Increased Platelet S100A8/S100A9 Associated with Vasculitis in Granulomatosis with Polyangiitis (GPA)

Abstract Granulomatosis with polyangiitis (GPA), formerly known as Wegener's Granulomatosis, is characterized by vasculitis that predominantly affects small- and medium-sized blood vessels in the sinuses, lungs, and kidneys. In addition to vascular inflammation, GPA is also characterized by an increased risk of thrombosis. The role of platelets in GPA pathogenesis remains incompletely understood. We aimed to better understand the changes in platelet gene expression and function in patients with GPA. Forty-two patients diagnosed with GPA (n=9 with active GPA and n=33 with GPA in remission) and 25 healthy, age-, gender-, and race-matched donors were enrolled. Patients with GPA showed typical disease manifestations, with an average Birmingham Vasculitis Activity Score of 1.6 (Mean±SD 1.6±3.5). One sixth of GPA patients (7/42) had a history of thrombosis. When stimulated with thrombin receptor activating peptide (TRAP, 50nM), platelets from patients with GPA showed significantly increased expression of P-selectin as compared to healthy controls (P-selectin+% Mean±SEM: Healthy 15.50±1.84 vs GPA 25.71±16.05, P<0.05). This suggests increased platelet activation in GPA, consistent with previous findings of increased platelet aggregation in vitro in GPA. In addition, released chemokines sCD40L and platelet-derived growth factor (PDGF) by activated platelets were increased in patients with GPA when we measured the cytokines in the platelet poor plasma using the Miliplex human cytokine Assay [sCD40L (ng/mL) Mean±SEM: Healthy 63.05±6.63 vs GPA 100.40±11.86, P<0.05, PDGF-AA (pg/mL) Mean±SEM: Healthy 137.50±46.52 vs GPA 357.30±79.65, P=0.052]. Next, we performed RNA-sequencing on platelets from GPA patients (n=8, 3 with active GPA disease and 5 in remission) and, for comparison, 4 healthy donors. We identified 75 genes that were significantly differentially expressed between GPA patients and healthy donors. The top 30 genes are listed in Figure 1A. S100A8 and S100A9 were the top two significantly differentially expressed transcripts in patients with GPA (Fig. 1B). These two genes encode proteins that form a heterodimer S100A8/S100A9 (commonly known as calprotectin) known to be increased in the plasma of GPA patients and associated with disease activity. Interestingly, platelets have not been identified as the cellular source of plasma calprotectin in GPA previously. Significantly increased RNA and protein expression of S100A8 and S100A9 in GPA patients was independently validated by qRT-PCR and immunoblot, respectively. The mRNA expression of S100A8 and S100A9 in platelets were significantly correlated with p-ANCA and anti-MPO antibodies, indicating platelet S100A8/S100A9 promotes neutrophil activation and inflammation (Mann-Whitney nonparametric test, P<0.05). As previously reported, plasma levels of calprotectin were also increased in GPA patients. To further evaluate if plateletS100A8/S100A9 mediates endothelial inflammation and vasculitis, we co-cultured platelets activated with thrombin (which increases S100A8/S100A9 secretion) with endothelial cells in the presence or absence of an anti-S100A8/S100A9 blocking antibody. Activated platelets triggered endothelial cell inflammation (e.g., increased expression of ICAM-1) that was significantly reduced when S100A8/S100A9 was blocked. In summary, the platelet transcriptome is altered in patients with GPA, with S100A8 and S100A9 being the top upregulated genes. Platelet functional responses are enhanced in patients with GPA, and our data suggests that increased plasma calprotectin levels in GPA patients may be platelet derived. Platelets and platelet S100A8/S100A9 appear to mediate vascular inflammation and thrombosis in GPA. Figure 1 Figure 1. Disclosures Rondina: Novartis: Research Funding; Platelet Biogenesis: Membership on an entity's Board of Directors or advisory committees; Acticor Biotech: Membership on an entity's Board of Directors or advisory committees; Platelet Transcriptomics: Patents & Royalties.

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Anti-Myeloma Activity of a Novel Free Radical Inducer Rrx-001

Blood ◽

10.1182/blood.v124.21.4712.4712 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4712-4712 ◽

Cited By ~ 5

Author(s):

Deepika Sharma Das ◽

Ze Tian ◽

Arghya Ray ◽

Durgadevi Ravillah ◽

Yan Song ◽

...

Keyword(s):

Oxidative Stress ◽

Clinical Trial ◽

Drug Resistance ◽

Board Of Directors ◽

Tumor Cells ◽

Cell Lines ◽

Advisory Committees ◽

Healthy Donors

Abstract Background and Rationale: Multiple Myeloma (MM) remains incurable despite the advent of novel drugs, highlighting the need for further identification of factors mediating disease progression and resistance. The bone marrow (BM) microenvironment confers growth, survival, and drug resistance in MM cells. Studies to date suggest an important role of BM hypoxia (low oxygenation) in MM cell survival, drug resistance, migration, and metastasis. Therapies targeting the MM cell in its BM milieu under hypoxic conditions may therefore achieve responses in patients resistant to various therapies. Recent studies led to the development of a novel aerospace-industry derived Phase 2 molecule RRx-001 with epigenetic and NO-donating properties. RRx-001 generates reactive oxygen and nitrogen species (RONS), which induces oxidative stress in tumor cells. Importantly, RRx-001 is also a potent vascular disrupting agent, which further provides rationale for utilizing RRx-001 as a therapeutic agent since tumor-associated angiogenesis is a characteristic of MM. A Phase I clinical trial has shown RRx-001 to have antitumor activity in heavily pretreated cancer patients and to be safe and well tolerated with no dose-limiting toxicities (Reid et al. J Clin Oncol 32:5s, 2014 suppl; abstr 2578). Here we examined the anti-MM activity of RRx-001 using in vitro and in vivo models of MM. Materials and methods: MM cell lines, patient MM cells, and peripheral blood mononuclear cells (PBMCs) from normal healthy donors were utilized to assess the anti-MM activity of RRx-001 alone or in combination with other agents. Drug sensitivity, cell viability, apoptosis, and migration assays were performed using WST, MTT, Annexin V staining, and transwell Inserts, respectively. Synergistic/additive anti-MM activity was assessed by isobologram analysisusing “CalcuSyn” software program. Signal transduction pathways were evaluated using immunoblotting. ROS release, nitric oxide generation, and mitochondrial membrane potential was measured as previously described (Chauhan et al., Blood, 2004, 104:2458). In vitro angiogenesis was assessed using matrigel capillary-like tube structure formation assays. DNMT1 activity was measured in protein lysates using EpiQuik DNMT1 assay kit. 5-methyl cytosine levels were analyzed in gDNA samples using methylflash methylated DNA quantification kit from Enzo life sciences; USA. For xenograft mouse model, CB-17 SCID-mice were subcutaneously inoculated with MM.1S cells as previously described (Chauhan et al., Blood, 2010, 115:834). Statistical significance of data was determined using a Student’st test. RRx-001 was obtained from RadioRx Inc., CA, USA; bortezomib, SAHA, and pomalidomide were purchased from Selleck chemicals, USA. Results: Treatment of MM cell lines (MM.1S, MM.1R, RPMI-8226, OPM2, H929, Dox-40 ARP-1, KMS-11, ANBL6.WT, ANBL6.BR, and LR5) and primary patient cells for 24h significantly decreased their viability (IC50 range 1.25nM to 2.5nM) (p < 0.001; n=3) without markedly affecting PBMCs from normal healthy donors, suggesting specific anti-MM activity and a favorable therapeutic index for RRx-001. Tumor cells from 3 of 5 patients were obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. Moreover, RRx-001 inhibits proliferation of MM cells even in the presence of BM stromal cells. Mechanistic studies show that RRx-001-triggered apoptosis is associated with 1) induction of DNA damage response signaling via ATM/p53/gH2AX axis; 2) activation of caspases mediating both intrinsic and extrinsic apoptotic pathways; 3) increase in oxidative stress through release of ROS and generation of NO; and 4) decrease in DNA methyltransferase (DNMT1) enzymatic activity and global methylation levels. Furthermore, RRx-001 blocked migration of MM cells and angiogenesis. In vivo studies using subcutaneous human MM xenograft models show that RRx-001 is well tolerated and inhibits tumor growth. Finally, combining RRx-001 with bortezomib, SAHA, or pomalidomide induces synergistic anti-MM activity and overcomes drug resistance. Conclusion: Our preclinical studies showing efficacy of RRx-001 in MM disease models provide the framework for clinical trial of RRx-001, either alone or in combination, to improve outcome in relapsed and refractory MM patients. Disclosures Richardson: Oncopeptides AB: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Oronsky:RadioRx Inc, : Employment. Scicinski:RadioRx Inc,: Employment. Chauhan:Triphase Accelerator: Consultancy. Anderson:Celgene: Consultancy; Millenium: Consultancy; Onyx: Consultancy; Gilead: Consultancy; Sanofi Aventis: Consultancy; BMS: Consultancy; Oncopep/Acetylon: Equity Ownership.

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Low-Dose Interleukin-2 Therapy Activates Circulating T Follicular Regulatory Cells and Suppresses Circulating T Follicular Helper Cells in Patients with Chronic Gvhd

Blood ◽

10.1182/blood.v130.suppl_1.74.74 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 74-74

Author(s):

Yusuke Kamihara ◽

Edouard Forcade ◽

John Koreth ◽

Hongye Liu ◽

Tomohiro Kubo ◽

...

Keyword(s):

B Cell ◽

Single Cell ◽

Board Of Directors ◽

Research Funding ◽

Low Dose ◽

Cell Mass ◽

Mass Cytometry ◽

Advisory Committees ◽

Healthy Donors

Abstract Introduction: T follicular helper (TFH) and T follicular regulatory (TFR) cells play important roles in the regulation of B-cell immunity. While TFH promote B cell functions in the germinal center (GC), TFR function as negative regulators of the GC response. Previous studies in murine models established that TFH and GC B cells are required for the development of chronic graft-versus-host disease (cGVHD). We previously reported that circulating TFH (cTFH) were more functionally activated in patients with active cGVHD compared with patients with no cGVHD. Low-dose IL-2 therapy has been shown to selectively expand CD4Treg and improve cGVHD symptoms. In the current study, we examined the effects of IL-2 therapy on cTFH and circulating TFR (cTFR) in patients with steroid resistant cGVHD. Methods: Single cell mass cytomtery (CyTOF) was performed on cryopreserved peripheral blood mononuclear cells (PBMC) from healthy donors and 17 adult patients with active cGVHD receiving daily low-dose IL-2 therapy (Koreth et al. Blood 2016). A panel of 35 metal-tagged monoclonal antibodies was used to simultaneously examine the phenotypic and functional effects of low-dose IL-2 on lymphocyte populations in vitro and in vivo. The analytic panel included 22 cell surface markers to identify distinct lymphocyte subsets and 13 intracellular markers to measure functional status and activation of specific signaling pathways. Before staining for surface and intracellular antigens, serial samples from individual patients were barcoded to ensure uniformity of analysis. viSNE was used to visualize of high-dimensional data on a two-dimensional map and quantify single cell mass cytometry data. Results: In PBMC from healthy donors, expression of CD25 (IL-2Rα), CD95, CTLA-4, BLIMP-1 and GITR was higher in cTFR compared with cTFH. To examine the response to IL-2 in vitro, PBMC from healthy donors were stimulated with IL-2 for 15 minutes (Figure 1A). At low IL-2 concentrations (1 to 10 IU/mL), phospho-STAT5 (p-STAT5) was selectively activated in cTFR compared with cTFH. At high IL-2 concentrations (100 to 1,000 IU/mL), p-STAT5 was activated in both cTFR and cTFH. To examine the response to IL-2 in vivo, we used mass cytometry to examine serial PBMC samples from cGVHD patients receiving daily low dose IL-2 therapy (1x106 IU/M2/day). Selective expansion of cTFR was noted after 1 week of treatment and cTFR expansion remained stable for the 12 week duration of therapy. Expanded cTFR increased expression of p-STAT5, FoxP3, BCL6, HLA-DR (Figure 1B) and CD25, CD95, CTLA-4, ICOS, Ki67 and Helios 1 week after starting IL-2. cTFR:cTFH ratio increased rapidly after starting low dose IL-2 and paralleled the increased Treg:Tcon ratio (Figure 1C). Activated TFH and TFR can be identified by expression of ICOS and PD-1. The expansion of ICOS+PD-1+ cTFR was evident after 1 week of IL-2 and remained elevated at the end of therapy. In contrast, ICOS+PD-1+ cTFH increased 1 week after starting IL-2 therapy but subsequently decreased and fell below baseline 6 and 12 weeks after starting IL-2 (Figure 1D). Activated ICOS+PD-1+ cTFR expressed higher levels of p-STAT5, BCL-6, FoxP3, HLA-DR and CD25 during low dose IL-2 therapy. In contrast, these functional markers were not increased in ICOS+PD-1+ cTFH during IL-2 therapy (Figure 1B). Conclusion: Single cell mass cytometry analysis revealed that daily low dose IL-2 therapy induces selective activation and increased expression of functional proteins in ICOS+PD-1+ cTFR. In contrast, activated ICOS+PD-1+ cTFH were suppressed during IL-2 therapy. The selective activation of cTFR and suppression of cTFH provide a mechanism whereby low dose IL-2 therapy can promote B cell tolerance as well as T cell tolerance in patients with cGVHD. Disclosures Forcade: Neovii: Other: Travel grant. Koreth: Amgen Inc.: Consultancy; Prometheus Labs: Research Funding; Kadmon Corp: Membership on an entity's Board of Directors or advisory committees; Millennium Pharmaceuticals: Research Funding; Takeda Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Nikiforow: Kite Therapeutics: Membership on an entity's Board of Directors or advisory committees. Armand: Infinity: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding; Otsuka: Research Funding; Tensha: Research Funding; Sequenta/Adaptive: Research Funding; Genmab: Consultancy; Affimed: Research Funding; Sigma Tau: Research Funding; Merck & Co., Inc.: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Roche: Research Funding. Cutler: Bristol-Myers Squibb: Consultancy; Pfizer: Consultancy; Kite: Consultancy; Pharmacyclics: Consultancy; Incyte: Consultancy; Astellas: Consultancy.

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CD19 Redirected CAR NK Cells Are Equally Effective but Less Toxic Than CAR T Cells

Blood ◽

10.1182/blood-2018-99-118005 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3491-3491 ◽

Cited By ~ 4

Author(s):

Concetta Quintarelli ◽

Simona Sivori ◽

Simona Caruso ◽

Simona Carlomagno ◽

Iolanda Boffa ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Board Of Directors ◽

Third Party ◽

Advisory Committees ◽

Car T Cells ◽

Healthy Donors ◽

Car T

Abstract Based on the clinical success observed in acute lymphoblastic leukemia (ALL) with chimeric antigen receptor engineered T (CAR T), we hypothesized that combining the specificity of a CAR with the innate allo-reactivity of KIR-mismatched NK cells might provide a powerful tool for adoptive cell therapy. The use of a third-party bank of CAR-NK cells offers the advantage of an immediate availability to be exploited in the allogenic setting and could be associated with a lower toxicity profile than CAR-T cells. In order to overcome regulatory and manufacturing hurdles associated with generation of CAR-NK cells, we developed a feeder-free culture resulting in a 3.2-log expansion after 20 days of culture. Specifically, natural cytotoxicity receptors (NCR) expressed on NK cells are stimulated in the presence of pleiotropic cytokines and expanded in GMP grade bioreactors. Expanded NK cells from healthy donors preserve a high percentage of CD56+ CD57- cells (85±13%), associated with high proliferative capability, and maintain the surface expression and the responsiveness of NCR and CD16. We proved that NK cells generated from 10 different healthy donors have high ability to recognize and eliminate different tumor types, including acute myeloid leukemia (AML) and ALL. After genetic modification with a retroviral vector encoding a CAR specific for CD19 antigen, transduction of activated NK cells averaged 38%±15% and the CAR.CD19 expression was stable over extended in vitro culture (60 days). Detailed phenotypic characterization of CAR-NK cells showed that CAR expression was not limited to the more mature NKG2A-/KIR+ cells, but rather was distributed across different NK subsets. We also demonstrated that NK and CAR-NK cells display significant anti-leukemia activity towards CD19+ leukemia and lymphoma cell lines (LCL 721.221, DAUDI and BV173) and primary blasts obtained from patients with B-cell precursor ALL (Bcp-ALL). Co-culture experiments using a 1:5 E/T ratio, showed that, while the anti-tumor activity was already remarkable with non-modified effector NK cells (60±30%, 71±33% and 54±23% of residual LCL 721.221, DAUDI and BV173 cells, respectively; p<0.05 vs T cells), it reached the highest level when CAR-NK cells were used as effectors (7±9%, 16±30% and 22±16% of residual LCL 721.221, DAUDI and BV173 cells, respectively; p<0.05 vs non-transduced NK cells). Importantly, INF-g production was significantly lower upon CAR-NK activation compared to CAR-T cells (DAUDI 384±194 ng/ml vs 1860±678 ng/ml, p=0.002). Functional analysis on primary Bcp-ALL blasts, demonstrate that CAR-NK cells exert high degree of leukemia control (on average 2.1±2% vs 5.4±1.6% with non-modified NK cells as effectors; p=0.04). An in vivo model of leukemia xenograft immunodeficient mice was used to evaluate whether CAR-NK cells are associated with a lower toxicity profile compared to CAR-T cells. While the in vivo antileukemia activity was superimposable between CAR-T and CAR-NK cells (mouse bioluminenscence at 20 days, 4.9x105 vs 6.6x105 photons/second, respectively; p=n.s. Figure A), mice treated with two i.v. infusions (day 0 and day 15) of 10x106 CAR.CD19 NK cells had a 100% overall survival (OS of 5 out of 5 mice) at 50 days compared to 20% of mice (1 out of 5) receiving 10x106 CAR.CD19 T cells (Figure B; p=0.01). Cytokine plasma level monitoring, performed on day +7 and +30 after effector cell infusion in the absence of leukemia persistence (as evidenced by a lack of bioluminescence signal), showed that mice engrafted with CD19+ leukemia and treated with CAR.CD19-NK cells have lower levels of circulating hIFN-g cytokine compared to mice treated with CAR.CD19-T cells at both day 7 (42±82 vs 330±346 ng/ml; p=0.05) and day 30 (0.9±0.7 vs 4148±667 ng/ml; p=0.05). These in vitro and in vivo data demonstrate the feasibility of clinical scale feeder-free expansion of non-modified NK cells and stably transduced CAR-NK cells. Both non-modified and gene-modified cells were capable of significant tumor killing, suggesting a multi-modal adoptive cell approach to treatment of leukemia. Since NK cells have been shown to be safely used in third-party setting (St. Jude Children's Research Hospital, USA; NCT00640796), we suggest that ex-vivo expanded, feeder-free NK cells can be universally applied for 'off-the-shelf' immuno-gene-therapy, and that their innate allo-reactivity can be safely harnessed to potentiate allogeneic cell therapy. Figure. Figure. Disclosures Locatelli: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees.

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Synergistic Anti-Myeloma Activity of a Proteasome Inhibitor Marizomib and IMiD® Immunomodulatory Drug Pomalidomide

Blood ◽

10.1182/blood.v124.21.2099.2099 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2099-2099

Author(s):

Deepika Sharma Das ◽

Durgadevi Ravillah ◽

Arghya Ray ◽

Yan Song ◽

Paul G. Richardson ◽

...

Keyword(s):

Board Of Directors ◽

Tumor Cells ◽

Cell Lines ◽

Proteasome Inhibitor ◽

Response To Treatment ◽

Low Doses ◽

Advisory Committees ◽

Healthy Donors

Abstract Background and Rationale: Proteasome inhibitor bortezomib is an effective therapy for the treatment of relapsed and refractory multiple myeloma (RRMM); however, prolonged treatment can be associated with toxicity, peripheral neuropathy and drug resistance. Our earlier studies showed that a novel proteasome inhibitor marizomib is distinct from bortezomib in its chemical structure, mechanisms of action, and effects on proteasomal activities (Chauhan et al., Cancer Cell 2005, 8:407-419). We also showed that marizomib triggers synergistic anti-MM activity in combination with lenalidomide (Chauhan et al., Blood 2010, 115:834-45). Pomalidomide, like lenalidomide, is an analogue of thalidomide with potent immunomodulatory activity, and has been approved by FDA for treatment of RRMM patients who have received at least two prior therapies including lenalidomide and bortezomib and showed disease progression on or within 60 days of completion of the last therapy. Approval of treatment is based on progression-free survival. Here we utilized in vitro and in vivo models of MM to examine the anti-MM activity of combined marizomib and pomalidomide. Materials and Methods:MM celllines, patient tumor cells, and peripheral blood mononuclear cells (PBMCs) from normal healthy donors were utilized to assess the anti-MM activity of marizomib and pomalidomide. Cell viability, apoptosis, and migration assays were performed using WST/MTT, Annexin V staining, and Transwell Inserts, respectively. Synergistic/additive anti-MM activity was analyzed by isobologram analysisusing “CalcuSyn” software program. Proteasome activity was measured, as previously described (Chauhan et al., Cancer Cell 2005, 8:407-419). In vitro angiogenesis was assessed using matrigel capillary-like tube structure formation assays. MM.1S-tumor-bearing mice were treated with vehicle control, marizomib, pomalidomide or marizomib plus pomalidomide at the indicated doses for 21 days on a twice-weekly schedule for marizomib and 4 consecutive days weekly for pomalidomide. Statistical significance was determined using a Student’s t test. Pomalidomide was purchased from Selleck chemicals, USA; and marizomib was obtained from Triphase Inc., USA. Results: MM cell lines (MM.1S, MM.1R, INA-6, RPMI-8226, Dox-40, U266, LR5, ANBL6.WT, and ANBL6.BR) and primary patient MM cells were pretreated with DMSO control or with pomalidomide for 24h; marizomib was then added for an additional 24h, followed by assessment of cell viability. A significant decrease in viability of all cell lines and patient cells was observed in response to treatment with combined low doses of marizomib and pomalidomide, compared with either agent alone. Isobologram analysis confirmed the synergistic anti-MM activity of these agents (CI < 1.0). Tumor cells from 5 of 7 patients were obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. Moreover, the cytotoxicity of combination therapy was observed in MM cell lines sensitive and resistant to conventional (dex, doxorubicin, melphalan) and novel (bortezomib) therapies. No significant decrease in viability of PBMCs from normal healthy donors was observed in response to treatment with combined low doses of marizomib and pomalidomide, suggesting selective anti-MM activity and a favorable therapeutic index for this combination regimen. Furthermore, marizomib plus pomalidomide inhibits proliferation of MM cells even in the presence of BM stromal cells. Mechanistic studies showed that marizomib plus pomalidomide-induced apoptosis was associated with: 1) activation of caspase-8, caspase-9, caspase-3, and PARP; 2) downregulation of Cereblon, IRF4, c-Myc, and Mcl-1; and 3) enhanced inhibition of chymotrypsin-like, caspase-like and trypsin-like proteasome activities versus single agent alone. Furthermore, combined low doses of marizomib and pomalidomide blocked migration of MM cells and angiogenesis. In vivo studies using a subcutaneous human MM xenograft models show that combined low doses of marizomib and pomalidomide are well tolerated, inhibit tumor growth, and prolong survival. Conclusion: Our preclinical studies in MM disease models support a clinical trial of combined marizomib and pomalidomide to improve outcome in patients with relapsed and refractory MM. Disclosures Richardson: Oncopeptides AB: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Trikha:Triphase Accelerator: Employment. Chauhan:Triphase Accelerator: Consultancy. Anderson:Celgene: Consultancy; Millenium: Consultancy; Onyx: Consultancy; Gilead: Consultancy; Sanofi Aventis: Consultancy; BMS: Consultancy; Oncopep/Acetylon: Equity Ownership.

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IL-2 Induces Selective Activation of Helios-Positive Regulatory T Cells and CD56bright NK Cells in Vitro and in Patients with Chronic Gvhd Receiving Low-Dose IL-2 Therapy

Blood ◽

10.1182/blood.v126.23.919.919 ◽

2015 ◽

Vol 126 (23) ◽

pp. 919-919 ◽

Cited By ~ 2

Author(s):

Masahiro Hirakawa ◽

Tiago R Matos ◽

John Koreth ◽

Edouard Forcade ◽

Jennifer Whangbo ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Board Of Directors ◽

Cd8 T Cells ◽

Low Dose ◽

Advisory Committees ◽

Low Concentrations ◽

Healthy Donors

Abstract Introduction: CD4+ FoxP3+ CD25+ regulatory T cells (Treg) play a central role in the maintenance of immune tolerance and prevention of chronic graft-versus-host disease (cGVHD) after allogeneic stem cell transplantation (SCT). Treg constitutively express high-affinity interleukin-2 (IL-2) receptors and murine models have established that IL-2 is a critical homeostatic regulator of Treg in vivo. We previously reported that daily administration of low-dose IL-2 in patients with cGVHD induces selective expansion of Treg and NK cells and results in clinical improvement in approximately 50% of patients. However, the mechanisms responsible for these selective effects and the influence of IL-2 therapy on other lymphocytes have not been established due to the limited resolution of traditional cell analytic methods such as flow cytometry. Methods: Single cell mass cytometry (CyTOF) with a panel of 33 markers was used to simultaneously examine the phenotypic and functional effects of low-dose IL-2 on lymphocyte populations in vitro and in vivo. The analytic panel included 22 cell surface markers to identify distinct T, B and NK cell subsets and 11 intracellular markers to measure functional status and activation of specific signaling pathways. viSNE, a cytometry analysis tool, was used to visualize high-dimensional cytometry data on a two-dimensional map. Results: In unstimulated lymphocytes from healthy donors, constitutive expression of CD25 (IL-2Ra) at high levels was restricted to Treg and CD56bright NK cells. Central memory (CM) and effector memory (EM) subsets of conventional CD4 T cells (Tcon) and CM CD8 T cells expressed low levels of CD25. Within the Treg population, the highest expression of CD25 was closely associated with expression of Helios transcription factor. Helios+ Treg also express higher levels of FoxP3, HLA-DR and CD95 and lower levels of BCL2 compared to Helios- Treg. To examine responses to IL-2, we stimulated peripheral blood mononuclear cells (PBMC) from healthy donors with IL-2 for 15 min in vitro (Figure 1). At low IL-2 concentrations (1 to 10 IU/ml), pSTAT5 was preferentially activated in Treg. Notably, pSTAT5 activation was more robust in memory Treg than naïve Treg and in Helios+ Treg than Helios- Treg. In addition, we observed activation of pSTAT5 in CD56bright NK cells at low concentrations of IL-2 (10 IU/ml). Higher IL-2 concentrations (100-1000 IU/ml) were required to activate pSTAT5 in Tcon, CD8 T cells and CD56dim NK cells. At high IL-2 concentrations, pSTAT5 was activated in all Treg, NK, Tcon and CD8 subsets. To examine the response to IL-2 in vivo, we examined PBMC from 14 patients with chronic GVHD receiving daily low-dose IL-2 using the same CyTOF panel of markers. Without additional in vitro stimulation, pSTAT5 expression was increased preferentially in Helios+ Treg. Peak pSTAT5 expression occurred 1 week after starting IL-2 and decreased with continued IL-2 therapy. Similarly, increased expression of FoxP3, CD25, HLA-DR and Ki67 occurred primarily in Helios+ Treg with peak expression at 1 week. At later time points during IL-2 therapy, changes in Treg included increased expression of CD95, CTLA4, PD-1, BIM and BCL2. Although there was no activation of pSTAT5 in CD4 Tcon and CD8 T cells, expression of PD-1 increased in effector memory subsets of Tcon and CD8 T cells 1 week after starting IL-2 therapy. Selective expansion of CD56bright NK cells was also noted, with peak activation at 1 week. No other changes were noted in Tcon, CD8 T cells and B cells. All changes observed during IL-2 therapy returned to baseline levels 4 weeks after treatment was stopped. However, examination of PBMC from 8 patients who received continuous daily low-dose IL-2 therapy for approximately 1 year showed that all of the changes noted above persisted during extended therapy. Conclusion: Comprehensive analysis of T, B and NK cells from healthy donors revealed that low concentrations of IL-2 result in selective activation of Helios+ Treg and CD56bright NK cells. Higher concentrations of IL-2 are required for activation of CD4 Tcon, CD8 T cells and CD56dim NK cells. Identical populations are activated in patients with cGVHD receiving daily low-dose IL-2 and these functional effects persist during extended IL-2 therapy. Although the function of Helios transcription factor is not well defined, Helios expression identifies those Treg most primed to respond to low concentrations of IL-2 in vitro and in vivo. Disclosures Armand: Infinity Pharmaceuticals: Consultancy; Merck: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding. Antin:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees. Soiffer:Gentium SpA/Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

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Phosphatidylserine-Exposing Extracellular Vesicles after Splenectomy Are Associated with Increased D-Dimers and Fibrin Generation in Hereditary Hemolytic Anemia

Blood ◽

10.1182/blood-2018-99-110918 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 630-630

Author(s):

Stephanie Van Straaten ◽

Chi Hau ◽

Najat Hajji ◽

Jill Verhoeven ◽

Roger Schutgens ◽

...

Keyword(s):

Board Of Directors ◽

Hemolytic Anemia ◽

Extracellular Vesicles ◽

Research Funding ◽

Hypercoagulable State ◽

Red Cell ◽

Advisory Committees ◽

Increased Risk ◽

D Dimer

Abstract Background Thrombosis is a common complication of hereditary hemolytic anemia (HHA). Etiology of a hypercoagulable state in patients with HHA involves inflammation and splenectomy, although the etiology of the latter is insufficiently established. Because the concentration of circulating extracellular vesicles (EVs) has been reported to increase after splenectomy, and because in patients with sickle cell disease (SCD) circulating EVs are associated with coagulation, we analyzed the concentration of circulating EVs and their procoagulant activity in plasma from splenectomized and non-splenectomized patients with HHA. Methods This is a cross sectional, observational study in adult patients with HHA (SCD, other hemoglobin disorders, red cell enzyme disorders, red cell membrane disorders). Blood samples were collected with a 21-gauge butterfly needle and collected in 9 mL citrate phosphate dextrose adenine (CPDA) vacutainers, without use of a tourniquet. The tubes were mixed gently and the time between blood collection and centrifugation was maximum one hour. EVs in platelet-depleted plasma were labeled for Heat Shock Protein 70, CD14 (monocyte-derived EVs), CD61 (platelet EVs), CD62e (endothelial EVs), CD62p (P-selectin-exposing platelet EVs), CD71 (reticulocyte EVs), CD144 (endothelial EVs), CD235a (erythrocyte EVs) and lactadherin (phosphatidylserine (PS)-exposing EVs), and measured with a dedicated flow cytometer for EVs (A60-micro, Apogee Flow systems; lower limit of detection 170-180 nm single EVs). The coagulant activity of EVs was studied by a fibrin generation test, which measures the EV-dependent clotting time of plasma. The time to fibrin formation (1/2max) was measured using optical densitometry (λ = 405 nm) and an arbitrary cut off of V1/2max <1,500s was used to consider FGT as positive. Samples with >25% difference between duplicates or from patients that used anticoagulant medication were excluded from analysis Results Ninety seven patients were included in the study. Baseline characteristics are shown in Table 1. FGT of 63 patients were included. Thirteen patients (21%) had a positive FGT. Patients with positive FGT had increased concentrations of circulating EVs (CD61, CD71, lactadherin: p=<0.001; HSP70, CD62p, CD61/CD62p, CD62e, CD235a: p=<0.05) compared to patients with a negative FGT. Of the patients with a positive FGT, 11 patients (85%) were splenectomized, versus 2 patients (28%) in the FGT-negative group (p=0.002, patients with SCD regarded as splenectomized). Splenectomized patients had increased concentrations of lactadherin-binding EVs (p<0.001), as well as increased concentrations of CD61- and CD61/CD62p-exposing EVs (p<0.001) and of CD235a- and CD71-positive EVs (p<0.01, Table 2). FGT V1/2max and D-dimer correlated with lactadherin-binding EVs (ρ=-0.631, p=<0.001, and ρ=0.331, p=0.001). Conclusion In this study we show that in HHA patients the plasma concentration of lactadherin-binding and thus PS-exposing EVs correlates with fibrin generation in vitro and plasma D-dimer concentration, indicating that EVs may be associated with the hypercoagulable state that is observed in patients with HHA. Splenectomized patients had higher concentrations of lactadherin-binding EVs, and their plasma samples were prone to clot, as shown by fibrin generation in vitro. As the spleen is the main organ removing PS-exposing cells, higher levels of PS-exposing EVs in such patients may be due to reduced clearance, which in turn may contribute to the increased risk of thrombosis in patients after splenectomy. Disclosures Schutgens: Novo Nordisk: Research Funding; Uniqure BV: Research Funding; Pfizer: Research Funding; CSL Behring: Research Funding; Bayer: Research Funding; Baxalta/Shire: Research Funding. van Wijk:Agios Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; RR Mechatronics: Research Funding. van Beers:RR Mechatronics: Research Funding; Bayer: Research Funding; Pfizer: Research Funding; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Specific Exosomal microRNA Are Differentially Expressed Between High and Low-Risk Myeloma Suggesting They Are Pathogenically Important

Blood ◽

10.1182/blood.v126.23.4189.4189 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4189-4189

Author(s):

Bangzheng Chen ◽

Gareth J Morgan ◽

Bart Barlogie ◽

Joshua Epstein

Keyword(s):

Bone Marrow ◽

High Risk ◽

Board Of Directors ◽

Low Risk ◽

Differentially Expressed ◽

University Of Arkansas ◽

Medical Sciences ◽

Advisory Committees ◽

Healthy Donors ◽

Serum Exosomes

Introduction Treating High Risk myeloma (HRMM) is an important clinical challenge and understanding the basis of its pathogenesis could offer new therapeutic options. At present very few clues exist as to what is crucially deregulated between HR and LR and micro RNA (miR) are prominent candidates. Since the first miRNA was discovered over 20 years ago, the important role of these small noncoding RNA molecules in multiple myeloma pathogenesis has been recognized. In addition to their function in regulating gene expression within their cell of origin, miR molecules encased in exosomes are also secreted into the circulation. In this setting exosomes can deliver their content to other cells throughout the body, and have been suggested to be a key factor mediating the interaction of the MM cells and their microenvironment. We have tested the hypothesis that miR content of exosomes in bone marrow serum of patients with myeloma have an impact on biological behavior and as such can distinguish between patients with GEP 70-defined high- and low-risk disease. Methods Exosomes were isolated from bone marrow serum using Invitrogen kits according to manufacturer's instructions. Aliquots from the resulting samples were analyzed using electron microscopy to confirm the presence of exosomes. The exosome preparations were spiked with the Caenorhabditis elegance miR mimetic Ce_miRNA39_1 as a control, and miR was subsequently isolated using miRNeasy Kit (Qiagen). The miR were converted to cDNA with polyA tailing, pre-amplified 10 cycles. Aliquots of the preamplified cDNA were loaded into the samples wells and primer pairs of 68 miR, reported to be differentially expressed in myeloma or other cancers, were loaded into the reagent wells of Fluidigm's 96x96 Dynamic Array IFCs (integrated fluidic circuit) and the arrays processed on a Fluidigm BioMark. Results were analyzed using the company's Real-Time PCR analysis software. The level of the 68 miRs were analyzed in preamplified exosomal miR cDNA from 13 healthy donors, and 76 untreated NDMM patients (54 low-risk, 22 high-risk as categorized by GEP 70 gene model). Results Electron microscopy analysis confirmed the presence of exosomes, sized between 50 and 150nm in all of the sample preparations. With a cutoff ratio of 1.5, 3 miRs were differentially expressed between HRMM and LRMM: miR-192, and 215 were present at a higher level in exosomes from LRMM (1.6, and 1.8 fold, respectively) than in HRMM, while miR 720 and 1308 were higher in HR MM (2.9 and 3.3 fold, respectively). Of the 4 differentially expressed miRs, 2 ( 192, and 1308) were 2-82 fold higher in MM bone marrow serum exosomes than in exosomes from the healthy donors, one (720) had equal levels, and one (miR-215) was not detected in healthy donor samples. Among the 68 miRs analyzed, 4 were differentially expressed between HR- and LRMM. miRNA 192 and 215 both target the MDM2/TP53 axis, and are lower in bone marrow serum exosomes from HRMM in comparison LRMM. In contrast, miR-720 and 1308 are higher in HRMM. In this context miR-720 inhibits tumor invasion in breast cancer and modulates proliferation in esophageal cancer; miR-1308 is a fragment of t-RNA, targets the apoptotic pathway, and has anti-apoptotic function. It has been reported that miR-137 is deregulated in solid tumors, and that overexpression can induce apoptosis in MM cell lines. It is interesting that it was present in bone marrow serum exosomes from only 2 low-risk myeloma patients and not in exosomes isolated from purified myeloma plasma cells from 12 high-risk patients. Conclusion These results indicate that exosomal microRNA are associated with the risk status of myeloma patients at presentation, either as a reflection of risk or as effectors. Their presence in protecting vesicles in the circulation indicates that miR have the capacity to modulate the properties of the microenvironment and myeloma cells in remote loci. To better elucidate the role of exosomal miR in the interaction of myeloma cells with the microenvironment it is important to determine the source of the exosomes. Figure 1. Figure 1. Disclosures Chen: University of Arkansas for Medical Sciences: Employment. Morgan:Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Weismann Institute: Honoraria; MMRF: Honoraria; CancerNet: Honoraria; University of Arkansas for Medical Sciences: Employment; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Barlogie:Multiple Myeloma Research Foundation: Other: Travel Stipend; Dana Farber Cancer Institute: Other: Travel Stipend; International Workshop on Waldenström's Macroglobulinemia: Other: Travel Stipend; ComtecMed- World Congress on Controversies in Hematology: Other: Travel Stipend; European School of Haematology- International Conference on Multiple Myeloma: Other: Travel Stipend; Celgene: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Myeloma Health, LLC: Patents & Royalties: Co-inventor of patents and patent applications related to use of GEP in cancer medicine licensed to Myeloma Health, LLC. Epstein:University of Arkansas for Medical Sciences: Employment.

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Integrated Transcriptomic and Proteomic Analyses of Inflammasome in Myelodysplastic Syndromes and Chronic Myelomonocytic Leukemia

Blood ◽

10.1182/blood-2019-128094 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2991-2991

Author(s):

María Zurdo ◽

Ana M Hurtado López ◽

Tzu Hua Chen-Liang ◽

Helios Martínez-Banaclocha ◽

Laura Palomo ◽

...

Keyword(s):

Board Of Directors ◽

Differentially Expressed Genes ◽

Myelodysplastic Syndromes ◽

Peripheral Blood ◽

Research Funding ◽

Chronic Myelomonocytic Leukemia ◽

Differentially Expressed ◽

Advisory Committees ◽

Myelomonocytic Leukemia

Background and aim: Inflammasome and pyroptosis overactivation have recently been associated as fundamental mechanisms in the ineffective hematopoiesis of myelodysplastic syndromes (MDS). Chronic myelomonocytic leukemia (CMML) shares histological and clinical characteristics with MDS but, within clinical differences, It stands out a high association with inflammatory/autoimmune diseases in which a disproportionate activation of inflamasome has been implicated. Our hypothesis is that CMML cases show a higher inflammasome activation with respect to the MDS subset, a relevant difference both in terms of potential therapeutic targets and pathogenic clues. The main objective is to confirm, describe and quantify these differences using high-performance and multi-gene/protein methods. Methods: We performed enhanced RNA-seq in bone marrow mononucleated cells of 27 CMML at diagnosis, 10 MDS and 9 controls (103 million average readings). We selected 116 genes related to the inflammasome and reviewed the differential expression between cases and controls. We evaluated by multiplex immunoassay the profile of 28 cytokines in peripheral blood in 35 CMML patients, 37 MDS and 8 controls. Subsequently, we studied whether these differentially expressed genes / cytokines showed differences in CMML depending on the mutational state of TET2, SRSF2 and ASXL1. Finally, we compared in vitro the degree of activation of inflamasome in the monocytoid component of 8 CMML patients versus 7 controls. Results: In the transcriptomic analysis of the inflamasome genes in patients with CMML, we found 30 of 116 differentially expressed genes compared with healthy controls. Of those 30 genes, 26 showed a pro-inflammatory function and, of them, 18 were up-regulated. Of the 4 differentially expressed genes with an anti-inflammatory function, 3 were significantly under-expressed in CMML patients. We highlight, due to the quantitative difference, the overexpression of two genes coding for monocyte chemotactic proteins, CCL7 and CCL2 (FC = 269.21, p = 0.032; FC = 11.79, p = 0.03) That pro-inflammatory transcriptional profile was not so evident in the cases of MDS: of the 29 differentially expressed genes with pro-inflammatory function, 18 were down-regulated. Subsequently, we designed a customized panel for proteomic analysis including 9 of the 30 differentially expressed genes in CMML. We found that, in a relevant percentage of cases, also proinflammatory cytokines derived from these differentially expressed genes were elevated (62.5%) in peripheral blood of patients, compared to healthy donors; pointing towards the key role of gene transcription in the definition of the pro-inflammatory sense of the proteomic dimension of inflammasome in CMML. Next, we found that those patients with CMML and somatic mutations of TET2 had a higher expression of CCL7 and CCL2 compared to patients with CMML wild type, with a tendency to significance in the first case and significant in the second (FC 11.9, p = 0.15; FC 7.8, p = 0.03). Finally, we conducted in vitro stimulation studies at diagnosis in patients with CMML confirming that the canonical activation of the NLRP3 inflammasome (increased production of IL-1β) is significantly enhanced with respect to control individuals. Conclusion: We describe for the first time, a hyperactivation in CMML, compared with MDS, of the components of the inflammasome. Hyperactivation associated in CMML to a gene transcriptional mechanism and related, in the case of the two most over-expressed genes, CCL2 and CCL7, to the presence of mutations in TET2. Our findings point to new therapeutic targets whose modulation could restore inefficient hemopoiesis and potential diagnostic and prognostic biomarkers in CMML. Disclosures Díez-Campelo: Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Jerez:Novartis: Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

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T Cell Immunity Toward Viral- and Tumor-Antigens Is Preserved in MDS Patients

Blood ◽

10.1182/blood-2019-126792 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4225-4225

Author(s):

Hussein Hamad ◽

Wingchi K Leung ◽

Spyridoula Vasileiou ◽

Shivani Mukhi ◽

Quillan Huang ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Healthy Donor ◽

Tumor Antigens ◽

Ex Vivo ◽

Advisory Committees ◽

Cell Immunity ◽

Healthy Donors ◽

T Cell Lines

Myelodysplastic syndromes (MDS) are a heterogeneous group of disorders characterized by bone marrow failure and a propensity to progress to acute myeloid leukemia (AML). A core component of the underlying pathogenesis in MDS is deregulation of inflammatory cytokines, such as tumor growth factor-β (TGFβ), which impact the function of immune cells and hence their capacity to mount anti-infective or anti-tumor responses. However, little is known about antigen-specific T cell function in patients with MDS. We hypothesized that virus-specific T cell (VST) function might be preserved in patients with MDS, and that the functional capacity of T cells reactive against tumor-associated antigens aberrantly overexpressed by clonal MDS cells such as Cyclin A1 (CCNA1) and Proteinase (PR3) might also be preserved and exploited for immunotherapeutic purposes. Following informed consent, we collected peripheral blood samples from 10 patients (pts) with MDS and 17 healthy donors. Most pts (9 out of 10) were transfusion dependent and 3 subsequently underwent an allogeneic HSCT. Table 1 summarizes the other clinical characteristics, karyotypic and mutational profile at the time of blood collection. Compared with T cells isolated from healthy donors, MDS patient-derived T cells had a similar CD4 to CD8 ratio (1.5-2.5:1 for healthy donors and 3:1 for MDS pts), but displayed a more exhausted profile at baseline (CD3+TIM3+: 1% in healthy donors and 5% in MDS pts) and produced higher levels of inflammatory cytokines [IFNγ (18±3pg/ml vs 36±16pg/ml, healthy donor vs MDS; p=0.12), and IL-8 (56±32 vs 704±446 pg/ml, p=0.01)]. Next, to assess the capacity of MDS pts to mount ex vivo functional virus-directed responses, we stimulated patient-derived PBMCs (n=5) with overlapping peptide libraries (pepmixes) spanning immunogenic AdV, CMV, EBV, BK and HHV-6 antigens. Similar to healthy donor-derived T cell lines (n=5, 3 specific for 4 viruses and 2 for 5 viruses), all 5 MDS patient-derived lines demonstrated specificity for one or more of the target viruses (1 for 5 viruses, 1 for 4, 2 for 3 and 1 for 1 virus) as observed by IFNγ ELISpot assay with comparable magnitude (range Adv: 43-730 vs 384-941 in healthy donors, CMV: 0-1599 vs 0-3002, EBV: 0-1486 vs 0-541, BK: 0-839 vs 38-275 and HHV6: 0-794 vs 5-407 SFU/2x105 cells, respectively). We next examined the feasibility of expanding autologous MDS-antigen directed T cell products (n=10) to determine whether an adoptive immunotherapeutic approach might be applicable for MDS treatment. Thus, we exposed patient-derived PBMCs to autologous dendritic cells (DC) loaded with pepmixes spanning 6 MDS-associated antigens (CCNA1, survivin, WT1, PRAME, PR3 and NYESO1). After 3 rounds of stimulation, the products obtained were predominantly CD3+ T cells (mean 88±1.3%) that were polyclonal (CD4: 46±5% and CD8: 41±4%) containing predominantly memory T cells (TEM: 36±6% TCM: 37±5% and Tnaïve =13±3%). Six lines (60%) showed specific recognition to at least one of the target antigens: 4 lines specific for PRAME, 1 for CCNA1, 1 for WT1 and 1 for NYESO1 (range 0-40, 0-184, 0-1386 and 0-179 SFU/2x105 cells, respectively by IFNγ ELIspot). T cell lines were capable of specifically secreting multiple effector cytokines in response to targets (TNFα: 12% and IFNγ: 16% in response to PRAME in a representative patient-derived T cell line). Furthermore, this line was capable of killing PRAME+ targets in a 4hr 51Cr release assay [60% specific lysis, E:T 20:1]. In conclusion, functional virus-directed T cell immunity in patients with MDS is preserved, potentially explaining the lower rates of viral reactivation seen in these patients compared with other infections. Moreover, T cells specific for MDS-expressed tumor antigens can also be successfully expanded ex vivo from patients. Taken together this raises the possibility of applying an adoptive immunotherapeutic approach for the treatment of MDS. Disclosures Ramos: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Research Funding. Leen:Allovir: Consultancy, Other: Cofounder, Ownership Interest; Marker Therapeutics: Consultancy, Other: Cofounder, Ownership Interest.

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VTE Rates and Safety Analysis of Newly Diagnosed Multiple Myeloma Patients Receiving Carfilzomib-Lenalidomide-Dexamethasone (KRD) with or without Rivaroxaban Prophylaxis

Blood ◽

10.1182/blood-2019-124403 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1835-1835 ◽

Cited By ~ 4

Author(s):

Katrina M Piedra ◽

Hani Hassoun ◽

Larry W. Buie ◽

Sean M. Devlin ◽

Jessica Flynn ◽

...

Keyword(s):

Multiple Myeloma ◽

Venous Thromboembolism ◽

Board Of Directors ◽

Research Funding ◽

High Dose ◽

Newly Diagnosed ◽

Advisory Committees ◽

Cancer Trial ◽

Oncology Research ◽

Increased Risk

Introduction Immunomodulatory agents (IMiD's) are associated with an increased risk of venous thromboembolism (VTE), particularly when combined with high dose steroids. Studies evaluating the use of lenalidomide-bortezomib-dexamethasone (RVD) and carfilzomib-lenalidomide-dexamethasone (KRD) in the frontline setting for multiple myeloma (MM) have reported a 6% and 24% incidence of thrombosis, respectively, despite primary thrombotic prophylaxis with aspirin (ASA) (Richardson, et al. Blood. 2010; Korde, et al. JAMA Oncol 2015). Recent data, including the Hokusai VTE Cancer Trial, have suggested that safety and efficacy of direct oral anticoagulants (DOACs) are preserved in the setting of treatment of solid malignancy-associated thrombosis (Raskob, et al. N Engl J Med. 2018; Mantha, et al. J Thromb Thrombolysis. 2017). Despite this data, there is limited experience and use of DOACs in prevention of thromboses in the setting of hematologic malignancies, specifically MM. After careful review of literature, since early 2018, we changed our clinical practice and routinely placed newly diagnosed MM (NDMM) patients receiving KRD at Memorial Sloan Kettering Cancer Center (MSKCC) on concomitant rivaroxaban 10 mg once daily, regardless of VTE risk stratification. In the following abstract, we present VTE rates and safety data for newly diagnosed MM patients receiving RVD with ASA vs. KRD with ASA vs. KRD with rivaroxaban prophylaxis. Methods This was an IRB-approved, single-center, retrospective chart review study. All untreated patients with newly diagnosed MM, receiving at least one cycle of RVD or KRD between January 2015 and October 2018 were included. The period of observation included the time between the first day of therapy until 90 days after completion of induction therapy. Patients were identified by querying the pharmacy database for carfilzomib or bortezomib administration and outpatient medication review of thromboprophylaxis with rivaroxaban or ASA. VTE diagnoses were confirmed by ICD-10 codes and appropriate imaging studies (computed tomography and ultrasound). Descriptive statistics were performed. Results During the observation period, 241 patients were identified to have received RVD or KRD in the frontline (99 RVD with ASA; 97 KRD with ASA; 45 KRD with rivaroxaban). Baseline characteristics were well distributed among the three arms, with a median age of 60 (30-94) in the RVD ASA arm, 62 (33-77) in the KRD ASA arm, and 60 (24-79) in the KRD rivaroxaban arm. Patients had International Staging System (ISS) stage 3 disease in 13% (N=13), 9.3% (N=9), and 11% (N=5) of the RVD ASA, KRD ASA, and KRD rivaroxaban arms, respectively. Median weekly doses of dexamethasone were higher in both KRD arms, 40 mg (20-40) vs. 20 mg (10-40) in the RVD ASA arm. The average initial doses of lenalidomide were 22 mg in the RVD ASA arm compared to 25 mg in both the KRD ASA and KRD rivaroxaban arms. After querying the pharmacy database, no patients were identified to have a history or concomitant use of erythropoietin stimulating agent (ESA) use. Treatment-related VTE's occurred in 4 patients (4.0%) in the RVD ASA arm, 16 patients (16.5%) in the KRD ASA arm, and in 1 patient (2.2%) in the KRD rivaroxaban arm. Average time to VTE was 6.15 months (Range 5.42, 9.73) after treatment initiation in the RVD ASA group, while it was 2.61 months (Range 0.43, 5.06) in the KRD ASA group and 1.35 months in the KRD rivaroxaban group. Minor, grade 1 bleeding events per the Common Terminology Criteria for Adverse Events (CTCAE) were identified in 1 (1.1%) patient in the RVD ASA arm, 5 (5.2%) patients in the KRD ASA arm, and 1 (2.2%) patient in the KRD rivaroxaban arm. Conclusion More efficacious MM combination therapies have been found to increase the risk of VTE when using ASA prophylaxis, indicating better thromboprophylaxis is needed. We found patients receiving ASA prophylaxis with KRD were more likely to experience a VTE and these events occurred earlier compared to patients receiving ASA prophylaxis with RVD. Importantly, the rate of VTE was reduced to the same level as ASA prophylaxis with RVD when low-dose rivaroxaban 10 mg daily was used with KRD, and without necessarily increasing bleeding risk. Our retrospective data support the development of prospective clinical trials further investigating DOAC use in thromboprophylaxis for NDMM patients receiving carfilzomib-based treatments. Figure Disclosures Hassoun: Novartis: Consultancy; Janssen: Research Funding; Celgene: Research Funding. Lesokhin:BMS: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Janssen: Research Funding; GenMab: Consultancy, Honoraria; Serametrix Inc.: Patents & Royalties; Genentech: Research Funding; Juno: Consultancy, Honoraria. Mailankody:Juno: Research Funding; Celgene: Research Funding; Janssen: Research Funding; Takeda Oncology: Research Funding; CME activity by Physician Education Resource: Honoraria. Smith:Celgene: Consultancy, Patents & Royalties, Research Funding; Fate Therapeutics and Precision Biosciences: Consultancy. Landgren:Theradex: Other: IDMC; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Other: IDMC; Sanofi: Membership on an entity's Board of Directors or advisory committees; Adaptive: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. OffLabel Disclosure: Off-label use of rivaroxaban for outpatient prophylaxis of venous thromboembolism (VTE) will be explicitly disclosed to the audience.

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