Novel Evidence That the Pannexin 1 Channel Is Involved in Adenosine Triphosphate (ATP) Release from Cells for Optimal Mobilization of Hematopoietic Stem Progenitor Cells, and the Pannexin 1 SNP 5 (Rs3020015) T/C Polymorphism Characterizes Poor Mobilizer Status in Patients

Background . Pannexins have been shown to act predominantly as large transmembrane channels connecting the intracellular and extracellular space, allowing the passage of ions and small molecules, such as adenosine triphosphate (ATP), between these compartments. Pannexin 1 channels are involved in the release of ATP from cells and have been shown to be involved in the early stages of the innate immune response through an interaction with the P2X7 purinergic receptor. Recently, we provided evidence that activation of innate immunity in BM after administration of G-CSF produces "sterile inflammation" in the BM microenvironment and activates the pannexin channel to release ATP, which through binding to the P2X7 receptor leads to the mobilization of HSPCs (Leukemia 2018, 32:1920-1931). Corroborating our observation, it has been recently reported that the presence of the Gln460Arg SNP polymorphism within the P2X7 receptor gene in HSPCs, which is always co-inherited with Ala348Thr to form the gain-of-function haplotype 4, resulted in a significant increase in CD34+ HSPC mobilization (Leukemia 2018; 32:2724-2726). To shed more light on the novel concept that pannexin-1-released ATP, which interacts with P2X7, plays a crucial role in the egress of HSPCs from BM into peripheral blood (PB), we focused on the role of pannexin 1 in the mobilization process. Hypothesis. We hypothesized that pannexin 1 deficiency would negatively impact mobilization of HSPCs. Materials and Methods. First, we mobilized mice with G-CSF or AMD3100 in the presence of pannexin-1-blocking peptide. Following mobilization, we measured i) the total number of white blood cells (WBCs) and ii) the number of circulating clonogenic colony-forming unit granulocyte/macrophage (CFU-GM) progenitors and Sca-1+c-kit+lineage- (SKL) cells circulating in PB. Next, we analyzed five types of polymorphisms in the human pannexin 1 gene (SNP1-Rs1138800 A/C, SNP2-Rs7928030 G/C, SNP3-Rs12294985 C/T, SNP4-Rs127933348 A/G, and SNP5-Rs3020015 T/C) and correlated them with good or poor mobilization status of the patients. Patients in our studies were mobilized in the Bone Marrow Transplant Unit, Warsaw Medical University with G-CSF using a standard mobilization protocol, and poor mobilizers were identified as patients who were not able to mobilize the required number of CD34+ cells according to standard criteria. In our studies DNA from patient blood samples was isolated and fragments of DNA amplified and subsequently sequenced. Our patients in the good- and poor-mobilizer groups were matched for age, sex, and basic disorders. Results. We found that mice with blocked pannexin 1 channels mobilized HSPCs significantly less efficiently. More importantly, our patient data revealed that ~60% of patients that turned out to be poor HSPC mobilizers (n=20) displayed the pannexin 1 polymorphism SNP5 (Rs3020015) T/C. This polymorphism was observed in only 1 out of 26 good-mobilizer patients. Conclusions. Our results further support an important role for ATP-mediated purinergic signaling in the mobilization of HSPCs. Moreover, the pannexin 1 SNP5Rs3020015 T/C polymorphism may serve as a diagnostic tool to identify poor mobilizers. This investigation shed more light on the molecular pathways involved in the egress of HSPCs from BM into PB and will help to design better mobilization protocols in the case of poor mobilizers. Disclosures No relevant conflicts of interest to declare.

Advanced Osteolytic Lesions (OL), Mobilization and Collection of Hematopoietic Progenitor Cells (HPC) in Multiple Myeloma (MM)

Introduction: Presence of advanced OL was recently reported as a risk for poor mobilization in patients with multiple myeloma who had poor HPC collections (Jung et al. J Clin Apheresis 2014; Apr 25. doi: 10.1002). We sought to confirm this finding and also whether poor collection correlated with low peripheral blood CD34+ cell numbers as evaluated by flow cytometry. Patients and Methods: Patients: We performed a retrospective study of patients who underwent autologous HPC collection at our institution between 2005 and 2012 to identify poor mobilizers and mega-mobilizers in a 2:1 ratio for data analysis. We defined poor mobilizers as those who required maximal plerixafor support (4 days) for collection, and mega-mobilizers as those who collected >30 x 106 CD34+ cells/kg in 2 days. We found 79 poor mobilizers, but removed 9 from data analysis because the collection variables of plerixafor timing and G-CSF dose differed from the others, leaving 64 myeloma (MM) and 6 non-myeloma plasma cell dyscrasias (NMPCD) patients for analysis: 41 male, 29 female, age range 43–86 (median 67.5). There were 37 mega-mobilizers: 36 MM, 1 NMPCD: 21 male, 16 female, age range 40–73 (median 61). Cumulative CD34+ cells/kg during leukapheresis and peak peripheral CD34+ cell counts were recorded. Apheresis: Apheresis was initiated using a central venous catheter when the predicted CD34+ cell collection for 30 L of blood processed was at least 1 x 106/kg using a predictive formula (Rosenbaum et al. Cytotherapy 2012; 14(4): 461-6). The volume of blood processed each day was based on the same predictive formula, and ranged from 5 to 30L. Cells were collected on a COBE ® Spectra apheresis machine, software version 7.0, using 1000 mL anticoagulant citrate dextrose (ACD) and 5000 units heparin for anticoagulation at an inlet:anti-coagulant ratio of 31:1, and an inlet flow rate of 150 mL/min with anti-coagulant infused at 5 mL/min. The collection flow rate was set at 1.5 mL/min and 10 mL ACD was added to the component at processed volumes of 10 L, 20 L and 30 L. An infusion of 2 g calcium chloride in 250 mL normal saline (0.9% sodium chloride) ran at 85 mL/h. Flow cytometry: CD34+ cells in peripheral blood and HPC products were quantified by flow cytometry using the ISHAGE protocol. Statistics: Mean peripheral blood CD34+ cells/µL and mean CD34+ cells/kg collected were calculated separately for the mega-mobilizer + poor mobilizer combined group, mega-mobilizer and poor mobilizer groups. All patients were subcategorized into those with ≤10 and >10 OL, and means for CD34+ cells/kg collected and peripheral blood CD34+ cells/uL were compared separately between the ≤10 and >10 OL groups using two-tailed Student’s t-tests and p-values evaluated for significance. Results: For all patients combined (mega + poor mobilizers) there were no significant differences in either peripheral CD34+ cells/µL or mean total CD34+ cells/kg collected between the ≤10 and >10 OL subgroups. Mean CD34+ cells/µL peripheral blood was 276 and 250 for the ≤10 and >10 OL groups, respectively (p=0.73), with means of 27.7 and 23.6 CD34+ × 106 CD34+ cells/kg collected (p=0.41). For the mega-mobilizers there was no significant difference in mean peripheral blood CD34+ cells/µL between the OL (</=10 and >10) groups (722 vs. 709, respectively; p=0.92) or in total CD34+/kg collected (55.8 and 53.8, respectively; p=0.78). For the poor mobilizers there was no significant difference in mean peripheral CD34 cells/µL between the ≤10 and >10 OL groups (27 and 20, respectively; p=0.10); however, there was a statistically significant difference in total number of CD34+ cells/kg collected, 11.9 and 8.4 ×106 CD34+ cells/kg, respectively (p=0.02). Conclusion: No significant difference was seen in mobilization as judged by peripheral blood CD34+ cells/ µL in mega-mobilizers or poor separately or combined, but a difference in the total number of CD34+ cells collected was seen in poor mobilizers. We suggest this difference results from variables in collection protocols, as we have previously shown that both mobilization and collection variables impact total CD34+ cells collected by apheresis (Abuabdou et al 2013; J Clin Aph Dec 18. doi: 10.1002). Disclosures Barlogie: Celgene: Consultancy, Patents & Royalties, Research Funding; Millenium: Consultancy, Patents & Royalties, Research Funding.

Use of Plerixafor in Predicted and Proven Poor Mobilizer (according to GITMO criteria). Experience of Policlinico Universitario Tor Vergata

Abstract 4415 BACKGROUND. Autologous stem cell transplantation (ASCT) of PBSCs has become a widely applied treatment for Multiple Mieloma (MM), non- Hodgking's lymphoma (NHL) and Hodgking's lymphoma (HL). Successful engraftment correlates with the number of CD34 hemopoietic progenitors cells infused. However, a part of MM or lymphoma patients (5% to 40%) fail to mobilize adequate numbers of PBSCs and thus cannot undergo to ASCT. The success of PBSCs mobilization is usually assessed by the total number of CD34+ stem cells collected, with a cutoff of 2.0–2.5 ×106 CD34+ cells/kg recipient body weight being considered as a minimum requirement for transplant. Poor mobilization of PBSCs is a major limitation to ASCT. Recently GITMO Working Group worked to define operational criteria for the identification/prediction of the poor mobilizer (PM) patients (Olivieri et al. 2011). Plerixafor, a CXCR4 chemochine antagonist, has been showed to improve significantly PBSC mobilization in PM patients. We present our experience using Plerixafor in PM patients classified according to GITMO criteria. METHODS. Between September 2009 and June 2012, a total of 17 patients (9F-8M) were enrolled. The diagnosis were: 10 MM (5F-5M), 1HL (1M), 6 NHL (4F-2M). The median age was 57 (range 15–66). 7 patients (3MM, 4NHL) were defined “Proven PM” and 10 patients (7MM, 2NHL, 1HL) “Predicted PM” according to GITMO criteria. The mobilization protocol included G-CSF, administered at a dose of 10μg/kg daily on 4 consecutive days. In the evening of the fourth day, patients received subcutaneous plerixafor at a dose of 0,24 mg/kg. Apheresis was initiated on the fifth day, 10–12 h after plerixafor and 1 h after G-CSF administration. Apheresis and daily administration of G-CSF and plerixafor continued until the patient collected enough CD34+ cells for auto- HSCT (> 2 ×106/kg; max 7 plerixafor injections if required). PBSC collection was initiated if peripheral CD34+ cells count was >10μl. A successful mobilization was defined as a total yeld of > 2×106/kg. RESULTS. 13 patients (76,5%) collected the minimum number of CD34 cells > 2×106/kg. The diagnosis were: 8MM, 1HL,1 NHL. 7 patients (2NHL; 4 MM; 1 LH; 7 predicted) were able to collect > 5×106/Kg. Only 4 patients (3 MM; 1 LNH; 4 proven) failed the mobilization because the numbers of cells CD34 were < 10μL and these patients did not undergo to apheresis procedures. The collection target of 2×106/Kg was reached in a median of 2 apheresis session (range 1–3). The technical characteristics of the procedures were (median value): blood volume processed 12 L (range 9–14), total CD34+/Kg collected 3,06 × 106(range 2,21-8,62), procedure efficiency 47,5% (range 35,3–79), duration of the procedure 261 minutes (range 210–309). Plerixafor was well tolerated and mild side effects were: reactions in the injection site, gastrointestinal disturbs, muscle pain. During administration of plerixafor we did not observe any significant laboratory abnormalities of liver or renal function. CONCLUSION. Unsuccessful mobilization represents an important limitation to ASCT in lymphoma and MM. In our experience plerixafor allowed to collect an appropriate amount of CD34 also in patients defined “proven PM” significantly reducing the percentage of patients that could not undergo ASCT (target value obtained in 43% of “proven PM”). Confirming the recent literature plerixafor is well tolerated with minimal side effects. We retrospectively applied GITMO criteria for PM patients and our experience, although limited, confirm that the use of a correct definition of PM allows the appropriate use of new mobilizing agents like plerixafor increasing significantly the therapeutic options also in patients who had no possibilities to receive an ASCT with the traditional mobilizing therapy. Disclosures: No relevant conflicts of interest to declare.