Impact of Pathogen Reduction Technology Treatment and Storage in New Generation Platelet Additive Solutions (PAS) on Platelet Function.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2115-2115
Author(s):  
Gines Escolar ◽  
Miguel Lozano ◽  
Patricia Molina ◽  
Susanne Marschner ◽  
Raymond Goodrich ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Function ◽  
Research Funding ◽  
Ex Vivo ◽  
Quality Parameters ◽  
Slight Reduction ◽  
Flow Cytometry Analysis ◽  
Adhesive Properties ◽  
Pathogen Reduction ◽  
New Generation

Abstract Abstract 2115 Poster Board II-92 New strategies for preparing platelet concentrates (PCs) are investigating the combination of pathogen reduction technologies (PRT) with storage of platelets in additive solutions (PAS) containing phosphate buffer. While these approaches have several advantages by reducing adverse effects, improving biological safety and increasing plasma availability, there is a reasonable concern on how these strategies could affect platelet function. In the present study we have evaluated the effect of Marisol® PRT treatment (CaridianBCT Biotechnologies, LLC) in combination with storage in PAS-III (Intersol; Baxter Healthcare Corp) or PAS-IIIM (SSP+; MacoPharma) on analytical and functional characteristics of apheresis PCS. PCs (2.5-3.4×109plts/ml) were split into three aliquots. One was kept untreated and stored in plasma as a control (CON-PPP). One was PRT-treated and stored in the corresponding PAS (PRT-PASIII or PRT-IIIM) with a 35% plasma carryover to achieve a final concentration of 0.7-1.5 × 109 plt/ml. The last aliquot was stored in the corresponding PAS (CON-PASIII or CON-PASIIIM), not subjected to PRT treatment. PCs were stored under standardized conditions and evaluations were performed on days 0, 5 and 7. Cell quality parameters (pH, swirl, lactate and glucose), flow cytometry analysis of major platelet glycoproteins and activation dependent antigens were assessed. Adhesive and aggregating functions of platelets were evaluated using an ex vivo perfusion model with flowing reconstituted blood recirculating through damaged vascular segments. A slight reduction in platelet counts was noticed on day 7 compared to day 0 in PRT-treated PCs (p<0.05), however no statistical differences were observed between the different study groups. All measured cell quality parameters were within ranges previously published. Flow cytometry analysis revealed comparable levels of the analyzed glycoproteins for all conditions evaluated. Only moderate reductions in the expression of GPIb were observed on day 7 of storage in all groups except for CON-PASIIIM. A progressive increase in the expression of P-selectin was observed in both controls and PRT-treated PCs during storage. Lysosomal integral membrane protein (LIMP) expression was increased in PRT-PASIII and PRT-PASIIIM and CON-PASIII, but not CON-PASIIIM PCs. PRT-PASIII PCs showed the highest levels of annexin V binding after 7 days of storage. Studies under flow conditions showed comparable levels of adhesion and aggregation to subendothelium for PRT-treated platelets stored in PAS solutions at 5 days of storage. A detailed morphometric analysis revealed slightly enhanced adhesive functions in CON-PASIII on day 0 and moderate reductions in adhesive properties in PRT-PASIII, but not PRT-PASIIIM PCs after 7 days of storage. Our investigations show that cell quality parameters, glycoproteins levels and platelet functions were preserved in PRT-treated concentrates and stored in new generation PAS for up to 5 days. The relevance of our experimental data on the in vivo performance of PCs exposed to PRT and stored in new generation PAS deserve further investigation in the clinical setting. Disclosures: Escolar: Caridian BCT Technologies: Consultancy, Research Funding. Marschner:Caridian BCT Technologies: Employment. Goodrich:Caridian BCT Technologies: Employment. Galan:Caridian BCT Technologies: Research Funding.

scholarly journals The Small Gtpase Rap1 in Platelets Is Critical for Arterial but Not Venous Thrombosis in Mice

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2131-2131
Author(s):  
Jean Mwiza ◽  
Robert H Lee ◽  
David Paul ◽  
Lori A Holle ◽  
Brian C Cooley ◽  
...  
Keyword(s):  
Shear Stress ◽  
Venous Thrombosis ◽  
Platelet Function ◽  
Whole Blood ◽  
Research Funding ◽  
Ex Vivo ◽  
Vena Cava ◽  
Small Gtpase ◽  
Alternative Mechanism ◽  
Clot Contraction

Abstract Background: Platelets and their main adhesion receptors, integrins, are critical in hemostasis and arterial thrombosis, i.e., in situations involving severe insult to the vasculature and elevated shear stress, respectively. We recently demonstrated that integrin activation under both of these conditions depends on the small GTPase Rap1 directly activating the integrin adapter protein, Talin1. Our studies further suggested that the Rap1-talin1 axis is less important for platelet function at sites of inflammation, i.e., in situations of mild endothelial insult and low shear stress. Aim: To investigate the contribution of the platelet Rap1-Talin1-integrin signaling axis in the pathogenesis of venous thrombosis (VT). Methods: The following mice with documented deficiencies in platelets were subjected to the inferior vena cava (IVC) stenosis VT model: Rap1amKO (Rap1a fl/fl pf4-Cre+), Rap1bmKO (Rap1b fl/fl pf4-Cre+), or both isoforms (Rap1a fl/flRap1b fl/fl pf4-Cre+ [Rap1dKO]), and Talin1mKO (Tln1 fl/flpf4-Cre+). Thrombus weight was determined 48 hours after flow restriction. Clot contraction and tissue plasminogen activator (tPA)-mediated lysis of whole blood clots were studied ex vivo to characterize effects on clot consolidation and stability . Results: Compared to control mice, thrombus weight was markedly reduced in Talin1mKO mice (13.1±2 and 2.2±1.2 mg, p&lt;0.05), but not Rap1dKO or single knockout mice. Ex vivo clot contraction was significantly impaired in whole blood from Talin1mKO (clot weight: control 13.35±3.4 mg vs. 34.63±4.2 mg), Rap1amKO (control 18.0±3.9 mg vs. 25.6±6.0 mg), Rap1bmKO (control 18.0±3.9 vs 23.0±6.6 mg) and in Rap1dKO (control 18.03±3.9 mg vs. 30.02±4.6 mg). Clot weights were not significantly different between Rap1dKO and Talin1mKO mice. However, ex vivo clot lysis assays revealed that compared to controls, clots formed in whole blood from Talin1mKO mice lysed faster, whereas clots from Rap1dKO did not. Conclusions: Platelet Talin1 is critical for VT in mice, and this role is partially independent of its best-known upstream regulator, Rap1. Future studies will identify the alternative mechanism allowing for Rap1-independent Talin1 signaling and platelet function in VT and how these signaling affect various phases of VT. Disclosures Wolberg: Takeda: Research Funding; Bristol Myers Squibb: Research Funding; CSL Behring: Consultancy.

scholarly journals DNA Methyltransferase Inhibitors Increase Expression of CD38 and Enhance Daratumumab Efficacy in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5618-5618 ◽  
Author(s):  
Priya Choudhry ◽  
Margarette C. Mariano ◽  
Arun P Wiita
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Cell Lines ◽  
Research Funding ◽  
Plasma Cells ◽  
Cpg Island ◽  
Ex Vivo ◽  
Single Agent ◽  
Cd38 Expression

Abstract Introduction: The anti-CD38 monoclonal antibody Daratumumab is highly effective against multiple myeloma, is well tolerated, and has high single agent activity as well as combination effects with lenalidomide-dexamethasone as well as bortezomib-dexamethasone. Patient response to daratumumab monotherapy is highly correlated with pretreatment levels of CD38 expression on MM plasma cells (Nijhof et al, Leukemia (2015) 29:2039) and CD38 loss is correlated with daratumumab resistance (Nijhof et al, Blood (2016) 128:959). As a result, there is significant interest in elucidating the regulation and role of CD38 in MM. Recently, All Trans Retinoic Acid (ATRA), a known small molecule inducer of CD38 in myeloid cells, as well as the FDA-approved histone deacetylase inhibitor panobinostat, were both demonstrated to induce CD38 in MM plasma cells leading to increased lysis by daratumumab. Examining ENCODE data, we found the presence of a CpG island at the first exon of CD38. We hypothesized that removing methylation sites from this CpG island may de-repress CD38 transcription and lead to increased CD38 protein at the cell surface in MM plasma cells. Therefore, here we studied the role of DNA methyl-transferase inhibitors (DNMTis), currently FDA-approved for treatment of myelodysplastic syndrome, as agents to potentiate daratumumab therapy. Methods: We treated MM cell lines (RPMI-8226, MM.1S, XG-1, KMS12-PE) with two different DNMTis, 5-Azacytidine and decitabine, and assessed CD38 cell surface expression by flow cytometry. Similarly, we treated MM patient bone marrow aspirates ex vivo and assessed induction of CD38 expression in the CD138 positive population by flow cytometry. We analyzed CD38 mRNA levels and total CD38 protein levels by qRT-PCR and western blotting respectively. ATRA was used as a positive control in all experiments. We further tested the functional effect of DNMTi treatment on MM cell lines using an Antibody Dependent Cell Cytotoxicity (ADCC) assay. Briefly, live treated cells were incubated overnight with daratumumab and NK92-CD16 transgenic cells at and E:T ratio of 20:1, and lysis was measured using CytoTox-Glo (Promega). Results: Flow analysis revealed that DNMTi treatment induces a 1.2-2 fold increase in CD38 surface protein expression in a dose-dependent manner across MM cell lines. DNMTi treatment consistently yielded similar or higher increases in CD38 expression than that seen in ATRA- or panobinostat-treated cells. Despite significantly lower single-agent cytotoxicity, we found that decitabine led to similar surface CD38 induction as 5-Azacytidine. By RT-qPCR, 5-Azacytidine increased CD38 mRNA expression ~3 fold versus DMSO control, compared to ~2 fold mRNA increase with ATRA. In functional ADCC assays, DNMTi treatment also led to greater lysis than ATRA. Furthermore, the combination of both DNMTi and ATRA was additive, leading to the greatest lysis by NK cells. In contrast, in ex vivo-treated patient samples, ATRA induced greater CD38 expression than 5-Azacytidine on malignant plasma cells. However, this result is expected since MM plasma cells from patients typically do not proliferate in standard ex vivo culture, and active DNA replication is a requirement for successful DNMT inhibition based on known mechanism of action. In patients, however, we anticipate that continual plasma cell proliferation will lead to effective increases in CD38 after DNMTi treatment, as found in MM cell lines here. Summary and Conclusions: Our results here demonstrate that CD38 expression in MM cells is regulated by DNA methylation and targeting DNMTs with small molecule inhibitors leads to increased vulnerability to Daratumumab treatment. We propose that combination treatment with DNMTi and Daratumumab can lead to higher efficacy of daratumumab in daratumumab-naïve MM, as well as reversal of daratumumab-resistance. These combinations should be tested in clinical trials. Disclosures Wiita: Sutro Biopharma: Research Funding; TeneoBio: Research Funding.

scholarly journals Drug Combinations Co-Targeting Myeloid Cell Leukemia-1 (Mcl-1) Protein Can Overcome Microenvironmentally-Induced Multi-Drug Tolerance in Non-Hodgkin Lymphomas

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2649-2649
Author(s):  
Kallesh D. Jayappa ◽  
Craig A. Portell ◽  
Vicki L. Gordon ◽  
Goutham Narla ◽  
Timothy P. Bender ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Research Funding ◽  
Ex Vivo ◽  
Drug Tolerance ◽  
Drug Combinations ◽  
Ex Vivo Model ◽  
Selective Toxicity ◽  
Apoptotic Proteins

Abstract Ibrutinib (IBR), an inhibitor of Bruton's Tyrosine Kinase (Btk) and venetoclax (VEN), an inhibitor of Bcl-2 have been used in Chronic Lymphocytic Leukemia (CLL), but single agent responses to these drugs are often incomplete and not durable. We previously reported synergistic cytotoxicity for the IBR+VEN combination and have initiated a trial to test that combination (NCT02419560). However, we noted extensive variability in response to IBR+VEN between patient samples ex vivo, suggesting intrinsic tolerance even to this combination. CLL cells with an "activation" phenotype (CD5+/19+/69+) that occurs from interactions with the microenvironment were less sensitive to IBR+VEN, implicating the cancer microenvironment as an inducer of drug tolerance. This was supported by our finding that CLL cells display decreased sensitivity to IBR+VEN in co-culture with agonists and cells that emulate the cancer cell microenvironment. The combination of CpG-oligodeoxynucleotides (ODN), sCD40L, and IL10 ("agonist mix") induced near complete loss of sensitivity to IBR+VEN and upregulated Mcl-1 and Bcl-xL expression in CLL cells. However, Mcl-1 or Bcl-xL inhibitors alone were weakly effective in these cells, suggesting that exploitation of these targets requires drug combinations. Here we report a combination drug screen with an Mcl-1 inhibitor in a CLL microenvironment model ex vivo and novel drug combinations that overcome multi-drug tolerance. CLL patient PBMCs were cultured in the microenvironment model consisting of HK follicular dendritic cells, HUVEC, Jurkat T cells expressing CD40L, and CpG-ODN. Markers characteristic of activation and drug tolerance in vivo were assessed in CLL (CD5+/19+) cells using flow cytometry. We found increased expression of Ki67, CD69, and anti-apoptotic proteins Mcl-1 and Bcl-xL and decreased CXCR4 expression, as expected upon microenvironmental stimuli in vivo.Also, CLL cells from 2 IBR-treated patients were still responsive to agonist-induced drug tolerance, suggesting Btk inhibition may be less effective in blocking these microenvironmental stimuli in vivo. By comparing the ratio of anti-apoptotic proteins with cognate pro-apoptotic proteins, we noted targetable dependence of CLL cells for diverse pro-survival proteins (Mcl-1 and Bcl-xL) in our model. Flow cytometry analysis demonstrated that inhibitors of Mcl-1 (S63845), Bcl-xL (A1155643), or Bcl-2 (VEN) when used as single agents failed to induce significant apoptosis in CLL cells cultured in our model, suggesting induction of multi-drug tolerance. Drug combinations inhibiting Mcl-1 with Bcl-xL, Bcl-2, or Bcl-xL and Bcl-2 have effectively overcome multi-drug tolerance. As direct and simultaneous inhibition of multiple pro-survival proteins could be fatal to healthy cells, we carried out a Mcl-1-anchored combinatorial drug screen with inhibitors of other survival pathways in 3 CLL patient PBMCs cultured in our ex vivo model to identify drug combination(s) that induce selective toxicity in multi-drug tolerant CLL cells. Cytotoxicity was determined by analyzing cleaved PARP and dead cell staining in CLL and healthy T (CD3+/5+) cells by flow cytometry. Results showed that inhibitors of apoptotic proteins (A1155643, VEN, ABT737), IRAK4 (CA-4948, compound 26, and AS2444697), intracellular TLRs (chloroquine), Hsp90 (ganetespib), CDK (ribociclib), proteasome (bortezomib), Btk (IBR), AKT (MK2206), and HDAC (SAHA and panobinostat) or activator of PP2A (DBK1532 and NZ8061) were synergistically toxic with Mcl-1 inhibitor (S63845) in CLL cells. By comparing toxicity in CLL and healthy T cells, drug combinations targeting Mcl-1 with proteasome, IRAK4, TLRs, Btk, Hsp90, and CDK showed selective toxicity in CLL cells, indicating a potential therapeutic window for these combinations. Drug combinations targeting Mcl-1 with other apoptotic proteins were highly toxic to healthy T cells. In conclusion, CLL cells in our ex vivo model display characteristics of microenvironmentally-induced multi-drug tolerance. A similar phenotype was noted using post IBR therapy samples, suggesting IBR alone may not be effective in overcoming this multi-drug tolerance in vivo. Drug combinations targeting Mcl-1 and proteasome, IRAK4, TLRs, Btk, Hsp90, or CDK selectively overcame multi-drug tolerance in CLL cells. Thus, these combinations may be effective in patients showing intrinsic tolerance to multiple drugs. Disclosures Portell: TG therapeutics: Research Funding; AbbVie: Research Funding; Infinity: Research Funding; Genentech/Roche: Consultancy, Research Funding; Acerta: Research Funding; BeiGene: Research Funding; Kite: Research Funding; Amgen: Consultancy. Narla:University of Michigan: Patents & Royalties: Small molecule PP2A activators. Williams:Takeda: Research Funding; TG Therapeutics: Consultancy; Seattle Genetics: Consultancy; Verastem: Consultancy; Juno: Consultancy; Sandoz: Consultancy; Celgene: Consultancy, Research Funding; Novartis: Research Funding; Astra-Zeneca: Consultancy; Abbvie: Consultancy; Pharmacyclics: Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Kite: Consultancy; Gilead: Consultancy, Research Funding.

scholarly journals Ex Vivo Assessment of Tnb-383B, a Bcma-Bispecific Antibody, Against Primary Tumor and Endogenous T Cells from Relapsing Multiple Myeloma Patients

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1940-1940 ◽  
Author(s):  
David M. Foureau ◽  
Manisha Bhutani ◽  
Myra Robinson ◽  
Fei Guo ◽  
Duy Pham ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
T Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Ex Vivo ◽  
Negative Control ◽  
Advisory Committees ◽  
Measured Variable ◽  
Dose Dependent

Abstract INTRODUCTION: The human B-Cell maturation antigen (BCMA) is a surface marker that is highly expressed on plasma cells and has been recognized as a novel target in multiple myeloma (MM). TNB-383B is a fully human bispecific monoclonal IgG4 antibody. TNB-383B consists of 2 heavy and 1 light chain(s) paired through knob-in-hole technology. The first heavy chain and a kappa light chain form the paratope to recognize and bind human CD3. The second heavy chain is comprised of two identical VH domains in sequence and targets human BCMA with high affinity and avidity. Herein, we describe the ability of TNB-383B to mediate killing of patient-derived tumor cell lysis by endogenous T-cells was assessed ex vivo. METHODS: Bone marrow mononuclear cells (BMMCs) were isolated by density gradient centrifugation from 7 relapsed MM patients enrolled in an IRB-approved biospecimen collection protocol. Freshly isolated BMMC subsets were characterized by flow cytometry, specifically plasma cell (PC) / cytotoxic T cell (CTL) distribution, PC BCMA expression and PC viability were determined. BMMCs were then incubated for 24h (± 2h) with TNB-383B, or negative control, at concentrations ranging from 0.001-1μg. Following incubation, PC lysis, viability, BCMA expression, as well as CTL distribution and degranulation were assessed by flow cytometry. Tukey's sequential trend test was performed for each measured variable, utilizing ANOVA models and contrast statements, to detect linear dose response trends to TNB-383B or negative control treatments. Additionally, a parametric model (EMax) was used for each measured variable to estimate dose response curves, interpolating between tested doses. Two-way factorial ANOVA was utilized to compare the main effects of E:T ratio (or PC phenotype) and dose level and the interaction effect between E:T ratio and dose level on measured variables. RESULTS: Dose-dependent PC lysis was triggered by TNB-383B at concentrations as low as 0.001μg (p=0.0102) while no significant loss of PC was observed with negative control (Figure). This effect was coupled with significant CTL degranulation as expressed by increased CD107a mean fluorescence intensity (MFI) specific to TNB-383B treatment (p=0.0153 at 1μg). Although apoptotic rates (7-AAD+, Annexin V+) of the remaining PC tend to increase among TNB-383B treated compared with isotype control-treated cells, this trend was not significant. As opposed to CTL degranulation, CTL proliferation was not significantly triggered by TNB-383B but was significantly increased when BMMCs were exposed to negative control antibody (p=0.0057 at 0.001 μg). BMMC containing effector to target (E:T) ratio >10 contained lower viable (7-AAD-) PC and higher apoptotic PC counts compared with BMMC specimen with E:T ratio <10 (p<0.001). Using CD45 expression as a surrogate marker of PC maturation and BCMA expression, CD45+ PC displayed higher BCMA expression than CD45- PC (p=0.0189) and were more sensitive TNB-383B-induced killing (p<0.001). Noticeably, overall BCMA expression pre/post TNB-383B exposure remained unaltered. CONCLUSION: Taken together, our findings demonstrate TNB-383B triggers primary PC lysis and CTL degranulation in a dose-dependent fashion ex vivo. The ex vivo bispecific monoclonal antibody assay employed in this study allowed us to identify underlying biological drivers of PC killing efficacy by TNB-383B and may provide a valuable preclinical platform to screen bispecific antibodies and clinical platform to identify mechanism of primary or acquired resistance to the drug. Enrollment of patients with relapsed/refractory MM into a phase I clinical trial with TNB-383B is expected in early 2019. Figure. Figure. Disclosures Foureau: Teneobio Inc.: Research Funding. Pham:Teneobio Inc.: Employment. Force Aldred:Teneobio Inc.: Employment. Buelow:Teneobio Inc.: Employment. Voorhees:Oncopeptides: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: served on an IRC; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Other: served on an IRC; BMS: 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, Other: served on an IRC; Amgen Inc.: Speakers Bureau; TeneoBio: Consultancy, Membership on an entity's Board of Directors or advisory committees. Usmani:Abbvie, Amgen, Celgene, Genmab, Merck, MundiPharma, Janssen, Seattle Genetics: Consultancy; Amgen, BMS, Celgene, Janssen, Merck, Pharmacyclics,Sanofi, Seattle Genetics, Takeda: Research Funding.

An Innovative High-Throughput Ex Vivo Drug Assay Incorporating the Native Microenvironment Reveals a Novel Mechanism of Action of Idelalisib in CLL

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2485-2485
Author(s):  
Joan Ballesteros ◽  
Lydia Scarfo ◽  
Mattias Mattsson ◽  
Aliki Xochelli ◽  
Pamela Ranghetti ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Viability ◽  
B Cell ◽  
High Throughput ◽  
Dose Response ◽  
Research Funding ◽  
Ex Vivo ◽  
Response Curves ◽  
Ex Vivo Assay

Abstract The microenvironment (ME) critically promotes progression of chronic lymphocytic leukemia (CLL), favoring leukemic cell survival and proliferation as well as inducing drug resistance. We aimed at reproducing the effects of ME stimuli for the development and optimization of an ex vivo assay that would enable to predict in vivo drug efficacy for agents interfering with ME protective effects e.g. the novel BCR inhibitors. To this purpose, we exploited the following 2 new approaches: 1) the Exvitech® proprietary automated flow cytometry-based platform by Vivia Biotech that enables evaluation of up to 20.000 wells and conditions per sample, and 2) Viviaxs Precision Medicine Native Environment approach that utilizes the whole blood sample rather than isolated leukocytes. We present this assay optimization using primary CLL samples and its validation when exposing CLL cells to the registered PI3Kd inhibitor, Idelalisib, for which only a weak pro-apoptotic effect has been reported, besides the known tissue mobilization activity in vivo. Cryopreserved peripheral blood (PB) mononuclear cells were provided from CLL patients in need of treatment. In order to more closely reproduce the complexity of the in vivo ME, the following elements were evaluated in different combinations: (i) 3 backbone stimulations, previously reported to improve, to different extent, CLL viability and proliferation: CD40L+CpG, CD40L+IL21, CpG+IL2; (ii) "Native Environment", defined as the plasma & erythrocyte/granulocyte fraction of a Ficoll gradient, already shown to improve ex vivo drug testing (Bennet et al. Clin Lymphoma Myeloma Leuk. 2014;14:305-18): the two fractions were added to thawed CLL samples and were obtained from fresh samples of normal donor PB or bone marrow as well as from CLL patients at different stages of disease; (iii) the stroma cell line H5S, added at different ratios (1:10 or 1:100); (iv) both human and bovine fetal serum (at 10 or 20% total volume); (v) stimulatory B cell factors, including IL-21, soluble CD40L, BAFF, and B cell receptor stimulation (anti-IG). CLL cell viability and proliferation was then tested and, although CLL cells from PB are notorious for a low proliferative index and tend to die quickly ex vivo by apoptosis, we achieved a median of 30±3% proliferation (assessed by the CFDA dye) and 60±5% viability (assessed by Annexin V staining) in cryopreserved progressive CLL samples. These results were obtained with the combination of the following assay conditions: CpG+IL2, HS5 (at 1:100 ratio), human serum 10%, and "native environment" from PB of CLL samples (pooled samples to prevent interpatient variability). We then tested the dose responses of Idelalisib in 16 cryopreserved progressive CLL samples and found little effect on the non-proliferative CLL fraction (Fig, 1A), suggesting a limited direct pro-apoptotic activity of the drug. In contrast, potent inhibition of proliferation with median potency (EC50) of 14 nM was observed (Fig 1B). The efficacy was nearly complete leaving a median of 5% resistant CLL cells that proliferated at the highest doses of Idelalisib. In conclusion, we report a novel ex vivo assay that enables high-throughput pharmacological characterization of compounds and combinations, optimized for CLL cells by incorporating ME stimuli and thereby more accurately simulating in vivo interactions. The increased cell viability and proliferation achieved with this innovative assay offers improved opportunities for ex vivo pharmacology, in particular unraveling a hitherto unknown anti-proliferative mode of action for Idelalisib, a drug interfering with the interaction of CLL cells with the ME. Figure 1. Dose response curves of Idelalisib incubated for 96 h with 16 CLL samples in the new Microenvironment Native Environment assay. The effect on non-proliferative (A) and proliferative (B) CLL cells identified using flow cytometry as subpopulations with different CFDA staining is shown. Figure 1. Dose response curves of Idelalisib incubated for 96 h with 16 CLL samples in the new Microenvironment Native Environment assay. The effect on non-proliferative (A) and proliferative (B) CLL cells identified using flow cytometry as subpopulations with different CFDA staining is shown. Disclosures Ballesteros: Vivia Biotech: Employment. Primo:Vivia Biotech: Employment. Robles:Vivia Biotech: Employment. Gorrochategui:Vivia Biotech: Employment. Munugalavadla:Gilead Sciences: Employment. Stamatopoulos:Gilead Sciences: Research Funding; Janssen Pharmaceuticals: Research Funding. Quéva:Gilead Sciences: Employment, Equity Ownership. Ghia:AbbVie: Consultancy; Pharmacyclics: Consultancy; Gilead: Consultancy, Research Funding, Speakers Bureau; Adaptive: Consultancy; Acerta Pharma BV: Research Funding; GSK: Research Funding; Roche: Consultancy, Research Funding; Janssen: Consultancy.

scholarly journals High Correlation Clinical Responses to 1st Line Acute Myeloid Leukemia Treatment with an Ex Vivo Native Environment Precision Medicine Test

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4837-4837
Author(s):  
Pau Montesinos ◽  
Joan Ballesteros ◽  
David Martínez-Cuadrón ◽  
Joaquín Martínez-López ◽  
Josefina Serrano ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Board Of Directors ◽  
Roc Curve ◽  
Predictive Value ◽  
Research Funding ◽  
Ex Vivo ◽  
Clinical Correlation ◽  
Advisory Committees ◽  
Patient Response ◽  
Predictive Values

Abstract Background: We have overcome the limitations of 40 years of ex vivo testing. The aim of this study is to determine the ability of Vivia's novel test to predict the complete remission (CR) rates after induction chemotherapy with cytarabine (Ara-C) and idarubicin (Ida) in 1st line AML. Material and Methods: Bone marrow samples from adult patients diagnosed with de novo AML in Spanish centers from the PETHEMA group were included. Whole marrow samples maintaining their Native Environment were incubated for 48h in well plates containing Ara-C, Ida, or their combination. Pharmacological responses are calculated using population models. Induction response was assessed according to the Cheson criteria (2003). Patients attaining a CR/CRi were classified as responders and the remaining as resistant. Results: 390 patient samples were used to calculate the dose response (DR) curves for Ara-C alone, Ida alone, and their synergism. For clinical correlation we used 142 patients with median 56 years. The strongest clinical predictor was the Area Under the Curve (AUC) of the DR of Ara-C, and the AUC of IDA. The GAM models revealed a significant relationship between the AUC of the concentration-effect curves of both, idarubicin and, particularly, Ara-C, with greater values associated to higher probabilities of post-induction resistance. The fitted Generalized Additive Method predictions of expected values for each patient were in turn related to overall survival when a discrimination value to define positive and negative test results that prioritized specificity over sensitivity was chosen based on equaling positive and negative predictive values (Fig 1A). Prioritizing specificity over sensitivity reflects the higher cost of false positive over false negative decisions: only in very rare instances, an effective treatment would be erroneously negated to a sensitive patient at the expense of overlooking a number of resistant patients. However, the later patients could take their chances on re-induction therapy. While for diagnostics sensitivity and specificity should both be optimized, for Personalized Medicine the positive and negative predictive values should be optimized preferentially because they define the patient response correlation. Fig 1B shows a table illustrating the correlation between clinical outcome (columns) and the test predictions (lines). From a diagnostic criteria (columns), clinically resistant patients (1st column) are not well predicted with a Sensitivity of 51%, while clinically sensitive patients (2nd column) are very well predicted with a Specificity of 94%. From a Precision Medicine criteria (Lines), patients predicted resistant (1st line) and well predicted with 80% positive predictive value, similar to patients predicted sensitive (2nd line) well predicted with 79% Negative Predictive Value. The test does not properly identify 23/142 that are clinically resistant and the test predicts as sensitive (bottom left quadrant right panel). This mismatched subgroup mimics the problems from molecular markers where a resistant clone present in a minority of leukemic cells cannot be detected yet drives the patient response. However, this group mismatch does not prevent a good correlation with the test predicted outcomes. Flow cytometry identified 2 clones in 75% of these 23 samples, and we revised all samples analyzing each of 2 clones separately whenever they were present. Results did not change by this clonal analysis, suggesting flow cytometry may not identify resistant clones. Future improvements of the test adding 16 concentrations to the dose response curves may be able to detect the presence and parameters of these resistant clones driving patient response. Conclusions: This novel test is able to predict the clinical response to Ida + Ara-C induction with overall correlation and predictive values of 80%, higher than ever achieved. It is significantly higher than the current clinical response rate of 66.7%. Thus this novel test may be valuable information to guide 1st line patient therapy. Figure 1. ROC curve and clinical correlation Figure 1. ROC curve and clinical correlation Disclosures Ballesteros: Vivia Biotech: Employment. Cordoba:Celgene: Research Funding. Ramos:GlaxoSmithKline: Honoraria; Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria; Celgene Corporation: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria. Gaspar:Vivia Biotech: Employment. Gorrochategui:Vivia Biotech: Employment. Rojas:Vivia Biotech: Employment. Gomez:Vivia Biotech: Employment. Hernández:Vivia Biotech: Employment. Robles:Vivia Biotech: Employment.

scholarly journals The Ribosomal Biogenesis Inhibitor CX-5461 Is an Anti-Cancer Therapeutic That Increases Platelet Count in Mice and in Humans

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2360-2360
Author(s):  
Amandeep Kaur ◽  
Katherine M Hannan ◽  
Samina Nazir ◽  
Kylee H Maclachlan ◽  
Gretchen Poortinga ◽  
...  
Keyword(s):  
Platelet Count ◽  
Board Of Directors ◽  
Platelet Function ◽  
Research Funding ◽  
Ex Vivo ◽  
Liver Carcinoma ◽  
Advisory Committees ◽  
Platelet Counts ◽  
Platelet Spreading ◽  
Disease Free

Background: The ribosomal biogenesis inhibitor CX-5461 is a first-in-class selective inhibitor of ribosomal RNA gene transcription with single-agent antitumor activity against advanced hematologic cancers and synergistic activity when combined with front-line and emerging agents (Devlin et al. 2015, Sornkom et al. 2018, Maclachlan et al. 2018). It has predictable pharmacokinetics and a safety profile allowing prolonged dosing (Khot et al, Cancer Discov 2019). Unexpectedly, CX-5461 treatment increases platelet count by ~50% in tumour diseased mice; an effect that was maintained for up to 6 months of treatment. Aims: We investigated mechanisms that may contribute to increased platelet counts following CX-5461 treatment in a liver carcinoma mouse model or disease-free mice and evaluated blood test data from patients with haematological malignancies treated with CX-5461. Methods: We evaluated blood cell numbers in blood from vehicle- or CX-5461 treated mice with liver carcinoma, and blood cell numbers, bone marrow megakaryocytes, platelet receptors, immature platelets, platelet function, thrombopoietin (TPO) and inflammatory cytokine levels in disease-free C57BL/6 KaLwRij mice treated with 35 mg/kg CX-5461 or vehicle (n=15). Human platelet receptors and function were assessed in CX-5461-treated platelet-rich plasma by flow cytometry, platelet spreading assays and light transmission aggregometry. We evaluated temporal platelet count, and the platelet activation marker soluble glycoprotein (GP) VI in patient plasma samples who had received a single dose of 25-250 mg/m2 CX-5461 (n=16) with 1-way ANOVA. Results: In a model of liver carcinoma, mice with disease displayed significantly elevated (2.3-fold) platelet counts after 3 months treatment with biweekly injections of 35 mg/kg CX-5461 compared with vehicle-treated diseased mice (p = 0.0013). Disease-free mice treated for 7 or 14 days (3 or 6 x 35 mg/kg doses) with CX-5461 showed ~60% increase in platelet count; in comparison white and red cells were mildly suppressed. The CX-5461-mediated platelet increase at d7 (p<0.0005) was reversible within 1 week. At d14 (p<0.01, 1.7-fold increase in platelet count) a significant increase in megakaryocytes (p<0.05) and immature platelets (p<0.01) was observed. CX-5461 treatment had no effect on plasma TPO, platelet lifespan or platelet GPVI or GPIbα levels. Integrin αIIb was significantly elevated. Inflammatory cytokines interleukin (IL)-6 (p<0.05) and TNFα (p<0.01) increased at d7 in plasma. After pre-treatment for 30 min with CX-5461 ex vivo, human platelet function (aggregation and platelet spreading on collagen and fibrinogen) and receptor levels were normal. In 8/16 patients receiving CX-5461, increases of up to 34% in platelet counts were measured at day 15 of CX-5461 treatment, with no change in sGPVI. Conclusions: CX-5461 treatment of mice for two weeks increased platelet counts, megakaryocyte number and immature platelet fraction by an unknown mechanism however IL-6 has been reported by others to enhance megakaryopoeisis. Brief CX-5461 treatment in a group of patients with advanced haematological malignancy resulted in small increases in platelet count with retained platelet activation and treatment ex-vivo demonstrated platelet function to be unaffected. Future work will explore the link between CX-5461 treatment and megakaryopoiesis and thrombopoiesis, including the potential for CX-5461 to ameliorate drug-induced thrombocytopenia. Disclosures Harrison: Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: investigator on studies, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Hannan:Pimera: Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Evaluation of Platelet Function Defects in Patients with Immune Thrombocytopenia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1021-1021
Author(s):  
Elena Monzón Manzano ◽  
María Teresa Alvarez Román ◽  
Andres Ramirez Lopez ◽  
Elena G Arias-Salgado ◽  
Paula Acuña ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Sialic Acid ◽  
Platelet Function ◽  
Research Funding ◽  
Immune Thrombocytopenia ◽  
Healthy Controls ◽  
Activation Markers ◽  
Platelet Production ◽  
Neuraminidase Activity ◽  
Fibrinogen Receptor

Abstract Background: Primary immune thrombocytopenia (ITP) is a megakaryocytic (MK)/platelet-specific autoimmune disorder characterized by platelet count &lt;100×10 9/L with or without bleeding manifestations, and diagnosed by exclusion of other causes of thrombocytopenia. It is widely accepted the involvement of platelet autoantibodies on deterioration of platelets from patients with ITP. Moreover, an enhanced activity of neuraminidase may also reduce sialic acid from glycoside residues on platelet surface, especially from the highly glycosylated von Willebrand factor (vWF) receptor. Because controversial results regarding the functionality of platelets from ITP patients can be found in literature, we aimed to determine platelet ability to be stimulated by agonists. Moreover, we aimed to determine the way anti-platelet auto- antibodies (abs) and neuraminidase activity may affect the function of platelets derived from MKs of healthy controls. Methods: This observational, prospective and transversal study included 42 patients with chronic primary ITP and 55 healthy controls. Platelet fibrinogen and vWF receptors and activation markers (PAC1 binding to activated fibrinogen receptor and exposure of P-selectin after agonists treatment), were evaluated by flow cytometry. Presence of Antibodies (abs) against platelet's glycoproteins in ITP serum was analysed with a Luminex based assay (LifecodesPak Lx). Neuraminidase (NEU) activity in serum was determined with the substrate 20-(4-methylumbelliferyl)-a-D-N-(MUNANA). Human CD34 + cell-enriched population was obtained with CliniMACS (MiltenyiBiotec) from G-CSF mobilized peripheral blood of a healthy donor. For MK differentiation, CD34 + cells were cultured 12 days in StemSpan™ Serum-Free Expansion Medium II (SFEM II) with 50ng/ml of recombinant human thrompoietin. Then, 10% of serum from healthy controls (4) or ITP patients (4) were added to the culture of mature MKs and incubated for 3 days. Phenotypic analysis of MKs and culture derived-platelets was carried out using abs against CD34, CD41, CD42a and CD42b.Platelet-like particles were considered as CD41-positive events with a size (FSC) and granularity (SSC) scatter properties similar to blood platelets. Culture-derived platelets were stimulated with 100 µM TRAP and 10 µM ADP and activation markers were analyzed by flow cytometry. Results: Expression of fibrinogen receptor on platelets from ITP patients were similar to those from healthy controls but showed a reduced capacity to be activated. Impairment in platelet degranulation measured as exposition of P-selectin after agonist's stimulation was also observed in platelets from these patients (Figure 1). Of note, surface content of CD42b subunit of vWF receptor was reduced (Figure 1). To determine whether diminished platelet function might be due to a plasma component, we induced platelet production from MK of healthy controls as referred in Methods. Abs against platelets and neuraminidase activity were determined in serum samples. Serum from 4 healthy controls or from 4 ITP patients (1 with anti-CD42b, 1 with anti-GPIa-IIa and 2 with undetectable abs) were added to MKs culture. No differences existed in MK differentiation and platelet production between MKs incubated with serum from healthy controls or from ITP patients, but similarly as observed in platelets from ITP patients, MK-derived platelets had an impaired ability to be activated (Table 1). Platelets derived from MKs incubated with ITP serum with anti-platelet abs had also a diminished exposure of CD42b (73±8% of controls). Moreover, neuraminidase content of these samples was slightly higher than that from ITP samples without abs (130 vs 100 % of controls). Conclusion: Platelets from ITP patients had a diminished ability to be stimulated. In vitro study showed that megakaryopoiesis was normal in presence of ITP serum, but released platelets had a lower ability to be activated. Involvement of abs in this effect cannot be ruled out despite we detected abs only in 2 of the tested sera because efficiency of method to detect these abs is ~ 50%. On the other hand, reduced levels of CD42b might be due to the increased activity of neuraminidase. Reduction of sialic acid from CD42b might initiate its metalloproteinase-mediated cleavage or change affinity of the ab used for its detection. Research funded by ISCIII-Fondos FEDER PI19/00772 and Platelet Disorder Support Association Figure 1 Figure 1. Disclosures Alvarez Román: Pfizer: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding; Biomarin: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; CSL-Behring: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Novo-Nordisk: Consultancy, Honoraria, Research Funding. García Barcenilla: Roche: Speakers Bureau; Takeda: Speakers Bureau; Bayer: Speakers Bureau; SOBI: Speakers Bureau. Canales: Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria; Gilead/Kite: Consultancy, Honoraria; Eusa Pharma: Consultancy, Honoraria; Incyte: Consultancy; Karyopharm: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Sanofi: Consultancy; iQone: Honoraria; Sandoz: Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Speakers Bureau. Jiménez-Yuste: Grifols: Consultancy, Honoraria, Research Funding; NovoNordisk: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; BioMarin: Consultancy; Sobi: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding. Butta: Novo-Nordisk: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Roche: Speakers Bureau; CSL-Behring: Research Funding.

Phospho-Specific Flow Cytometry of Fixed Whole Blood During AML Clinical Trials to Monitor In Vivo FLT3 Inhibition in Leukemic Blasts,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3508-3508
Author(s):  
Alexander E Perl ◽  
Grace R Jeschke ◽  
Takashi Sato ◽  
Shiro Akinaga ◽  
Niranjan S. Rao ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Flow Cytometry ◽  
Whole Blood ◽  
Peripheral Blood ◽  
Initial Dose ◽  
Research Funding ◽  
Ex Vivo ◽  
Blood Samples ◽  
Flt3 Inhibitors

Abstract Abstract 3508 Although first-generation FLT3 inhibitors may have had limited anti-leukemic effects due to suboptimal target inhibition, newer drugs such as AC220 and KW-2449 have substantially greater in vitro potency and bioavailability. Ex vivo assays such as the plasma inhibition assay (PIA) are useful to estimate free drug bioavailability, but direct confirmation of biochemical FLT3 inhibition in leukemic blasts in vivo has proven more challenging to employ systematically for drug development. Here we report the development of a fixed whole blood intracellular flow cytometry platform to measure real-time signal inhibition during a clinical trial of the second-generation FLT3 inhibitor KW-2449. Methods: Anticoagulated blood samples were aliquoted into FACS tubes within four hours of collection; a subset was exposed to signaling inhibitors (KW-2449, rapamycin × 30 min.) or activators (phorbol ester/PMA or FLT3 ligand/FL × 10 min.) to establish dynamic controls. Following incubation, samples were formaldehyde-fixed, red cells were lysed with the permeabilizing agent triton X-100, and specimens were stored at −20C in glycerol medium. Subjects' samples from all time points were simultaneously thawed, denatured with ice-cold methanol, and stained with a single cocktail of antibodies. Blasts were identified by CD45 and side scatter (SSC) and confirmed by multiple surface markers (CD33, CD34, CD117, HLA-DR, etc.). Positive gates for phospho-proteins were created by comparing blasts in stimulated and unstimulated conditions and/or autofluorescence (FMO) controls. Results: Despite adequate controls, flow demonstrated limited changes in FLT3-ITD+ blasts' pSTAT5 signal following either FL stimulation or ex vivo KW-2449 treatment of these peripheral blood primary samples. This contrasted with the FLT3-ITD+ cell line Molm14, in which FLT3 inhibition reduced pSTAT5. However, the PI3K/AKT/mTOR downstream target ribosomal protein S6 (S6) was consistently observed to be constitutively phosphorylated in both Molm14 cells and peripheral blood FLT3-ITD+ AML blasts. pS6 in all FLT3-ITD+ samples markedly augmented with ex vivo FL, and decreased following ex vivo KW-2449 treatment. We therefore serially monitored S6 phosphorylation during therapy on a phase 1/2 trial of KW-2449. In this clinical trial, subjects were treated with KW-2449 every 6–8 hours, due to the drug's relatively short half life. 10 subjects (9 FLT3-ITD+, 1 FLT3-WT) provided serial blood samples for analysis. All FLT3-ITD+ subjects had blasts identifiable by morphology and immunophenotype. Samples with as few as 500 blasts/uL were informative for pS6. In all cases, blasts showed dynamic changes in pS6 in response to ex vivo FL. As previously described using intracellular flow cytometry, pS6 in primary AML samples was heterogeneous, and, at basal state, frequently only demonstrable in a subset of blasts. We observed constitutive S6 phosphorylation in 8/9 subjects' leukemic cells. The mean percentage of blasts with constitutive pS6 was 21% (median 7%, range 5–70%). To directly quantify FLT3 kinase inhibition in vivo, we serially monitored pS6 in blasts by flow prior to and following their initial oral KW-2449 dose. In 8/8 patients with baseline constitutive S6 phosphorylation, blood obtained two hours following the initial dose showed marked reduction in the percentage of pS6+ blasts to a mean of 3.8% (median 1.3% range 0.1 to 20%). This reflected an 83% mean reduction in the percentage of pS6+ blasts. PIA was performed in 8/9 of FLT3-ITD+ subjects and confirmed that potent FLT3-inhibitory concentrations were present 2 hours after a single dose of KW-2449 (mean reduction from baseline of 79% for pFLT3 and 88% for pSTAT5). Two subjects' samples were followed serially by flow cytometry throughout the dosing interval. One showed sustained inhibition (consistent with concurrent PIA), while in the other, pS6 returned to baseline within 4–6 hours of the initial dose (concurrent PIA not done). Summary: We confirm that PI3K/AKT/mTOR is a major downstream pathway of FLT3 signaling in primary AML samples. We further demonstrate the feasibility of intracellular flow cytometry for S6 phosphorylation to monitor the biochemical efficacy of FLT3 inhibitors in patients. Studies are underway to correlate biochemical FLT3 inhibition by flow cytometry with clinical response/resistance to KW-2449 and other FLT3 inhibitors. Disclosures: Sato: Kyowa Hakko Kirin Co., LTD: Employment. Akinaga:Kyowa Hakko Kirin Co., LTD: Employment. Rao:Kyowa Hakko Kirin Co., LTD: Employment. Levis:Kyowa Hakko Kirin Co., LTD: Research Funding; Ambit Biosciences: Consultancy. Carroll:Kyowa Hakko Kirin Co., LTD: Research Funding.

Sign in / Sign up

Export Citation Format

Share Document