Peripheral Blood Stem Cell Products Research Articles

Outcomes of Patients Receiving Allogeneic Peripheral Blood Stem Cell Products Mobilized with Intravenous Plerixafor

Abstract 314We have previously reported mobilization data of hematopoietic stem and progenitor cells (HSPCs) in donors treated with IV plerixafor in a phase I/II open label trial (Rettig et al. Blood 2010 116: a2261). Here we report the final outcomes of donors and recipients of the phase II portion of the trial evaluating the use of 0.32 mg/kg plerixafor IV as a single agent for stem cell mobilization. We hypothesized, based on preclinical studies in the mouse, that IV plerixafor would have more rapid and prolonged mobilization and increase stem cell yields so that failure to collect ≥2×106 CD34+ /Kg recipient weight in one apheresis would be reduced from 33% (seen with 240 ug SC plerixafor) to 11%. We also hypothesized based on our prior observations of increased plasmacytoid dendritic cells (pDCs) in plerixafor mobilized products (Rettig et al. Blood 2009 114: a32), that peripheral blood stem cell products would be enriched in pDCs which may lead to improved immunity in the recipient after transplant and reduced incidence of CMV. Methods.In this phase II open label trial donors were mobilized with 0.32 mg/Kg IV plerixafor (over 30 minutes), followed by a 20 L apheresis starting 4 hours after injection. The primary outcome was failure to collect ≥2×106 CD34+/Kg actual recipient weight on day 1. Secondary outcomes included donor toxicity, successful collection in 2 aphereses, pharmacodynamics of mobilization, engraftment, graft-versus-host disease (GvHD), treatment related mortality (TRM), incidence of CMV, relapse free survival, and overall survival. The study was powered to detect a difference in failure rate from 33% to 11%. Sibling donors were 18 – 70 years of age and 6/6 HLA matched with recipients. Recipients were 18 – 67 years of age with a hematologic malignancy (AML n=14, NHL n=9, CLL n=3, MDS n=3, ALL n=2, CML n=1, and HD n=1). 33 recipients were transplanted (5 reduced-intensity and 28 myeloablative conditioning). A total of 27 donor recipient pairs were initially enrolled and 7 additional pairs of subjects were enrolled to replace 6 donors who failed to collect goal apheresis volume (>17.5L) due to technical problems related to initial apheresis collections. Median follow up of recipients is 248 days (range 37–695 days). Results.The primary outcome of failure to reach ≥2×10e6 CD34+/Kg recipient weight was analyzed on a per protocol basis. CD34 HSPC mobilization kinetics were previously reported for the phase I cohorts (ASH 2010). 29 donors were evaluable for the primary outcome. 34% (10/29) failed to reach the day 1 collection goal of ≥2×106 CD34/Kg recipient weight. 10% (3/29) failed to reach goal after 2 apheresis procedures. Toxicity in donors was mild with the most common reported side effects being grade 1 GI toxicity of abdominal bloating (30%) and grade 1 bradycardia (30%). All recipients engrafted and there was no graft failure. The median time to neutrophil engraftment (ANC>500K/uL × 2 days) was 14 days, with 31/33 engrafting by day 21. The median time to platelet engraftment (Platelets>50K/uL × 2 days) was 25 days, with 23/33 engrafting by day 30. The incidence of grade II-IV acute GvHD was 7/33 (21%). The incidence of grade III-IV acute GvHD was 4/33 (12%). The cumulative incidence of chronic GvHD in those living beyond 100 days was 7/25 (28%) and 4/25 (16%) had severe disease. The incidence of CMV viermia >10,000 copies in at risk recipients (defined as serology positive in donor or recipient) was 3/20 (15%). The incidence of CMV disease was 1/20 (5%). Median overall survival was 258 days, with 43% one-year survival. Median relapse free survival was 238 days. The day 100 TRM was 1/33 (3%). Summary.IV administration of plerixafor was well tolerated by donors. However, based on our prior experience with subcutaneous dosing with 0.24mg/kg (Devine et al Blood, 2008) there was no improvement in the percent of normal donors who successfully collected ≥2×106 CD34+/Kg after a single IV infusion of 0.32 mg/Kg plerixafor (67% vs. 66% respectively). Failure to reach collection goal in one apheresis procedure remains high with single agent plerixafor regardless of route of administration. Incidence of CMV viremia was low and could possibly be related to increased plasmacytoid dendritic cells in the product. The incidence of aGvHD was lower than historical controls for G-CSF mobilized peripheral blood transplantation and deserves further evaluation in future prospective trials involving CXCR4 antagonists such as plerixafor. [Display omitted] [Display omitted] Disclosures:Schroeder:Genzyme: Research Funding. Rettig:Genzyme: Honoraria. Uy:Genzyme: Consultancy, Speakers Bureau. DiPersio:Genzyme: Honoraria.

Risk factors for microbial contamination of peripheral blood stem cell products

Despite the well-known contamination rates and presence of microbial agents in stem cell products, the risk factors affecting microbial contamination have not been well described. In a 12-year period, we retrospectively reviewed culture results of peripheral blood stem cell products with the intent of identifying risk factors for microbial contamination. Microbial contamination was detected in 28 (5.7%) products of the postprocessing period and in 18 (3.66%) products of the postthawing period. Large-volume leukapheresis (LVL; odds ratio [OR], 5.85; 95% confidence interval [CI], 1.52-22.49; p = 0.01) and high numbers of stem cell culture sampling (OR, 1.4; 95% CI, 1.03-1.91; p = 0.03) were found to be risk factors for postprocessing bacterial contamination. The presence of postprocessing bacterial contamination was a risk factor for postthawing (OR, 28.89; 95% CI, 6.67-125.15; p < 0.001) and posttransplant (OR, 3.25; 95% CI, 1.24-8.50; p = 0.01) microbial growth. In transplants that were performed using contaminated products, the same pathogen was detected in 20% of patients and different pathogens were found in 35% of patients. Cultures should be carefully monitored in LVL products and in samples with high numbers of cultures performed. Growth of different bacterial pathogens must be considered in transplants that are performed with contaminated products.

Limiting the Daily Total Nucleated Cell Dose of Cryopreserved Peripheral Blood Stem Cell Products for Autologous Transplantation Improves Infusion-Related Safety with No Adverse Impact on Hematopoietic Engraftment

Cryopreserved peripheral blood stem cell (PBSC) products can induce a number of infusion-related adverse reactions, including life-threatening cardiac, neurologic, and other end-organ complications. Preliminary analyses suggested limiting the daily total nucleated cell dose infused might decrease the incidence of these adverse effects. A policy change implemented in December 2007, limiting the total nucleated cell (TNC) dose to <1.63 × 10(9) TNC/kg/day, allowed us to assess the impact of this intervention on infusion-related safety, infusion schedules, engraftment, and costs in cohorts of patients undergoing autologous stem cell transplants (ASCTs) 2 years before (325 ASCTs in 288 patients) and 2 years after the policy change (519ASCTs in 479 patients). The percentage of autologous transplant patients requiring multiple day infusions increased from 6% to 24%. Concurrently, the incidence of infusion-related grade 3-5 severe infusion-related adverse events (SAEs) decreased significantly, from 4% (13 of 325) prepolicy change to 0.6% (3 of 519) postpolicy change (P < .0004). Multiday infusions were not associated with increased time to neutrophil or platelet engraftment or the costs of transplantation. We conclude that limiting the daily TNC dose improved the safety of this procedure without compromising engraftment or increasing the costs of the procedure.

Microbial contamination of autologous peripheral blood stem cell products: incidence, clinical outcome, quality control and management strategies

Microbial screening is part of quality control for autologous peripheral blood stem cell (PBSC) manipulation. The incidence of culture positive products varies widely. The clinical significance of infusing culture positive products is uncertain, with no consensus on the interventions required. Microbial cultures of 606 consecutive autologous PBSC products from 278 patients between 1995 and 2005 were retrospectively analysed to determine the incidence of culture positivity and classified according to likely clinical significance. Clinical outcomes of patients who received culture positive products were reviewed. Using the baseline probability of contamination, a quality control method to detect a systematic source of contamination during product manipulation was developed. Eleven (1.8%) of 606 products were culture positive in eight (2.9%) patients. Ten products yielded coagulase negative Staphylococcus species and one Corynebacterium accolens. One patient had catheter sepsis during collection. Five patients received culture positive products, with prophylactic antibiotics when there was known coagulase negative Staphylococcus species. None had clinical evidence of infection following product infusion. The incidence of culture positive products was comparable to published series. Only skin commensals were isolated. Infusion of skin commensal positive products appears safe with antibiotic prophylaxis. Using local and published experience, clinical and laboratory policies were developed for culture positive products.

Cytomegalovirus Viral Load and Virus-Specific Immune Reconstitution after Peripheral Blood Stem Cell versus Bone Marrow Transplantation

Peripheral blood stem cell (PBSC) products contain more T cells and monocytes when compared with bone marrow (BM), leading to fewer bacterial and fungal infections. Cytomegelovirus (CMV) viral load and disease as well as CMV-specific immune reconstitution were compared in patients enrolled in a randomized trial comparing PSBC and BM transplantation. There was a higher rate of CMV infection and disease during the first 100 days after transplantation among PBSC recipients (any antigenemia/DNAemia: PBSC, 63% vs BM, 42%, P = .04; CMV disease: PBSC, 17% vs BM, 4%, P = .03). By 2 years, CMV disease rates were similar. The early increase in CMV events correlated temporarily with lower CMV-specific CD4(+) T helper and CD8(+) cytotoxic T lymphocyte function at 30 days after transplantation in PBSC recipients. By 3 months after transplantation and thereafter, CMV-specific immune responses were similar between BM and PBSC recipients. In conclusion, higher CMV infection and disease rates occurred in PBSC transplant recipients early after transplantation. These differences may be because of a transient delay in CMV-specific immune reconstitution following PBSC transplantation.

Nonleukemic myeloid dendritic cells obtained from autologous stem cell products elicit antileukemia responses in patients with acute myeloid leukemia

Dendritic cell (DC)-based immunotherapeutic protocols are being developed to treat acute myeloid leukemia (AML). So far, DCs for clinical use are obtained from leukemic blasts or from monocytes, after 6 to 10 days of ex vivo culture. However, DC precursors are easily driven to DCs in short-term culture. We tested if DC precursors contained in peripheral blood stem cell (PBSC) products obtained from AML patients can be used to induce antileukemia responses. PBSCs obtained from 30 consecutive AML patients were tested. Myeloid DCs (MDCs) were purified by immunomagnetic selection and screened for cytogenetic and/or molecular abnormalities by fluorescence in situ hybridization (FISH) or polymerase chain reaction (PCR) assays. MDCs were matured and pulsed with autologous blast lysates and tested for stimulatory capability against AML cells. A median of 0.62 × 10(6) MDCs (range, 0.04-3.25)/mL were quantified in PBSC products. Isolated MDC expressed Class I and II HLA but CD86, CD54, and CCR5 partially. By FISH or PCR assay, these MDCs lacked cytogenetic or molecular abnormalities detected in leukemia cells at diagnosis. MDCs achieved a maturated stage (mature-MDCs) after 24-hour ex vivo culture with tumor necrosis factor-α and autologous blast lysates. These mature-MDCs were capable of stimulating autologous peripheral blood effectors to exert cytotoxicity against autologous leukemia cells and HL-60 cell line. We conclude that PBSCs obtained for autologous stem cell transplantation can constitute a novel source of MDCs to design feasible vaccination trials.

Validation of short‐term handling and storage conditions for marrow and peripheral blood stem cell products

Allogeneic hematopoietic stem cell transplants from unrelated donors are routinely used in the treatment of patients with hematologic malignancies. These cellular products are often collected off-site and require transport from the collection site to transplantation centers. However, the effects of transport conditions and media on stem cell graft composition during short-term storage have not been well described. Five bone marrow (BM), four filgrastim-mobilized peripheral blood stem cell (PBSC), and four nonmobilized peripheral blood mononuclear cell (PBMNC) products were collected from healthy volunteer donors and stored at 4 or 20°C for up to 72 hours in 10% PlasmaLyte A plus anticoagulants such as 10% acid citrate dextran-A (ACD-A) and/or 10 IU/mL heparin. Products were evaluated at 0, 24, 48, and 72 hours for cellular content, viability, and metabolic activities. BM products maintained equivalent cell viability when stored at either 4 or 20°C over 72 hours, but cell viability was better maintained for PBSC products stored at 4°C. The mean viable CD34+ cell recovery for PBSC and BM products stored over 72 hours at 4°C was higher than 75%. Significantly lower CD34+ cell and colony-forming unit recoveries were seen in PBSC products but not BM products stored at room temperature. Faster lactic acid accumulation was observed in PBMNC and PBSC products stored without ACD-A. Seventy-two-hour storage of BM, PBSC, and PBMNC products at refrigerated temperature maintains optimal cell viability and recovery. Anticoagulation with ACD-A is preferred over heparin to reduce lactic acid accumulation in the product media.

Clinical Grade Isolation of Regulatory T Cells From G-CSF Mobilized PBSC Improves with Initial Depletion of Monocytes.

AbstractAbstract 1176We have recently demonstrated that G-CSF mobilized peripheral blood stem cells (PBSC) CD4+CD25+FoxP3+ cells (Tregs) prevent anti-CD34+ hematopoietic stem cells T cell alloreactivity in-vitro and co-transplantation of CD34+ cells and Tregs does not affect human stem cell engraftment in NOD/SCID mice (Mahmud D et al. Biol Blood Marrow Transplant, 2010). Since only a small number of Tregs can be isolated from normal peripheral blood we examined whether PBSC can be a useful source of Tregs for future clinical trials. Five leukapheresis products from healthy donors who received rh G-CSF at 10 ug/kg daily for 5 days were processed using the CliniMACS instrument (Miltenyi Biotec, Auburn, CA). CliniMACS CD34 reagent was initially used to isolate CD34+ cells. To isolate Tregs a 2-step procedure was initially utilized. A cocktail of clinical grade CD14, CD8 and CD19 reagents was mixed with the CD34- cells and depletion of monocytes, cytotoxic T cells and B cells was obtained by using the Depletion 2.1 program. The CD4+ cells were then enriched in Tregs by positive selection of CD25+ cells using a clinical grade CD25 reagent (Miltenyi). Because PBSC contain large amount of myeloid cells, and particularly monocytes, this clinical scale 2-step strategy was compared with a 3-step method that included an initial negative selection of CD14+ monocytes, followed by negative selection of CD8+ and CD19+ cells and a positive selection of CD25+ cells. Prior to isolation, the average proportion of CD4+CD25+ cells in PBSC was 0.77±0.26% in 5 separate PBSC products. After the 2-step process the proportion of CD4+CD25+ cells was 35±33% (n=3) vs 72±1% after the 3-step process. Therefore, utilizing the 3-step approach a better yield of Tregs was observed (10 vs 60%). Intracellular expression of FoxP3 was on average 74% in CD4+CD25+ cells obtained with a 3-step process. Contamination of different cell subsets in the final products enriched in Tregs was largely superior following the 2-step as compared to 3-step isolation method. Contaminating monocytes were, on average, 43 vs 5.7%, and contaminating CD8 and CD19+ cells were 12 vs 1.7% and 0.9 vs 0.3%, respectively. In the two procedures using a 3-step approach the final absolute number of Tregs isolated from products containing on average 30 × 109 mononuclear cells, was 95 and 93 × 106, respectively. These findings obtained using clinically available reagents and device, suggest that depletion of monocytes may improve the purity of Treg cell population isolated from PBSC. PBSC may represent a valuable source of Tregs for future clinical trials. Disclosures:No relevant conflicts of interest to declare.

HLA-C Antigen Mismatch Is Associated with Worse Outcome in Unrelated Donor Peripheral Blood Stem Cell Transplantation

The association between HLA matching and outcome in unrelated-donor peripheral blood stem cell (PBSC) transplantation has not yet been established. In the present study, a total of 1933 unrelated donor-recipient pairs who underwent PBSC transplantation between 1999 and 2006 for acute myelogenous leukemia, acute lymphoblastic leukemia, myelodysplastic syndrome, or chronic myelogenous leukemia and received high-resolution HLA typing for HLA-A, -B, -C, -DRB1, -DQA1, and -DQB1 were included in the analysis. Outcomes were compared between HLA-matched and HLA-mismatched pairs, adjusting for patient and transplant characteristics. Matching for HLA-A, -B, -C, and -DRB1 alleles (8/8 match) was associated with better survival at 1 year compared with 7/8 HLA-matched pairs (56% vs 47%). Using 8/8 HLA-matched patients as the baseline (n = 1243), HLA-C antigen mismatches (n = 189) were statistically significantly associated with lower leukemia-free survival (relative risk [RR], 1.36; 95% confidence interval [CI], 1.13-1.64; P = .0010), and increased risk for mortality (RR, 1.41; 95% CI, 1.16-1.70; P = .0005), treatment-related mortality (RR, 1.61; 95% CI, 1.25-2.08; P = .0002), and grade III-IV graft-versus-host disease (RR, 1.98; 95% CI, 1.50-2.62; P < .0001). HLA-B antigen or allele mismatching was associated with an increased risk for acute GVHD grade III-IV. No statistically significant differences in outcome were observed for HLA-C allele (n = 61), HLA-A antigen/allele (n = 136), HLA-DRB1 allele (n = 39), or HLA-DQ antigen/allele (n = 114) mismatches compared with 8/8 HLA-matched pairs. HLA mismatch was not associated with relapse or chronic GVHD. HLA-C antigen-mismatched unrelated PBSC donors were associated with worse outcomes compared with 8/8 HLA-matched donors. The study's limited power due to small sample size precludesconclusions about other mismatches.

How do I collect and process stem cells?

How do I collect and process stem cells?

Clinical relevance of germ cell cancer cells detected by real time PCR in apheresis products of poor risk patients undergoing high dose chemotherapy.

4579 Background: The clinical relevance of germ cell tumor (GCT)-specific mRNA in peripheral blood stem cell (PBSC) harvests of germ-cell cancer patients undergoing high dose chemotherapy (HDCT) is controversial, possibly caused by inappropriate PCR conditions with inadequate sensitivity and specificity and small numbers of patients analyzed. We re-evaluated this issue in a large number of PBSC-products using a novel, GCT-specific quantitative PCR assay. Methods: PBSC-harvests of 140 patients treated with HDCT for relapsed or platinum-refractory GCT were tested for the presence contaminating tumor cells using a highly tumor cell-specific PCR protocol, which amplified the un-conserved 5’-untranslated region (5’UTR) of the germ cell tumor-specific alkaline phosphatase (gcap) gene, but not alkaline phosphatase mRNA, the latter being also present in the peripheral blood of healthy donors. In order to further avoid false positive results by amplification of pseudogenes, all mRNA's were subjected to repeated DNAse digestion before entering cDNA synthesis. Results: The novel 5’UTR gcap specific PCR enabled the quantitative detection of gcap mRNA with an unprecedented specificity and good sensitivity of 10^-5. A total of 140 PBSC apheresis products were screened, with sufficient mRNA and clinical data available from 113 patients. The median time to progression (TTP) was significantly shorter in patients with gcap-positive (n=66) as compared to gcap-negative PBSC products (n=44) (7.0 months versus not reached, p=0.01). With a median follow up of 8 years, the median overall survival was shorter in patients with gcap-positive as compared to gcap-negative PBSC products (20 month vs. not reached). This difference did not reach statistical significance (p=0.08). Conventional qualitative gcap-PCR failed to separate cohorts with different prognosis, underscoring the importance of a validated quantitative PCR assay to address the clinical impact of PBSC tumor cell contamination in GCT. Conclusions: A quantitative and gcap-specific 5’UTR PCR assay detecting gcap-mRNA in PBSC-harvests is a strong adverse prognostic factor in GCT patients undergoing HDCT. No significant financial relationships to disclose.

Quantitative Real-Time PCR Detection of Wilms' Tumor Gene (WT1) Transcript in Autologous Peripheral Blood Stem Cell (PBSC) Products Predict the Risk of Acute Myeloid Leukaemia (AML) Relapse After Autologous Transplantation (ASCT).

Abstract 4690 Introductionwhen allogeneic transplantation is not feasible, ASCT is an alternative option as post-remission therapy for patients (pts) with AML, as it can prolong their disease-free survival (DFS). Relapse is the main cause of AML treatment failure after initial complete response; AML relapse after ASCT is partly due to contamination with leukemic blasts of the PBSC products, although neither morphological nor genetic evidence of disease are detected in the bone marrow before leukapheresis. Thus, identification and quantification of a reliable minimal residual disease (MRD) marker in the collected PBSC could be relevant in determining the relapse risk after ASCT. The WT1 gene is overexpressed in leukemic blasts of most AML cases; several studies have shown that quantification with RT-PCR of WT1 in the bone marrow and peripheral blood of AML pts in complete remission has prognostic value. We have determined the WT1 transcript levels in autologous PBSC of AML pts autografted for complete remission (CR) consolidation; preliminary results and data interpretation are here presented. Aimto evaluate quantitative WT1 transcript levels in autologous PBSC collections and to compare results with outcome in AML patients who received an ASCT as consolidation of CR, at our Institute. Patients and Methods9 pts, period 06/2006-03/2009, median age 68 years (range 41-76). At diagnosis cytogenetic prognostic risk was intermediate for all pts. All pts were in morphological and genetic CR at the time of PBSC collection and before ASCT. Eight patients were in first and 1 in second CR. PBSC collection by leukapheresis (COBE Spectra cell separator): median count of CD34+ 9.75 ×106/kg (3.79-32). Conditioning regimen: Treosulfan 30 mg/sqm, Fludarabine 150 mg/sqm and Cytarabine 5 g/sqm. Median count of CD34+ cells infused: 5×106/kg (range 3.3-8.5×106/kg). RT-PCR quantification of WT1 transcript was performed using TaqMan technology starting from 1 μg of RNA extracted from mononucleated cells of fresh (4) or cryopreserved (5) PBSC samples. The housekeeping gene ABL was used as the control gene for these quantifications with WT1 level being normalised to 104 copies of ABL per sample. We used the Mann-Whitney-U-test to determine if median WT1 levels in the PBSC products of relapsed and not-relapsed pts was statistically different. Than we used the log-rank test to compare RFS and median WT1 value in the PBSC products. Resultsat last follow-up 4 pts relapsed and 5 were still in CR. The WT1 levels in the autologous PBSC of the 4 relapsed pts were 89.96, 193.87, 779.43 and 839.63, of the 5 CR pts were 8.40, 16.96, 36.45, 74.89 and 82.49. Overall median WT1 in the PBSC products was 82,49 copies. The median WT1 levels in the PBSC products of relapsed and not-relapsed pts were 486.65 (89.96–839.63) and 36.35 (8.40–82.49) copies, respectively; this difference was statistically significant (p<0.05). Overall, median relapse free survival (RFS) from ASCT was 534 (93-1096) days. Median RFS was 360 days for pts with a WT1 level > 82,49 copies (n=4), and has not been reached for pts with a WT1 level ' 82,49 (n=5) (p=ns). Conclusionsthese results suggest that RT-PCR quantification of the WT1 transcript in autologous PBSC could predict AML relapse in pts who receive ASCT in CR. Higher WT1 levels should reflect higher PBSC product contamination with leukemic blasts, indicating an increased risk of relapse after ASCT. These last pts could probably benefit of other strategies than ASCT. We conclude that, if our preliminary data will be confirmed in a larger number of pts, RT-PCR quantification of WT1 transcripts should be used for MRD detection and quantification in autologous PBSC, before proceeding to ASCT; according to our preliminary data the cut-off level could be about 80 WT1 copies to discriminate which pts should receive the ASCT and which should not. Disclosures:No relevant conflicts of interest to declare.

Collection of Autologous Peripheral Blood Stem Cells Using the COBE Spectra Is Associated with Higher CD34+ Yields and Requires Less Collection Time Than the Fresenius AS104 Cell Separator.

Abstract 2158Poster Board II-135We retrospectively analyzed yields and characteristics of autologous peripheral blood stem cell (PBSC) products collected using either a COBE Spectra (CS) or Fresenius AS104 (F) cell separator. CS and F collections were performed according to manufacturer's protocol.One hundred and fifty nine adult patients (pts) collected between January 2006 and July 2009 at our center were randomly selected for analysis. Eighty nine pts had multiple myeloma (MM) and 72 pts other cancers, including non-Hodgkin's lymphoma (n=42), Hodgkin's disease (n=20), testicular cancer (n=5), neuroblastoma (n=1), acute lymphocytic leukemia (n=1), or acute promyelocytic leukemia (n=1). Pts were collected on either CS (n=85) or F (n=74), and mobilized with G-CSF alone (n=47), G-CSF and Plerixafor (P) (n=26), or G-CSF and Chemotherapy (Ch) (n=66). Data from pts who failed to mobilize were not included in the analysis. Results are summarized in table. All p-values were calculated using two-tailed, heteroscedastic student T-test; statistical significance was defined as p < 0.05.COBE MediansStandard DeviationFresenius MediansStandard DeviationNumber of Products165180Number of Patients8574Weight at Collection (kg)82.7±18.079.6±16.0p = 0.459Volume Processed (L)18.00±2.4018.05±2.75p = 0.488Volume Collected (ml)241±56.8402±72.0p < 0.001Product WBC (K/μl)163.3±136.0155.85±29.39p < 0.001Total Cells4.07E+10±3.98E+102.17E+10±1.26E+10p < 0.001% MNC75±2385±11p < 0.001Peripheral CD34 / μl24±4425±79p = 0.050Absolute Product CD34 / μl726.73±1325.94264.63±781.01p < 0.001Product / Peripheral CD34 Ratio24.87±10.9010.91±6.64p < 0.001Product CD34/kg (10 6)2.02±4.671.40±4.15p = 0.152Hours per collection4.25±0.655.20±0.88p < 0.001Number of Collections / pt2±12±1p = 0.550CD34 cells/kg × 106infused3.85±3.203.85±2.24p = 0.273CD34 yields are calculated as the ratio of the absolute CD34 count/μl of the PBSC product to the pt's peripheral blood CD34 / μl count taken on the day of collection. Pt age, weight on day of collection, number of collections per pt, volume processed per collection, disease type (MM versus other) and mobilization strategy did not differ between patients collected with CS or F. Products collected on the CS had significantly higher total WBC when compared to the F (163.3 ± 136 K/μl vs. 55.7 ± 29.2 K/μl, p < 0.001), but had lower percentages of mononuclear cells (75% ± 23% vs. 85% ± 11%, p < 0.001); this relationship persisted when patients were divided by disease type or mobilization strategy. CD34 / kg counts of PBSC products were not significantly different in any cohort, but absolute CD34 count of the PBSC product was significantly higher in every disease type and mobilization strategy on the CS (all pts, 727 ± 1326 cells/μl vs. 265 ± 789 cells/μl, p < 0.001). CD34 yields were also significantly higher on the CS than the F across all disease types and mobilization strategies (all pts, 24.9 ± 10.9 vs. 11.2 ± 7.4, p < 0.001). Analysis of peripheral blood CD34 counts/ μl and product CD34 count/ μl showed a stronger correlation in pts mobilized with G-CSF + P (R2 = 0.8735) versus G-CSF + Ch (R2 = 0.5492) or G-CSF alone (R2 = 0.5). Hours for collection were lower on the CS than the F, regardless of disease type (4.25 ± 0.65 hrs vs. 5.2 ± 0.98 hrs, p < 0.001). No difference in WBC or platelet engraftment after transplantation was observed between the cell separators. Adverse effects on infusion were similar except, 3 of 169 pts experienced seizures upon infusion of PBSC product; all three were collected on the CS and all three had product WBC counts of 590 K/μl or above (compared to a median of 163.3 K/μl for the total MM/CS population).In conclusion, collection of PBSC using the CS is associated with higher CD34 yields and requires less collection time than collections with the F cell separator. Disclosures:No relevant conflicts of interest to declare.

Pegylated Recombinant Murine GM-CSF Is a Potent Mobilizer of Murine Bone Marrow Progenitors, Synergizes with BIO5192 and Plerixafor (AMD3100), and Skews Mobilized Cells to a Tolerogenic Phenotype.

Abstract 2432Poster Board II-409Granulocyte-macrophage colony stimulating factor (GM-CSF) is a naturally occurring acidic glycoprotein growth factor that regulates proliferation and differentiation of hematopoietic stem and progenitor cells. Inflammation results in the release of GM-CSF from numerous cell types including T-cells, macrophages, and neutrophils. Murine GM-CSF (mGM-CSF), unlike human GM-CSF, has a very short half-life in vivo. Pegylation of mGM-CSF (peg-mGM-CSF) has been shown to prolong its half-life.There are numerous reports of GM-CSF use in humans to mobilize stem cells. It was observed by our group that patients who were transplanted with stem cell products mobilized by GM-CSF had a lower incidence of acute GVHD (Devine et al. BBMT 2005; 36:531). The reason for this is unclear. A recent report suggested that regulatory T-cells (T-regs) express the low affinity alpha subunit of the mGM-CSF receptor and can be expanded ex vivo in the presence of GM-CSF and independent of IL-2 (Kared et al. Blood 2008;112:2575). We hypothesized that peg-mGM-CSF may expand T-regs in vivo and act synergistically with the CXCR4 antagonist Plerixafor (AMD3100) and a small molecule inhibitor of VLA-4, BIO5192, to generate optimal peripheral blood stem cell products enriched in hematopoietic progenitors and T-regs.Peg-mGM-CSF was manufactured in a bioreactor as previously described (Sainathan et al. Prot Express and Purification 2005;44:94). C57/Bl6 mice were injected with peg-mGM-CSF 5ug IP, daily x 4 days then bled and analyzed for CFU progenitor assays (CFU-C) and FACS analysis. The same mice were then injected with AMD3100 (5mg/kg SC) with or without BIO5192 (1mg/kg IV) and serially bled at 30 minutes, 1 hour, 3 hours, and 6 hours. CFU-C were performed and blood and spleen were analyzed for T-cell and dendritic cell subsets by FACS. We observed that peg-mGM-CSF was a potent mobilizer of hematopoietic progenitors and resulted in leukocytosis (mean difference 17.98 K/uL +/- 2.15, 95%CI 13.4 - 22.53, p<0001, n=6) and splenomegally. Peg-mGM-CSF increased CFU-C by 26.5 fold compared to PBS treated mice (n=6, 95% CI = 10.25-42.73). Peg-mGM-CSF had synergistic effects on progenitor mobilization when combined with AMD3100 (89.8 fold, 95% CI 61.6-118, n=6) or BIO5192 (94 fold, 95% CI 46.5-141.6, n=6) compared to PBS treated mice. The combination of peg-mGM-CSF, AMD3100 and BIO5192 resulted in 119.7 fold (95% CI 63.7-175.6, n=6) increase in progenitor mobilization over PBS treated mice and peaked at 30 minutes. In addition, peg-mGM-CSF increased circulating CD4+ FoxP3+ T-regs from a mean of 10.66% +/- 2.65 to 23.86% +/- 4.7 at 5 days (n=15, p=0.001 by paired t-test). Splenic T-regs were also increased from 16.09% +/- 2.7 to 31.56% +/- 4.34 (n=9, p=0.001 by paired t-test). This effect was sustained out to 4 days after the last dose of peg-mGM-CSF. Furthermore, there was no significant change in thymic T-regs, and this effect was not significantly affected by the addition of AMD3100 or BIO5192. The function of these peg-mGM-CSF T-regs, as measured in a MLR was found to be equivalent to T- regs from PBS treated control mice (n=3, p=0.27, by paired t-test). Interestingly, analysis of the spleens from animals on day 5 after peg-mGM-CSF demonstrated an increase in absolute CD11c+ cells, as expected, and a 4 fold increase in the absolute number of plasmacytoid dendritic cells as determined by CD11c+ SiglecH+ staining. Additional studies are underway to assess the functional impact of murine peripheral blood stem cells mobilized with peg-mGM-CSF in combination with small molecule inhibitors of the CXCR4 and VLA-4 axes on engraftment and graft versus host disease in a murine acute GVHD parent to F1 model. Disclosures:DiPersio:Genzyme Corporation: Honoraria.

Gene Expression Changes in CD34+ Cells Precede Development of Therapy-Related Leukemia (t-MDS/AML) After Autologous Hematopoietic Cell Transplantation (aHCT) for Hodgkin (HL) or Non-Hodgkin Lymphoma (NHL).

Abstract 677t-MDS/AML is a lethal complication of aHCT for HL and NHL. The pathogenesis of t-MDS/AML is unclear. We are addressing this gap by conducting a prospective study of patients undergoing aHCT for HL/ NHL at City of Hope, with serial collection of biospecimens from pre-aHCT to 5 years after aHCT (Figure). In this report we describe alterations in gene expression (analyzed using Affymetrix HG-U133 plus 2.0 arrays) in CD34+ hematopoietic stem and progenitor cells (HSPC) associated with development of t-MDS/AML. Patients who developed t-MDS/AML after aHCT (”cases”) were compared with patients who did not develop t-MDS/AML after aHCT (“controls”: matched for primary diagnosis, age, race/ethnicity, and time since aHCT). CD34+ cells were selected from specimens obtained at the following time points: 1) pre-aHCT: peripheral blood stem cell (PBSC) product; and 2) at time of t-MDS/AML for cases and comparable time for controls: bone marrow (BM). Conditional logistic model was used to identify differences in gene expression between cases and controls in: 1) PBSC samples (18 patients who subsequently developed t-MDS/AML [cases]; 37 who did not [controls]); 2) BM samples at time of t-MDS/AML (12 cases; 21 controls); and 3) changes in gene expression from PBSC to t-MDS/AML (Figure). Gene Set Enrichment Analysis (GSEA) showed that PBSC from patients that later developed t-MDS/AML showed significant downregulation of gene sets related to mitochondria and oxidative phosphorylation, ribosomes, tRNA synthesis, proteasome, late progenitors and cell cycle (FDR<10-5for each) and upregulation of genes related to G-protein coupled receptors (GPCR, FDR=0.003) and hematopoietic regulation (CEBP, HOX, Hedgehog, FDR=0.003, 0.04, 0.06 respectively). These pathway alterations were confirmed by Ingenuity and Gene Ontology analysis. BM CD34+ cells obtained at t-MDS/AML showed downregulation of gene sets for cell cycle, p21 and p53 signaling and late progenitors (FDR<10-5 for each) and upregulation of gene sets for GPCR and cell communication/adhesion (FDR<10-5). Analysis of changes in gene expression from PBSC to t-MDS/AML revealed that compared to the controls, the cases demonstrated increased expression of mitochondrial and ribosomal gene sets and reduced expression of DNA damage response and cell cycle regulation gene sets. These results show that specific abnormalities in gene expression associated with t-MDS/AML are present in HSPC long before the development of clinical disease, and that other gene expression abnormalities occur later in the course of disease development. Indeed 22 of the top 50 upregulated and 20 of the top 50 downregulated gene sets at t-MDS/AML were represented within the top 50 up- and down-regulated sets prior to aHCT in the PBSC sample. A gene set comprised of genes differentially expressed between cases and controls at t-MDS/AML was significantly enriched amongst genes differentially expressed in PBSC (NES= -1.74, P = 0.003, FDR=0.024), further demonstrating that gene expression abnormalities associated with t-MDS/AML were present at the time of PBSC collection. To explore the hypothesis that reduced mitochondrial oxidative phosphorylation may lead to enhanced generation of reactive oxygen species (ROS) and oxidative damage, we measured ROS levels in PBSC in untreated cells and after VP16 exposure. ROS level were increased at baseline in PBSC CD34+ cells from t-MDS/AML cases compared to controls (P=0.003) and ROS elimination after VP16 exposure was reduced (P=0.05, 2 hours after VP-16 treatment). The observed gene expression changes support a model for t-MDS/AML development in which 1) reduced mitochondrial oxidative phosphorylation leads to increased ROS levels and increased damage to HSPC following therapeutic exposures, and 2) subsequent loss of DNA damage response and cell cycle regulatory mechanisms in damaged HSPC leads to the emergence of t-MDS/AML. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Comparison of Clinical Outcomes of Patients with Relapsed Acute Promyelocytic Leukemia Induced with Arsenic Trioxide and Consolidated with Either an Autologous Stem Cell Transplant or an Arsenic Trioxide–Based Regimen

In patients with relapsed acute promyelocytic leukemia (APL), the best consolidation regimen following induction of remission with arsenic trioxide (ATO) remains to be defined. Since January 2000, 37 patients with relapsed APL were treated at our center. The median age was 34 years (range, 6-57 years), and there were 20 males (54.1%). The median duration of first remission was 20.3 months (range, 2.9-81.2 months). Relapse was treated with single-agent ATO in 22 patients (59.5%), ATO+ATRA in 5 patients (13.5%), and ATO+ATRA + anthracycline in 10 patients (27%). Thirty-three patients (89%) achieved molecular remission after induction and a consolidation course. Fourteen patients opted to undergo autologous stem cell transplantation (SCT), and the remaining 19 patients received monthly cycles of ATO as a single agent (n=13) or ATO+ATRA (n=6) for 6 months. At a median follow-up of 32 months, the 5-year Kaplan-Meier estimate of event-free survival (EFS) was 83.33% +/- 15.21% in those who underwent autologous SCT versus 34.45% +/- 11.24% in those who did not (P=.001; log-rank test). Following remission induction with ATO-based regimens in patients with relapsed APL, consolidation with autologous SCT is associated with a significantly superior clinical outcome compared with ATO- and ATO+ATRA-based maintenance regimens.

Increased plasma EPO and MIP-1α are associated with recruitment of vascular progenitors but not CD34(+) cells in autologous peripheral blood stem cell grafts

Increased levels of endothelial-like vascular progenitor cells (VPCs) in peripheral blood stem cell (PBSC) products have been associated with reduced transplant-related toxicity following autologous hematopoietic stem cell transplantation. In this study, a panel of angiogenic and inflammatory plasma proteins were quantitatively analyzed in patients undergoing PBSC collection for autologous hematopoietic stem cell transplantation to identify profiles associated with greater VPC recruitment. A panel of 16 candidate plasma factors were quantified using multianalyte fluorescence and/or enzyme-linked immunosorbent assay. VPC clusters were enumerated using a standard cell culture assay. Thirty-six patients (mean age=51 years, 42% female) had plasma collected at baseline prior to PBSC mobilization and on the day of PBSC collection. Only erythropoietin (EPO) levels increased significantly on the day of PBSC collection in comparison with baseline plasma levels (2.2-fold increase; p=0.003). Interleukin-2, -10, epidermal growth factor, interferon-alpha, and angiopoietin-1 all decreased significantly between baseline and the day of PBSC collection (p<0.02). The remaining cytokine levels did not change appreciably (p=NS). The cohort was divided into "low" graft VPCs (<2.0 x 10(3)/kg) and "high" graft VPCs (>or=2.0 x 10(3)/kg) and cytokine levels were compared between the groups. At baseline, increased levels of macrophage inflammatory protein-1 alpha (MIP-1 alpha) were associated with increased graft VPCs (p=0.05) while higher EPO concentrations on the day of PBSC collection predicted higher graft VPC levels (p=0.02). These cytokines were not associated with CD34(+) cell mobilization. The association of different plasma proteins with graft VPC and CD34(+)-cell levels suggests that mobilization of vascular and hematopoietic progenitors occurs through independent mechanisms. Patients with low levels of MIP-1 alpha at baseline may be candidates for interventions aimed at increasing graft VPC levels. Strategies that increase plasma EPO concentrations may be most promising to augment the regenerative properties of PBSC products.

Consensus criteria for sensitive detection of minimal neuroblastoma cells in bone marrow, blood and stem cell preparations by immunocytology and QRT-PCR: recommendations by the International Neuroblastoma Risk Group Task Force.

Disseminating disease is a predictive and prognostic indicator of poor outcome in children with neuroblastoma. Its accurate and sensitive assessment can facilitate optimal treatment decisions. The International Neuroblastoma Risk Group (INRG) Task Force has defined standardised methods for the determination of minimal disease (MD) by immunocytology (IC) and quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) using disialoganglioside GD2 and tyrosine hydroxylase mRNA respectively. The INRG standard operating procedures (SOPs) define methods for collecting, processing and evaluating bone marrow (BM), peripheral blood (PB) and peripheral blood stem cell harvest by IC and QRT-PCR. Sampling PB and BM is recommended at diagnosis, before and after myeloablative therapy and at the end of treatment. Peripheral blood stem cell products should be analysed at the time of harvest. Performing MD detection according to INRG SOPs will enable laboratories throughout the world to compare their results and thus facilitate quality-controlled multi-centre prospective trials to assess the clinical significance of MD and minimal residual disease in heterogeneous patient groups.

Postthaw clotting of peripheral blood stem cell products due to insufficient anticoagulant

The amount of acid citrate dextrose formula A (ACD-A), which is a commonly used anticoagulant in leukopheresis, has to ensure both the safety of the donor and guarantee the integrity of the peripheral blood stem cell (PBSC) product until its transplant. Two recent consecutive cases of postthaw PBSC product clotting initiated a look-back investigation of the ACD-A percentage in leukopheresis products collected in our facility. The data indicated a significant difference between the average amount of ACD-A in prefreezing products collected during 2006 (11.4%) and in products collected during 2007 and 2008 (8.8% and 8.7%, respectively). These findings and the fact that the two clotted products had less than 7% ACD-A indicated that insufficient amount of anticoagulant might contribute to their clotting. This investigation prompted us to modify our collection and thawing procedures to prevent similar events in the future.

Improved post-thaw recovery of peripheral blood stem/progenitor cells using a novel intracellular-like cryopreservation solution

Peripheral blood stem cells (PBSC) have become the preferred stem cell source for autologous hematopoietic transplantation. A critical aspect of this treatment modality is cryopreservation of the stem cell products, which permits temporal separation of the PBSC mobilization/collection phase from the subsequent high-dose therapy. While controlled rate-freezing and liquid nitrogen storage have become 'routine' practice in many cell-processing facilities, there is clearly room for improvement as current cryopreservation media formulations still result in significant loss and damage to the stem/progenitor cell populations essential for engraftment, and can also expose the patients to relatively undefined serum components and larger volumes of dimethylsulfoxide (DMSO) that can contribute to the morbidity and mortality of the transplant therapy. This study compared cryopreservation of PBSC in a novel intracellular-like, fully defined, serum- and protein-free preservation solution, CryoStor (BioLife Solutions Inc.), with a standard formulation used by the Fred Hutchinson Cancer Research Center (FHCRC). Briefly, human PBSC apheresis specimens were collected and 5 x 10(7) cells/1 mL sample vial were prepared for cryopreservation in the following solutions: (a) FHCRC standard, Normosol-R, 5% human serum albumin (HAS) and 10% DMSO; and (b) CryoStor CS10 (final diluted concentration of 5% DMSO). A standard controlled-rate freezing program was employed, and frozen vials were stored in the vapor phase of a liquid nitrogen freezer for a minimum of 1 week. Vials were then thawed and evaluated for total nucleated cell count (TNC), viability, CD34 and granulocytes by flow cytometry, along with colony-forming activity in methylcellulose. The PBSC samples frozen in CryoStor CS10 yielded significantly improved post-thaw recoveries for total viable CD34(+), colony-forming units (CFU) and granulocytes. Specifically, relative to the FHCRC standard formulation, cryopreservation with CS10 resulted in an average 1.8-fold increased recovery of viable CD34(+) cells (P=0.005), a 1.5-fold increase in CFU-granulocyte-macrophage (GM) numbers (P=0.030) and a 2.3-fold increase in granulocyte recovery (P=0.045). This study indicates that use of CryoStor for cryopreservation can yield significantly improved recovery and in vitro functionality of stem/progenitor cells in PBSC products. In addition, it is important to note that these improved recoveries were obtained while not introducing any extra serum or serum-derived proteins, and reducing the final concentration/volume of DMSO by half. Further in vitro and in vivo studies are clearly necessary; however, these findings imply use of CryoStor for cryopreservation could result in improved engraftment for those patients with a lower content of CD34(+) cells in their PBSC collections, along with reducing the requirement for additional apheresis collections and decreasing the risk of adverse infusion reactions associated with higher exposure to DMSO.