Mononuclear Cell Collection Research Articles

Exenatide and Saxagliptin Do Not Attenuate NFkB-Induced Inflammation in Prediabetic Humans

Incretin mimetic medications acutely downregulate inflammation via the NfkB pathway during the fasting state in humans with type 2 diabetes mellitus. Free fatty acids (FFAs) activate TLR4, stimulating NFkB and the production of IL-6, which downregulates insulin signaling via SOCS3. We investigated this pathway in the acute postprandial state—utilizing a high-fat meal and incretin mimetic therapies—in humans with prediabetes. Sixteen humans with prediabetes (age 50 ± 2 years, BMI 32.5 ± 0.4 kg/m2, HbA1c 5.96 ± 0.05%) participated in a randomized, crossover, double-blinded trial comparing exenatide, saxagliptin, and placebo. Blood was drawn at baseline (fasting) and 2 hours for the collection of peripheral blood mononuclear cells (PBMCs) and plasma. After baseline blood draw, subjects were given study medication followed by high-fat test meal. Procedures were repeated for all study arms. Subsequently, seven of these subjects (age 51 ± 2 years, BMI 33.3 ± 0.6 kg/m2, HbA1c 5.86 ± 0.17%) participated in an open-label extension study after the administration of exenatide extended-release (EXR) for six weeks. The above procedures were repeated. In the randomized trial, plasma FFA decreased in all groups (p<0.05), though the magnitude of decrease was smaller for exenatide. Plasma IL6 levels increased in all groups except the saxagliptin (p<0.05). Western blots of PBMCs showed no significant changes in levels of NFkB, TLR4, or SOCS3. Plasma insulin did not change in the exenatide group but increased in other groups. In the extension study with EXR, plasma FFA decreased between 0 and 2 hours (p<0.05), and to a greater extent than the prior exenatide group. Plasma IL6 levels decreased (p<0.05) and insulin levels increased (p<0.05). Western blots showed no significant changes in levels of NFkB, TLR4, or SOCS3. These data show that six weeks of EXR significant reduced postprandial FFA levels and IL-6 without any noted changes in NFkB pathway between 0 and 2 hours. Disclosure A.D. Gutierrez: Speaker's Bureau; Self; AstraZeneca, Med Learning Group. K. Bermudez: None. V. Hamidi: None. K. Riggs: None. S. Coverdale: None. A. Dursteler: None. N.K. Kumar: None. M. Ruscheinsky: None.

Evaluation of the new continuous mononuclear cell collection protocol versus an older version on two different apheresis machines.

Cell separators are routinely used to collect CD34+ blood stem cells in the context of customized stem cell transplantation procedures. The Spectra Optia (Terumo BCT) is a novel development of the precursor instrument, the Cobe Spectra (Terumo BCT). In this report, 146 autologous and 42 allogeneic donors undergoing apheresis on the Cobe Spectra using the mononuclear cell (MNC) program 4.7 or on the Spectra Optia using the new continuous mononuclear cell (cMNC) program 11.2 are compared. Viability of cells and collection efficacy within the apheresis products was comparable for autologous and allogeneic products collected with the MNC or cMNC method. However, we found a reduced duration of the apheresis procedure and lower hematocrit within the apheresis products when using the cMNC in autologous and allogeneic donors. Moreover, allogeneic donors collected substantially more CD34+ cells per kilogram of body weight when using the cMNC method. Differences in platelets before and after apheresis were substantially smaller in this cohort when compared to the cohort collected with the MNC method. Neutrophil and platelet engraftment after autologous or allogeneic transplantation with a product collected with the MNC procedure was comparable to a transplantation with a product processed according to the cMNC method. Comparison of the MNC (Cobe Spectra) and the cMNC (Spectra Optia) methods demonstrated an equal performance and outcome. However, advantages were present using the cMNC method with respect to apheresis duration and hematocrit within the apheresis product (autologous/allogeneic donors) and numbers of CD34+ cells collected, especially in allogeneic donors.

199 - An automated method of enrichment of lymphocytes from apheresis products for cellular engineering

Adoptive Cell Therapy (ACT) using lymphocytes and/or monocytes from apheresis collections has had a profound effect on cancer treatment. The success of T lymphocyte cell engineering protocols for chimeric antigen receptor (CAR) gene expression is dependent upon the quality and purity of the starting material. Contamination with platelets, red blood cells (RBC) and other cell types has been determined to adversely affect genetic modifications. We demonstrate an automated method, using a functionally-closed system and GMP-grade Ficoll Hypaque, of mononuclear cell preparation that is highly enriched in lymphocytes. Apheresis products were collected using the Spectra Optia® Apheresis System with continuous-flow mononuclear cell collection (CMNC) from sixteen individuals (9 healthy donors and 7 multiple myeloma patients). All products were subjected to ficoll separation using the Haemonetics® Cell Saver® 5+ Blood Recovery System. We developed a protocol using a mathematical algorithm for collection of the relevant cell fraction(s) in order to standardize the process to achieve consistent results among trained technical staff and to compensate for donor-to-donor variability associated with the starting material. Table I demonstrates exceptional recovery post-ficoll treatment of the apheresis product of WBCs (82%), lymphocytes (85%) and monocytes (87%) with a significant reduction of neutrophils (44%) and platelets (47%). Importantly, the final cell product was essentially devoid of RBC (HCT <2%) and neutrophils (3%) and highly enriched in lymphocytes (73%). No significant differences were observed in procedures from healthy normal donors versus patients, with the exception of the lymphocyte composition of the final product which was 78 ± 8% vs 67 ± 12% (p= 0.05), respectively. We have utilized this protocol to achieve superior results compared to historic methodologies for preparation of apheresis products for cell engineering of CAR-T cells, dendritic cell vaccines and purified T regulatory cells. Given the high expense of manufacturing ACT products, addition of an extra step of ficoll separation prior to further cell engineering, using an automated GMP-compliant method, greatly enhances the probability of minimizing subsequent manufacture failures due to unwanted cell contamination. Table ICell recovery and composition following automated ficoll separation of apheresis products with Cell Saver®5+. N = 16 Post Ficoll Cell Recovery (%) WBCs Neutrophils Lymphocytes Monocytes Platelets Average ± SD 82 ± 9 56 ± 30 85 ± 8 87 ± 14 53 ± 42 Cell Composition Final Product (%) HCT Neutrophils Lymphocytes Monocytes MNCs Average ± SD 1.3 ± 0.7 3.0 ± 3 73 ± 11 24 ± 10 96 ± 3 Open table in a new tab

Evaluation of a new continuous mononuclear cell collection procedure in a single transplant center cohort enriched for AL amyloidosis patients

BackgroundThe Spectra Optia continuous mononuclear cell (CMNC) program is newly available, and herein validated in a single-center cohort enriched with AL amyloidosis patients to collect a target CD34+ yield of 2.5 × 106 cells/kg within 2 days. MethodsConsecutive autologous transplant patients in 2016 are included. Patients undergo leukapheresis with Optia CMNC and Spectra v4.7 over a 2-day cycle. Data collection includes collection efficiency, adverse events and engraftment kinetics. Results36 leukapheresis procedures on 18 patients are included. The diagnoses are AL amyloidosis (9), myeloma (7), lymphoma (2), and scleroderma (1). Median age is 60; 12 are men. Plerixafor was employed pre-emptively in 6 cycles. Median blood CD34+ on Day 1 of leukapheresis was 46 cells/uL. Median number of blood volumes processed on Day 1 was 3.1. All collection cycles were completed within 2 days; only one in a heavily pretreated lymphoma patient did not reach the target requiring a second mobilization attempt. Mean collection efficiencies were comparable between the two devices. There were 2 adverse events: tubing rupture on the Optia; and one case of hypotension. All 18 patients underwent high-dose chemotherapy: median cell dose infused was 7.7 × 106 CD34+ cells/kg. Median days to neutrophil and platelet engraftment were 10 and 13 respectively. ConclusionThe Optia CMNC collection protocol is safe and effective in a small single-center autologous stem cell transplant cohort enriched for high-risk patients with AL amyloidosis and cardiac involvement. Caution is needed for tubing setup because there is less cumulative experience with Optia.

Continuous Mononuclear Cell Collection (cMNC) protocol impact on hematopoietic stem cell collections in donors with negative collection predictors

BackgroundRecently, novel protocol utilizing Continuous Mononuclear Cell Collection (cMNC) have been introduced for leukapheresis. We compared the efficacy of cMNC with an older protocol – mononuclear cell collection (MNC) for CD34+ cell collection in unrelated donors with negative stem cell collection predictors. Material and methodsRetrospective data from a series of 258 consecutive unrelated hematopoietic stem cell donors was included in this single-center study (80 donors collected with cMNC and 178 with MNC). The donors with poor predictors for collection such as low number of circulating CD34+ cells and/or weight disproportion were assigned to the cMNC arm. ResultsThe cMNC protocol yielded a higher number of CD34 + cells per donor body weight (7.63 × 106/kg vs 6.82 × 106/kg, p = 0.027). One apheresis was sufficient for collection of target cell number in 89% individuals from both groups despite negative predictors in the cMNC group. In donors with CD34 + cell count <100/μL and a body weight disproportion between donor and recipient one apheresis was sufficient in 83% of donors in cMNC group and in 58% in MNC group (p = 0.0345) with collection efficiency CE2% values of 61% for cMNC and 62% for MNC (p = 0.77). ConclusioncMNC protocol is more efficient in donors with low pre-apheresis CD34+ cell count and weight disproportion between donor and recipient. This suggests that the use of cMNC in unrelated donors could possibly further improve the results of HSC collections.

Lymphocyte apheresis for chimeric antigen receptor T-cell manufacturing in children and young adults with leukemia and neuroblastoma.

The first step in the production of chimeric antigen receptor T cells is the collection of autologous T cells using apheresis technology. The procedure is technically challenging, because patients often have low leukocyte counts and are heavily pretreated with multiple lines of chemotherapy, marrow transplantation, and/or radiotherapy. Here, we report our experience of collecting T lymphocytes for chimeric antigen receptor T-cell manufacturing in pediatric and young adult patients with leukemia, non-Hodgkin lymphoma, or neuroblastoma. Apheresis procedures were performed on a COBE Spectra machine using the mononuclear cell program, with a collection target of 1 × 109 total mononuclear cells per kilogram. Data were collected regarding preapheresis and postapheresis blood counts, apheresis parameters, products, and adverse events. Ninety-nine patients (ages 1.3-25.7 years) and 102 apheresis events were available for analysis. Patients underwent apheresis at a variety of absolute lymphocyte cell counts, with a median absolute lymphocyte count of 944 cells/μL (range, 142-6944 cells/μL). Twenty-two patients (21.6%) had absolute lymphocyte counts less than 500 cells/μL. The mononuclear cell target was obtained in 100% of all apheresis harvests, and chimeric antigen receptor T-cell production was possible from the majority of collections (94%). Mononuclear cell collection efficiency was 65.4%, and T-lymphocyte collection efficiency was 83.4%. Ten patients (9.8%) presented with minor adverse events during the 102 apheresis procedures, with one exception of a severe allergy. Mononuclear cell apheresis for chimeric antigen receptor T-cell therapy is well tolerated and safe, and it is possible to obtain an adequate quantity of CD3+ lymphocytes for chimeric antigen receptor T-cell manufacturing in heavily pretreated patients who have low lymphocyte counts.

Low-Volume Leukapheresis in Non-Cytokine-Stimulated Donors for the Collection of Mononuclear Cells

Background: There is an increasing demand for products containing mononuclear cells (MNCs) for cellular immune therapy. Hence, leukapheresis is increasingly performed in healthy volunteer donors. Methods: We evaluated 147 low-volume leukapheresis procedures from 77 healthy non-cytokine-stimulated donors. Complete blood counts (CBCs) of the donors were measured before and directly after the procedures as well as from the MNC products. Follow-up CBCs were collected from donors within 21 days. Results: The product hematocrit within a range from 1.2 to 6.0% did not correlate with the collection efficiency of any cell population or the granulocyte and platelet yield. There was a strong correlation between the CBC values before leukapheresis and the cell yield of lymphocytes and monocytes as well as a perfect negative correlation between cell recruitment and cell loss in all cell populations. Furthermore, we observed a significant decrease in the CBC values in all cell populations directly after leukapheresis, which recovered within a mean of 16.1 days (SD ± 2.1 days) and even showed a significant increase in granulocytes and platelets. Conclusion: Low-volume leukapheresis is feasible for the collection of MNCs in which the product hematocrit is negligible for the collection efficiency, cell yield, or contamination of residual cells under operational settings recommended by the manufacturer. Our data suggests that cell recruitment is regulated by the number of cells removed, which may also be the stimulus to induce granulo- and thrombopoiesis within the first days after leukapheresis.

161 - Safety, clinical efficancy, and cost-effectiveness of mononuclear cell collections for autologous immunotherapies: First experience from a private cell collection facility

Background & Aim Within the EU the collection of mononuclear cells (MNC) as starting source for the manufacturing of autologous cell therapies are mainly performed in hospitals or hospital-associated apheresis centers. We report about the challenges to perform the leukapheresis procedure (LA) at a private held medical practice, with specific emphases on safety, cell collection efficiency, and cost-effectiveness. Methods, Results & Conclusion We reviewed the records of altogether 60 outpatients and 100 healthy donors who underwent a total of 220 unstimulated LA procedure at Cyto-Care, a private held medical practice/ certified cell collection facility located in Vienna, Austria. All patients participated in various industry-sponsored clinical P I-III trials; the study sponsors were responsible for the manufacturing of the active cell product. Disease entities were mainly prostatic cancer (75%) and ovarian cancer (20%). Based on differences in the study protocols LA was performed either one-time (41%), two-times (27%) or three-times (32%), with an interval of at least 2 weeks between repeated collections. All patients successfully completed the apheresis course. Because of poor venous access, 3 out of 60 patients (5%) required a short-term femoral catheter insertion. There were no serious side effects in patients who required a femoral catheter, or in patients with repeated LA procedures. Side effects of the LA procedure mainly consisted on mild hypocalcaemia-related symptoms in 16% of patients. A follow-up survey one week after completion of the LA revealed no infectious complications, and no patient required hospitalization. Median cell yield collected per single apheresis was 1.5 × 10e10 WBC consisting of 1.3 × 10e10 MNC. MNC cell yields remained stable even in repeated LA collections. All cell products were successful transformed into an active cellular product. Analysis of the cost structure showed that the total cost of care was 32% lower in the setting of a private collection center compared to hospital-based apheresis centers. Leukapheresis performed in a private medical practice/ certified cell collection facility is safe and effective, with low rates of complications and high levels of patient satisfaction. This service model is cost-effective and can help to reduce the cost of manufactured goods in the production of innovative cellular products.

Optimizing autologous nonmobilized mononuclear cell collections for cellular therapy in pediatric patients with high-risk leukemia.

The manufacturing of cellular products for immunotherapy, such as chimeric antigen receptor T cells, requires successful collection of mononuclear cells. Collections from children with high-risk leukemia present a challenge, especially because the established COBE Spectra apheresis device is being replaced by the novel Spectra Optia device (Optia) in many institutions. Published experience for mononuclear cell collections in children with Optia is lacking. Our aim was to compare the two collection devices and describe modified settings on the Optia to optimize mononuclear cell collections. As a quality initiative, we retrospectively collected and compared data from mononuclear cell collections on both devices. Collected data included patient's clinical characteristics; collection parameters, including precollection lymphocyte/CD3 counts, total blood volumes processed, runtimes, and side effects (including complete blood count and electrolyte changes); and product characteristics, including volumes and cell counts. Collection efficiencies and collection ratios were calculated. Twenty-six mononuclear cell collections were performed on 20 pediatric patients: 11 with COBE and 15 with Optia. Adequate mononuclear cell products were successfully collected with a single procedure from all patients except one, with mean calculated mononuclear cell collection efficiency that was significantly higher from Optia collections compared with COBE collections (57.9 ± 4.6% vs 40.3 ± 6.2%, respectively; p = 0.04). CD3-positive yields were comparable on both machines (p = 0.34) with significantly smaller blood volumes processed on Optia. Collected products had larger volumes on Optia. No significant side effects attributed to the procedure were noted. Mononuclear cell apheresis using the Optia device in children is more efficient and is as safe as that with the COBE device.

Comparison of Amicus and COBE Spectra for allogenic peripheral blood stem cell harvest: Study from tertiary care centre in India

IntroductionMost common source of stem cell graft for both autologous and allogenic haematopoietic transplants are peripheral blood haematopoietic progenitor stem cells. Adequate collection of the CD34+ cells and safety of the allogenic donor during the leukapheresis are of prime importance to an apheresis physician. Our retrospective analysis is a comparison between of two platforms namely, COBE Spectra and Amicus, for CD34+ mononuclear cell collection. Material and methodThe study included the data of GSCF (Granulocyte-Colony-Stimulating Factor) mobilized allogenic PBSC collections at our centre from January 2015 to June 2016. The apheresis platforms used were COBE Spectra and Amicus. Blood cell counts were done using LH750 Beckman Coulter (Florida, Miami, USA). CD45+ & CD34+ cell counts were done using BD FACS Canto-II Flow-Cytometer by ISHAGE guidelines. ResultsA total of 170 PBSC (100 COBE Spectra & 70 Amicus) harvests were done on 143 donors, of which 116 completed the collection in a single session and 27 required a second session. Demographic details and pre harvest peripheral blood counts for both the groups did not show any statistical differences. Amicus processed higher blood volume with higher ACD exposure and procedure time compared to COBE Spectra. Higher platelets loss was with COBE Spectra harvests with higher product volumes collection. Collection efficiency (CE2), collection ratio, CD34+ cells dose was similar on both the platforms. RBC contamination, absolute lymphocyte and monocytes counts were significantly higher with Amicus harvest product compared with COBE Spectra. A total of 14 (8.2%; citrate toxicity) adverse reactions were reported out of 170 allogenic PBSC collections. Discussion/conclusionOur study suggests that both Amicus and COBE Spectra platforms offer comparable results for allogenic PBSC collections. Amicus offers a concentrated PBSC product with lesser volume and platelets loss but higher RBC contamination.

168 - Optimizing Autologous Mononuclear Cell Collections for Cellular Therapy Using the Novel Optia Apheresis Device in Pediatric Patients with High Risk Leukemia

168 - Optimizing Autologous Mononuclear Cell Collections for Cellular Therapy Using the Novel Optia Apheresis Device in Pediatric Patients with High Risk Leukemia

Comparison of the Terumo BCT MNC and Cmnc Protocols for Peripheral Blood Stem Cell Collections

Background: Terumo BCT recently offered a new method of peripheral blood stem cell (PBSC) collection using the Spectra Optia¨, an apheresis instrument. The new protocol includes a continuous mononuclear cell collection (CMNC) as opposed to an older version, the mononuclear cell collection (MNC), which involves an additional step where product pools through a cell separation insert. Our institution has used both methods and the purpose of this study was to compare the CMNC to the MNC protocol, including run times and PBSC product characteristics.Methods: A retrospective review and comparison of parameters from 120 collection procedures using the MNC insert and 60 collection procedures using the CMNC insert was done using the t-test. Data from patients/donors (including 20 allogeneic donors) as well as procedure details including run time, flow cytometry marker for stem cells (CD34)-positive (CD34+) throughput, CD34+ collection efficiency (CE%), platelet loss (plt loss/total blood volume [TBV]), and collection product characteristics were included in the analysis.Results: The MNC donor group included 12 allogeneic donors, which is comparable to the 8 allogeneic donors in the CMNC group. Donor weight and patient weight was not significantly different between the two groups. Pre-procedure laboratory values (WBC, percentage of lymphocytes [lymph%], percentage of monocytes [MNC%], and platelet count) were also similar between the two groups.Run time was found to be significantly shorter using the CMNC protocol compared to the MNC protocol. Product volume was also significantly lower in the CMNC group compared to the MNC group. Although the volume was lower, the CMNC product had significantly higher white blood cell count (WBC), MNC%, and lymph% when compared to the MNC product. The CD34+ throughput was significantly higher in the CMNC group than the MNC group. The CD34+ CE% was found to be slightly increased in the CMNC group, though not significantly. The platelet loss was nearly identical in both protocols when normalized for total blood volume. Product hematocrit (HCT%) was significantly higher using the CMNC protocol; however, the red blood cell volume never exceeded 20 mL due to the lower product volume with the CMNC protocol. The numerical results are summarized in the Table.Conclusion: The CMNC protocol collects a smaller volume of a purer product when compared to the MNC protocol with comparable platelet and red blood cell loss. Staff members who perform apheresis procedures are pleased by the shorter run time. [Display omitted] DisclosuresRoig:Terumo BCT: Employment.

Final Report of Phase 1 Study of the DOT1L Inhibitor, Pinometostat (EPZ-5676), in Children with Relapsed or Refractory MLL-r Acute Leukemia

Introduction: MLL-rearranged (MLL-r) acute leukemia in children is characterized by young age at presentation and a poor overall prognosis despite multi-agent chemotherapy. Aberrant fusion proteins involving the MLL histone methyltransferase (HMT) recruit another HMT, DOT1L, to a multi-protein complex leading to aberrant methylation of histone H3 lysine 79 (H3K79) at MLL target genes. This results in enhanced expression of critical genes for hematopoietic differentiation, including HOXA9 and MEIS1, and has been established as a key mechanism for leukemogenesis in MLL-r leukemias (Krivstov, 2007). Pinometostat is a small molecule inhibitor of DOT1L with sub-nanomolar affinity and >37,000 fold selectivity against non-MLL HMTs. Treatment of MLL-rearranged cells and xenograft models with pinometostat led to reduced histone 3 lysine 79 methylation (H3K79me2), decreased MLL target gene expression and selective leukemia cell kill (Daigle, 2013). Here we report the final results of the pinometostat phase 1 trial in children with relapsed/refractory (R/R) MLL-r acute leukemia.Methods: An open label dose escalation study of pinometostat was performed in patients (pts) aged 3 months to 18 years (yr) with R/R MLL-r leukemia (NCT02141828). Pinometostat was administered via continuous intravenous infusion (CIV) until disease progression or unacceptable toxicity. Pts were assigned to one of two aged-based dose escalation schemas developed from simulations of pediatric exposures using a previously reported physiologically-based PK (PBPK) model (Waters, 2014). All patients underwent serial collection of PK and peripheral blood mononuclear cells (PBMC). Leukemic blasts were isolated from PBMCs using flow cytometry and quantified for H3K79me2 levels by ChIP-Seq.Results: 18 pts were enrolled on study with 9 pts dosed at 70 mg/m2/day and 7 pts at 90 mg/m2/day in the older age cohort (1 to 18 yr) plus 2 pts dosed at 45 mg/m2/day in the younger age cohort (<1 yr).Grade ≥3 treatment-emergent adverse events (TEAEs) regardless of attribution reported in >20% of pts were: febrile neutropenia; anemia; leukopenia; thrombocytopenia; hypokalemia; respiratory failure; lymphopenia; neutropenia. Drug-related TEAEs reported in >15% of pts were: anemia; thrombocytopenia; leukopenia; rash; lymphopenia; hypocalcemia; hypophosphatemia; neutropenia; ALT elevation; nausea; vomiting.Dose-limiting toxicities (DLT) were: apnea (70 mg/m2); elevated transaminases (2 at 90 mg/m2) in the >1 year of age cohort, thus defining 70 mg/m2 as the recommended phase 2 dose (RP2D) in older pts. A RP2D was not determined in pts < 1 year of age. Median duration of treatment was 23 days (range 7- 53 days). Pinometostat induced transient decreases in peripheral or marrow leukemic blasts in 7/18 pts, however, these reductions did not meet formal thresholds for objective response.Adjusted mean plasma pinometostat concentrations during the infusion in children >1 yr at 70 and 90 mg/m2/d doses (1151 ng/mL) was comparable to mean steady-state concentrations observed in adult patients at the 80 mg/m2/d and 90 mg/m2/d doses (1320 ng/mL and 1410 ng/mL, respectively) and was within the range of 1000-1600 ng/mL at 90 mg/m2/d in ≥ 1 yr predicted by earlier PBPK modeling results. CSF concentrations of pinometostat were below the lower limit of quantification of 1 ng/mL (n = 8) or very low (< 12 ng/mL) (n = 4), suggesting negligible CSF exposure. H3K79me2 ChIP-Seq on leukemic blasts demonstrated that pinometostat induced reductions in methylation at MLL-r target genes (e.g. HOXA9 and MEIS1) of ≥ 80 % at all post dose time points (15 and 28 days) and doses (70 & 90 mg/m2) tested consistent with DOT1L inhibition.Conclusions: In pediatric pts with R/R MLL-r pinometostat has an acceptable safety profile with a RP2D defined as 70 mg/m2 CIV in children > 1 yr. Pinometostat dose/exposure relationships were comparable between adults and children > 1 yr. Pharmacodynamic evidence of DOT1L inhibition was observed in leukemic blasts. Transient reductions in peripheral or bone marrow blasts were detected in ~40 % of pts, however no objective responses were observed. Based on the biological activity observed and evidence of combination benefit of pinometostat with standard of care and novel agents in preclinical MLL-r models (Daigle 2015 and Klaus 2015) further clinical investigation of pinometostat combinations is warranted. [Display omitted] DisclosuresO'Brien:Seattle Genetics: Research Funding; Celgene: Other: travel expenses for required site investigator meeting, October 2015;; steering committee member for pediatric AML trial, Research Funding; Epizyme: Research Funding; Amgen: Other: participated in one pediatric advisory board for blinatumomab in May 2015, paid consultant fee and travel expenses, Research Funding. Blakemore:Epizyme: Employment. Daigle:Epizyme: Employment. Suttle:Epizyme: Employment. Armstrong:Epizyme, Inc: Consultancy; Vitae Pharmaceuticals: Consultancy; Imago Biosciences: Consultancy; Janssen Pharmaceutical: Consultancy. Ho:Epizyme: Employment, Equity Ownership.

Increased Efficacy of Chemomobilization with Intermediate Dose Cytarabine + G-CSF Compared to G-CSF Alone for Patients with Multiple Myeloma: Results of a Prospective, Randomized Trial

INTRODUCTIONHigh-dose therapy with autologous stem cell support (autologous stem cell transplantation, autoSCT) remains the standard of care for eligible patients with multiple myeloma (MM). Planned tandem autoSCT which can also be considered for all transplant candidates requires collection of at least 5 x 10^6 CD34+ cells/kg. Randomized trials comparing chemomobilization with use of cyclophosphamide (CY) + G-CSF to G-CSF alone did not demonstrate clear advantage of addition of CY to growth factor. In recent years, intermediate-dose cytosine arabinoside (ID-AraC) + G-CSF has been proposed, showing high mobilization potential, as demonstrated in retrospective analysis (Giebel et al, Bone Marrow Transplant 2013, 48: 915-21). The goal of this prospective, randomized trial (ClinicalTrials.gov identifier: NCT01908621) was to compare the efficacy of ID-AraC + G-CSF with G-CSF alone in patients with MM referred for double autoSCT. METHODSThe inclusion criteria were set as follows1) the diagnosis of MM, 2) age 18-65 years, 3) at least partial remission achieved after one or more lines of therapy including six or more cycles containing components like thalidomide, lenalidomide , bortezomib, or melphalan, 4) planned tandem autoSCT procedure.The proportion of patients with stem cell yield of at least 5 × 10^6 CD34+ cells/kg was the primary study end-point.Mobilization regimens were as follows1) ID-AraC arm - AraC 2 x 0.4 g/m2 on days 1,2 (total 1.6 g/m2) + filgrastim 10 ug/kg/day starting from day 5; 2) G-CSF arm - filgrastim 10 μg/kg/day for five consecutive days. Leukaphereses were started when the level of circulating CD34+ cells in peripheral blood (PB) reached at least 10/uL and were continued for the maximum period of three days or until reaching the target CD34+ cell yield. Leukaphereses were performed using Spectra Optia Apheresis System, Mononuclear Cell Collection Procedure (Therumo BCT Inc., Lakewood, CO, USA), processing 2 total blood volumes. RESULTSBetween March 2013 and March 2016, 44 and 46 patients were randomly assigned to ID-AraC and G-CSF study arm, respectively. Patients in the ID-AraC arm were younger (median age 56 years, range (33-65) for ID-AraC and 60 years (37-65) for G-CSF, p=0.04). The groups did not differ in terms of MM remission status at mobilization (p=0.3), the number of preceding lines of chemotherapy (p=0.5) and the frequency of preceding radiotherapy (p=0.4).The level of CD34+ cells in PB required to start leukaphereses was achieved in all patients in the ID-AraC arm and in 44 (96%) of them in the G-CSF arm, p=0.2.In the ID-AraC group, 43 patients (98%) collected at least 5 x 10^6 CD34+ cells/kg compared to 32 patients (70%) in the G-CSF group (p=0.0003). In a multivariate model adjusted for age, the use of G-CSF alone was independently associated with increased risk of mobilization failure (Odds ratio = 17.5; 95% CI = 2.1-144.9; p=0.007).The median peak number of circulating CD34+ cells was 346 (11-1044)/µL vs. 40 (1-326)/µL (p<0.000001), while the median number of collected CD34+ cells was 20.2 (2.9-59.4) x10^6/kg vs. 5.9 (0-11) x10^6/kg, respectively (p<0.000001). A single apheresis was sufficient to achieve the threshold number of harvested CD34+ cells in 37 of cases (86%) after ID-AraC+G-CSF compared to 13 (41%) after G-CSF alone (p=0.00008). The median day of the first apheresis was 13 (range, 12-15; SD=0.7) after ID-AraC. CONCLUSIONSMobilization with ID-AraC + G-CSF is associated with significantly higher efficacy than G-CSF alone. In the studied group it allowed for collection of CD34+ cell number adequate for tandem autoSCT in almost all MM patients, usually with a single leukapheresis. This study provides the first evidence coming from prospective, randomized trial for the advantage of chemomobilization over G-CSF monotherapy in terms of proportion of patients achieving CD34+ yield sufficient for autoSCT. DisclosuresNo relevant conflicts of interest to declare.

Continuous CD34+ cell collection by a new device is safe and more efficient than by a standard collection procedure: results of a two‐center, crossover, randomized trial

Peripheral blood stem cells (PBSCs) collected from granulocyte-colony-stimulating factor (G-CSF)-mobilized donors are routinely used for hematopoietic stem cell transplantation. Most PBSC collections worldwide have used the COBE Spectra (COBE) platform that is being replaced by the Spectra Optia (OPTIA). This study compared the PBSC collection performance and safety of the OPTIA using a single-step, continuous mononuclear cell (CMNC) collection procedure and the standard COBE MNC procedure. A prospective, noninferiority, randomized, open-label, crossover, multicenter study was conducted in G-CSF-mobilized donors randomized to undergo MNC collection on Days 5 and 6. The primary endpoint was CD34+ cell collection efficiency (CE1%) with a noninferiority margin of 10%. The secondary endpoint was CD34+ cell CE2%. Product purity and safety were also assessed. Twenty-three healthy donors (87% male) participated in the study. Mean (±SD) CD34+ CE1% was 84.4% (±16.4%) and 66.2% (±15.3%) for the OPTIA and COBE, respectively (p < 0.001 for noninferiority and superiority). Mean (±SD) CD34+ CE2% was 62.4 (±11.6) and 48.4 (±11.2) for the OPTIA and COBE, respectively (p < 0.001 for superiority). Granulocyte and platelet (PLT) contamination were lower in OPTIA-collected products. There were no unexpected adverse events (AEs) and no significant differences in the incidence of AEs between study arms. PLT loss was less with the OPTIA than with the COBE. The OPTIA CMNC collection procedure is safe and effective for the collection of CD34+ cells in G-CSF-mobilized donors and was superior to the COBE for CE1% and CE2%, collecting approximately 19 and 16% higher, respectively.

Comparison of two automated mononuclear cell collection systems in patients undergoing extracorporeal photopheresis: a prospective crossover equivalence study.

Extracorporeal photopheresis (ECP) is an effective cell therapy employed in several diseases, including graft versus host disease (GVHD) and organ rejection. When ECP is performed using an off-line technique, mononuclear cell (MNC) collection by leukapheresis is necessary for further manipulation (addition of 8-methoxypsoralen and ultraviolet A irradiation before reinfusion to the patient). We report the results of the first crossover equivalence study on yield and purity of MNCs collected from patients undergoing ECP with two different automated systems: MNC and CMNC (working with intermittent and continuous-flow collection, respectively), released by Terumo BCT. Fifty-one patients (15 males and 36 females) with GVHD or chronic lung allograft dysfunction were consecutively enrolled and randomly assigned to MNC collection alternatively by the CMNC or MNC system within each ECP cycle (two procedures) in two consecutive cycles. ECP procedures were performed using the off-line technique, according to our internal protocol, processing 1.5 blood volumes. A total of 204 ECP procedures were evaluated. The MNC system showed a higher MNC concentration capacity than the CMNC (mean difference, -13.46; 95% confidence interval, -21.05 to -5.47; p < 0.001). Collection efficiency was higher with the CMNC system as well as total MNC bag content, while MNC bag purity was equivalent for both systems. Platelet loss was higher with the CMNC. Equivalence was shown for MNC purity and anticoagulant infused to the patient. CMNC and MNC systems offer different advantages in different clinical conditions, and both are safe and efficient in collecting MNCs for ECP.

Therapeutic apheresis

Apheresis is a procedure to separate and collect or remove a particular blood component. Apheresis may be used for collecting a therapeutic dose of component, remove the circulating amount of harmful component/substances, or collect a particular blood cell/precursor from a patient for reinfusion. Therapeutic plasma exchange (TPE) is a therapeutic procedure in which the plasma is removed and replaced with a replacement solution. It is used primarily for the removal of pathological immunoglobulin. Red cell exchange is a procedure in which the patient's red cells are removed and replaced with the donor red cells. It is usually used in the case of sickle cell disease to prevent stroke. Mononuclear cell collection is a procedure used primarily to collect peripheral blood haemopoietic progenitor cells (HPC) for autologous or allogeneic transplantation. As the most frequently performed procedures, they will be described in some detail in this article. Therapeutic leucapheresis and thrombocytapheresis are less frequently performed and will be mentioned briefly.

Comparison of influenza vaccine regimens in pediatric patients:immunity and efficacy in 2014–15

Abstract Currently, there are two types of influenza vaccines available to children and adolescents, Influenza Inactivated Vaccine (IIV) which is administered intramuscularly, and Live Attenuated Influenza Vaccine (LAIV) which is administered intranasally. The present study was designed to compare the efficacy of the two vaccines in pediatric patients in eliciting immune responses and protecting against infection in the 2014–15 influenza season. To conduct the study, patients ranging from ages 3 to 17 years were recruited for vaccination and evaluation. Patients were segregated by age (3 to 8 vs. 9 to17) and vaccine (IIV vs. LAIV). Peripheral blood samples were collected between the months of August and December, 2014 and processed for collection and storage of serum, plasma and peripheral blood mononuclear cells (PBMC). Each child who agreed to participate in the study was prescreened, consented and provided blood samples at three time points: D0 (blood drawn prior to vaccination), D7 and D21 following influenza vaccination. At the end of the vaccination season and once all enrolled patient samples had been collected, antibody responses were evaluated for reactivity to the vaccine strains as well as against additional circulating or heterogonous strains of influenza. Early in the 2014–15 season, it became apparent that the LAIV strain in the vaccine was not well matched to the circulating strain of influenza, resulting in less-than-effective immunity. Therefore, our analyses focused on IIV only. Our results showed that all participants in the study demonstrated higher titers and increased breadth of reactivity to diverse influenza strains when vaccinated with IIV, regardless of prior vaccination history.

Genome-Wide Changes in Peripheral Gene Expression following Sports-Related Concussion.

We conducted a prospective study to identify genome-wide changes in peripheral gene expression before and after sports-related concussion (SRC). A total of 253 collegiate contact athletes underwent collection of peripheral blood mononuclear cells (PBMCs) before the sport season (baseline). Sixteen athletes who subsequently developed an SRC, along with 16 non-concussed teammate controls, underwent repeat collection of PBMCs within 6 h of injury (acutely). Concussed athletes underwent additional sample collection at 7 days post-injury (sub-acutely). Messenger RNA (mRNA) expression at baseline was compared with mRNA expression acutely and sub-acutely post-SRC. To estimate the contribution of physical exertion to gene changes, baseline samples from athletes who subsequently developed an SRC were compared with samples from uninjured teammate controls collected at the acute time-point. Clinical outcome was determined by changes in post-concussive symptoms, postural stability, and cognition from baseline to the sub-acute time-point. SRC athletes had significant changes in mRNA expression at both the acute and sub-acute time-points. There were no significant expression changes among controls. Acute transcriptional changes centered on interleukins 6 and 12, toll-like receptor 4, and NF-κB. Sub-acute gene expression changes centered on NF-κB, follicle stimulating hormone, chorionic gonadotropin, and protein kinase catalytic subunit. All SRC athletes were clinically back to baseline by Day 7. In conclusion, acute post-SRC transcriptional changes reflect regulation of the innate immune response and the transition to adaptive immunity. By 7 days, transcriptional activity is centered on regulating the hypothalamic-pituitary-adrenal axis. Future efforts to compare expressional changes in fully recovered athletes with those who do not recover from SRC could suggest putative targets for therapeutic intervention.

Extracorporeal photopheresis versus standard treatment for acute graft-versus-host disease after haematopoietic stem cell transplantation in paediatric patients.

Acute graft-versus-host disease (aGvHD) is a major cause of morbidity and mortality after haematopoietic stem cell transplantation (HSCT) occurring in 8% to 59% of the recipients. Currently, the therapeutic mainstay for aGvHD is corticosteroids. However, there is no established standard treatment for steroid-refractory aGvHD. Extracorporeal photopheresis (ECP) is a type of immunomodulatory method amongst different therapeutic options that involves ex vivo collection of peripheral mononuclear cells, exposure to the photoactive agent 8-methoxypsoralen and ultraviolet-A radiation, and re-infusion of these treated blood cells to the patient. The mechanisms of action of ECP are not completely understood. This is an updated version of a Cochrane review first published in 2014. To evaluate the effectiveness and safety of ECP for the management of aGvHD in children and adolescents after HSCT. We searched the Cochrane Register of Controlled Trials (CENTRAL) (Issue 9, 2015), MEDLINE (PubMed) and EMBASE (Ovid) databases from their inception to 23 September 2015. We searched the reference lists of potentially relevant studies without any language restrictions. We searched eight trial registers and four conference proceedings on 29 September 2015. Randomised controlled trials (RCTs) comparing ECP with or without standard treatment versus standard treatment alone in paediatric patients with aGvHD after HSCT. Two review authors independently performed the study selection. We resolved disagreement in the selection of trials by consultation with a third review author. We identified no additional studies in the 2015 review update, in total leading to no studies meeting the criteria for inclusion in this review. The efficacy of ECP in the treatment of aGvHD in paediatric patients after HSCT is unknown and its use should be restricted within the context of RCTs. Such studies should address a comparison of ECP alone or in combination with standard treatment versus standard treatment alone. The 2015 review update brought about no additions to these conclusions.