Stem Cell Mobilization Using Parathyroid Hormone Analog: A Single Institutional Review

Objective: Hematopoietic stem cell mobilization increases the release of immature and maturing hematopoietic cells from the marrow into the blood circulation. For successful hematopoietic stem cell transplantation an adequate number of stem cells must be mobilized and collected. For autologous stem cell transplants, a product bag CD34+ count of > 5.0 x 106 CD34/kg is a target; however, patients have been successfully transplanted with doses as low as 2.0 x 106 CD34/kg. Some patients are "poor mobilizers" and standard protocols do not result in adequate numbers of circulating CD34 cells to collect. Risk factors associated with poor stem cell mobilization include increasing age, underlying diagnosis, low premobilization platelet count, history of increasing cycles, and regimens of chemotherapy. Teriparatide, a parathyroid hormone (PTH) analog has been used in "poor mobilizers." Two patients at our institution received the drug as part of an additional mobilization strategy. Method: Medical records of patients who had stem cell mobilization were reviewed. Two patients who failed routine mobilization protocol received PTH as part of an additional mobilization regimen. Clinical outcomes, collection, and engraftment data were reviewed. Result: Patient 1 had a diagnosis of Hodgkin Lymphoma and failed to mobilize adequately on the first attempt using filgrastim and plerixafor with peripheral blood CD34 counts of 1, 4, and 3 resulting in cancellation of stem cell collection. For the second mobilization attempt, teriparatide was added to the regimen. Peripheral blood CD34 counts improved to 8, 6, and 2 resulting in three collections with a total of 2.23 x 106 CD34/kg for reinfusion. Engraftment data showed 14 days for neutrophils and 17 days for platelets. The patient is 6 months post-transplant with no major morbidities reported, currently in maintenance therapy, and has not recurred. Patient 2 had a diagnosis of multiple myeloma and failed to mobilize on filgrastim and plerixafor with peripheral blood CD34 counts of 2, 2, and 2 resulting in collections with a total of 0.6 x 106 CD34/kg for reinfusion. For the second mobilization attempt, peripheral blood CD34 counts of 2, 2, 2, and 0 resulting in collections with a total of 0.822 x 106 CD34/kg for reinfusion. For the third mobilization attempt, teriparatide was added to the regimen. Peripheral blood CD34 counts improved to 8 and 4 resulting in collections with a total of 1.8 x 106 CD34/kg for reinfusion. Patient expired one month after collection without reinfusion. Conclusion: Two patients who failed standard mobilization for stem cell collection at our institution received teriparatide as part of an additional stem cell mobilization regimen. Adequate doses of stem cell products for transplant were collected. One patient was reinfused and subsequently engrafted appropriately. Teriparatide can be used in the setting of poor mobilization. Disclosures No relevant conflicts of interest to declare.

Unexpected Novel Findings That Caspase-1-KO Mice Are Poor Mobilizers and Engraft Poorly with Wild Type Bone Marrow Cells - Indicating a Presence of an Autocrine Feedback Loop Involving Interleukin 1b and Interleukin 18 Signaling That Potentiates Nlrp3 Inflammasome Activity, Both in HSPCs and in the BM Microenvironment for Optimal Stem Cell Trafficking

Background . Our recent results indicate that Nlrp3 inflammasome expressed in hematopoietic stem/progenitor cells (HSPCs) and in the bone marrow (BM) microenvironment are required for optimal mobilization of murine cells (Leukemia 2020 Jun;34(6):1512-1523, Stem Cell Rev Rep. 2019 Jun;15(3):391-403). Moreover, Nlrp3 inflammasome expressed in these cellular contexts are also required for normal homing and engraftment after hematopoietic transplantation (Stem Cell Rev and Rep (2020) in press. https://doi.org/10.1007/s12015-020-10005-w). Consistent with these results, Nlrp3-KO mice are poor mobilizers, cells from Nlrp3-KO mice engraft poorly, and, as transplant recipients, Nlrp3-KO mice show a delay in hematopoietic recovery after transplantation of normal BM cells. Activation of Nlrp3 inflammasomes leads to the release in caspase-1-dependent manner from HSPCs and cells in the BM microenvironment of two potent pro-inflammatory cytokines, interleukin 1b (IL-1b) and interleukin 18 (IL-18). Hypothesis. Based on the aforementioned results, we envisioned that the activated Nlrp3 inflammasome caspase-1 autocrine feedback loop involving IL-1bor IL-18 signaling potentiates HSPCs trafficking by amplifying Nlrp3 inflammasome activation in HSPCs and the BM microenvironment.Materials and Methods. We employed normal control and caspase-1-KO mice for mobilization and homing/engraftment experiments. Mice were mobilized with G-CSF or AMD3100 in the absence or presence of administered IL-1b or IL-18, and we measured i) the total number of white blood cells (WBCs) and ii) the number of clonogenic colony-forming unit granulocyte/macrophage (CFU-GM) progenitors and Sca-1+c-kit+lineage- (SKL) cells circulating in PB. Next, caspase-1-KO cells were transplanted into lethally irradiated wild type animals, while caspase-1-KO mice were transplanted with wild type BMMNCs. We then evaluated homing and engraftment by measuring the number of PKH27-labeled cells, the number of clonogenic progenitors 24 hours after transplantation, and the numbers of day-12 CFU-S colonies and day-12 CFU-GM progenitors in the BM of recipient mice. In control experiments, to provide proof of our hypothesis, we also mobilized Nlrp3-KO mice with AMD3100 or G-CSF in the presence of administered IL-1b and IL-18. Results. We found that IL-1b or IL-18 alone mobilizes HSPCs and, significantly, that caspase-1-KO mice were poor mobilizers. Radiation chimera experiments revealed that this pro-mobilizing defect is dependent on the lack of caspase-1 in hematopoietic cells. To our surprise, administration of IL-1b and IL-18 did not improve G-CSF- or AMD3100-induced mobilization in Nlrp3-KO animals. We also found that both of these pro-inflammatory cytokines activate Nlrp3 inflammasomes in an Myd88-dependent manner. Moreover, HSPCs from caspase-1-KO mice show defective migration in response to a BM-released SDF-1 gradient and other supportive homing chemoattractants (sphingosine-1 phosphate; S1P and extracellular adenosine triphosphate; eATP). Finally, caspase-1-KO mice recipients were also engrafted poorly with normal HSPCs. To explain this phenomenon, we observed that the Nlrp3 inflammasome-caspase-1-IL-1b axis is important in upregulating SDF-1 and other key HSPC chemoattractants in the BM microenvironment conditioned for transplantation. Conclusions. We demonstrate for the first time that an Nlrp3 inflammasome-caspase-1-IL-1b/IL18 autocrine feedback mechanism operating in HSPCs and the BM microenvironment is involved in the normal trafficking of HSPCs during mobilization and homing/engraftment. This research demonstrates the unexpected role of caspase-1, which is situated downstream of the Nlrp3 inflammasome and directs the release of the potent pro-inflammatory cytokines IL-1b and IL18 that by feedback mechanism, in turn, activate Nlrp3 inflammasomes in HSPCs and the BM microenvironment, which is required for optimal trafficking of HSPCs. These results are important for understanding the novel casapase-1-mediated autocrine mechanisms involved not only in pharmacological mobilization but also in the inflammatory response of the organism to external challenges, such as infection and tissue/organ damage. Disclosures No relevant conflicts of interest to declare.

Administration of Plerixafor in Poor Mobilizers Allows Mobilization of Haematopoietic Progenitors: Clonogenic Potential and Engraftment Analysis

BACKGROUND Plerixafor (PLX) blocks the binding of stromal cell-derived factor to CXCR4, resulting in hematopoietic stem cell (HSC) release from the bone marrow. It has been successfully used as a mobilizing agent in poor mobilizers (PM). The best strategy is probably the "pre-emptive" (on demand) use, as it allows an "on time" identification of PMs, preventing collection failure and need for further mobilization. Usually a cut-off of < 10/ μL CD34+ cell count at leukocyte recovery is used for PLXa dministration, while a minimum level is not defined, as mobilization may be obtained even with very low values. An important issue that has not been extensively explored so far is represented by the biological characteristics of the yields collected with PLX, especially clonogenicity, and its clinical counterpart, represented by the engraftment times observed after transplant. In this report we retrospectively analysed our data on the use of PLX in PMs, evaluated its efficacy and focused on the clonogenicity of collected stem cells and engraftment post auto transplant (ASCT). METHODS We collected data on PM patients mobilized with PLX between 2011 and 2019:Clinical data: mobilization and collection performance, transplant rate and engraftment times (days to PMN > 500/uL).Biological data: Clonogenicity of HSC collected after PLX, measured by hematopoietic progenitor semi-solid cultures according to Stem Cell Technology. These clinical and biological data were then compared to those obtained in patients mobilized without PLX and in allogeneic donors who received G-CSF in the same period. RESULTS From January 2011 to June 2019 PLX was used in 73 mobilization cycles, performed in 64 patients. Patients characteristics are described in Table 1. 105 doses of PLX were given during 73 mobilization cycles (1,44 doses/cycle) and 93 collections performed (1,27/ cycle). Circulating CD34+ pre PLX administration were in median 6,81/µl (range: 0,27-21), while after treatment 26,20/µl (range: 4,8-155,4). Using a cut-off of ≥ 10/ μL CD34+, a successful mobilization was achieved in 67/73 cycles (success rate 91,7%). Overall 59 out of 64 patients achieved the collection target (92,2%) at any time and 54/64 have been transplanted (84,4%), the other 10 not yet due to mobilization failure (n=2), insufficient yield (n=3), clinical unfitness (n=2) or because too early (n=3). A median of 4 HPC bags were reinfused (range: 2-12) and median time to WBC engraftment was 10 days (8-21). Engrafment times are in line with those of MM and NHL patients mobilized without PLX in the same period of time (median: 10, range: 8-12) at our Institution. Interestingly, 33 out of 35 pts mobilized with PLX (94,2%), compared to 442/449 (98,4%) engrafted in ≤11 days. We analyzed the cellular composition of yields and clonogenicity of HSC collected in 3 different groups:Group A: PLX mobilization (n=93)Group B: chemo + G-CSF without PLX (n=755)Group C: G-CSF mobilization in allogeneic donors (n=206) We failed to demonstrate a statistical difference between clonogenicity in group A and B (p=0,691), while clonogenicity was slightly but significantly higher in group C compared to group A and B (p< 0,001). Data are shown in tables 2,3 and 4. CONCLUSION In our cohort of patients, administration of PLX in PM resulted in successful mobilization of HPCs with good clonogenicity and engraftment potential. The use of PLX allowed an high proportion of patients to undergo ASCT. Disclosures No relevant conflicts of interest to declare.

Evaluation of the Role of Plerixafor in Peripheral Blood Progenitor Cell Mobilization and Collection in Autologous Donors with Multiple Myeloma and Non-Hodgkin Lymphoma

Purpose Autologous hematopoietic stem cell transplantation is commonly used as treatment for patients with Multiple Myeloma (MM) or Non-Hodgkin Lymphoma (NHL). The mobilization and collection of peripheral blood progenitor cells (PBPC) to reach the required cell dose for transplantation is problematic for some patients when standard mobilization agents such as G-CSF, alone or combined with chemotherapy, are used. These patients are considered poor mobilizers with < 20 CD34+ cells /μl on the planned day of collection and the optimal strategy to collect a sufficient number of PBPC in poor mobilizers is still not fully known. Plerixafor, a CXCR4 inhibitor, when added to the standard mobilization regimen, has been shown to improve mobilization and increase the probability of collecting a PBPC graft > 2 X106CD34 /kg in standard mobilizers. In our center, Plerixafor became available in October 2012. However, how best to use plerixafor to improve care for poor mobilizers while ensuring the most cost-effective use of health care resources for this patient population remains an important question. To begin answering this question, we evaluated the impact of plerixafor usage in our population of poor mobilizers in our institution using a historical cohort for comparison. Material and method The population of poor mobilizers in the setting of MM or NHL (defined as < 20 CD34 / μl on the planned day of collection) between January 2009 and December 2017 at Hôpital de l'Enfant Jesus (HEJ) du CHU de Québec was retrospectively studied and analyzed. To ensure that all poor mobilizers were included, we screened all flow cytometry CD34+ cell count results < 50 /μl on the planned day of collection and reviewed all the charts of patients with < 20 CD34 / μl regardless of whether they were collected or not. This chart review allowed a comprehensive description of the characteristics of our poor mobilizers, a comparison of the capacity to collect an adequate PBPC with or without plerixafor and to evaluate its impact on different variable with regard to PBPC collection and subsequent engraftment. The data were compiled in an ACCESS database using a retrospective review of digital patient charts. The data was analyzed by the Centre de Recherche du CHU de Quebec (CRCHUQ). Data were stratified by disease type and CD34 level on the planned day of PBPC collection. Results 207 patient charts were reviewed; 70 poor mobilizers with 95 mobilizations attempts, 30 of them using plerixafor, were included. The baseline characteristics of both groups were similar as well as the length of hospitalization for transplant and relapse free survival at one year. The addition of Plerixafor to our management of poor mobilizers did not significantly increase the proportion of patients proceeding to autologous PBPC transplant (80,4 vs 75% p=0,7599). During the mobilization attempts, the maximal CD34 /μl reached was similar in both groups (18,86 vs 15,4 p=0,1541). Plerixafor was associated with a superior collection efficiency (% of CD34 collected) (40,85 vs 60,91% p=0,007), but this can be explained by a greater use of central venous catheters for collection in this group (70 vs 95,1% p = 0,0275). Data stratified by initial diagnosis and CD34 level on the planned day of PBPC collection (<10 c/μl versus [10c/μl - 20c /μl [) showed the same tendency. The collection duration was not significantly reduced in the plerixafor group, but there was a trend for a shorter mean duration of 22 minutes also explained by a greater use of central venous catheters in this group. The preplanned subgroups analyses have shown the same trend when stratified by diagnosis, with a reduction of 53 minutes in the multiple myeloma subgroup with a greater total CFU-GEM / kg per collect (46,35 vs 70,55 p=0,0163). Conclusion The use of plerixafor at HEJ did not improve significantly the proportion of poor mobilizers reaching an adequate PBPC graft and did not improve transplant outcomes compared to a historical control. Better strategies are needed to improve care for true poor mobilizers. The optimal use of plerixafor needs to be further studied. Table. Disclosures Blais-Normandin: Sanofi: Other: unrestricted research grant. Laroche:Sanofi: Other: unrestricted research grant.

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.

Novel Evidence That Extracellular Nucleotides and Nucleosides Regulate the Expression of Heme Oxygenase 1 (HO-1) in Opposite Ways in Hematopoietic Stem/Progenitor Cells (HSPCs), Which Explains Why ATP Enhances Mobilization of HSPCs, While Its Metabolite Adenosine Inhibits This Process

Background . One of the problems with the mobilization of hematopoietic stem/progenitor cells (HSPCs) in the clinic is that a significant number of patients are poor mobilizers. In order to develop more efficient mobilization strategies, we have to better understand the mobilization process at the molecular and cellular levels. We have reported that HSPCs express heme oxygenase 1 (HO-1), which is a negative regulator of the complement cascade (ComC), which in turn is required for stem cell mobilization (Stem Cell Rev. 2015, 11:110-8, Leukemia. 2017, 31:446-458). Moreover, very recently we became interested in the novel role of extracellular nucleotides (EXNs) and nucleosides in this process (Leukemia 2018, in press, doi: 10.1038/s41375-018-0122-0). The most important EXN is adenosine triphosphate (ATP), which is involved in intracellular energy transfer but if released from activated cells into the extracellular space becomes an important signaling molecule involved in purinergic signaling. Secretion of ATP from activated cells occurs in a pannexin channel-dependent manner and is additionally augmented by a positive regulatory loop after ATP binding to the P2X7 purinergic receptor. We recently found that the ATP concentration during the mobilization process increases to micromolar concentrations in the BM microenvironment. Moreover, we found that in the extracellular BM space ATP is processed by the CD39 and CD73 ectonucleotidases to ADP, AMP, and finally to adenosine, which is also an important mediator of purinergic signaling. Hypothesis. We hypothesized that EXNs and purinergic signaling via extracellular ATP and its metabolite adenosine are involved in triggering the mobilization of HSPCs and that this effect is mediated by the expression of HO-1 in HSPCs. Materials and Methods. To address this question, 2-month-old P2X7-/-, CD39-/-, and CD73-/-mice as well as their normal wild type (WT) littermates were mobilized with G-CSF or AMD3100. 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. The secretion of ATP from BM cells was inhibited by employing the pannexin 1 blocking drug probenecid or a synthetic pannexin 1 blocking peptide. In parallel, we evaluated i) the expression of HO-1 in HSPCs by employing RQ-PCR and western blot analysis and ii) the activation of the ComC by C5a ELISA. Results. Here we provide evidence that ATP, as an EXN secreted in a pannexin-1-dependent manner from BM cells, triggers activation of the ComC and initiates mobilization of HSPCs. Inhibition of the pannexin 1 channel by probenecid or a pannexin 1 blocking peptide inhibited this process. Furthermore, mobilization of HSPCs was augmented in a P2X7 receptor-dependent manner, evidenced by the finding that P2X7-/- mice were poor mobilizers. Furthermore, ATP is processed to adenosine in the extracellular space by CD39 and CD73 ectonucleotidases, and to our surprise we observed that CD73-deficient mice mobilize more HSPCs than their control wild type (WT) littermates, which indicates a novel negative role for adenosine in the mobilization process. This finding was confirmed by injecting mice with adenosine during AMD3100 administration. Finally, we found that ATP and adenosine modulate the mobilization process by regulating in opposite ways HSPC expression of HO-1, which is a negative regulator of mobilization. Therefore, while ATP downregulates expression of HO-1 in HSPCs and enhances mobilization, adenosine upregulates its expression and negatively affects the egress of HSPCs from BM into PB. Conclusions. We demonstrate for the first time that purinergic signaling involving ATP and its metabolite adenosine regulate the mobilization of HSPCs in an HO-1-dependent manner. While ATP triggers and promotes this process, adenosine has an inhibitory effect. The opposite effects of ATP and adenosine on the mobilization process can be explained by their different effects on the regulation of HO-1 expression in HSPCs. Finally, administration of ATP as a signaling molecule (together with G-CSF or AMD3100), inhibition of CD73 or HO-1 by small-molecule antagonists may provide the basis for more efficient mobilization strategies. Disclosures No relevant conflicts of interest to declare.