Hematopoietic Cell Mobilization Research Articles

Novel Small Molecule VLA-4 Inhibitors Optimized for Extended Hematopoietic and Progenitor Cell Mobilization

Background: Hematopoietic stem and progenitor cell (HSPC) transplant is an essential treatment for a variety of blood disorders and malignancies, and more recently, for gene editing therapies such as sickle cell anemia. A key step in this procedure is the effective mobilization of a large quantity of primitive donor HSPCs to ensure a durable and long lasting engraftment capable of developing into all blood cell lineages. The most commonly used regimen for donor mobilization is a 5-day course of Granulocyte Colony Stimulating Factor (G-CSF). However, G-CSF does not effectively mobilize a sufficient number or quality of stem cells in all donors, can have unwanted side effects, and is contraindicated in sickle cell disease. Therefore, it is desirable to identify an alternative treatment that is safe, rapid and cost effective. The cell surface integrin receptor VLA-4 (a4b1) and the chemokine receptor CXCR4 are essential for HSPC homing and retention in the bone marrow. Inhibition of these receptors rapidly mobilizes HSPCs within an hour, as opposed to the multi-day process using G-CSF. Small molecule inhibitors of VLA-4 have been reported previously, but most have poor pharmacokinetic properties and/or weak inhibition of VLA4. These inhibitors rapidly mobilize HSPCs into the peripheral blood of mice after a single s.c. injection, but their effect is short lived, yielding an inadequate number of HSPCs necessary for stem cell transplantation or for gene therapy. Therefore, VLA4 inhibitors with improved pharmacokinetic properties imparting extended and greater HSPC mobilization after a single dose is desirable. Methods: We designed and synthesized multiple novel VLA-4 inhibitor molecules and tested their potency using soluble VCAM-1 binding assays. Inhibitors determined to be most potent with improved aqueous solubility were then tested in vivo in DBA mice for extended HSPC mobilization beyond 4 hours after a single SC injection, alone and in combination with the CXCR4 inhibitors. Results: We developed two main core molecules that each incorporate key VLA4 receptor binding motifs critical for potent inhibition of VLA4 binding to VCAM-1. Our first generation of inhibitors were quite potent and rapidly mobilized HSPCs, but their effect was short lived and yielded insufficient numbers of HSPCs for gene editing and engraftment studies. Most had poor solubility and very short plasma half-lives after s.c. injection in mice. We designed later generation analogues that strategically exploited a region of each optimized core, allowing for covalent addition of polyethylene glycol (PEG) chains of increasing length without decreasing potency. We determined that a minimum PEG length of 24 PEG units was required for extended HSPC mobilization in mice. WU-106, one of our VLA-4 inhibitors containing 24 PEG units, exhibited excellent aqueous solubility, longer plasma half-life after s.c. dosing, and mobilized 2-fold more murine HSPCs for a longer time than previously published best-in-class VLA-4 inhibitors or our first generation VLA4 inhibitor leads. Importantly, a synergistic effect on HSPC mobilization was achieved when WU-106 was co-administered with CXCR4 inhibitors (Plerixafor or BL-8040). Further optimization by the addition of longer PEG chains, even up to 40KD, retained sub nM potency and extended mobilization beyond 24 hours after a single injection. Our 20kD PEG analogues attached to our two different core molecules (WU-125 and WU-133) allowed for dosing of the drug in the evening, with maximum mobilization still present the next morning, thereby enabling HSPC collection to initiate soon (2-4 hours) after CXCR4 inhibitor dosing in the morning. The quantity of HSPCs obtained using this staggered dosing regimen was greater than simultaneous administration of the VLA-4 and CXCR4 inhibitors ( Fig. 1). Furthermore, our novel, long-acting PEG analogues demonstrated superior HSPC mobilization compared to an alpha 4-targeted antibody (anti-CD49d) in mice ( Fig.2). Conclusion: We have developed novel, potent PEGylated small molecule VLA-4 inhibitors that have excellent aqueous solubility and pharmacokinetics. The kinetics and magnitude of HSPC mobilization by our PEGylated inhibitors are superior to other previously described VLA-4 inhibitors and an antibody to alpha-4(anti-CD49d).

Combination of Propranolol and a Novel CXCR4 Antagonist Burixafor (GPC-100) for Enhanced Hematopoietic Cell Mobilization

Burixafor (GPC-100), a novel CXCR4 antagonist, has shown safe and effective mobilization of hematopoietic stem and progenitor cells (HSPC) in a phase I clinical study. In multiple myeloma patients, GPC-100 combined with granulocyte-colony stimulating factor (G-CSF) elicited a significant increase in circulating HSPCs that was comparable with the historical results from G-CSF plus the CXCR4 antagonist plerixafor. GPC-100 is currently being investigated for HSPC mobilization in combination with the FDA-approved ß-adrenergic receptor (ß-AR) blocker propranolol in a phase II clinical trial for autologous stem cell transplant (ASCT) in multiple myeloma. Here, we present preclinical findings indicating that the addition of propranolol to GPC-100 alone or GPC-100 and G-CSF combination can address the unmet needs of patients undergoing mobilization. Additionally, we show an enhanced and unique pattern of lymphoid cell mobilization that may have applications in adoptive cell therapies. Compared to chemotherapy, ASCT significantly improves survival in multiple myeloma patients. Because HSPCs constitute a very small fraction (~ 0.1%) of circulating mononuclear cells, it is critical to mobilize a sufficient number of HSPC into peripheral blood during the ASCT process. Current therapies for HSPC mobilization include G-CSF as a monotherapy or combined with plerixafor. However, mobilization failure is experienced by 15-50% patients, leading to potential loss of ASCT as a treatment option and significant toxicity from repeated mobilization attempts. Moreover, G-CSF is contraindicated in sickle cell disease and may exacerbate autoimmune diseases. Therefore, our goal is to identify novel strategies that will not only address the mobilization failure, but also explore non-G-CSF options for adequate mobilization. G protein-coupled receptors like CXCR4 can form heteromers with other receptors and result in altered signaling pathways. Using Proximity Ligation Assay in Namalwa (lymphoma) cells, we show that endogenously expressed CXCR4 and ß 2AR form heteromers, which are absent in CXCR4 knockout cells. In cell-based assays, co-activation of both receptors with their respective ligands, CXCL12 and epinephrine, leads to a synergistic increase in calcium flux (Figure 1) as well as ß-arrestin recruitment, indicating functional consequences of the heteromerization. Complete blockade of this enhanced signaling is only achieved with co-inhibition by both antagonists. Like CXCR4, ß 2AR is also expressed by HSPCs, and in lymph nodes was shown to heteromerize with CXCR4 to increase lymphocyte retention. In multiple myeloma patients, propranolol upregulated stem-cell like gene signature. Therefore, we sought to understand the effects of propranolol on HSPC mobilization by GPC-100. In mice, single injection of GPC-100 effectively mobilized hematopoietic stem cells characterized as Lin negSca1 +cKit +(LSK) cells as well as mature Lin negCD34 + progenitor cells by 2-4-fold, respectively. Seven-day pretreatment with propranolol enhanced LSK cell mobilization by GPC-100 resulting in a 4-fold increase in circulating HSPCs (Figure 2). Addition of propranolol to G-CSF and GPC-100 led to 8-17-fold increases in mobilization of long-term repopulating HSCs (CD34 negLSK and CD150 +LSK), which were greater than the 4-7-fold increases elicited by G-CSF and G-CSF with plerixafor. Additionally, propranolol enhanced GPC-100 induced mobilization of white blood cells, neutrophils, and lymphocytes (Figure 2). Immune cell subset analyses revealed that GPC-100 induced a 3-fold increase in circulating CD8 +T cells, which was increased to 7-fold when combined with propranolol (Figure 2). For comparison, plerixafor increased CD8 + T cell mobilization by only 2-fold. GPC-100 induced mobilization of NK and B cells was also improved by propranolol. These pre-clinical data support our phase II clinical trial currently in progress (NCT05561751). In the first arm of this two-arm trial, GPC-100 is combined with orally administered propranolol, and in the second arm, G-CSF is added to this combination. We anticipate that our clinical studies can fill the gap in the current standard of care without adding a significant financial burden or co-morbidities. Furthermore, enhanced mobilization of CD8+ T cells can provide extended opportunities for improved adoptive cell therapies.

GPC-100, a novel CXCR4 antagonist, improves in vivo hematopoietic cell mobilization when combined with propranolol.

Autologous Stem Cell Transplant (ASCT) is increasingly used to treat hematological malignancies. A key requisite for ASCT is mobilization of hematopoietic stem cells into peripheral blood, where they are collected by apheresis and stored for later transplantation. However, success is often hindered by poor mobilization due to factors including prior treatments. The combination of G-CSF and GPC-100, a small molecule antagonist of CXCR4, showed potential in a multiple myeloma clinical trial for sufficient and rapid collection of CD34+ stem cells, compared to the historical results from the standards of care, G-CSF alone or G-CSF with plerixafor, also a CXCR4 antagonist. In the present study, we show that GPC-100 has high affinity towards the chemokine receptor CXCR4, and it potently inhibits β-arrestin recruitment, calcium flux and cell migration mediated by its ligand CXCL12. Proximity Ligation Assay revealed that in native cell systems with endogenous receptor expression, CXCR4 co-localizes with the beta-2 adrenergic receptor (β2AR). Co-treatment with CXCL12 and the β2AR agonist epinephrine synergistically increases β-arrestin recruitment to CXCR4 and calcium flux. This increase is blocked by the co-treatment with GPC-100 and propranolol, a non-selective beta-adrenergic blocker, indicating a functional synergy. In mice, GPC-100 mobilized more white blood cells into peripheral blood compared to plerixafor. GPC-100 induced mobilization was further amplified by propranolol pretreatment and was comparable to mobilization by G-CSF. Addition of propranolol to the G-CSF and GPC-100 combination resulted in greater stem cell mobilization than the G-CSF and plerixafor combination. Together, our studies suggest that the combination of GPC-100 and propranolol is a novel strategy for stem cell mobilization and support the current clinical trial in multiple myeloma registered as NCT05561751 at www.clinicaltrials.gov.

Steady-state versus chemotherapy-based hematopoietic cell mobilization after anti-CD38-based induction therapy in newly diagnosed multiple myeloma.

The incorporation of anti-CD38 monoclonal antibodies (mAb) in induction regimens of newly diagnosed transplant-eligible multiple myeloma (MM) patients has been established as a new standard. However, the optimal strategy of stem cell mobilization in this context is not yet clear. From May 2020 till September 2022, we retrospectively reviewed patients receiving anti-CD38 mAb-based induction therapy followed by stem cell mobilization either in a steady-state protocol (SSM) using 10 μg/kg granulocyte colony-stimulating factor (G-CSF) for 5 days or in a chemotherapy-based protocol (CM) using 1-4 g/m2 cyclophosphamide and G-CSF. Overall, 85 patients (median age 61 years) were included in the analysis. In total, 90 mobilization attempts were performed, 42 with SSM and 48 with CM. There was no significant difference in the median concentration of CD34+ cells in peripheral blood (PB) prior to apheresis between SSM and CM (61/μL vs. 55.4/μL; p = .60). Cumulative CD34+ yields did not differ between the groups with median of 6.68 and 6.75 × 106 /kg body weight, respectively (p = .35). The target yield (≥4 × 106 CD34+ cells/kg body weight) was reached in 88% (CM) and 86% (SSM), with a high proportion even after a single apheresis session (76% vs. 75%). Plerixafor was found to be more frequently used in SSM (52%) than in CM (23%; p < .01). A total of 83 patients underwent autologous transplantation and all were engrafted. Stem cell collection in patients undergoing anti-CD38-based induction therapy is feasible with either CM or SSM, although SSM more frequently requires plerixafor.

Non-interventional Study Evaluating the Mobilization of Stem Cells by Plerixafor Before Salvage Autologous Stem Cell Transplant in Relapsed Multiple Myeloma (IFM-2015-03)

IntroductionDespite the implementation of new therapeutic agents, management of relapsed multiple myeloma (MM) remains a challenge. Salvage autologous hematopoietic cell transplant (AHCT) remains a valid therapeutic option for eligible patients who achieve prolonged response after a first AHCT. However, a second graft is not always available, and these patients may need a second mobilization.Patients and MethodsThis prospective, non-interventional, multicenter study aimed to collect data on the feasibility of salvage AHCT using a plerixafor-based hematopoietic cell mobilization in relapsed MM, according to the plerixafor label in France. Adult patients with relapsed MM eligible for a second AHCT and mobilized using granulocyte- colony stimulating factor (G-CSF) and plerixafor were included.ResultsOf the 23 patients, 17 achieved a successful hematopoietic cell mobilization and 13 were able to proceed to a second AHCT. Median age was 62.9 years (min–max 51–71). Ten patients (77%) were male. Eleven (85%) received AHCT as a third-line treatment or more. Median time between first and second AHCT was 5.4 years (range, 2.6–16.3). Among 18 evaluable patients, mobilization was successful for 17 (94%) of them [95% CI 84–100], with no reported side effects. Among the 13 patients who underwent salvage AHCT, the median time to engraftment was 14 days (min–max 11–29). One-year progression-free and overall survival were 88.9% [95% CI 43.3–98.4] and 100%, respectively.ConclusionThis study demonstrated that plerixafor allows safe and efficient mobilization in relapsed MM patients who are candidates for a salvage AHCT.Trial RegistrationNCT02439476 Registered 8 May 2015, https://clinicaltrials.gov/ct2/show/NCT02439476.

NCCN Guidelines® Insights: Hematopoietic Cell Transplantation, Version 3.2022.

The NCCN Guidelines for Hematopoietic Cell Transplantation (HCT) provide an evidence- and consensus-based approach for the use of autologous and allogeneic HCT in the management of malignant diseases in adult patients. HCT is a potentially curative treatment option for patients with certain types of malignancies; however, recurrent malignancy and transplant-related complications often limit the long-term survival of HCT recipients. The purpose of these guidelines is to provide guidance regarding aspects of HCT, including pretransplant recipient evaluation, hematopoietic cell mobilization, and treatment of graft-versus-host disease-a major complication of allogeneic HCT-to enable the patient and clinician to assess management options in the context of an individual patient's condition. These NCCN Guidelines Insights provide a summary of the important recent updates to the NCCN Guidelines for HCT, including the incorporation of a newly developed section on the Principles of Conditioning for HCT.

Long-term follow-up of autologous peripheral blood hematopoietic stem cell transplantation for refractory lupus nephritis-a series study of 20 patients.

Autologous hematopoietic cell transplantation (ASCT) improves immunologic homeostasis in autoimmune diseases. ASCT-treated refractory lupus nephritis (LN) has been reported. Nevertheless, the long-term outcome of patients with refractory LN after ASCT remains unknown. This study reports the outcomes of 20 refractory lupus patients with 10-year of follow-up after receiving ASCT. Twenty-two patients with LN refractory to immunosuppressive therapy were enrolled. Twenty patients were examined closely and two cases died within 100 days after ASCT. Hematopoietic cell mobilization with cyclophosphamide and granulocyte colony-stimulating factor (G-CSF) was followed by collection of CD34+ positively selected cells. The conditioning regimen consisted of intravenous cyclophosphamide, rabbit antithymocyte globulin, methylprednisolone, and G-CSF. All immunosuppressive therapies were discontinued at the start of mobilization and corticosteroids were tapered rapidly after ASCT. Data was collected from 22 patients with refractory LN treated by ASCT. 59% were female, duration of lupus before ASCT was 46 (33-71) months, and median duration of follow-up after ASCT was 89.5 (56-108) months. 20 long-term followed up patients had an average follow-up time of 92 months (63.25-109.5). Eighteen patients achieved complete remission, one patient reached partial remission, one patient without remission started peritoneal dialysis at month 12, and one patient received short-term renal replacement therapy before ASCT started hemodialysis at 84 months after transplantation. Nine patients relapsed 10 times during the follow-up, and three patients received rituximab. Two patients relapsed during pregnancy after complete response and the Apgar scores of infants were 9 and 10, respectively. All nine patients received glucocorticoids and immunosuppressive medication after relapse and responded again. The 10-year overall survival, 10-year disease-free survival rate, and 10-year renal survival were 100%, 35%, and 90%, respectively. The rate of relapse was 45%. Complications included hypocytosis, infection, B-type insulin resistance syndrome, and monoclonal immunoglobulinemia. This study suggests ASCT is effective and safety in treating refractory LN and is beneficial to improve their long-term outcomes.

Comparison Between Standard and High Dose of G-CSF for Mobilization of Hematopoietic Progenitors Cells in Patients and Healthy Donors.

A standard dose of 10 µg/kg/day granulocyte colony stimulating factors (G-CSF) is currently recommended for hematopoietic progenitor cells (HPCs) mobilization. Our aim was to analyze whether certain patients or healthy donors could benefit from high dose of G-CSF.We performed a retrospective multicenter analysis of HPCs mobilization procedures (2015-2020) in patients and healthy donors. Those who received standard dose of G-CSF (10 µg/Kg/day for 4 days to patients and healthy donors) and those that received higher dose (24 µg/Kg/day for 4 days to patients and 16 µg/Kg/day for 4 days to healthy donors) were compared.496 individuals were included (201 standard dose and 295 higher dose). Between standard or higher dose, we did not find significant differences in median number of mobilized CD34+ cells/mL, neither among healthy donors (77 100 vs 75 500 respectively, P=.895), nor in patients (34 270 vs 33 704 respectively, P=.584). Additionally, among those with the same underlaying pathology the comparison between standard and higher dose did not showed differences. High G-CSF dose was not associated with a less frequent incidence of poor mobilizers (<20 000 CD34+ cells/mL) neither in healthy donors (1 [1.3%] vs 0; P=.218) nor patients (30 [24.4%] vs 32 [18.1%]; P=.165). Multivariate analysis showed that age, gender, and G-CSF dose did not influence median number of mobilized CD34+ cells/mL in healthy donors or patients. However, the underlying pathology among patients significantly influenced the CD34+ cells mobilization. In healthy donors, cellular blood count showed significantly higher leukocytes and platelets count with G-CSF high-dose, while in patients just a higher platelets count was found. To conclude, high dose of G-CSF compared to standard dose did not show significant benefit in terms of mobilization of CD34+ cells in healthy donors or in patients, also without a decrease in the incidence of poor mobilizers.

Long-term follow-up after lymphodepleting autologous haematopoietic cell transplantation for treatment-resistant systemic lupus erythematosus.

Autologous haematopoietic cell transplantation (AHSCT) improves immunologic dysfunction in patients with SLE. However, the curative potential of this therapy remains uncertain. This study reports outcomes in SLE patients receiving a lymphodepleting, reduced intensity regimen for AHSCT in SLE. Eight patients with SLE refractory to treatment, including i.v. cyclophosphamide (CYC), were enrolled. Five had LN and three CNS involvement as primary indications for transplant. Haematopoietic cell mobilization with CYC, G-CSF and rituximab was followed by collection of CD34+ positively selected cells. The conditioning regimen consisted of concurrent administration of CYC, fludarabine and rituximab. All immunosuppressive medications were discontinued at the start of mobilization and CS were rapidly tapered after the transplant. Five of eight patients achieved a complete response, including a decline in the SLEDAI to zero, which was sustained in four patients for a median of 165 months (range 138-191). One patient achieved a partial response, which was followed by relapse at month 18. Two patients with nephritis and underlying comorbidities in most organs had early deaths from infection and multiorgan failure. AHSCT resulted in profound lymphodepletion, followed by expansion of Treg cells and repopulation of naive T and B cells. Patients with a complete response showed a sustained suppression of the SLE-associated IFN-induced gene signature, marked depletion of memory and plasmablast B cells and resultant sustained elimination of anti-dsDNA antibody. Durable clinical and serologic remissions with suppression in the IFN gene signature can be achieved in refractory SLE following lymphodepleting AHSCT. ClinicalTrials.gov, https://clinicaltrials.gov, NCT00076752.

Alterations in microRNA Expression during Hematopoietic Stem Cell Mobilization.

Simple SummaryLymphoproliferative disorders comprise a heterogeneous group of hematological malignancies characterized by abnormal lymphocyte proliferation. Autologous hematopoietic stem cell transplantation plays a very important role in the treatment of lymphoproliferative diseases. The key element in this process is the effective mobilization of hematopoietic cells from the marrow niche to the peripheral blood. Mobilization of HSC is regulated by many factors, out of which miRNAs present in the hematopoietic niche via targeting cytokines, and signaling pathways may play an important regulatory role. This study investigated the expression of selected miRNAs in patients with multiple myeloma, Hodgkin’s lymphomas, and non-Hodgkin’s lymphomas undergoing mobilization procedures. The aim of the study was to evaluate the expression of hsa-miR-15a-5p, hsa-miR-16-5p, hsa-miR-34a-5p, hsa-miR-126-3p, hsa-miR-146a-5p, hsa-miR-155-5p, and hsa-miR-223-3p during the mobilization procedure, and to assess their role in mobilization efficacy. The level of miRNAs was tested at two time points before the initiation of mobilization and on the day of the first apheresis. Our results suggest that the investigated miRNAs, especially hsa-miR-146a-5p, may influence the efficacy of HSC mobilization.microRNAs play an important role in the regulation of gene expression, cell fate, hematopoiesis, and may influence the efficacy of CD34+ cell mobilization. The present study examines the role of hsa-miR-15a-5p, hsa-miR-16-5p, hsa-miR-34a-5p, hsa-miR-126-3p, hsa-miR-146a-5p, hsa-miR-155-5p, and hsa-miR-223-3p in the course of hematopoietic stem cell mobilization. The numbers of CD34+ cells collected in patients with hematological malignancies (39 multiple myelomas, 11 lymphomas) were determined during mobilization for an autologous hematopoietic stem cell transplantation. The miRNA level was evaluated by RT-PCR. Compared to baseline, a significant decline in hsa-miR-15a-5p, hsa-miR-16-5p, hsa-miR-126-3p, hsa-miR-146a-5p, and hsa-miR-155-5p was observed on the day of the first apheresis (day A). An increase was observed only in the expression of hsa-miR-34a-5p. On day A, a negative correlation was found between hsa-miR-15a-5p and hsa-miR-146a-5p levels and the number of CD34+ cells in peripheral blood. A negative correlation was observed between hsa-miR-146a-5p and the number of collected CD34+ cells after the first apheresis. Good mobilizers, defined according to GITMO criteria, demonstrated a lower hsa-miR-146a-5p level on day A than poor mobilizers. Patients from the hsa-miR-146a-5p “low expressors” collected more CD34+ cells than “high expressors”. Our results suggest that the investigated miRNAs, especially hsa-miR-146a-5p, may influence the efficacy of HSC mobilization.

Systems Approach to Discovery of Therapeutic Targets for Vein Graft Disease: PPARα Pivotally Regulates Metabolism, Activation, and Heterogeneity of Macrophages and Lesion Development.

Vein graft failure remains a common clinical challenge. We applied a systems approach in mouse experiments to discover therapeutic targets for vein graft failure. Global proteomics and high-dimensional clustering on multiple vein graft tissues were used to identify potential pathogenic mechanisms. The PPARs (peroxisome proliferator-activated receptors) pathway served as an example to substantiate our discovery platform. In vivo mouse experiments with macrophage-targeted PPARα small interfering RNA, or the novel, selective activator pemafibrate demonstrate the role of PPARα in the development and inflammation of vein graft lesions. In vitro experiments further included metabolomic profiling, quantitative polymerase chain reaction, flow cytometry, metabolic assays, and single-cell RNA sequencing on primary human and mouse macrophages. We identified changes in the vein graft proteome associated with immune responses, lipid metabolism regulated by the PPARs, fatty acid metabolism, matrix remodeling, and hematopoietic cell mobilization. PPARα agonism by pemafibrate retarded the development and inflammation of vein graft lesions in mice, whereas gene silencing worsened plaque formation. Pemafibrate also suppressed arteriovenous fistula lesion development. Metabolomics/lipidomics, functional metabolic assays, and single-cell analysis of cultured human macrophages revealed that PPARα modulates macrophage glycolysis, citrate metabolism, mitochondrial membrane sphingolipid metabolism, and heterogeneity. This study explored potential drivers of vein graft inflammation and identified PPARα as a novel potential pharmacological treatment for this unmet medical need.

淋巴瘤自体造血干细胞动员和采集中国专家共识（2020年版）

淋巴瘤自体造血干细胞动员和采集中国专家共识（2020年版）

Risk Stratified Tandem Vs Single Autologous Stem Cell Transplantation for Multiple Myeloma Yields Equivalent Survival

The standard of care for patients with newly diagnosed multiple myeloma who are transplant eligible is induction therapy followed by conditioning with high dose melphalan and autologous hematopoietic cell transplant (HCT). While the value of a single HCT (S-HCT) is established in standard-risk myeloma (SRM), there is controversy about whether tandem transplantation would improve outcomes such as progression-free survival (PFS) or overall survival (OS) in high-risk myeloma (HRM) patients. In this IRB approved retrospective study, the records of 343 multiple myeloma patients, who were referred by community oncologists and underwent HCT at Virginia Commonwealth University from 2008 to 2015, were reviewed. Patients were classified into different disease risk groups and OS and PFS determined. HRM was defined as including at least one of the following prognostic factors at diagnosis: t(4;14), t(14;16), t(14;20), del17p13, or gain of 1q by FISH; del 13 or aneuploidy by karyotyping; and ISS stage 3; patients without these features and with standard risk attributes were classified as SRM, while any patients with missing attribute information were classified as having undetermined risk myeloma (URM). In the study population, there were 135 SRM, 100 HRM, and 104 URM patients. Median age of the entire cohort was 60 years (76-34). The patients underwent hematopoietic cell mobilization and collection with filgrastim ± plerixafor and were conditioned with high-dose Melphalan (140-200 mg/m2). When possible, patients with SRM underwent single HCT (S-HCT) and those with HRM, tandem HCT (T-HCT) based on risk classification, response to prior therapy, insurance mandate and patient/physician preference. In those undergoing T-HCT, the median time to second HCT was 141 days. Evaluating the entire study population, and comparing patients who underwent a T-HCT (median follow up 82 mo.) vs. a S-HCT (85 mo.), there was no significant difference in OS (adjusted HR 1.1, 95% CI: 0.7-1.8; Cox proportional hazard model, adjusted for age, gender and risk group) (Figure 1) and PFS (HR 0.9, 95% CI: 0.6-1.2) between these two treatment groups. Of the 135 SRM patients, 20 underwent T-HCT; there was no significant difference in OS (HR = 1.2, 95% CI 0.5-2.9) and PFS (HR = 0.9, 95% CI = 0.5-1.7) in these patients compared to those undergoing S-SCT. Of the 100 HRM patients, 46 underwent T-HCT (P &amp;lt;0.01 c/w SRM &amp; URM for T-HCT assignment). Again, there was no significant difference observed in OS (HR = 1.3, 95% CI = 0.7 - 2.5) (Figure 2) and PFS (HR = 1.0, 95% CI = 0.6 - 1.8) in the T-HCT recipients as compared to S-SCT amongst the HRM patients. Similar outcomes were observed in the URM patients. There were no differences observed between S-HCT and T-HCT outcomes in patients with deletion of chromosome 17p or in those with ISS stage 3 disease in OS (HR = 1.7, 95% CI = 0.8 - 3.8) and PFS (HR = 1.0, 95% CI: 0.5, 1.9). Overall, HRM patients had a trend for worse, albeit not significantly different, OS (HR 1.4, 95% CI: 0.9-2.2), as well as PFS (HR 1.1, 95% CI 0.7-1.6) compared to SRM; URM demonstrated a similar trend vis a vis SRM. This retrospective review of survival in SRM and HRM transplanted at a single center demonstrates that in the era of induction and maintenance therapy with novel agents, tandem transplantation does not improve OS or PFS in either SRM or HRM patients compared to a single transplant. Standard of care for newly diagnosed multiple myeloma patients who are transplant eligible should remain induction therapy with novel agents followed by single autologous HCT and maintenance therapy. Disclosures No relevant conflicts of interest to declare.

Presence of Chip-Mutated Autologous Hematopoietic Cells in Mobilized Peripheral Blood Products Is Associated with Shorter Progression-Free Survival after Autologous Transplants for Multiple Myeloma

Multiple Myeloma (MM) remains a cancer of terminally-differentiated plasma cells that reside predominantly within the bone marrow. Malignant plasma cells are nurtured by a permissive microenvironment that favors tumor progression, drug resistance and disease relapse. Recurrent somatic mutations in hematopoietic stem cells, the source of major components in bone marrow niche, lead to age-associated clonal hematopoiesis of indetermined potential (CHIP) and has been associated with inferior survivals among individuals without malignant hematologic disorders. It is possible that these mutations in non-MM cells would affect remission time after transplant. We performed whole exome sequencing to identify CHIP-associated mutations within the autograft utilized to rescue hematopoiesis after high dose melphalan and autologous hematopoietic cell transplant (HCT). We then correlated the presence of CHIP with the outcome of MM patients (pts) following autologous HCT. Methods. MM pts (N=101) that underwent HCT between 2006 and 2017 were studied. DNA was extracted from 1 ml of cryopreserved mobilized hematopoietic cell product. Targeted sequencing was then performed using Tempus xE whole exome platform (Tempus, Chicago, IL) with variable allele frequency (VAF) ≥0.1. Progression-free survival (PFS) and overall survival (OS) were defined as the time from transplantation until event of interest, with censoring at time of last follow up. Cox regression was used for time-to-event outcomes; hazard ratios (HR) and 95% confidence intervals (CI) were reported. P-values were two-sided and those &amp;lt;0.05 were considered statistically significant. Results. The median age was 60.1 years at the time of HCT. 58 pts were male and 43 female; 29 pts were African-American, 1 Asian/Indian and 71 were Caucasian. Seventy five (75%) and 42 (43%) pts had prior exposure to lenalidomide or cyclophosphamide, respectively. Thirty two pts (32%) underwent hematopoietic cell mobilization with GCS-F and plerixafor, 38 pts (38%) with cyclophosphamide and 31 pts (31%) with GCS-F alone. Forty seven pts (47%) had at least one CHIP-associated mutation. The median number of CD34+ yield in the first day was 6.7 x 10e6/kg for pts with CHIP mutations and 5.64 x 10e6/kg for pts without any CHIP mutation (p = 0.4). The presence or absence of CHIP mutations did not impact the yield of CD34+ stem cells collected (Fig. 1). The majority of mutations were either missense variants or mRNA splicing variations (Fig. 2), most commonly SF3B1, ASXL1, TET2 and ASXL2. CHIP mutations were associated with increased age (p= 0.021), but there was no association with prior history of previous cancer, smoking, cytogenetically-defined high risk disease, pre-HCT hemoglobin level or red blood cell mean corpuscular volume. There was also no significant difference between the transplant course between the two groups in terms of neutrophil and platelet engraftment or length of hospital stay. Median follow up was 32 months. During follow up 3 pts develop MDS/AML (2 pts in the CHIP group and one in no CHIP group). Coronary artery disease was the cause of death in one patient in CHIP cohort versus none in the CHIP negative cohort. Myeloma progression was the major cause of death in CHIP and No CHIP cohorts (66% vs. 60%, p=0.48). Median PFS was 19 months in CHIP cohort vs. 39 months in No CHIP group (HR: 0.62, 95% CI: 0.51-0.92, p= 0.001). Median OS has not been reached in the No CHIP group while it was 56 months in CHIP group (HR: 0.78, 95% CI: 0.62-1.04, P=0.09). In multivariate analysis, presence of CHIP remained a significant predictor of worse PFS after adjusting for age, maintenance therapy, high risk disease, performance status, triplet vs. doublet pre-SCT therapy, melphalan dose and MM genetic risk. (Table 2). Conclusion: Here we showed high frequency of large clonal hematopoiesis clones (i.e., VAF ≥0.1) in autograft of MM pts. The presence of this mutations does not impact stem cell yield or transplant course, however it impacts PFS independent of age and other unfavorable factors. Our data highlights high frequency of SF3B1; the role of this mutant in modulating apoptotic factors in myeloma microenvironment should be investigated. Disclosures Malek: Celgene: Consultancy; Amgen: Speakers Bureau; Adaptive: Consultancy; Janssen: Speakers Bureau; Medpacto: Research Funding; Sanofi: Consultancy; Takeda: Consultancy. Caimi:ADC Therapeutics: Research Funding; Celgene: Speakers Bureau; Fate Therapeutics: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kite Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Caldwell:Tempus Inc.: Employment.

621-P: Cardiovascular Mortality and Orthostatic Hypotension in Type 2 Diabetic Patients (T2D) with Lower Limb Lesions—Italian Diabetic Patients' Association-Section of Treviso Project

Introduction: Neuroischemic (NI) diabetic foot (DF) is an advanced complication leading to limb amputations and cardiac mortality. We contributed to demonstrate that bone marrow (BM) T2D neuropathy impairs vascular regenerative hematopoietic cell mobilization. We here hypothesize that autonomic neuropathy (DAN) and pathological orthostatic hypotension (reducing systolic and diastolic blood pressure -BP at least 30 and 10 mm Hg) convert to cardiovascular death in T2D patients with NI-DF. Subjects and Methods: We performed DAN and postural hypotension tests in 204 surgical-treated neuropathic T2D without (87 N) or with (117 NI) critical limb ischemia (pO2 &amp;lt;30 mm Hg). In peripheral blood (PB) (204) and BM (65) samples we evaluated: percentage of haematopoietic precursor CD34+, VEGF receptor KDR+ (PreC) and neurokinin-1 receptor on BM-CD34+ cells (BM-NK1R+preC) by flow cytometry; circulating P substance neuropeptide by ELISA. Results: We observed 46 NI cardiac deaths (DD) and 158 alive patients (AD) after prospective 5 years. DD were older but comparable for diabetes age, BMI, HbA1c, post-revascularization oximetry and TUC lesion grade. All T2D had autonomic failure but only DD were orthostatically hypotensive (-BP -35±2 and -21± 3 mm Hg p&amp;lt;0.01 vs. AD) with different BM/PB preC incremental ratio (8.8±4 vs. 1.5 ±0.3 106 events p&amp;lt;0.0003 vs. AD), similar circulating P levels, reduced BM-NK1R+preC (14.5±8.5 vs. 36.4±5 % p&amp;lt;0.04). Both -BP correlate with BM/PB preC (p&amp;lt; 0.04) but not with BM-NK1R+preC. Conclusions: We prospectively confirm that autonomic failure impacts on vascular homeostasis and cardiac mortality in T2D with prevalent NI-DF. Now we demonstrate that pathological orthostatic hypotension, a simple clinical measure, could contribute to persistent ischemic bone marrow signal in neuroreceptor reduced BM-derived stem cells, worsening BM vascular regenerative capacity and cardiac performance. Disclosure M. Sambataro: None. L. Sambado: None. G. Spinetti: None. E. Seganfreddo: None. E. Trevisiol: None. M. Marcon: None. P. Stefani: None. A. Furlan: None. M. Cacciatore: None. A. Paccagnella: None. Funding Italian Diabetic Patients' Association of Treviso and Oderzo

The role of CXCR2 in acute inflammatory responses and its antagonists as anti-inflammatory therapeutics.

CXCR2 is key stimulant of immune cell migration and recruitment, especially of neutrophils. Alleviating excessive neutrophil accumulation and infiltration could prevent prolonged tissue damage in inflammatory disorders. This review focuses on recent advances in our understanding of the role of CXCR2 in regulating neutrophil migration and the use of CXCR2 antagonists for therapeutic benefit in inflammatory disorders. Recent studies have provided new insights into how CXCR2 signaling regulates hematopoietic cell mobilization and function in both health and disease. We also summarize several CXCR2 regulatory mechanisms during infection and inflammation such as via Wip1, T-bet, P-selectin glycoprotein ligand-1, granulocyte-colony-stimulating factor, and microbiome. Moreover, we provide an update of studies investigating CXCR2 blockade in the laboratory and in clinical trials. Neutrophil homeostasis, migration, and recruitment must be precisely regulated. The CXCR2 signaling pathway is a potential target for modifying neutrophil dynamics in inflammatory disorders. We discuss the recent clinical use of CXCR2 antagonists for controlling inflammation.

Ephrin ligands and Eph receptors contribution to hematopoiesis.

Hematopoietic stem and progenitor cells reside predominantly in the bone marrow. They supply billions of mature blood cells every day during life through maturation into multilineage progenitors and self-renewal. Newly produced mature cells serve to replenish the pool of circulating blood cells at the end of their life-span. These mature blood cells and a few hematopoietic progenitors normally exit the bone marrow through the sinusoidal vessels, a specialized venous vascular system that spreads throughout the bone marrow. Many signals regulate the coordinated mobilization of hematopoietic cells from the bone marrow to the circulation. In this review, we present recent advances on hematopoiesis and hematopoietic cell mobilization with a focus on the role of Ephrin ligands and their Eph receptors. These constitute a large family of transmembrane ligands and receptors that play critical roles in development and postnatally. New insights point to distinct roles of ephrin and Eph in different aspects of hematopoiesis.

In vivo engineering of bone tissues with hematopoietic functions and mixed chimerism

Synthetic biomimetic matrices with osteoconductivity and osteoinductivity have been developed to regenerate bone tissues. However, whether such systems harbor donor marrow in vivo and support mixed chimerism remains unknown. We devised a strategy to engineer bone tissues with a functional bone marrow (BM) compartment in vivo by using a synthetic biomaterial with spatially differing cues. Specifically, we have developed a synthetic matrix recapitulating the dual-compartment structures by modular assembly of mineralized and nonmineralized macroporous structures. Our results show that these matrices incorporated with BM cells or BM flush transplanted into recipient mice matured into functional bone displaying the cardinal features of both skeletal and hematopoietic compartments similar to native bone tissue. The hematopoietic function of bone tissues was demonstrated by its support for a higher percentage of mixed chimerism compared with i.v. injection and donor hematopoietic cell mobilization in the circulation of nonirradiated recipients. Furthermore, hematopoietic cells sorted from the engineered bone tissues reconstituted the hematopoietic system when transplanted into lethally irradiated secondary recipients. Such engineered bone tissues could potentially be used as ectopic BM surrogates for treatment of nonmalignant BM diseases and as a tool to study hematopoiesis, donor-host cell dynamics, tumor tropism, and hematopoietic cell transplantation.

Результаты мобилизации, афереза и аутореинфузии гемопоэтических стволовых клеток у детей с нейробластомой: роль мониторинга количества CD34+ клеток в периферической крови

Результаты мобилизации, афереза и аутореинфузии гемопоэтических стволовых клеток у детей с нейробластомой: роль мониторинга количества CD34+ клеток в периферической крови

The unresolved role of systemic factors in bone metastasis

Systemic factors including cytokines, cell-free nucleic acids, microvesicles, and platelets are appreciated as important regulators of adenocarcinoma progression. Research findings using pre-clinical mouse models have revealed that many such systemically acting factors are either secreted by or responsive to peripheral tumors and impact bone and bone marrow (collectively referred to as the bone microenvironment) to initiate processes that ultimately govern disease progression, even in the absence of detectable bone metastases. In some cases, cancer-driven modulation of the bone microenvironment involves mobilization of bone marrow hematopoietic and mesenchymal cells into the circulation that are subsequently recruited into peripheral tissues and tumors. In other cases, systemic factors alter bone marrow cell (BMC) differentiation and/or gene expression to render the BMCs pro-tumorigenic even prior to their mobilization into the circulation. Given their effect on the bone microenvironment, it stands to reason that such systemic factors might also influence metastases in the bone; however, this hypothesis remains to be comprehensively tested. Here, we briefly review what is known, and not known, about systemic factors that regulate the bone microenvironment and thereby influence bone metastases. We also pose a number of currently unanswered questions in this active area of research. A better understanding of systemic processes that influence bone metastasis should aid discovery of therapeutic approaches that aim to eradicate or reduce disease burden in the bone, which is the cause of significant patient mortality and morbidity and is currently incurable.