Peripheral Blood Stem Cells CD34 Research Articles

No Influence of Peripheral Blood CD34+ and CD3+ Graft Cell Counts on Outcomes after Reduced-Intensity Conditioning Transplantation Using Post-Transplant Cyclophosphamide

IntroductionAlthough associated with a higher incidence of acute GVHD, peripheral blood stem cells (PBSC) are increasingly used for haploidentical allogeneic transplantation with post-transplant cyclophosphamide (PTCY). Data reporting whether or not the composition of the PBSC graft impacts outcomes of patients receiving PTCY are still scarce.Materials and MethodsThis retrospective study included all adults allografted in our department who received a PBSC allotransplant after reduced-intensity conditioning (RIC) with PTCY. Grafts originated from a matched or haploidentical donor and recipients received a Baltimore-based or a Clo-Baltimore (where fludarabine is replaced by clofarabine)-based RIC regimen. All patients received cyclosporine + mycophenolate mofetyl and PTCY as GVHD prophylaxis. CD34+ and CD3+ graft cell contents were considered to study their potential impact on the following outcomes: OS, DFS, GRFS, acute grade 2-4 and 3-4 GHVD and chronic GVHD, early immune reconstitution (IR, lymphocytes and monocytes counts at days+30 and +100 post-transplant, CD4+, CD8+, NK and B cells at day+100 post-transplant).ResultsBetween November 2013 and May 2017, 77 patients met the inclusion criteria. There were 48 males and 29 females with a median age of 58 years (range: 22-71) and a median follow-up for alive patients of 29.2 months (range: 8.4-53.2). Initial diagnoses were mainly acute myeloid (n=21) or lymphoid (n=6) leukemia, lymphoma (n=13), myelodysplastic syndrome (SMD, n=12), myelofibrosis (n=9). Thirty-eight patients were in complete remission at time of transplant (CR1 n=23; CR2 n=12, CR3 n=3) while 22 had active disease and 17 were in partial remission. Donors were sibling in 6 cases, matched unrelated (MUD) in 14, 9/10 mismatched unrelated in 1 and haploidentical in 56. Forty patients received a Baltimore RIC regimen and 37 a Clo-Baltimore RIC regimen. Analyses were performed in July 2018.Median infused CD34+ and CD3+ graft cell counts, based on recipients' weight, were 7.8 106/Kg (range: 1.45-14.24) and 22.23 107/kg (range: 1.95-66.75), respectively. All patients but three engrafted, the latter being 1 patient transplanted with a haplodonor, 1 with a MUD and 1 who died of infection during induction. Two-year OS, DFS and GRFS were 62.6% (52-74), 51.5% (40-63) and 36.6% (27-49), respectively. The incidences of grade 2-4 and 3-4 acute GVHD were 46.7% and 14.2%, respectively, while the incidence of moderate + severe chronic GVHD was 14%. Relapse occurred in 26 patients and non-relapse mortality (NRM) was 15.5%.Baltimore vs Clo-Baltimore patients shared similar median infused CD34+ and CD3+ graft cell counts and outcomes. The same was observed for patients allografted with a haplodonor or not, except for the incidence of grade 2-4 acute GVHD which was significantly higher for the haplo group at 57.1% vs 19% (p=0.006). This difference was first attributed to the higher CD3+ cell content infused in this group (median: 24.05 vs 18.85 107/kg, p=0.04). However, using the median of CD3+ cell graft content as threshold, the incidence of acute grade 2-4 GVHD was not different between low vs high groups when considering haplo patients. This suggests that it was rather the type of donor that did influence acute GVHD occurrence and that PTCY is of particular interest for GVHD prophylaxis when using matched donors.Partitioning the patients according to the median level of CD34+ cells, there was no difference in terms of 2-year OS (57.1% vs 60.7%, p=0.53), DFS (44.5% vs 55.6%, p=0.47) , GRFS (31.7% vs 44.3%, p=0.32), acute grade 2-4 (59% vs 39%, p=0.13) and 3-4 GVHD (17% vs 6.4%, p=0.29), and moderate + severe chronic GVHD (19.5% vs 20%, p=0.57). Similarly, partitioning patients according to median graft CD3+ cell content, outcomes were similar for 2-year OS (64.8% vs 55.8%,p=0.45), DFS (46.3% vs 55.8%, p=0.62), GRFS (36.3% vs 40.4%, p=0.63), acute grade 2-4 (44.7% vs 56.4%, p=0.42) and 3-4 GVHD (10.5% vs 15.3%, p=0.76), and moderate + severe chronic GVHD (14% vs 17.6%, p=0.91). Finally, early IR was not influenced by CD34+ and CD3+ graft cell contents.Conclusion: PBSC CD34+ and CD3+ graft cell contents have no impact on survivals, GVHD incidence nor early IR after a RIC allotransplant using PTCY as GVHD prophylaxis. As a consequence, there is no need to manipulate the graft nor cap the stem cells dose to be infused. These data have to be confirmed prospectively on a larger cohort of patients. DisclosuresGastinne:Millennium/Takeda: Honoraria. Moreau:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Pegylated Filgrastim Versus Filgrastim for Stem Cell Mobilization in Multiple Myeloma After Novel Agent Induction

BackgroundThe current standard of care for transplant-eligible myeloma patients is novel agent-based induction, followed by high-dose chemotherapy and autologous stem cell rescue. Chemo-mobilization of peripheral blood CD34+ stem cells (PBSCs) with pegylated filgrastim (pegfilgrastim), a sustained-duration formulation of filgrastim, has been used as an alternative to filgrastim in several studies involving heterogeneous cohorts of lymphoma and multiple myeloma (MM) patients and shown to be equivalent in PBSC yield and cost-effectiveness. The present study focused on the efficacy of pegfilgrastim in PBSC mobilization compared with filgrastim exclusively after novel agent-based induction in a homogeneous group of MM patients. Patients and MethodsWe analyzed the data from 89 patients with MM treated at 2 transplant centers in Singapore who had received novel agent-based induction chemotherapy, PBSC mobilization with vinorelbine/cyclophosphamide, high-dose melphalan conditioning, and autologous stem cell rescue. Of the 89 patients, 61 were included in the pegfilgrastim group and 28 in the filgrastim group, with a similar median age and disease characteristics. PBSC harvesting was performed at a similar median time of 9.51 ± 0.84 days for both, and the peak peripheral blood CD34+ stem cell count was 19.90 × 106/kg for pegfilgrastim and 32.50 × 106/kg for filgrastim (95% confidence interval, −4.36 to 0.70 × 106/kg). ResultsNo significant difference was found in the median PBSC collection between the 2 groups (pegfilgrastim, 7.90 × 106/kg vs. filgrastim, 10.10 × 106/kg; P = .16). ConclusionThe present study has demonstrated that a single dose of pegfilgrastim is comparable to filgrastim in terms of the timing and efficacy of PBSC harvest and could potentially spare the patient 6 days of filgrastim injections. In addition, ours is the first study to compare these growth factors using vinorelbine/cyclophosphamide as mobilization chemotherapy.

Study on the efficacy of CD34+ cells combined with sarpogrelate hydrochlotride tablets for the reconstruction of the blood supply of thromboangiitis obliterans

Objective To investigate the effect of autologous peripheral blood stem cell transplantation combined with CD34+ cells and oral sarpogrelate hydrochloride for the treatment of vascular reconstruction and blood supply on the thromboangiitis obliterans. Methods Thromboangiitis obliterans (TAO) of 262 patients with 262 lower limbs were divided into stem cells CD34+ cells transplantation combined with sarpogrelate hydrochloride group (group A) with 100 lower limbs, CD34+ stem cell transplantation group (group B) with 91 lower limbs, and sarpogrelate hydrochloride oral group ( group C) with 71 lower limbs. The degree of lower limb local blood flow variability were calculated at the three levels of skin, blood vessels and blood by preoperative and postoperative use of multi-function monitoring instrument, Doppler detector, transcutaneous oxygen pressure monitor, and digital subtraction angiography (DSA), respectively. Results ⑴ The degree of shank, foot cyanosis, cool skin, and pain was relieved significantly in groups A and B than in group C for 1 month after the treatment (P<0.05). ⑵ Intermittent claudication distance, the skin temperature of the lower leg and foot to patients in the groups A and B than in group C, with a significant difference (F=7.01, F=7.04, P<0.05) for 3 months after the treatment. ⑶ Among the patients with amputation, 10 cases were in group A, 16 cases in group B, and 31 cases in group C for 6 months after the treatment. ⑷ Transcutaneous oxygen pressure was increased from (30.43±4.31)mmHg to (37.21±9.01)mmHg (F=5.69, P<0.05), ankle brachial index from (0.32±0.23) to (0.91 ± 0.16) (F=6.71, P<0.05), the volume of blood flow index from the photoelectric (0.22±0.04) to (0.83±0.13 (F=5.69, P<0.05), oxygen saturation from (42.41±9.61)% to (79.61±20.34)% (F=5.74, P<0.05), and DSA score (0.23±0.03) increased to (1.35±0.23) (F=7.14, P<0.05), which was significantly higher than group B and group C (F=7.01, F=7.04, F=7.12, F=7.08, F=7.15, P<0.05) for one year after treatment. Conclusions Treatment of peripheral blood stem cells CD34+ cell transplantation combined with oral sarpogrelate hydrochloride can significantly improve the vascular regeneration and its blood supply in TAO lower extremity limb. Key words: Peripheral blood stem cell transplantation; Antigens, CD34/AD; Platelet aggregation inhibitors/AD; Thromboangiitis obliterans/TH; Blood circulation

Donor Experiences of Second Marrow or Peripheral Blood Stem Cell Collection Mirror the First, but CD34+ Yields Are Less

Little is known about the experiences of individuals donating peripheral blood stem cells (PBSCs) or marrow for a second time. To study this, unrelated donors making a second donation through the National Marrow Donor Program between 2004 and 2013 were evaluated. Experiences of second-time donors giving marrow (n = 118: first donation was PBSC in 76 and marrow in 42) were compared with those making only 1 marrow donation (n = 5829). Experiences of second-time donors giving PBSCs (n = 602) (first donation was PBSCs in 362; marrow in 240) were compared to first-time PBSC donors (n = 16,095). For donors giving a second PBSC or marrow donation there were no significant differences in maximum skeletal pain, maximum symptoms measured by an established modified toxicity criteria, and recovery time compared with those who donated only once. Notably, the yield of marrow nucleated cells and PBSC CD34+ cells with second donations was less. As previously noted with single first-time donations, female (PBSCs and marrow) and obese donors (PBSCs) had higher skeletal pain and/or toxicity with a second donation. PBSC donors who experienced high levels of pain or toxicity with the first donation also experienced high levels of these symptoms with their second donation and slower recovery times. In conclusion, for most donors second donation experiences were similar to first donation experiences, but CD34+ yields were less. Knowledge of the donor's first experience and stem cell yields may help centers decide whether second donations are appropriate and institute measures to improve donor experiences.

Higher dose of CD34+ peripheral blood stem cells is associated with better survival after haploidentical stem cell transplantation in pediatric patients.

Haploidentical stem cell transplantation (SCT) is increasingly used to treat pediatric patients with malignant or nonmalignant hematological disorders. The CD34+ dose of bone marrow or peripheral blood stem cells (PBSCs) has been shown to be an important determinant of the transplant outcome in adults under various preparative regimens. However, knowledge of the effect of the CD34+ dose in pediatric haploidentical SCT is limited. We analyzed the data of 348 pediatric patients (aged 2-18years) with acute or chronic leukemia, myelodysplastic syndrome (MDS), and other hematological disorders that received a transplant between 2002 and 2012. The results of multivariate analysis showed that PBSC CD34+ counts greater than 1.01×106 kg-1 improved platelet engraftment, improved overall survival, and reduced nonrelapse mortality. In contrast, a higher PBSC CD34+ dose did not affect the incidence of acute or chronic graft-versus-host disease, including engraftment syndrome. These data suggest that a PBSC CD34+ dose greater than 1.01×106 kg-1 is optimal for pediatric haploidentical SCT.

Low-Dose Cyclophosphamide Plus Granulocyte Colony-Stimulating Factor As an Efficacious and Safe Peripheral Blood Stem Cell Mobilizing Regimen in Patients with Hematologic Malignancies

▪Introduction:Collection of adequate numbers of Hematopoietic Stem Cells (HSCs) is a prerequisite for proceeding to autologous bone marrow transplant. However, various studies have showed that approximately 5% to 40% of patients do not meet the minimum threshold of 2×106 CD34+ cells/kg that is associated with timely engraftment and better outcomes. There is little consensus over the optimal mobilization regimen for procurement of peripheral blood CD34 + stem cells (PBSCs). Studies comparing mobilization using high-dose cyclophosphamide(HD-CY) {5-7 g/m2} or intermediate dose cyclophosphamide (ID-CY) {3-4g/m2} plus G-CSF with low dose cyclophosphamide (LD-CY) {1-2g/m2) plus G-CSF have reported higher total PBSC yield with the former but at the cost of higher toxicity [Hiwase et al, Cytotherapy 2007]. Hence the significance of our study to evaluate the efficacy and safety of LD-CY plus G-CSF in the era of novel induction therapies, takes precedence in terms of optimal resource utilization.Methods:We retrospectively analyzed mobilization efficacy and need for supportive care in Myeloma and Lymphoma patients that received LD-CY (2g/m2) plus G-CSF (10µg/kg/day) as the preferred mobilization regimen. Patients treated with novel induction regimens only, with or without radiation were included. LD-CY was given on day 1 with MESNA. G-CSF was started Day +5 from LD-CY and continued until the completion of apheresis. We started to measure peripheral CD34+ (pCD34) when patient's white blood cell count recovered to >1000/µL. When the pCD34 count was ≥10/uL, apheresis was started.Results:Out of 21 patients analyzed, 14 (67%) were Myeloma, predominantly IgG subtype (71%) and 7(33%) Lymphoma predominantly Non-Hodgkins type (71%). Our study population comprised of 53% females, 81% Caucasians with median age of 59 years (30-66). 28.5% patients had radiation treatment in the past. Mean/Median lines of chemotherapy previously received were 2/1 (1-5).71% patients received Lenalidomide (57%) / Thalidomide (14%) based regimens for induction therapies. 52% patients had complete response at the time of chemo mobilization.Successful mobilization (defined as total CD34+ cells collected >2x106/kg) was significantly achieved in 95% patients with mean/ median collection of 12.4/11.5 x106 CD34+ cells/kg (20 out of 21 patients, One sample T- test with significance level, alpha =0.025 [one sided ]showed T statistic 5.36 with p-value <0.0001). First apheresis yielded at least 2 x10 6/kg CD34+ cells in 76 % patients with a mean/median of 5.8/3.8 x106/kg CD34+ cells. 66% of patients were found to be good mobilizers, defined as patients collecting ≥5 x106 CD34+ cells/kg in ≤2 days. 81% patients collected ≥5x106 CD34+ cells/kg and 57 % patients collected ≥10 x106 CD34+ cells/kg. Mean/Median Peak pCD34count was 97.7/60 cells/µL(2-488). Median number of aphereses was 2 (mean 2.6) with the mean and median time to apheresis being 11 days (9-15). None of the patients needed inpatient hospitalization or intravenous antibiotics during mobilization or had any complication related to immunosuppression. 14% and 19% of patients needed packed red blood cell and platelet transfusions respectively during the mobilization period. One Myeloma patient (4.8%) that failed mobilization had previously received five lines of chemotherapy including lenalidomide/thalidomide containing regimens and had stable disease at the time of mobilization. Interestingly, 61% of myeloma patients that successfully mobilized had received lenalidomide containing induction regimen (mean/median no. of cycles- 3.9/4[1-7])Conclusion:Our analysis shows that LD-CY plus G-CSF is efficacious to mobilize sufficient number of stem cells (>2x106 CD34+ cells /kg) in Myeloma and Lymphoma patients treated with novel agents, including the challenging sub-group of lenalidomide treated myeloma patients, with no infectious complications or morbidity noted during the entire period of mobilization. Therefore, we believe LD-CY plus G-CSF may overcome the negative effects of prior lenalidomide exposure on PBSC mobilization [Mark et al, Biol Blood Marrow Transplant 2008] and is superior in terms of optimal resource utilization compared to novel plerixafor based regimens [Chaudhary et al, J Clin Apher 2013]. Prospective studies involving CY based regimens will help elucidate the optimal dose of chemo-mobilization. DisclosuresNo relevant conflicts of interest to declare.

Correlation Between Actual Stem Cells Collected By Apheresis and Predicted Stem Cells Based on Pre-Apheresis Peripheral Blood CD34+ Cell Counts

Introduction. Stem cell (SC) mobilization and collection from peripheral blood (PB) is a broadly used strategy in the setting of autologous stem cell transplantation (ASCT). An adequate correlation between CD34+ cell count in PB and CD34+ cell count in apheresis product is essential for the planning of the mobilization-apheresis process. Prediction of SC yield in the basis of peripheral blood stem cell (PBSC) number and blood volume processed (BVP) was proposed by Pierelli et al (Vox Sanguinis (2006)91:126-34).Objective. Evaluation of correlation between pre-apheresis PBSC count and CD34+ cell dose collected in the first apheresis procedure. Evaluation of correlation between predicted CD34+ yield by Pierelli's method and actual CD34+ yield collected in the first apheresis procedure.Patients and Methods. Eighty-four apheresis procedures performed to 79 patients in a single institution between 2008 and 2015 were retrospectively evaluated. Data analyzed included age, gender, body weight, diagnosis, date of first apheresis, pre-apheresis PB CD34+ counts, CD34+ cell dose collected in the first apheresis product and total BVP. Only patients in whom pre-apheresis PBSC count was available were included in this analysis. Median age was 52 years (16-66), 30 (38%) were females. Median body weight was 71 kg (47 - 120). Diagnosis was non-Hodgkin lymphoma (48%), multiple myeloma (24%), Hodgkin lymphoma (22%), acute myeloblastic leukemia (4%), amyloidosis (1%) and solid tumor (1%). Using the method described by Pierelli et al, predicted CD34+ yields were calculated (predicted CD34+ cells/kg= PB CD34+ cells/mL x 0.4 x BVP per kg (mL/kg). Correlation between variables was analyzed by Pearson's coefficient (r). An r value close to 1 indicates significant correlation between variables. An r value close to 0 indicates no statistical correlation. Predicted yields were classified in low (lower than 2 x 106 CD34+ cells / kg) (n=34), intermediate (2 to 5) (n=28) or high (higher than 5) (n=22), according with the conventional limits for SC dose in the setting of ASCT. Positive predictive value (PPV) of predicted yields was calculated as the proportion of actual yields falling into each category of predicted yields.Results. Median pre-apheresis PBSC count was 25 CD34+ cells / uL (5-310). Median BVP was 240 mL/kg (143-360). Median predicted CD34+ yield was 2.1x106 CD34+ cells/kg (0.5-29.41) and median actual CD34+ yield was 2.84 x106 CD34+ cells/kg (0.26-38.21). Pearson's correlation coefficient (r) between pre-apheresis PBSC and collected CD34+ cells was 0.842 (p=0.000). Pearson's correlation coefficient (r) between predicted and actual CD34+ was 0.836 (p=0.000). Median ratio actual CD34+ cells / predicted CD34+ cells was 1.10 (0.31-4.55). PPV of predicted yields higher than 5 x 106 CD34+ cells /kg was high (95%). Only 18% of cases with a predicted yield between 2 and 5 x 106 CD34+/kg had an actual CD34+ yield lower than 2 x 106 CD34+/kg.Conclusion. The results show a significant correlation between PBSC count and collected SC during the first apheresis procedure. A significant correlation between predicted and actual CD34+ yields reproduce what has been shown by others regarding the predictive power of this formula. The PPV of this method is higher for predicted CD34+ yields higher than 5 x 106 CD34+ cells/kg. DisclosuresNo relevant conflicts of interest to declare.

PBSC mobilization in lymphoma patients: analysis of risk factors for collection failure and development of a predictive score based on the kinetics of circulating CD34+ cells and WBC after chemotherapy and G‐CSF mobilization

Autologous stem cell transplantation (ASCT) is a potentially curative treatment of lymphoma, but peripheral blood stem cell (PBSC) mobilization fails in some patients. PBSC mobilizing agents have recently been proved to improve the PBSC yield after a prior mobilization failure. Predictive parameters of mobilization failure allowing for a preemptive, more cost-effective use of such agents during the first mobilization attempt are still poorly defined, particularly during mobilization with chemotherapy + granulocyte colony-stimulating factor (G-CSF). We performed a retrospective analysis of a series of lymphoma patients who were candidates for ASCT, to identify factors influencing PBSC mobilization outcome. Premobilization parameters-age, histology, disease status, mobilizing protocol, and previous treatments-as well as white blood cell (WBC) and PBSC kinetics, markers potentially able to predict failure during the ongoing mobilization attempt, were analyzed in 415 consecutive mobilization procedures in 388 patients. We used chemotherapy + G-CSF in 411 (99%) of mobilization attempts and PBSC collection failed (<2 × 10(6) CD34+ PBSC/kg) in 13%. Multivariable analysis showed that only a low CD34+ PBSC count and CD34+ PBSC/WBC ratio, together with the use of nonplatinum-containing chemotherapy, independently predicted mobilization failure. Using these three parameters, we established a scoring system to predict risk of failure during mobilization ranging from 2 to 90%, thus allowing a selective use of a preemptive mobilization policy.

Seizures During Infusion of Autologous Peripheral Blood Stem Cells Are Related to High White Blood Cell Concentration in the Peripheral Blood Stem Cell Product and Can Be Prevented by Dilution of Product with Autologous Plasma

Abstract 4386Seizures are rare during infusion of autologous peripheral blood stem cells (PBSC). We retrospectively analyzed 159 adult patients (pts.) collected consecutively between January 2006 and July 2009. Pts. were collected on either COBE Spectra (COBE) (n=85) or Fresenius AS 104 (Fresenius) (n=74) cell separators and mobilized with granulocyte colony stimulating factor (G-CSF) alone (n=47), G-CSF and Plerixafor (n=26), or G-CSF and chemotherapy (n=66). Pts. characteristics did not differ between the COBE and Fresenius cohorts, but there were differences in PBSC product (Table). Pts. collected with COBE had higher white blood cell (WBC) and total nucleated count (TNC) but lower mononuclear cell (MNC) percentage and cell viability than pts. collected with the Fresenius. Absolute CD34+ cells in the PBSC product, CD34+ cells / kg and total CD34+ cells / kg infused at transplant were not significantly different. CD34+ yields (calculated as the ratio of CD34+ cells /μl of the PBSC product to the patient’s peripheral blood CD34+ cells / μl taken on the day of collection) were significantly higher on the COBE than Fresenius. No serious adverse events occurred during PBSC infusion except 3 of 159 pts. developed seizures during infusion of PBSC; all collected on the COBE and all three had product WBC > 590 × 103/μl (compared to a median of 163.3 × 103/ μl for all other products)(Figure). Evaluation of pts. did not identify abnormalities in imaging studies, cerebrospinal fluid analysis, electrolytes, or past history which might explain etiology of seizures. No significant difference in WBC or platelet engraftment was observed in pts. collected with COBE or Fresenius.We then prospectively correlated WBC counts midway and at the end of PBSC collections. Fourteen pts. had 15 apheresis using the Fresenius. Mid- and post-WBC concentrations were 64 +/− 23 × 103/μl and 69 +/− 20 × 103/μl, respectively. Fifty-one pts. had 66 apheresis using COBE, with WBC counts obtained midway and at the end of collection of 287 +/− 150 × 103/μl and 273 +/− 144 × 103/μl, respectively. Mid-WBC accurately correlated with WBC at the end of the collection in both the COBE and Fresenius cohorts (r2 = 0.940 and r2 = 0.904, respectively). Using this information, we prospectively evaluated 65 pts. who underwent 80 PBSC collections in anticipation of an autologous (n=44) or allogeneic (n=7) stem cell transplant between June 2009 and January 2010. Collections for these pts. were performed using the COBE (n=66) or the Fresenius (n=15). Mid-WBC were obtained and products with mid-collection WBC concentration > 450 × 103/uL (n=29) had additional autologous plasma collected at the time of collection for final product dilution to < 450 × 103/uL prior to cryopreservation. Pts weight, volume of PBSC product and CD34+ cells/kg infused did not differ between the pts who received diluted PBSC product and those who did not. There were also no differences in either ANC (12 ± 1.3 days vs. 11.5 ± 1.3 days, dilution vs. non-dilution, p = 0.760) or in platelet engraftment (18 ± 3.7 days vs. 16 ± 2.7 days, dilution vs. non-dilution, p = 0.561). No serious adverse infusion effects were observed in either group. In conclusion, high number of WBC in COBE collections is a possible cause of PBSC infusion related seizures. No seizures were observed after dilution of PBSC with high WBC concentration.TIENT AND PRODUCT CHARACTERISTICSCOBE (±SD)Fresenius (±SD)Number of Products165180Number of Patients8574Age at collection56 ± 1456 ± 15Weight at Collection (kg)82.7 ± 17.979.5 ± 15.9Collections / Patient2 ± 12 ± 1Blood Volume Processed at end of Collection (L)18.0 ± 2.418.1 ± 2.7(*)Product Volume (ml)241 ± 56.8402 ± 72.0Peripheral WBC (103/ μl)36.6 ± 18.933.3 ± 24.5(*)Product WBC(103/ μl)163.3 ± 136.055.8 ± 29.3(*)TNC (1010)3.51 ± 1.861.95 ± 1.19(*)MNC (1010)2.36 ± 1.191.60 ± 0.09(*)MNC (%)75.0 ± 23.385.0 ± 10.8Volume prior to freezing(ml)100 ± 54100 ± 32(*)Post Freeze Viability (%)70 ± 1475 ± 10Peripheral CD34+/ μl24.0 ± 43.825.3 ± 79.1(*)Product CD34+/μl726.7 ± 1325.9264.63 ± 781.0(*)Product / Peripheral CD34+24.87 ± 10.9010.91 ± 6.64Absolute Product CD34+ cells (108)1.77 ± 3.521.14 ± 3.35Product CD34+/kg (106)2.02 ± 4.671.39 ± 4.15Total CD34+ cells infused (106 / kg)3.85 ± 3.203.85 ± 2.24(*) = p values < 0.05 [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Purified T-depleted, CD34+ peripheral blood and bone marrow cell transplantation from haploidentical mother to child with thalassemia

AbstractFetomaternal microchimerism suggests immunological tolerance between mother and fetus. Thus, we performed primary hematopoietic stem cell transplantation from a mismatched mother to thalassemic patient without an human leukocyte antigen–identical donor. Twenty-two patients with thalassemia major were conditioned with 60 mg/kg hydroxyurea and 3 mg/kg azathioprine from day −59 to −11; 30 mg/m2 fludarabine from day −17 to −11; 14 mg/kg busulfan starting on day −10; and 200 mg/kg cyclophosphamide, 10 mg/kg thiotepa, and 12.5 mg/kg antithymocyte globulin daily from day −5 to −2. Fourteen patients received CD34+-mobilized peripheral blood and bone marrow progenitor cells; 8 patients received marrow graft–selected peripheral blood stem cells CD34+ and bone marrow CD3/CD19-depleted cells. T-cell dose was adjusted to 2 × 105/kg by fresh marrow cell addback at the time of transplantation. Both groups received cyclosporine for graft-versus-host disease prophylaxis for 2 months after transplantation. Two patients died (cerebral Epstein-Barr virus lymphoma or cytomegalovirus pneumonia), 6 patients reject their grafts, and 14 showed full chimerism with functioning grafts at a median follow-up of 40 months. None of the 14 patients who showed full chimerism developed acute or chronic graft-versus-host disease. These results suggest that maternal haploidentical hematopoietic stem cell transplantation is feasible in patients with thalassemia who lack a matched related donor.

Peripheral blood stem cell support for multiple cycles of dose intensive induction therapy is feasible with little risk of tumor contamination in advanced stage neuroblastoma: A report from the Childrens Oncology Group

Poor outcome in Stage 4 neuroblastoma may be improved with increased dose intensity of therapy. We investigated the feasibility of sequential collection and infusion of peripheral blood stem cells (PBSCs) as hematopoietic support for non-myeloablative dose intensive induction chemotherapy given every 21-28 days. Twenty-two children with Stage 4 neuroblastoma (>or=1 year of age) received two cycles of high-dose cyclophosphamide (4 g/m(2)), doxorubicin (75 mg/m(2)), and vincristine (2 mg/m(2)) followed by three cycles of interpatient dose escalating carboplatin (Dose Level 0 = 800 mg/m(2); Dose Level 1 = 1,000 mg/m(2)), high-dose cyclophosphamide (4 g/m(2)), and etoposide (600 mg/m(2)). PBSC were harvested following cycle 2, 3, and 4 in Cohort 1 and infused after each subsequent cycle. In Cohort 2, PBSC were harvested after cycle 2 and split into three aliquots for infusion. Dose limiting toxicity (DLT) and ability to administer cycles within 28 days was assessed. Sufficient PBSC (>or=2 x 10(6) CD34 cells/kg per infusion) were collected from 17/21 eligible patients with minimal toxicity and no detectable neuroblastoma cells by immunocytology. Carboplatin at 1000 mg/m(2) resulted in DLT of delayed platelet recovery >28 days in 4/8 patients. Despite de-escalation to 800 mg/m(2), platelet DLT occurred in 4/7 Cohort 1 and 3/7 Cohort 2 patients. As defined in this protocol, doses of carboplatin were not tolerable with the PBSC dose administered. However, it was feasible to collect sufficient PBSC from small neuroblastoma patients to use as hematopoietic support with minimal risk of tumor contamination and toxicity.

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.

Harvesting of Peripheral Blood Stem Cells Using Plerixafor (MOZOBIL) and Granulocyte-Colony Stimulating Factor in Patients Who Had Never Undergone Stem Cell Collection.

Abstract 4236 IntroductionThe collection of peripheral blood stem cells (PBSC) can be tedious and expensive. Moreover, PBSC yields can be severely compromised in patients who previously received chemotherapy. The use of plerixafor (MOZOBIL) for PBSC mobilization has been shown to significantly improve CD34 stem cell yields in patients whom PBSC collections have been adequate. However, this requires the patient to undergo further PBSC harvesting sessions; adding to the patient's inconvenience, exposure to adverse events and costs. We therefore investigated whether using plerixafor at the outset of PBSC mobilization would improve patient acceptability; reduce PBSC collection days and accordingly the patient's costs. Patients and MethodsThree adult patients who had never undergone PBSC harvesting were mobilized using subcutaneous (SC) granulocyte-colony stimulating factor (G-CSF) 10 μg/kg daily starting 4 days prior to PBSC harvesting; plus SC plerixafor 240 μg/kg 10 hours prior to PBSC harvesting. Both G-CSF and plerixafor were continued for as long as was required for PBSC collections. Two patients had multiple myeloma (MM) and were both in stringent complete remission (sCR). The third patient suffered from adult-onset spinomuscular atrophy (SMA) and stem cells were required for possible re-infusion therapy. Importantly, the bone marrows of all 3 patients were assessed to be normal prior to PBSC mobilization and collection. The target PBSC CD34 yield was 2 million CD34 cells/kg per PBSC collection. ResultsAs can be seen, the CD34 yields (expressed as x million CD34 cells/kg) exceeded expectations:PatientDiagnosisCell SeparatorDay 1Day 2Day 3Patient #1MMCOBE Spectra2.794.354.21Patient #2MMCOBE Spectra3.433.12not donePatient #3SMASPECTRA Optia8.105.565.50All patients tolerated the procedure very well and were discharged within 24 hrs of completing the final PBSC collection. The only adverse event was grade 1 restless legs syndrome in Patient #1 following the first injection of plerixafor. It was improved with oral diazepam and did not recur. Both patients with MM reported a sense of “rejuvenation” 2-3 weeks after PBSC collection. All patients were pleased with the relatively complication-free procedure. ConclusionsUpfront collection of PBSCs using G-CSF/plerixafor is both highly effective and safe. There is great patient acceptability as well as much fewer PBSC collection days and an accompanying reduction in procedure costs. The added advantage of using the SPECTRA Optia cell separator is worth further investigations. Disclosures:Off Label Use: Plerixafor (MOZOBIL) - Use in patients who had never undergone stem cell collection..

Intentional induction of mixed haematopoietic chimerism as platform for cellular therapy after HLA-matched allogeneic stem cell transplantation in childhood leukaemia patients

Allogeneic peripheral blood stem cell CD34(+)-selected transplantation followed by donor lymphocyte infusion (DLI) to maximize graft-versus-leukaemia effect while avoiding graft-versus-host disease was investigated in 22 paediatric patients with acute myeloid leukaemia (n = 10) or acute lymphoblastic leukaemia (n = 12). Patients were grafted with a median (range) 6 x 10(6) (2-31 x 10(6))/kg CD34(+) cells and 1.1 x 10(4) (0.2-3.9 x 10(4)) CD3(+) cells. Seventy-five DLI were performed with no complications. Median time (range) to neutrophil and platelet engraftment was 13 (11-15) and 12 d (8-13) respectively. Probability of relapse and disease-free survival was 23 +/- 9% and 72 +/- 6% respectively (median follow-up of 15 months).

Optimal ex vivo expansion of neutrophils from PBSC CD34+ cells by a combination of SCF, Flt3-L and G-CSF and its inhibition by further addition of TPO

BackgroundAutologous mobilised peripheral blood stem cell (PBSC) transplantation is now a standard approach in the treatment of haematological diseases to reconstitute haematopoiesis following myeloablative chemotherapy. However, there remains a period of severe neutropenia and thrombocytopenia before haematopoietic reconstitution is achieved. Ex vivo expanded PBSC have been employed as an adjunct to unmanipulated HSC transplantation, but have tended to be produced using complex cytokine mixtures aimed at multilineage (neutrophil and megakaryocyte) progenitor expansion. These have been reported to reduce or abrogate neutropenia but have little major effect on thrombocytopenia. Selective megakaryocyte expansion has been to date ineffective in reducing thrombocytopenia. This study was implemented to evaluate neutrophil specific rather than multilineage ex vivo expansion of PBSC for specifically focusing on reduction or abrogation of neutropenia.MethodsCD34+ cells (PBSC) were enriched from peripheral blood mononuclear cells following G-CSF-mobilisation and cultured with different permutations of cytokines to determine optimal cytokine combinations and doses for expansion and functional differentiation and maturation of neutrophils and their progenitors. Results were assessed by cell number, morphology, phenotype and function.ResultsA simple cytokine combination, SCF + Flt3-L + G-CSF, synergised to optimally expand and mature neutrophil progenitors assessed by cell number, phenotype, morphology and function (superoxide respiratory burst measured by chemiluminescence). G-CSF appears mandatory for functional maturation. Addition of other commonly employed cytokines, IL-3 and IL-6, had no demonstrable additive effect on numbers or function compared to this optimal combination. Addition of TPO, commonly included in multilineage progenitor expansion for development of megakaryocytes, reduced the maturation of neutrophil progenitors as assessed by number, morphology and function (respiratory burst activity).ConclusionGiven that platelet transfusion support is available for autologous PBSC transplantation but granulocyte transfusion is generally lacking, and that multilineage expanded PBSC do not reduce thrombocytopenia, we suggest that instead of multilineage expansion selective neutrophil expansion based on this relatively simple cytokine combination might be prioritized for development for clinical use as an adjunct to unmanipulated PBSC transplantation to reduce or abrogate post-transplant neutropenia.

Mobilization of peripheral blood CD34 stem cells in a heavily pre-treated pediatric medulloblastoma patient using AMD3100 and G-CSF

Mobilization of peripheral blood CD34 stem cells in a heavily pre-treated pediatric medulloblastoma patient using AMD3100 and G-CSF

Peripheral blood stem cell CD34+ autologous transplant in relapsed follicular lymphoma.

To evaluate CD34+ selection of peripheral blood stem cells (PBSC) as a graft for autologous transplantation. Eight relapsing follicular lymphoma (FL) patients were submitted to CD34+ autologous stem cell transplantation (ASCT). All patients received at least two front line conventional therapies; mean time to treatment failure (TTF) was 4.5 months. Patients had disseminated stage III-IV disease after a median number of 2.1 relapses. Chemotherapy and G-CSF were used as mobilization for leukapheresis. CEPRATE SC concentrator (CellPro, Inc, Bothell, WA) was used to select CD34+ cells from leukapheresis products. With a mean of 1.8 leukaphereses per patient, 8.1 x 10(8) mononuclear cells (MNCs)/kg were collected. After the selection process, the median number of MNCs was 9.4 x 10(6)/kg; 4.3 x 10(6)/kg CD34+ cells and 17 x 10(4)/kg CFU-GM, with a purity of 83.7% and a viability of 89.2%. Mbr bcl2/IgH PCR analysis of 5 grafts showed that initial buffy-coat, and CD34- fractions were negative in 3 cases and positive in 2 cases (from whom selected CD34+ fraction remained positive in 1 case). After a conditioning regimen including total body irradiation, cyclophosphamide and etoposide, CD34+ selected cells were reinfused. All patients but one had successful engraftment, median time to WBC > 1 x 10(9)/l was 12 days and platelets > 50 x 10(9)/l 17 days. No severe infectious complications were seen. After transplant, with a minimum follow up of 2 years, 5 patients are still in complete remission (CR). Three patients have relapsed after 1 year of transplant with a mean TTF of 15.6 months. We conclude that PBSC CD34+ selection for ASCT was a safe technique, capable of reconstituting hemopoiesis without severe complications for high risk FL patients included in this study. The effects of tumor cell purging need to be evaluated in a larger series.