Carfilzomib and Bortezomib Containing Regimens Yield High Rates Of MRD Negative Autologous Hematopoietic Progenitor Cell (HPC) Grafts In Multiple Myeloma (MM) and High Risk Smoldering Myeloma (SMM)

Abstract Background Regimens incorporating modern anti-myeloma drugs, such as carfilzomib (CFZ) and bortezomib (BOR), produce rapid, deep and durable responses in newly diagnosed myeloma patients but their effect on collection of autologous HPC is not well known, including minimal residual disease (MRD) testing of stem cell grafts. Employing older induction regimens (such as VAD), less sensitive flow cytometry techniques detected circulating myeloma cells in 38-46% of autologous HPC grafts (Stewart, et al. JCO. 2001 and Bourhis, et al. Haematologica. 2007). We hypothesized that the use of modern CRd combination therapy including Carfilzomib (CFZ)-Lenalidomide (LEN)-Dexamethasone (DEX) would significantly lower the rates of HPC product contamination. Methods Thirty-six patients, including 29 with MM and 7 with high-risk SMM, underwent HPC mobilization and collection following induction with CRd (n=30), LEN-BOR-DEX (RVd, n=4), Cyclophosphamide-BOR-DEX (CyBorD, n=1) and Cyclophosphamide-BOR-Prednisone (CyBorP, n=1). For HPC mobilization, all patients received 5 days of filgrastim at 10-16 mcg/kg/dose. A combination of the patient’s weight and a peripheral blood CD34 count after 4 doses was used to determine the likelihood of collecting > 4 x106 CD34+ cells/ kg in a single apheresis procedure after a fifth filgrastim dose, according to a previously published algorithm from our institution. Only subjects predicted to require > 1 apheresis by the algorithm received Plerixafor (PLX) at 240 mcg/kg/dose on the fifth day along with the fifth filgrastim dose. HPC collection occurred on day 6, 8 hours after the last mobilizing agent(s) administration. Product contamination with myeloma cells (i.e. MRD status) was evaluated using multi-parameter flow cytometry with a minimum of 3 x 106 events obtained (sensitivity detection rate 1 x 10-5) to examine expression of 9 antigens by the plasma cells. Results The median age at mobilization was 56.2 years (range 40-73) and 19 (53%) were male. At the time of HPC collection, 20 (55%) patients were in sCR/CR/nCR, 11 (30%) had VGPR with 4 PR (11%) and 1 SD (3%). The mean CD34+ cells in the peripheral blood were 33/uL on day 5 and 55/uL on day 6 for the whole cohort. Thirteen (36%) patients did not need PLX. Interestingly, the mean CD34+ count dropped by a mean of 2% from D5 to D6 in patients not receiving PLX while, as expected, it increased by 304% in those who did. The median number of CD34+ cells collected was 6.86 million/kg (range 2.6-12.5) for the whole cohort, (6.6 million/kg without PLX and 7.52 million with PLX p=0.46). Thirty-three of 36 patients (92%) achieved a collection of > 4 million cells /kg in a single apheresis procedure. The 30 patients treated with CRd had a median of 5 (range = 3-7) prior cycles containing LEN with a median of 12 days (range 1-34) between mobilization and last LEN dose. Only 2 of 36 (5%) products were found to have evidence of tumor cell contamination (i.e. MRD positive) using sensitive multiparameter flow cytometry, one patient in PR after 6 cycles of CRd and a second patient in CR after 5 cycles of RVd. Conclusions Modern anti-myeloma therapies, such as CRd and RVd, allow adequate HPC collection in a single apheresis procedure in most cases and improve the quality of the HPC product with greatly reduced tumor cell contamination compared to historical controls. Indeed, 34/36 (94%) patients treated with modern anti-myeloma therapy collected an MRD negative HPC product. Future prospective studies are needed to assess whether autologous stem cell transplants (ASCT) using tumor-free HPC products collected in the era of modern induction therapies have better outcomes. Disclosures: No relevant conflicts of interest to declare.

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Mobilization of Peripheral Blood Stem Cells with Rituximab Plus ESHAP Combination Regimen in B-Cell NHL - Preliminary Results.

Blood ◽

10.1182/blood.v108.11.5443.5443 ◽

2006 ◽

Vol 108 (11) ◽

pp. 5443-5443

Author(s):

Min Kyoung Kim ◽

Shin Kim ◽

Seong Sook Lee ◽

Sun Jin Sym ◽

Dae Ho Lee ◽

...

Keyword(s):

Stem Cell ◽

High Risk ◽

B Cell ◽

Peripheral Blood ◽

B Cell Lymphoma ◽

Hematopoietic Progenitor Cell ◽

High Dose ◽

Optimal Response ◽

Cd34 Cells ◽

Pbsc Mobilization

Abstract Objectives: The mobilization chemotherapy should be directed toward the dual objectives of good antilymphoma activity and adequate PBSC mobilization. Previously, we reported the ESHAP plus G-CSF is an effective mobilization regimen in patients with relapsed and high risk NHL. The purpose of this study is to assess the efficacy and feasibility of autologous stem cell collection after mobilization with Rituximab plus ESHAP (etoposide, methylprednisolone, high-dose cytarabine, and cisplatin) combination therapy in B-cell Non-Hodgkin’s lymphoma. Patients and Methods: CD20 + B-cell NHL patients with either high risk, relapsed or refractory patients were eligible for the study. The regimen consisted of: Rituximab 375mg/m2 given on day 1, standard ESHAP therapy (etoposide 40mg/m2 IV/2hr day 2–5, methylprednisolone 500mg/m2 days 2–6, cytarabine 2g/m2 IV/3hr on day 5 and cisplatin 25mg/m2 CIV days 2–5). In all patients, the collection of PBSC was performed during recovery after giving G-CSF (10μ/kg/day) started on day 7. The first harvest was started only if the peripheral blood hematopoietic progenitor cell (HPC) count exceeded 5/μl. Results: Fifty-five mobilization procedures performed on 20 patients with a median of three apheresis (range 2–5) per patient. Median age was 37 years (range 15–65). At the time of PBSC mobilization, Seventeen patients were considered to be responsive (CR, PR and sensitive relapse). Aphereses were started on day 16 (range 13–18). The number of total MNCs (×106/kg) collected was 5.4 (range, 1.4–14.5) and the number of CD34+cells (× 106/kg) was 10.6 (range, 4.9–52.6). The median days of G-CSF usage was 11(range 9–15). Most non-hematologic adverse events were mild and reversible. Nineteen patients (95%) were achieved optimal response, which was defined as ≥ 5 × 106 CD34 cells/kg. In all, sixteen of the patients underwent high-dose chemotherapy. The median time to ANC ≥ 0.5 × 109/L was 10 days (range, 8–17). The median days to platelets ≥ 20 × 109/L was 12 (range, 7–27). Conclusion: Addition of Rituximab to ESHAP chemotherpy does not show any adverse effect in autologous stem cell mobilization and collection. R-ESHAP regimen is effective as a combined mobilization and second-line regimen for patients with pretreated B-cell lymphoma. Figure 1. Days to achieve optimal response Figure 1. Days to achieve optimal response

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Aldehyde Dehydrogenase (ALDH) Vs CD34 for Predicting Engraftment After Autologous Hematopoietic Progenitor Cell (autoHPC) Transplant

Blood ◽

10.1182/blood.v116.21.4441.4441 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4441-4441

Author(s):

Muthu Veeraputhiran ◽

Lakshmikanth Katragadda ◽

Bart Barlogie ◽

Michele H. Cottler-Fox

Keyword(s):

Flow Cytometry ◽

Progenitor Cells ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Correlation Coefficients ◽

Threshold Dose ◽

Hematopoietic Progenitor Cell ◽

Aldh Activity ◽

Cd34 Cells ◽

Fresh Products

Abstract Abstract 4441 Introduction: When the cell surface marker CD34 became available for enumerating HPC there was controversy as to whether it was useful for predicting time to engraftment after autologous peripheral blood HPC transplant (autoHPCT). It was demonstrated that a threshold dose of 2.0–2.5 × 106 CD34+ cells/kg in the product, measured before cryopreservation, predicted reliable engraftment within a reasonable period of time. CD34 is expressed on the surface of HPC ranging from primitive to committed progenitor cells. ALDH is highly expressed in primitive cells such as HPC, endothelial progenitor cells and mesenchymal stromal cells. ALDHbr cells are by definition viable, while measurement of viable CD34+ cells requires an additional assay. We tested the correlation of CD34 and ALDH activity in fresh HPC products and then examined the usefulness of these two assays for predicting time to WBC and platelet engraftment after autoHPCT. Materials and Methods: We identified 42 consecutive HPC apheresis products used for autoHPCT for which data on CD34+ numbers and viability as well as ALDHbr cell numbers were available. Data from 5 of these product were not used for predicting engraftment due to lack of engraftment data. Viability was assesed by flow cytometry on pre-cryopreservation (fresh) and post-thaw samples by looking at propidium iodide or 7-AAD uptake within the CD34+ population identified using the ISHAGE method. ALDH activity was measured by flow cytometry on pre-freeze samples using the manufacturers prescribed method. HPC were cryopreserved using a controlled rate freezer and 10% DMSO, then stored in liquid nitrogen. Data on time to WBC and platelet engraftment were determined by chart review. The patents received a median of 3.78 × 106 CD34+ cells/kg (range 2.43–8.59 × 106 CD34+ cells/kg) for autoHPCT. Regression analyses were performed to see if viable CD34 in the fresh product, and in the thawed product, correlated with ALDH and whether any of these measures correlate with time to WBC (ANC >500/mm3) or platelet engraftment (>20,000/mm3). Results: The correlation coefficients for ALDHbr vs viable CD34+ cells pre-cryopreservation (r=0.97; n=42) and for ALDHbr cells vs post-thaw CD34+ cells (r=0.96; n=42) were both significant. The percent of variation for ALDHbr cells to WBC engraftment (r2 = 2.2%; n=37) and for CD34+ cells both pre-freezing and post-thaw to WBC engraftment (r2 =1.9%; n=37) were not significant. The r2 for ALDHbr cells and platelet engraftment was 0.9%, the r2 for CD34+ cells and platelet engraftment was 1.1% pre-freeze (n=37) and was 0.9% post-thaw (n=37) were also not significant. Conclusion: Enumeration of HPC by ALDH correlates well with CD34, suggesting that the two assays are equivalent when analyzing autologous peripheral blood. Neither the number of ALDHbr cells or CD34+ cells in fresh products, nor the number of viable CD34+ cells in the thawed products, correlates with time to WBC and platelet engraftment. Since our patients received at least 2.4 × 106 CD34+ cells/kg, our observations support previous data showing no correlation between cell dose and time to engraftment if more than the HPC threshold dose for engraftment is given for transplant. Disclosures: No relevant conflicts of interest to declare.

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The Tetraspanin CD9 Regulates Engraftment and Mobilization of Human CD34+ Hematopoietic Stem/Progenitor Cells and Modulates VLA-4 Activity

Blood ◽

10.1182/blood.v126.23.1169.1169 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1169-1169

Author(s):

Kam Tong Leung ◽

Karen Li ◽

Yorky Tsin Sik Wong ◽

Kathy Yuen Yee Chan ◽

Xiao-Bing Zhang ◽

...

Keyword(s):

Stem Cell ◽

Progenitor Cells ◽

Peripheral Blood ◽

Scid Mouse ◽

Conflicts Of Interest ◽

Chimeric Mice ◽

Hematopoietic Stem ◽

Cell Engraftment ◽

Cd34 Cells

Abstract Migration, homing and engraftment of hematopoietic stem/progenitor cells depend critically on the SDF-1/CXCR4 axis. We previously identified the tetraspanin CD9 as a downstream signal of this axis, and it regulates short-term homing of cord blood (CB) CD34+ cells (Leung et al, Blood, 2011). However, its roles in stem cell engraftment, mobilization and the underlying mechanisms have not been described. Here, we provided evidence that CD9 blockade profoundly reduced long-term bone marrow (BM; 70.9% inhibition; P = .0089) and splenic engraftment (87.8% inhibition; P = .0179) of CB CD34+ cells (n = 6) in the NOD/SCID mouse xenotransplantation model, without biasing specific lineage commitment. Interestingly, significant increase in the CD34+CD9+ subsets were observed in the BM (9.6-fold; P < .0001) and spleens (9.8-fold; P = .0014) of engrafted animals (n = 3-4), indicating that CD9 expression on CD34+ cells is up-regulated during engraftment in the SDF-1-rich hematopoietic niches. Analysis of paired BM and peripheral blood (PB) samples from healthy donors revealed higher CD9 expressions in BM-resident CD34+ cells (46.0% CD9+ cells in BM vs 26.5% in PB; n = 13, P = .0035). Consistently, CD34+ cells in granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood (MPB) expressed lower levels of CD9 (32.3% CD9+ cells; n = 25), when compared with those in BM (47.7% CD9+ cells; n = 16, P = .0030). In vitro exposure of MPB CD34+ cells to SDF-1 significantly enhanced CD9 expression (1.5-fold increase; n = 4, P = .0060). Treatment of NOD/SCID chimeric mice with G-CSF decreased the CD34+CD9+ subsets in the BM from 79.2% to 62.4% (n = 8, P = .0179). These data indicate that CD9 expression is down-regulated during egress or mobilization of CD34+ cells. To investigate the possible mechanisms, we performed a VCAM-1 (counter receptor of the VLA-4 integrin) binding assay on BM CD34+ cells. Our results demonstrated that CD34+CD9+ cells preferentially bound to soluble VCAM-1 (17.2%-51.4% VCAM-1-bound cells in CD9+ cells vs 12.8%-25.9% in CD9- cells; n = 10, P ≤ .0003), suggesting that CD9+ cells possess higher VLA-4 activity. Concomitant with decreased CD9 expression, MPB CD34+ cells exhibited lower VCAM-1 binding ability (2.8%-4.0% VCAM-1-bound cells; n = 3), when compared to BM CD34+ cells (15.5%-37.7%; n = 10, P < .0130). In vivo treatment of NOD/SCID chimeric mice with G-CSF reduced VCAM-1 binding of CD34+ cells in the BM by 49.0% (n = 5, P = .0010). Importantly, overexpression of CD9 in CB CD34+ cells promoted VCAM-1 binding by 39.5% (n = 3, P = .0391), thus providing evidence that CD9 regulates VLA-4 activity. Preliminary results also indicated that enforcing CD9 expression in CB CD34+ cells could enhance their homing and engraftment in the NOD/SCID mouse model. Our findings collectively established that CD9 expression and associated integrin VLA-4 activity are dynamically regulated in the BM microenvironment, which may represent important events in governing stem cell engraftment and mobilization. Strategies to modify CD9 expression could be developed to enhance engraftment or mobilization of CD34+ cells. Disclosures No relevant conflicts of interest to declare.

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First Reported Use of a Biosimilar G-CSF in Allogeneic Stem Cell Mobilisation

Blood ◽

10.1182/blood.v118.21.4571.4571 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4571-4571

Author(s):

Nabih Azar ◽

Sylvain Choquet ◽

Alice Garnier ◽

Damien Roos-Weil ◽

Véronique Leblond

Keyword(s):

Body Weight ◽

Stem Cell ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Median Number ◽

Large Hospital ◽

Originator Product ◽

Healthy Donors ◽

Stem Cell Mobilisation ◽

Cd34 Cells

Abstract Abstract 4571 OBJECTIVES: Biosimilar granulocyte colony-stimulating factor (G-CSF) has been approved on the basis of comparable quality, safety and efficacy as the originator product. Approval of biosimilar G-CSF is in the same indications as the originator, including autologous and allogeneic peripheral blood stem cell (PBSC) mobilisation, for which it is being used throughout our large hospital group in Paris, France. Concerns have been raised by professional bodies over use of biosimilar G-CSF in allogeneic transplants. To our knowledge, this is the first reported use of biosimilar G-CSF in healthy donors for allogeneic transplantation. METHODS: Healthy related donors received biosimilar G-CSF (EP-2006, Sandoz Biopharmaceuticals) 10 μg/kg/day for PBSC mobilisation. G-CSF was administered on days 1–4 with the first leukapheresis performed on day 5. If the required number of CD34+ cells per recipient body weight (4 × 106 CD34+ cells/kg) was not collected, G-CSF administration was continued and a second PBSC collection was performed on day 6. Further G-CSF administration and a third leukapheresis was done on day 7 if required. RESULTS: Twelve healthy donors (7 male, 5 female; mean ± SD age 61.0 ± 7.8 years, range 51–73) received biosimilar G-CSF for PBSC mobilisation. Median donor body weight was 82 kg (range 55–115 kg). Median CD34+ cell count on day 5 was 75/ml (range 23–140). A single leukapheresis after 4 injections of biosimilar G-CSF was sufficient to collect the CD34+ cell dose required in six donors with peripheral blood (PB) CD34+ counts ranging from 80–140 ml. Six donors underwent a second leukapheresis after 5 days of biosimilar G-CSF, one of whom (a 73-year old female) underwent a third leukaphereses on day 7 (PB CD34+ at first apheresis 23 ml). Median number of CD34+ cells per recipient bodyweight was 5.5 × 106 cells/kg (range 2.6–8.9). Five donors reported bone pain (4 mild, 1 moderate severity) which was effectively treated with paracetamol. No other adverse events were reported. Blood measurements were normal in all donors when assessed one week after the end of mobilisation. These findings are consistent with our previous use of originator G-CSF (Neupogen®) in allogeneic stem cell mobilisation. CONCLUSION: These preliminary findings suggest biosimilar G-CSF is effective and well-tolerated in allogeneic stem cell mobilisation. Further studies are required to assess longer-term safety outcomes. The use of biosimilar G-CSF may provide important cost-savings when compared with the originator product. Disclosures: No relevant conflicts of interest to declare.

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Hematopoietic Progenitor Cell Counts Is a Useful Indicator To Determine Appropriate Time For Peripheral Blood Stem Cell Collection

Blood ◽

10.1182/blood.v122.21.2037.2037 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2037-2037

Author(s):

Sun-Young Kong ◽

Hyoeun Shim ◽

Se-Na Lee ◽

Jung-Hee Kong ◽

Hyeon-Seok Eom ◽

...

Keyword(s):

Multiple Myeloma ◽

Stem Cell ◽

Peripheral Blood ◽

Progenitor Cell ◽

Peripheral Blood Stem Cell ◽

Hematopoietic Progenitor Cell ◽

Hematopoietic Progenitor ◽

Non Hodgkin Lymphoma ◽

Cell Counts ◽

Cd34 Cells

Abstract Background The optimal peripheral blood stem cell (PBSC) collection is a key step for successful outcome in hematopoietic stem cell transplantation (HSCT). Many indicators including preharvest white blood cell (WBC), mononuclear cell (MNC), and CD34 positive cell counts have been used for deciding the adequate time for collection of PBSCs, but each indicator has limitations. Here we investigated hematopoietic progenitor cell (HPC) count as an indicator for PBSC collection. Methods: Data from 851 autologous PBSC collections from 233 patients at the National Cancer Center, Korea, were analyzed. The correlations between harvested CD34 cell counts with preharvest WBC, MNC, CD34 cell counts, and HPC were analyzed, as were correlations by disease and mobilizing agent. Also how the outcome for engraftment can be predicted based on HPC count was studied. Results: The median age of patients was 41 years (range 0.1-72 years). The most frequent diseases were multiple myeloma (n=64) and non-Hodgkin lymphoma (n=56). The correlation coefficient between collected CD34 cells and preharvest CD34 count was (r=0.669, p<0.001), followed by preharvest HPC count (r=0.419, p<0.001), preharvest MNC (r=0.190, p<0.001) and preharvest WBC (r=0.014, p=0.679). The most adequate cut-off value for obtaining >1x106 CD34+ cells/kg at first time of PBSC was 24.0 HPCs/μL with sensitivity and specificity of 67.7% and 74.3% respectively. The cutoff as 28.0 HPCs/μL was adequate for obtaining 2.0 x106 CD34+ cells/kg with sensitivity and specificity of 73.7% and 72.2% respectively. HPC was well correlated with CD34 in PBSC of patients with multiple myeloma (r=0.326, p=0.009), non-Hodgkin lymphoma (r=0.353, p=0.008), especially diffuse large B-cell lymphoma (r=0.810, p<0.001) and acute leukemia (r=0.998, p<0.001). HPC was a better indicator for non-cyclophosphamide (r=0.337, p<0.001) than cyclophosphamide-based chemomobilization (r=0.572, p=0.052). Infused number of HPCs did not affect the times to engraftment of platelets (p=0.896) and neutrophils (p=0.953), though CD34 count of infusion had positive effect on platelet engraftment (p=0.017). Conclusion: HPC count represented good correlation with CD34+ and high area under the curve. Considering advantages of ease for use and cost-effectiveness than those of CD34 count, HPC is a good surrogate marker to determine appropriate timing for PBSC. Disclosures: No relevant conflicts of interest to declare.

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Influence Of Race and Ethnicity On The Collection Of G-CSF Mobilized Peripheral Blood Stem Cells From Unrelated Donors, a Center For International Blood and Marrow Transplant Research Analysis

Blood ◽

10.1182/blood.v122.21.3273.3273 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3273-3273

Author(s):

Jack W. Hsu ◽

John R. Wingard ◽

Brent R. Logan ◽

Pintip Chitphakdithai ◽

Bronwen E. Shaw ◽

...

Keyword(s):

Stem Cell ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Collection Efficiency ◽

Univariate Analysis ◽

Categorical Variables ◽

Continuous Variables ◽

Cd34 Cells ◽

Unrelated Donors

Abstract Peripheral blood stem cell (PBSC) collection is increasingly used in allogeneic stem cell transplantation. However, a small percentage of healthy donors have a poor mobilization response to G-CSF. Very little information exists on the effect of donor race or ethnicity on PBSC mobilization. We analyzed 10776 unrelated donors from the National Marrow Donor Program (NMDP) who underwent G-CSF mobilized PBSC collection from 2006–2012. We investigated the effect of self-reported donor race/ethnicity on collection efficiency, defined as number of CD34+ cells/L (of donor blood processed), number of mononuclear cells (MNC)/L and CD34+ cells/MNC collected on the first day of apheresis. Categorical variables were analyzed by the Chi-square test and the Kruskal-Wallis test was used for continuous variables. A linear regression model was used to compare the various race/ethnic groups while controlling for potential confounding factors (such as age, BMI, gender, and year of apheresis). The result of our analysis is shown in Table 1. Univariate analysis revealed statistically significant differences in CD34+ cells/L, MNC/L and CD34+/MNC in all races analyzed. In general, African Americans (AA) had the highest collection efficiency while Caucasians had the lowest. Other races/ethnicities had collection efficiencies between the two groups. On multivariate analysis, statistically significant differences in CD34+ cell/L were seen in Hispanics, AA and Asian/Pacific Islanders (API), primarily in the obese (Hispanic, AA, API) and overweight (AA, API) donors. In the API group the differences in collection efficiency were predominately seen in males. No differences were seen between Caucasians and Native Americans. This study reveals significant racial/ethnic differences in the efficiency of collection of CD34+ cells in unrelated donors. Although these differences do not appear to interfere with the ability to collect adequate numbers of PBSC, it is currently unknown why they exist. This is an area for continued research. Disclosures: No relevant conflicts of interest to declare.

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Functional Studies of Ex Vivo Expanded Hematopoietic Stem Cells in Mice and Monkeys

Blood ◽

10.1182/blood.v126.23.1899.1899 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1899-1899

Author(s):

Yu Zhang ◽

Bin Shen ◽

Meng Qin ◽

Zhihua Ren ◽

Xinxin Ding ◽

...

Keyword(s):

Flow Cytometry ◽

Stem Cell ◽

Peripheral Blood ◽

Autologous Transplantation ◽

Hematopoietic Stem ◽

Cynomolgus Monkeys ◽

Human Cd34 ◽

Cd34 Cells ◽

Bone Morrow

Abstract Hematopoietic stem cell (HSC) transplantation has been widely applied for the treatment of malignant blood diseases. However, obtaining sufficient HLA-matched stem/progenitors for cell transplantation is an obstacle for clinical applications. We reported here that an optimal cytokine cocktail in a modified IMDM basal medium was developed that contained stem cell factor, Flt-3 ligand, thrombopoietin, interleukin 3, G-CSF and GM-CSF. Up to 7.3 folds of expanded CD34+ cells with 66.3% CD34+ of whole cells were obtained after 4 days' culture from human umbilical cord blood. Colony-forming unit (CFU) assays showed that expanded CD34+ cells retained the same renewal ability as the pre-expanded counterparts. To test the repopulating ability of the expanded CD34+ in vivo, sixteen NOD/SCID mice were divided to four groups and injected with saline (group 1), 0.4 million pre-expanded CD34+ cells (group 2), 0.4 million 4-day expanded CD34+ cells (group 3), and 2.9 million expanded CD34+ cells (group 4), respectively. Multi-lineage differentiations in the peripheral blood were assessed by flow cytometry with antibodies against a panel of human cell surface markers. In week 3, human CD34+ cells were decreased below 1% in groups 2 and 3, and 1.717%±0.65% in group 4. Whereas, human CD45+ was increased up to 3.831%±1.54%, 3.108%±1.18% and 10.408%±3.27% for groups 2, 3 and 4, respectively. The other human CD41+, CD71+ and CD15+ were also increased in groups 2-4. No expression of any human cell lineage markers was detected in group 1, indicating that expanded human CD34+ cells possessed the repopulating viability of HSCs in vivo. Furthermore, in week 12, the human CD34+ cells were re-isolated from the bone morrow of the mice (one mouse from each group). The isolated human CD34+ cells were again transfused into new NOD/SCID mice for the secondary transplantation. In week 6, human CD45+, CD15+ and CD19+ were observed from the bone morrow cells of sacrificed mice. On the other hand, human CD45+, CD15+ and CD19+ were also detectable in bone morrow cells for all remaining mice in week 24, suggesting that the expanded CD34+ cells could be successfully engrafted into mice in a long term. In addition, the cytokine cocktail was further evaluated for its safety and efficacy in primates. The CD34+ cells were isolated from the peripheral blood of cynomolgus monkeys and expanded for about 8 folds were obtained on day 9. Harvested CD34+ cells were transducted with the gene of green fluorescent protein (GFP). These cynomolgus monkeys (n=11) were administered with cyclophosphamide via intravenous injection at a dose of 50 mg/kg/day for two days. The myelo-suppressed monkeys were randomly divided into three groups as follows: a control group treated with saline (n=3), a group with autologous CD34- cells (n=3), and a group treated with GFP-labeled, expanded autologous CD34+ cells (n=5), respectively. After autologous transplantation, routine blood tests and flow cytometry analysis were performed to determine the proportion of GFP+ cells in the peripheral blood. The flow cytometry analysis revealed that the white blood cells (WBC), neutrophil (NEU) and platelets (PLT) in peripheral blood of cynomolgus monkeys were completely recovered to the normal levels on days 12, 11 and 10 post autologous transplantation of expended CD34+ cells, respectively. For the control groups, WBC, NEU and PLT returned to the normal on days 22, 22 and 12 for the saline treatment and on days 20, 20 and 12 for the CD34- group, respectively. Similarly, the lymphocytes of cynomolgus monkeys were recovered completely on day 20 post autologous CD34+ cell transplantation compared with the saline control (day 25) and the CD34- group (day 22). On day 30 after the autologous transplantation, the GFP+ ratio in CD45+ populations was around 2% in the peripheral blood. GFP+ cells (ranging from 1.8% to 4.1%) were also detected in bone morrow of cynomolgus monkeys. All primates transplanted with the expanded autologous CD34+ cells have survived for 18 months without any noticeable abnormalities. In conclusion, our results indicate that expanded CD34+ cells can be safely and efficiently used for repopulating stem cell compartment in mice and primates, underscoring the potential applications in the clinic. Furthermore, the results from successful autologous transplantation of cynomolgus CD34+ cells strongly suggest a possible application for personalized treatment of blood diseases. Disclosures Qin: Biopharmagen. corp: Employment. Ren:Biopharmagen corp: Employment.

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CD138-Independent Strategy for Detecting Residual and Circulating Myeloma Plasma Cells

Blood ◽

10.1182/blood.v124.21.2077.2077 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2077-2077

Author(s):

Barbara Muz ◽

Feda Azab ◽

de la Puente Pilar ◽

Jacob Paasch ◽

Justin King ◽

...

Keyword(s):

Flow Cytometry ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Plasma Cells ◽

Conflicts Of Interest ◽

Residual Disease ◽

Positive Control ◽

The Other ◽

Myeloma Cells ◽

New Strategy

Abstract INTRODUCTION: Flow cytometry has been used extensively to detect MM cells in the bone marrow (BM), micro-residual disease and circulating myeloma cells. Accumulating literature defines MM cells as CD138+/CD38+ for the primary gating of plasma cells; however, several studies demonstrated the presence of clonogenic CD138-negative MM cells, and that hypoxia can decrease the expression of CD138 in MM cells. We propose a novel set of biomarkers to detect MM cells regardless of their CD138 expression or hypoxic status. METHODS and RESULTS: We have tested the effect of hypoxia on the expression of the MM markers, CD138 and CD38, and found that hypoxia decreased the expression of CD138, therefore it cannot be used as a universal marker for MM cells. Hypoxia did not alter the expression of CD38 in MM cells; however, CD38 is a general leukocyte marker and cannot be used alone to identify MM cells, since it is expressed on multiple other leukocytes including T-cells, B-cells, monocytes, NK cells, and dendritic cells. Therefore, we negatively selected these cells by flow cytometry using the specific markers for each of these populations including CD3, CD19, CD14, CD16, and CD123; respectively. Therefore, we detected MM cells as CD38+/CD3-/CD19-/CD14-/CD16-/CD123-. We used CD38 antibody conjugated with APC and FITC-conjugated antibodies for all the other markers, thus MM cells were defined as APC+/FITC- population. To compare traditional method (CD138-based) with our strategy to detect hypoxic and normoxic MM cells, MM cell lines were stained with a cocktail of CD38-APC, FITC-antibodies, and CD138-V450, and analyzed by flow cytometry. The use of CD138+ as a universal marker for MM cells detected 85-100% of the normoxic cells, and only 60-75% of the hypoxic MM cells. While APC+/FITC- strategy detected close to a 100% of the MM cells independent of the cells’ normoxic/hypoxic status or expression of CD138. The ability of the new strategy to detect hypoxic and normoxic MM cells in the peripheral blood was tested by staining hypoxic and normoxic MM cells with cell-tracker Calcein-Red-Orange (as a positive control), spiked 104 MM cells into 106 mononuclear cells from a healthy donor (1% MM in total), and the percentile of Calcein-Red-Orange+ (as a positive control), APC+/FITC-, and CD138+ populations, were analyzed by flow cytometry. Calcein-Red-Orange staining showed exact 1% of MM cells detected in total mononuclear cells for both hypoxic and normoxic MM cells; detection with CD138+ showed 0.95% for normoxic and 0.45% for hypoxic cells; and detection of MM cells using the APC+/FITC- strategy showed 1.05% for normoxic and 1.1% for hypoxic MM cells. Hence, the new strategy detects MM cells selectively and independently of their CD138 expression or hypoxic status. We have used the APC+/FITC- strategy to detect MM cells in BM CD138-negative fractions of 20 MM patients. The APC+/FITC- strategy was able to detect a range of 1.6-44% myeloma cells in the CD138-negative population of BM mononuclear cells isolated from MM patients. Moreover, we have assessed the clonality of this population using APC+/FITC- in the CD138-negative fractions of MM patients. We found that the clonality of the APC+/FITC- population was similar to the clonality of the original disease (CD138+ cells) in 70% of the cases. The other 30% of the cases showed very low involvement of myeloma population in the BM and showed Kappa/Lambda ratio within normal range. Analyzing the prevalence of circulating MM cells in the peripheral blood from 12 MM patients showed that all patients had a higher number of circulating MM cells as detected by APC+/FITC- strategy compared to CD138+, and the fold change ranged from 1.5 to 86 times. CONCLUSION: We found that CD138 cannot be used as a universal marker to detect MM cells. Moreover, we developed a novel strategy to detect MM cells independent of their CD138 expression or hypoxic status; and we used CD38+/CD3-/CD19-/CD14-/CD16-/CD123- population as an alternative set of biomarkers to detect MM cells. This strategy was able to detect a clonal MM cell population in the CD138-negative fraction of BM mononuclear cells isolated from MM patients, as well as in the peripheral blood. Currently, we are exploring the ability of this strategy to predict relapse in MM patients whose BM was defined a CD138-negative. More investigation to characterize this population and the role in tumor recurrence and drug resistance in MM is warranted. Disclosures No relevant conflicts of interest to declare.

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Utility of Plerixafor In Addition to Chemotherapy and G-CSF Mobilization Regimens

Blood ◽

10.1182/blood.v116.21.4443.4443 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4443-4443

Author(s):

Farrukh Awan ◽

David Deremer ◽

Elaine Mebel ◽

Samith Thomas Kochuparambil ◽

Anand P. Jillella

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Chemotherapeutic Agents ◽

Phase Iii ◽

Cell Transplant ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Significant Difference

Abstract Abstract 4443 Introduction: Various chemotherapeutic agents particularly cyclophosphamide (CY) are utilized in combination with growth factors in an attempt to increase the number of stem cells available for collection in the peripheral blood. Plerixafor (P) is a reversible antagonist of CXCR4 and interrupts its interaction with SDF-1. This results in a rapid release of hematopoietic stem cells from the marrow to the circulation. Recent pivotal phase III trial data has established the efficacy of P in combination with G-CSF (G) in patients who had failed prior attempts at stem cell collection. However, there is limited data about the utility of plerixafor in patients who are being mobilized with chemotherapy and G. Method: In this single institution study of uniformly treated patients we describe our experience with the use of P as a salvage option in patients who fail to optimally mobilize CD34+ cells (>5 × 106 CD34+ cells/kg). Patients received CY (3-4 g/m2) followed by GCSF (10 mcg/kg) from day 1 to day 10. Thirteen patients (6 NHL, 4 MM, 2 Hodgkin lymphoma, 1 Ewings sarcoma) received salvage P from 2008–2010. Their outcomes were compared with 10 matched, historic controls mobilized with (CY n=8; CY + etoposide n=1; CY + topotecan n=1) plus G-CSF (10mcg/kg/d) identified from our institutional database. Data was collected on mobilization and transplant outcomes and analyzed utilizing SPSS version 13.0. Patients receiving P were closely matched to historic controls (CY+G). Result: Both groups were similar with regards to age, gender, disease type, prior therapies and performance status (p>0.05 for all). Patients in the P arm received a median of 2.5 doses (range 1–8). The mean CD34+ count was 21.5cells/ul in the P arm and 32.5 cells/ul in the CY+G arm (p=0.2). Similarly, no significant difference was observed in the average number of apheresis sessions in the P vs. CY+G arms (4.2 vs. 4.4, p=0.8) or the total number of CD34+ stem cells collected (4.0×106/kg vs. 3.9×106/kg, p=0.9). However, 7 out of the 13 patients who received P did have an increase of >10 CD34+ cells/ul in their peripheral blood. Utilizing a cut-off of 5×106 CD34+/kg, 3 (23%) patients in the P arm and 3 (30%) patients in the CY+G arm had a successful harvest. Three NHL patients required >4 doses of P, but all eventually collected >2 × 106 CD34+ cells/kg. Neutrophil and platelet engraftment dynamics were similar in both groups of patients. Median time to neutrophil engraftment was 10 days for both groups, p=0.8, and to platelet engraftment was 22 days vs. 20.5 days, p=0.1, respectively for P vs. CY+G. Conclusion: Our limited single-center retrospective case-controlled outcomes data, suggests that when compared with CY+G, the addition of P as a salvage agent does not significantly improve mobilization outcomes. Further evaluation is needed to combine P with CY+G in terms of optimal timing and potentially dosing of chemotherapy agents utilized. We suggest that the combination P+G would provide better potential outcomes such as improved collection and less hospitalization and reduce the use of chemo-mobilization prior to an Autologous Hematopoietic Stem Cell Transplant. Disclosures: No relevant conflicts of interest to declare.

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Mobilization, Harvesting and Selection of Peripheral Blood Stem Cells in Patients with Autoimmune Diseases Undergoing Non-Myeloablative Autologous Hematopoietic Stem Cell Transplantation.

Blood ◽

10.1182/blood.v106.11.1980.1980 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1980-1980

Author(s):

Laisvyde Statkute ◽

Larissa Verda ◽

Yu Oyama ◽

Marcelo Villa ◽

Thomas Shook ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Autoimmune Diseases ◽

Peripheral Blood ◽

Hematopoietic Stem ◽

Positive Correlation ◽

Cd34 Cells ◽

The Mean ◽

Stem Cell Collection ◽

Selection Of

Abstract We have analyzed peripheral blood stem cell (PBSC) mobilization, harvesting and selection properties in 128 patients with severe autoimmune diseases undergoing non-myeloablative autologous hematopoietic stem cell transplantation (HSCT) (50 patients with systemic lupus erythematosus (SLE), 43 - with multiple sclerosis (MS), 15 - with Crohn’s disease (CD), 8 - with scleroderma (Scl), and 12 - with others). Female/male ratio and mean age (range) were 90/38 patients, and 34 (14 to 59) years old, respectively. Mobilization regimen included cyclophosphamide 2g/m2 and G-CSF 10 mcg/kg (except for SLE patients 5 mcg/kg). Forty one patients underwent stem cell collection using Baxter CS300, 78 patients - Spectra, and for 9 patients both apheresis machines were utilized. The mean number of aphereses was 1.8 (range 1–10). Patients with SLE required the largest number of apheresis sessions (mean 2.4), comparing to patients with CD (mean 1.9), Scl (mean 1.4), MS (mean 1.3). Five patients additionally required bone marrow harvest for collection of adequate numbers of stem cells. One patient failed to reach CD34+ cell number of 1.0x106/kg, therefore did not proceed to HSCT. The mean number of CD34+ cells in each apheresis unit was 6.07+−6.96x106/kg (the highest of 9.22+−8.52x106/kg in patients with MS, and the lowest of 3.93+−4.48x106/kg in patients with SLE). Ninety eight patients underwent stem cell selection with CEPRATE SC (N=18), Isolex 300iv1.12 (N=2) or Isolex 300iv2.5 (N=78) stem cell concentrator. The mean purity of selected products was 74.3% (the highest of 81.1% attained in patients with Scl); mean recovery of CD34+cells was 61.2%. T cell reduction by average of 3.7 logs was achieved. The mean number of infused CD34+ cells was 7.24+−5.5x106/kg. The highest mean number of CD34+ cells/kg were infused to patients with MS (9.04+−6.74x106/kg), the lowest - to patients with SLE (5.78+−4.13x106/kg). We found a moderate positive correlation between peripheral blood (PB) CD34+ cells/ul and PB WBC/ul (R=0.34, p<0.05), PB platelets/ul (R=0.51, p<0.05) and a strong positive correlation between PB CD34+ cells/ul and the number of CD34+ cells/kg/apheresis (R=0.67, p<0.05). A weak positive correlation was observed between the number of infused CD34+cells/kg and faster WBC engraftment (ANC>500) and platelet engraftment (platelet count>20K). There was no toxicity observed in our patient population during peri-mobilization period except for 1 patient with SLE who died of disseminated mucormycosis 1 week after stem cell collection. Mobilization and selection of PBSC are safe and efficient in patients with severe autoimmune diseases undergoing non-myeloablative autologous HSCT.

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