graft type, but the effects of PBPC on survival in patients with SAA are not known. Two hundred and forty nine patients transplanted in 2000–2007 received either BM (n=186) or PBPC (n=63) from unrelated adult donors matched at HLA-A, -B, -C, -DRB1. Median follow up was 3 years. Compared with recipients of BM, PBPC recipients were older (median age: 19 vs. 35 years), transplanted in 2006–2007 (37% vs. 46%), more likely to be male (47% vs. 65%), receive non-TBI containing transplant conditioning (fludarabine + cyclophosphamide or busulfan or melphalan ± anti-thymocyte globulin; 34% vs. 55%) and tacrolimus-containing GVHD prophylaxis (29% vs. 57%). Among patients who received TBI-containing regimens, approximately 90% of BM and PB recipients received TBI 200 cGy. There were no differences in patient performance score at HSCT, immunosuppressive treatment prior to HSCT and interval from diagnosis to HSCT (median time to HSCT was 13 months in BM recipients and 11 months, in PBPC recipients). As shown after HLA-matched sibling transplantation, the cumulative incidence of neutrophil recovery (30.5 × 109/L at day-28) and platelet recovery (≥20 × 109/L) was similar in recipients of BM (92% and 83%, respectively) and PBPC (95% and 90%, respectively). The day-100 probability of grades 2–4 acute GVHD was higher after PBPC than after BM (51% vs. 33%, p=0.01). The 3-year probability of chronic GVHD was higher after PBPC than after BM, in patients older than 20 years at HCT (60% vs. 41%, p=0.001) but not in younger patients (23% vs. 26%, p=NS). In multivariate analysis, the risk of mortality was higher after PBPC than after BM (HR 1.79, p=0.02). Other factors that predicted mortality included poor performance score (HR 1.95, p=0.009) and non-TBI transplant conditioning regimens (HR 1.96, p=0.006). Although patient age (>20 vs. ≤20 years) was significantly associated with chronic GVHD there was no significant effect of age on mortality (HR 1.21, p=0.47). The 3-year probability of overall survival adjusted for performance score and transplant conditioning regimen was 76% for BM and 60% for PBPC recipients (p=0.02). These data suggest that: 1) outcomes after URD HSCT for SAA are now similar to outcomes observed in MRD HSCT, 2) BM is the preferred graft source when considering unrelated donor HCT in patients with SAA and 3) the data also support low dose TBI in the preparative regimen for transplant conditioning. Disclosures:No relevant conflicts of interest to declare.

Comparison of Outcomes after Transplantation of G-CSF–Stimulated Bone Marrow Grafts versus Bone Marrow or Peripheral Blood Grafts from HLA-Matched Sibling Donors for Patients with Severe Aplastic Anemia

We compared outcomes of patients with severe aplastic anemia (SAA) who received granulocyte-colony stimulating factor (G-CSF)-stimulated bone marrow (G-BM) (n = 78), unstimulated bone marrow (BM) (n= 547), or peripheral blood progenitor cells (PBPC) (n = 134) from an HLA-matched sibling. Transplantations occurred in 1997 to 2003. Rates of neutrophil and platelet recovery were not different among the 3 treatment groups. Grade 2-4 acute graft-versus-host disease (aGVHD) (relative risk [RR] = 0.82, P = .539), grade 3-4 aGVHD (RR = 0.74, P = .535), and chronic GVHD (cGVHD) (RR = 1.56, P = .229) were similar after G-BM and BM transplants. Grade 2-4 aGVHD (RR = 2.37, P = .012) but not grade 3-4 aGVHD (RR = 1.66, P = .323) and cGVHD (RR = 5.09, P < .001) were higher after PBPC transplants compared to G-BM. Grade 2-4 (RR = 2.90, P < .001), grade 3-4 (RR = 2.24, P = .009) aGVHD and cGVHD (RR = 3.26, P < .001) were higher after PBPC transplants compared to BM. Mortality risks were lower after transplantation of BM compared to G-BM (RR = 0.63, P = .05). These data suggest no advantage to using G-BM and the observed higher rates of aGVHD and cGVHD in PBPC recipients warrants cautious use of this graft source for SAA. Taken together, BM is the preferred graft for HLA-matched sibling transplants for SAA.

Post-Thaw Viability of Cryopreserved Hematopoietic Progenitor Cell Grafts Influences Patients' Predisposition towards Infectious Complications in the Post-Transplant Period.

Abstract 4666Cell viability in peripheral blood progenitor cell (PBPC) grafts and its influence on the clinical course following transplantation was evaluated in 81 consecutive transplantations (72 autologous, 9 allogeneic) performed in patients with hematological diseases. Viability of cells in PBPC grafts, assessed by trypan blue dye exclusion test immediately upon collection was 98.6±3.5%, after addition of dimethyl sulfoxide (DMSO) 73.3±21.8%, and post-thaw 65.2±16.1%. It did not differ significantly between patients with different diagnoses, gender, age, type of priming used, dose of G-CSF administered or number of CD34+ cells collected. However, grafts stored for more than 60 days showed lower post-thaw viability compared to the ones thawed in the 60 days following cryopreservation (56.6±15.2% vs. 67.6±15.5%, p=0.04). A strong, statistically significant correlation was found between pre-cryopreserved CD34+ cell dose and hematopoietic recovery in patients, expressed as time to reach ANC>1×109/l and platelets >20×109/l (R2=0.14 p=0.001, R2=0.12 p=0.005, respectively). Occurrence of febrile neutropenia and total number of febrile days did not differ significantly between patients depending on the dose of CD34+ cells infused. On the contrary, post-thaw graft viability did not influence engraftment time, but there was a predisposition towards infectious complications in the post-transplant period in patients receiving grafts with lower percentage of viable cells. These patients developed febrile neutropenia more often (72.2% vs. 50% of patients, p=0.05) and had more febrile days (2.4±2.6 vs. 1.5±2.3, p=0.05) following transplantation. Our results demonstrate that PBPC grafts are capable of long term engraftment regardless of the graft storage time or percentage of viable cells post-thaw, which confirms the robustness of CD34+ cells during the freeze/thaw procedures carried out in daily clinical practice. Granulocyte concentration in PBPC grafts could have an influence on infectious complications following transplantation. This is an intriguing finding that deserves further investigation on a larger number of patients, less heterogeneous in terms of underlying disease, type of transplantation or conditioning regimen used. Disclosures:No relevant conflicts of interest to declare.

The Effect of the Composition of Unrelated Donor Bone Marrow and Peripheral Blood Progenitor Cell Grafts on Transplantation Outcomes

To test the hypothesis that the outcome of hematopoietic stem cell (HSC) grafts is at least partially determined by the cellular composition of the graft, the National Marrow Donor Program (NMDP) analyzed the correlation of cellular phenotypes of unrelated grafts with graft outcome. Samples from 94 bone marrow (BM) and 181 peripheral blood progenitor cell (PBPC) grafts for transplantations at 40 U.S. transplant centers between 2003 and 2005 were analyzed at a single immunophenotyping reference laboratory. Samples were shipped from transplant centers upon receipt of graft. Graft cellular composition included analysis of leukocyte total cell numbers, and subsets of myeloid [CD34(+), CD34(+) CD38(-)], lymphoid [CD3(+), CD3(+) CD4(+), CD3(+) CD8(+)], and activated lymphoid cells [CD3(+) CD25(+), CD3(+) CD69(+), CD3(+) HLA-DR(+)] coexpressing CD3(+). There was substantial variability in the cellular composition of BM and PBPC grafts before and after graft processing by red blood cell (RBC) removal or plasma depletion in preparation for transplant. With BM grafts, cellular composition was not associated with hematopoietic recovery, graft-versus-host disease (GVHD), or survival. With PBPC grafts, survival rates were higher with CD34(+)>5 x 10(6)/kg, 59% compared to 34% with CD34(+)< or =5 x 10(6)/kg at 1 year. Platelet recovery was higher with PBPC containing CD3(+) CD8(+) >8 x 10(7)/kg. Neutrophil recovery or GVHD could not be predicted by any cellular subsets of PBPC grafts. Although survival was superior with PBPC grafts containing >5 x 10(6) CD34(+)/kg, an optimal graft mix of myeloid, lymphoid, and activated lymphoid subsets was not identified.

Long‐term cryopreservation of autologous stem cell grafts: a clinical and experimental study of hematopoietic and immunocompetent cells

Autologous stem cell transplantation(ASCT) is used in the treatment of several malignancies.Harvesting sufficient peripheral blood progenitor cells (PBPCs) for a potential second autotransplantation at the time of relapse several years after diagnosis is becoming an increasingly common practice. Cryopreserved PBPCs were prepared with different concentrations of dimethyl sulfoxide (DMSO; 2, 4, 5, and 10%) and stored for at least 5 years before the recovery of CD34+ cells and various T- and natural killer (NK)-cell subsets were analyzed by flow cytometry. Furthermore,clinical variables for myeloma patients having a second autotransplantation with long-term-stored autografts were evaluated. The number of viable CD34+ cells in longterm-stored grafts was higher when autografts were cryopreserved with 4 or 5% than with 2 and 10%DMSO. The number of viable CD34+ cells was reduced by 13.9% after 5 years of cryostorage in 5% DMSO.Lymphocyte viability was also higher with 4 or 5%DMSO. However, the frequencies of several T-cell subsets showed DMSO-dependent differences,whereas NK-cell subsets did not. Furthermore, after a second autotransplantation with long-term-stored PBPC grafts at the time of myeloma relapse (median storage time, 42 months) all 17 patients reached neutrophil counts exceeding 0.5 x 10⁹/L and platelet counts exceeding 20 x 10⁹/L within 15 days. There was no difference in engraftment between patients receiving autografts preserved with 5 and 10% DMSO. PBPC autografts can safely be stored for at least 5 years in 5% DMSO and used for ASCT.

Reduced hemoglobin on day of peripheral blood progenitor cell collection is associated with low graft content of vascular progenitors and increased toxicity after autologous hematopoietic stem cell transplantation

Tissue damage after hematopoietic stem cell transplantation (HSCT) occurs as a result of high-dose chemotherapy and radiation. The aim was to determine the importance of pretransplant anemia on toxicity and red blood cell (RBC) transfusion requirements after autologous HSCT. A total of 350 patients undergoing autologous HSCT were included in the analysis. Patient factors and pretransplant laboratory values of possible relevance were assessed in multivariate regression analysis. Reduced hemoglobin (Hb) on the first day of peripheral blood progenitor cell (PBPC) collection was significantly associated with increased organ toxicity after HSCT, as measured by the Seattle criteria. Lower Hb levels at baseline before transplantation, but not at PBPC collection, were significantly associated with increased RBC transfusion requirements. In a second cohort of 28 patients, higher Hb levels on the day of PBPC collection were significantly associated with increased levels of endothelial-like vascular progenitor cells in PBPC grafts. Our observations suggest that higher Hb levels on the day of PBPC collection may be a marker of reduced toxicity associated with HSCT and increased vascular progenitors in PBPC collections. Further, baseline anemia before transplant may reflect an unfavorable hematopoietic microenvironment that leads to increased RBC transfusion requirements.

Contamination of autologous peripheral blood progenitor cell grafts predicts overall survival after high-dose chemotherapy in multiple myeloma

Despite of the introduction of novel treatment modalities for multiple myeloma, high-dose chemotherapy with hematopoietic stem-cell rescue is still considered the standard of care for eligible patients <65 years of age. As we have previously reported, stem-cell grafts regularly contain quantities of plasma cells measurable by flow cytometry. However, the pathogenetic significance of this finding remains unknown. Multiple myeloma patients (n = 60) were mobilized with chemotherapy and filgrastim. Peripheral blood stem cell grafts were obtained by standard leukapheresis, and the number of CD38++/CD138+ cells/kg was determined by flow cytometry. Plasma cell contamination above a threshold of 4.5 x 10(5) plasma cells/kg was considered "high", whereas lower quantities of plasma cells or absent plasma cells in the graft were considered "low". Progression-free survival: the median statistical progression-free survival was 33.5 months (range 11-99 months) in the high-contamination group (n = 16) versus 47 months (range 8-148 months) in the low-contamination group (n = 44). This difference turned out not to be statistically significant (P = 0.15). However, the difference was highly significant regarding overall survival with 53 months (range 11-119 months) in the high-contamination group and with 114 months (range 8-158 months) in the low-contamination group (P = 0.012). Patients with >4.5 x 10(5) plasma cells/kg contaminating the peripheral blood stem cell graft received after high-dose chemotherapy have a significantly reduced overall survival. Whether high contamination of grafts with plasma cells might reflect residual in vivo tumor mass prior to stem cell transplantation and a generally more aggressive behavior of malignant myeloma cells in these patients, or whether reinfused plasma cells contribute to an unfavorable course of disease remains to be determined.

Graft Composition and Outcomes in Unrelated Donor Transplantation.

We examined the effect of graft composition parameters including total white blood cell (WBC), myeloid cell dose (CD34+, CD34+/CD38+) lymphoid cell dose (CD3+, CD4+, CD8+), and activated lymphoid cells expressing activation antigens (CD25, CD69, HLA-DR) in 123 bone marrow (BM) and 198 peripheral blood progenitor cell (PBPC) transplants from unrelated donors (URD) between July 2003-March 2005. Most recipients (85% BM, 94% PBPC) had a hematologic malignancy. Samples were shipped from 33 participating centers for analysis at a central laboratory. Most BM grafts (67%) were processed: 24% T-cell depleted, 26% red blood cell (RBC) depleted, 12% plasma depleted and 5% mononuclear cell concentrated. Fewer PBPC grafts (29%) were processed: 19% plasma removal, 9% CD34+ selection and 1% RBC depletion. All samples had acceptable viability (≥70%). CD34 recovery was high for BM grafts after plasma or RBC depletion (84% and 62%, respectively) but low after T-cell depletion (26%). CD34 recovery was high for plasma depleted PBPC (83%). Plasma or RBC depletion had minimal effect on lymphoid cell content of either BM or PBPC, while T- cell depletion or CD34 selection resulted in 3–4 log10 depletion of CD3+ and HLA-DR+ lymphoid cells. We did not observe differences in transplant outcomes among those receiving processed vs. non-processed BM or PBPC grafts. The overall or subset cellular composition of BM grafts was not associated with the likelihood of hematopoietic recovery, acute and chronic graft-versus-host disease (GVHD) or 100-day survival. After adjusting for other clinically significant factors, risks of overall mortality were significantly lower when BM grafts contained ≥2.36 × 106/kg CD25 cells (relative risk [RR] 0.55, p=0.048) and ≥8.20 × 104/kg CD 34+/38+ (RR 0.58, p=0.043); no other subsets were associated with overall survival. With PBPC grafts, day-28 neutrophil recovery was higher with grafts containing ≥3.30 × 106/kg CD69 cells (97% vs. 89%, p=0.036). Platelet recovery by day-90 was higher with PBPC grafts containing: ≥5.44 × 106/kg CD34+ cells (87% vs. 74%, p=0.027), ≥2.20 × 108/kg CD3+ cells (87% vs. 74%, p=0.028) and ≥8.15 × 107/kg CD8+ cells (88% vs. 73%, p=0.011). The likelihood of acute GVHD was not associated with the composition of PBPC grafts, but chronic GVHD was higher with PBPC grafts containing ≥2.20 × 108/kg CD3+ cells (RR 1.66, p=0.029). Early mortality rates were lower with PBPC grafts containing ≥8.15 × 107/kg CD 8+ cells (RR 0.47, p=0.025) and overall mortality rates were lower with PBPC grafts containing ≥5.44 × 106/kg CD 34+ cells (RR 0.58, p=0.003) and ≥8.15 × 107/kg CD 8+ cells (RR 0.67, p=0.029). These data suggest that adequate CD25 and CD34+/38+ are necessary for optimal survival after URD BM and adequate CD34+ and CD8+ after PBPC transplantation. Further analysis of the ideal graft composition associated with transplant outcome will be needed to plan graft engineering manipulations to improve patients' survival and could be particularly relevant for PBPC transplants which are associated with higher chronic GVHD.

Worse outcome and more chronic GVHD with peripheral blood progenitor cells than bone marrow in HLA-matched sibling donor transplants for young patients with severe acquired aplastic anemia

AbstractWe analyzed the outcome of 692 patients with severe aplastic anemia (SAA) receiving transplants from HLA-matched siblings. A total of 134 grafts were peripheral blood progenitor cell (PBPC) grafts, and 558 were bone marrow (BM) grafts. Rates of hematopoietic recovery and grades 2 to 4 chronic graft-versus-host disease (GVHD) were similar after PBPC and BM transplantations regardless of age at transplantation. In patients older than 20 years, chronic GVHD and overall mortality rates were similar after PBPC and BM transplantations. In patients younger than 20 years, rates of chronic GVHD (relative risk [RR] 2.82; P = .002) and overall mortality (RR 2.04; P = .024) were higher after transplantation of PBPCs than after transplantation of BM. In younger patients, the 5-year probabilities of overall survival were 73% and 85% after PBPC and BM transplantations, respectively. Corresponding probabilities for older patients were 52% and 64%. These data indicate that BM grafts are preferred to PBPC grafts in young patients undergoing HLA-matched sibling donor transplantation for SAA.

In Multiple Myeloma (MM) CAMPATH-1 Antibodies “In the Bag” Reduce the Early Toxicity of Stem Cell Transplantation from Graft vs. Host Disease (GvHD) and Seem To Be Associated with Improved Long Term Outcome.

Introduction: Although allogeneic stem cell transplantation remains the only curative approach in patients with MM, its role remains controversial. Early high treatment related mortality, particularly from infections, GvHD and later, from disease recurrence remains substantial challenges. This study reviewed the outcome of consecutive patients who received T-cell depleted grafts from HLA identical siblings.Patients and Methods: Patients with symptomatic MM (stage II, n= 4 & III, n=17) who had an HLA identical sibling were initially treated until response with anthracycline or steroid based combinations. Three patients had, in addition, undergone autografting. Individuals who received cytokine mobilized peripheral blood progenitor cell (PBPC) grafts were also prescribed therapeutic serum levels of cyclosporine for 90 days post transplantation. The objective of the study was to determine transplant related mortality OS and DFS.Results: Twenty one patients with a median age of 44 (range 37– 56) years who had responded to chemotherapy (CR or VGPR) received stem cell enriched grafts from HLA identical siblings. Median performance status was 1 (0 – 2). Five individuals had significant organ dysfunction from effects of the disease. At presentation median albumin and β2MG blood levels were 30 g/L and 3.2 ng/mL, respectively. Myeloablative conditioning was radiotherapy (n= 12) or chemotherapy (n= 9) based. GvHD prophylaxis consisted of T-cell depletion with CAMPATH-1G (n=7) or H (n=14) antibodies “in the bag”. BM had all been treated with ex vivo with CAMPATH-1G (median 20 mg) while PBPC grafts in 14 patients were incubated with CAMPATH-1H (median 10 mg). Patients received a median of 23 ×104/kg CFU-GM in 0.87 BM mononuclear cells or 9.22 ×108/kg PBPC (CD34+: 4.46 ×106/kg). Median time to engraftment was 12 days. Two patients developed GvHD (grade 2) and 3 limited chronic form (1 progressed from acute). The 1 year non relapse mortality was 19%. Six patients suffered disease recurrence. One refused further therapy. Three of 5 responded to DLI and 2 remain in unsustained remission. Fatal events appeared lower in patients receiving chemotherapy based conditioning (mortality 11% vs. 50; p= 0.06), exposure of stem cells to CAMPATH-1H (15% vs. 62.5%; p= 0.04) and a lower median CAMPATH-1 dose given (p= 0.01). Cox analysis confirmed that lower CAMPATH-1 dose was associated with improved outcome. Fourteen (67%) patients survive at a median of 1101 days (range 385–5309) and 62% are disease free.Conclusions: In this cohort of chemotherapy responsive patients with advanced myeloma, ex vivo T-cell depletion of allogeneic grafts was associated with good protection from GvHD and 19% 1-year transplant related mortality. Low grades of GvHD post transplantation were associated with a favourable impact in the long term survival.

In Vitro Culture During Retroviral Transduction Improves Thymic Repopulation and Output After Total Body Irradiation and Autologous Peripheral Blood Progenitor Cell Transplantation in Rhesus Macaques

Immunodeficiency after peripheral blood progenitor cell (PBPC) transplantation may be influenced by graft composition, underlying disease, and/or pre-treatment. These factors are difficult to study independently in humans. Ex vivo culture and genetic manipulation of PBPC grafts may also affect immune reconstitution, with relevance to gene therapy applications. We directly compared the effects of three clinically relevant autologous graft compositions on immune reconstitution after myeloblative total body irradiation in rhesus macaques, the first time these studies have been performed in a large animal model with direct clinical relevance. Animals received CD34(+) cell dose-matched grafts of either peripheral blood mononuclear cells, purified CD34(+) PBPCs, or purified CD34(+) PBPCs expanded in vitro and retrovirally transduced. We evaluated the reconstitution of T, B, natural killer, dendritic cells, and monocytes in blood and lymph nodes for up to 1 year post-transplantation. Animals receiving selected-transduced CD34(+) cells had the fastest recovery of T-cell numbers, along with the highest T-cell-receptor gene rearrangement excision circles levels, the fewest proliferating Ki-67(+) T-cells in the blood, and the best-preserved thymic architecture. Selected-transduced CD34(+) cells may therefore repopulate the thymus more efficiently and promote a higher output of naïve T-cells. These results have implications for the design of gene therapy trials, as well as for the use of expanded PBPCs for improved T-cell immune reconstitution after transplantation.

A comparison of postengraftment infectious morbidity and mortality after allogeneic partially T cell–depleted peripheral blood progenitor cell transplantation versus T cell–depleted bone marrow transplantation

Postengraftment infections are a major cause of transplant-related morbidity and mortality following allogeneic hematopoietic stem cell transplantation (allo-SCT). Allogeneic peripheral blood progenitor cell transplantation (PBPCT) is associated with faster hematopoietic recovery compared to bone marrow transplantation (BMT) and unmanipulated PBPCT may be associated with fewer postengraftment infections. We set out to evaluate and compare the incidence, cause, and outcome of postengraftment infections following HLA-identical sibling T cell-depleted PBPCT vs T cell-depleted BMT between days 30 and 365 posttransplant. Forty recipients of peripheral blood progenitor cells (PBPC) and 47 recipients of bone marrow (BM) were included. The two groups of patients were comparable with respect to their baseline characteristics. PBPC grafts contained significantly more CD34+ cells and PBPCT was associated with significantly faster neutrophil and lymphocyte recovery as compared to BMT. PBPC recipients experienced more chronic graft-vs-host disease (GVHD; 55% vs 34%; p=0.02). The number of definite and clinical infections per 100 patient days was comparable between recipients of PBPC and BM with similar contribution of causative microorganisms. At one year post SCT, 68% of PBPC recipients had experienced at least one CTC grade 3-4 infection vs 65% of BM recipients. Treatment-related mortality at one year from transplantation was 34% after PBPCT vs 30% after BMT, and no difference in infection-related mortality was observed. Postengraftment infectious morbidity and mortality were comparable between recipients of PBPC and BM despite a higher CD34+ cell content of PBPC grafts and faster lymphocyte recovery after PBPCT, which may in part be explained by the higher incidence of chronic GVHD.

Reduced risk of acute GVHD following mobilization of HLA-identical sibling donors with GM-CSF alone

We retrospectively reviewed the results of transplanting peripheral blood progenitor cell (PBPC) allografts from HLA-matched sibling donors mobilized using various hematopoietic cytokines. Patients had received allografts mobilized with Granulocyte colony-stimulating factor (G-CSF) (G, N = 65) alone, G plus Granulocyte-macrophage colony stimulating factor (GM-CSF) (G/GM, N = 70), or GM-CSF alone at 10 or 15 microg/kg/day (GM, N = 10 at 10 microg/kg/day and 21 at 15 microg/kg/day). The CD34+ and CD3+ cell content of grafts were significantly lower following GM alone compared to G alone (P < 0.001 and 0.04, respectively). Nonhematopoietic toxicity observed in donors precluded dose escalation of GM-CSF beyond 10 microg/kg/day. Hematopoietic recovery was similar among all three groups. Grades II-IV acute graft-versus-host disease (GVHD) was observed in only 13% of patients in the GM alone group compared to 49 and 69% in the G alone or G/GM groups, respectively (P < 0.001). In a multivariate analysis, receipt of PBPC mobilized with GM alone was associated with a lower risk of grades II-IV acute GVHD (hazard ratio 0.21; 95% CI 0.073, 0.58) compared to G alone or G/GM. There were no differences in relapse risk or overall survival among the groups. Donor PBPC grafts mobilized with GM-CSF alone result in prompt hematopoietic engraftment despite lower CD34+ cell doses and may reduce the risk of grades II-IV acute GVHD following HLA-matched PBPC transplantation.

Establishment and optimization of a flow cytometric method for evaluation of viability of CD34+ cells after cryopreservation and comparison with trypan blue exclusion staining

Trypan blue exclusion staining is probably the most frequently applied method (Method I) for assessment of viability in peripheral blood progenitor cell grafts after cryopreservation. Alternatively, a flow cytometry-based method (Method II) was established and optimized. In a first series of 22 autologous apheresis products, the influence of duration of antibody staining and red cell (RBC) lysis on viability was investigated. In a second series of 21 autologous and 1 allogeneic apheresis products, the effect of omitting the RBC lysis was evaluated. On the basis of the results of the first two series, 155 autologous and 57 allogeneic apheresis products were analyzed with Method I and the now optimized Method II. Halving the incubation times did not influence the viability of CD45+ or CD34+ cells. Omission of RBC lysis resulted in a significantly (p = 0.022) increased median viability of CD45+ cells (75.8% vs. 71.0%) without any influence on CD34+ cells. In the third series, the median viability of CD34+ cells (96.9%) was significantly (p < 0.0001) higher compared with the viability of CD45+ cells (76.2%) and the viability determined by Method I (86.5%). The viability of CD34+ cells was significantly higher compared with the viability of all white blood cells. The presented cytometry-based method is superior to the standard trypan blue method regarding the number of analyzable cells and documentation, regarding observer independence and standardization; it allows the analysis of the cells of interest for transplantation after minimal sample manipulation.

Ancestim (r-metHuSCF) in combination with filgrastim to rescue mobilization and collection of peripheral blood progenitor cells for autologous transplantation. Compassionate use in 288 patients at 62 sites in France (1998–2003)

6642 Background: Approximately 15% of patients (pts) eligible for HDC and peripheral blood progenitor cell (PBPC) transplantation, failed to generate an adequate PBPC graft. Methods: Ancestim (r-metHuSCF, Amgen, Thousand Oaks, CA) was delivered on a named-pt basis under the French Temporary Authorization for Use program to 288 pts with prior failure to achieve 20 CD34+ cells/μL peripheral blood (no leukapheresis attempt, n = 209 pts) or failure to collect an adequate PBPC yield (2x106 CD34+ cells/kg; n = 64 pts) (no. of prior failed attempts: 1 to 5)(median age 53 yrs [1 –70 yrs], n ≤ 18 yrs of age = 31) (lymphomas, 140 pts; myeloma, 84 pts; CLL, 21 pts; Ewing's sarcoma, 12 pts; neuroblastomas, 11 pts; other tumor types: 20 pts). Ancestim (20μg/kg/d) was combined with filgrastim alone (10μg/kg/d; median 7 d, 151 pts) or with chemotherapy and filgrastim (5μg/kg/d, median 12 d, 136 pts). Results: An autograft appropriate to support HDC (2x106 CD34+ cells/kg) was obtained from 106 (37%) of 288 pts after rescue with ancestim (72 of 209 pts with prior failure(s) to mobilize CD34+ cells to the peripheral blood; 29 of 64 pts with prior failure(s) of collection of an adequate CD34+ cell yield). In the 134 pts to undergo leukaphereses after the ancestim attempt, the median (range) CD34+ cell yield was 2.77 (0.03 to 39.05) x 106 cells/kg. To date, 85 pts have undergone transplantation. Hematological recovery was comparable to those obtained with filgrastim-mobilized PBPCs (platelets > 20 x 109/L : median 13 d; neutrophils > 0.5 109/L : median : 12 d). Only 2 pts have experienced anaphylactoid symptoms associated with systemic histamine release after inadvertent IV injection. Conclusions: The combination of ancestim and filgrastim allows to rescue an important number of pts who experienced prior failure of PBPC mobilization and/or collection, without any significant toxicity in this large multicenter series. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Amgen France; Amgen UK Amgen, Inc.

Ancestim (r-metHuSCF) in combination with filgrastim to rescue mobilization and collection of peripheral blood progenitor cells for autologous transplantation. Compassionate use in 288 patients at 62 sites in France (1998–2003)

6642 Background: Approximately 15% of patients (pts) eligible for HDC and peripheral blood progenitor cell (PBPC) transplantation, failed to generate an adequate PBPC graft. Methods: Ancestim (r-metHuSCF, Amgen, Thousand Oaks, CA) was delivered on a named-pt basis under the French Temporary Authorization for Use program to 288 pts with prior failure to achieve 20 CD34+ cells/μL peripheral blood (no leukapheresis attempt, n = 209 pts) or failure to collect an adequate PBPC yield (2x106 CD34+ cells/kg; n = 64 pts) (no. of prior failed attempts: 1 to 5)(median age 53 yrs [1 –70 yrs], n ≤ 18 yrs of age = 31) (lymphomas, 140 pts; myeloma, 84 pts; CLL, 21 pts; Ewing's sarcoma, 12 pts; neuroblastomas, 11 pts; other tumor types: 20 pts). Ancestim (20μg/kg/d) was combined with filgrastim alone (10μg/kg/d; median 7 d, 151 pts) or with chemotherapy and filgrastim (5μg/kg/d, median 12 d, 136 pts). Results: An autograft appropriate to support HDC (2x106 CD34+ cells/kg) was obtained from 106 (37%) of 288 pts after rescue with ancestim (72 of 209 pts with prior failure(s) to mobilize CD34+ cells to the peripheral blood; 29 of 64 pts with prior failure(s) of collection of an adequate CD34+ cell yield). In the 134 pts to undergo leukaphereses after the ancestim attempt, the median (range) CD34+ cell yield was 2.77 (0.03 to 39.05) x 106 cells/kg. To date, 85 pts have undergone transplantation. Hematological recovery was comparable to those obtained with filgrastim-mobilized PBPCs (platelets > 20 x 109/L : median 13 d; neutrophils > 0.5 109/L : median : 12 d). Only 2 pts have experienced anaphylactoid symptoms associated with systemic histamine release after inadvertent IV injection. Conclusions: The combination of ancestim and filgrastim allows to rescue an important number of pts who experienced prior failure of PBPC mobilization and/or collection, without any significant toxicity in this large multicenter series. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Amgen France; Amgen UK Amgen, Inc.