bluuebunny/crossref_metadata_2025_split huggingface dataset:
CAR-T Cells Are Serial Killers of Tumor Cells
Authors: Jenkins Misty , Davenport Alex , Cross Ryan , Yong Carmen , Ritchie David S. , Trapani Joe , Kershaw Michael , Darcy Phil , Neeson Paul 
Published: October 2019

Abstract
Chimeric antigen receptor (CAR) T cells have shown clinical efficacy in refractory B cell malignancies. Despite these exciting clinical results, our fundamental understanding of CAR-T cell biology is limited, possibly limiting the broader application of CAR-T cells to additional haemopoetic and solid cancers. To date, the mechanism of CAR-T immunological synapse formation with tumor cells and kinetics of subsequent serial killing by CAR-T cells has not been explored. Here, we investigated the kinetics of CAR-T cell activation and cytotoxicity, including immune synapse formation, and kinetics of tumor cell killing, closely comparing to activation and cytotoxicity via the T cell receptor (TCR). To address this, we developed a mouse model in which the CD8+ T cells (termed CAR.OT-I cells) co-expressed two antigen receptors, the clonogenic OT-I TCR, and a second generation CAR comprising a scFV to human HER2, CD28 and CD3ζ signaling domains. Effector CAR.OT-I cells were activated via their antigen receptors using either SIINFEKL-pulsed or HER-2 expressing tumor cells, the interactions between the effector CAR.OT-I cells and tumor cells were then assessed by time lapse live microscopy. CAR.OT-I cell activation via the endogenous TCR or the CAR did not affect tumor killing kinetics, except the time taken from CAR.OT-I activation to detachment (from the dying tumor cell) was significantly slower when the endogenous TCR was engaged. Subsequently, we showed for the first time, that CAR.OT-I cells have serial killing capacity, which is important to consider when therapeutic numbers of CAR-T cells are likely to be outnumbered by tumor targets. Individual CAR.OT-I cells killed multiple tumor cells, whether activated via the endogenous TCR or the CAR. We further explored whether these findings have implications for killing of tumor cells using low effector:target cell ratio in short versus long-term killing assays, chromium release and xCELLigence killing assays respectively. We observed, no matter which antigen receptor was activated, the effector CAR.OT-I cells were equivalent killers of tumor cells in short term assays (4-8 hours). However, over a period of 50 hours, CAR.OT-I cells activated via the CAR killed tumor cells at a lower rate than when activated via the TCR. This was due to CAR.OT-I CAR expression down-regulation from 20-50 hours. This study highlights that fundamental differences occur in the way CAR-T cells kill tumor cells, depending on how the effector CAR-T cell is activated. Furthermore, the study provides important insights for CAR-T cell activation in vivo with implications for single- or dual-receptor-focused CAR-T cell therapy and improved clinical benefit.

Disclosures
No relevant conflicts of interest to declare.
Targeting IRAK1/IRAK4 Signaling in Waldenstrom's Macroglobulinemia
Authors: Yang Guang , Liu Xia , Chen Jie , Xu Lian , Tsakmaklis Nicholas , Chen Jiaji , Patterson Christopher J , Castillo Jorge J , Cohen Philip , Tan Li , Buhrlage Sara , Gray Nathanael , Treon Steven P 
Published: October 2019

Abstract
Background: MYD88 L265P somatic mutations are highly prevalent in Waldenström's macroglobulinemia (WM) (NEJM 367(9):826-33). MYD88 L265P activates multiple downstream signaling pathways including BTK and IRAK1/IRAK4 that support malignant cell growth and survival (Nature 470(7332):115-9; Blood 122(7):1222-32). Ibrutinib targets BTK, and shows high overall and major clinical response rates, though no complete responses are observed indicating alternative survival signaling.
Methodology: Phospho-flow analysis of IRAK1, IRAK4, and BTK was performed in primary WM cells taken from untreated WM patients, and those on ibrutinib therapy. IRAK1 or IRAK4 knockdown experiments were performed using lentiviral shRNA transduction. Immunoprecipitation, western blot and phospho-flow studies were used to detect protein expression and phosphorylation in WM cells. Cell survival following IRAK4 or IRAK1 knockdown, ibrutinib and/or IRAK4/IRAK1 inhibitor (EMD Millipore) treatment was assessed by Annexin V staining, AlamarBlue® Cell Viability Assay or CellTiter-Glo® Luminescent Cell Viability Assays.
Results: Phospho-flow analysis of bone marrow lymphoplasmacytic cells taken from WM patients following > 6 months of continued ibrutinib treatment demonstrated highly active IRAK1 and IRAK4, but not BTK. These findings prompted us to dissect the relative impact of IRAK1 and IRAK4 in supporting WM cell survival. Using lentiviral transduction, we identified shRNAs that produced similar levels of protein reduction by western blot analysis for both IRAK1 and IRAK4. Compared to scrambled control vector, knockdown of IRAK1 or IRAK4 both produced decreased tumor cell survival in MYD88 mutated BCWM.1 and MWCL-1 cells. More pronounced apoptosis, as well as sustained reduction in tumor cell growth occurred following knockdown of IRAK1 versus IRAK4. Treatment of primary WM cells taken from untreated patients, patients on ibrutinib therapy, as well as MYD88 mutated WM cells lines with ibrutinib and a toolbox IRAK4/IRAK1 inhibitor resulted in more robust reductions in NFkB signaling, and at least additive tumor cell killing versus either agent alone.
Conclusions: MYD88 L265P mutated WM cells show greater dependence on IRAK1 versus IRAK4 directed signaling. IRAK1/IRAK4 signaling may contribute to persistent WM cell survival following ibrutinib treatment. Combined BTK and IRAK inhibition leads to augmented blockade of NFKB signaling and enhanced WM cell killing. The studies provide a framework for the development and investigation of IRAK inhibitors, alone and in combination with ibrutinib in WM patients.

Disclosures
No relevant conflicts of interest to declare.
Pulmonary Complications in Patients Treated with Tyrosine Kinase Inhibitors, a Systematic Review and Meta-Analysis
Authors: Chai-Adisaksopha Chatree , Calvin Diep , Hillis Christopher M 
Published: October 2019

Abstract
Background: In patients with chronic myeloid leukemia (CML), long-term use of tyrosine kinase inhibitors (TKIs) is associated with hematological and non-hematological complications. To investigate the pulmonary complications (pulmonary edema and pulmonary arterial hypertension [PAH]) in CML patients receiving TKIs, we conducted a systematic review and meta-analysis of clinical trials and observational studies.
Methods: We searched MEDLINE, EMBASE and the Cochrane library to May 2015. The studies were included if they enrolled CML patients, used at least one of imatinib, nilotinib, dasatinib, bosutinib and ponatinib, and reported pulmonary effusion or PAH as one of their outcomes. We performed a single proportion meta-analysis for the cumulative incidence of each outcome. Subgroup analysis was carried out based on type of TKI, phase of disease, dosage and line of treatment.
Results: Thirty-nine studies, involving 6562 patients were included in the meta-analysis. The overall cumulative incidence of pleural effusion was 5% (95% confidence interval [CI]; 4.2-5.8, I2=95.9%). Subgroup analysis according to type of TKI revealed that dasatinib was associated with highest incidence of pleural effusion, 26.2% (95%CI; 21.4-31.1) compared to imatinib (0.5%), nilotinib (0.1%), bosutinib (2.1%) and ponatinib (0.3%), Figure 1. Subgroup analysis demonstrated that accelerated/blastic phase, non-first line treatment and higher dose of dasatinib were associated with a higher risk of pleural effusion. There were 7 cohorts reporting PAH. The overall cumulative incidence of pleural effusion was 0.2 per 100 patient year (95% CI; -0.3, I2 =0%) for all TKIs, Figure 2.
Conclusions: Pleural effusion is a frequently found pulmonary complication among CML patients receiving dasatinib. However, PAH is uncommon for TKI treated patients. We recommend a low threshold for performing a chest x-ray in symptomatic patients on dasatinib. Because of the low incidence of PAH, we cannot recommend routine screening for PAH in the absence of highly suspicious symptoms.
Figure 1. Cumulative incidence of pleural effusion in CML patients treated with tyrosine kinase inhibitors Figure 1. Cumulative incidence of pleural effusion in CML patients treated with tyrosine kinase inhibitors Figure 2. Cumulative incidence of pulmonary artery hypertension in CML patients treated with tyrosine kinase inhibitors Figure 2. Cumulative incidence of pulmonary artery hypertension in CML patients treated with tyrosine kinase inhibitors

Disclosures
No relevant conflicts of interest to declare.
Therapeutic Immune Monitoring of Chronic Myeloid Leukemia Patients Treated with Tyrosine Kinase Inhibitors: Focus on CD4+ CD25+ t Cells
Authors: Lu Ziyuan , Xu Na , Zhou Xuan , Gao Guanlun , Li Lin , Huang Jixian , Li Yuling , Lu Qisi , He Bolin , Pan Chengyun , Xiaoli Liu 
Published: October 2019

Abstract
Background and Objectives: In clinical, conventional Tyrosine Kinase Inhibitors (TKIs) including imatinib, dasatinib, and nilotinib are remarkably effective forms of therapy for certain types of solid cancers as well as Ph+ leukemias. In addition to the BCR-ABL target oncoprotein, they also inhibit certain off-target kinases (Eph, c-KIT, TEC, SRC). Some TKIs affect immune reconstitution as well as the proliferation, function, and activation of T cells. Certain TKIs have been known to have an especially strong effect on CD4+CD25+ T cells, also known as regulatory T Cells (Tregs). There is currently a gap in the clinical data available about on this area of study.
Patients and methods: In this study, we collected 108 Peripheral Blood (PB) samples from patients in the Chronic Phase (CP) of Chronic Myeloid Leukemia (CML) at the time of diagnosis (n=31) and also the TKIs treatment. Groups consisted of individuals treated with TKIs like imatinib (n=12), dasatinib (n=11) and nilotinib (n=8), as well as healthy controls (n=15). We evaluated the quantity and function of Tregs from patients in the CML-CP at the time of diagnosis and during treatment with TKIs.
Results: It was found that at diagnosis, patients with CML had a similar proportion and absolute number of lymphocytes compared to healthy donors. After TKIs treatment, proportions and absolute numbers of total T cellsACD4+ T cells and Tregs decreased at different degree. Moreover, thedecrease would be more and more significant as time goes on.Our results indicated that although these three TKIs show similar inhibitory effects in the proportion and number of Tregs in vivo, they have differential effects on the functions of Tregs in vitro. The proliferation, suppression, and expression of suppressive cytokines (IL-4,IL-10 and TGF-β) as well as suppression-associated molecules (FoxP3, GITR, and CTLA-4) of Tregs decreased in groups treated with imatinib and dasatinib. The decrease was not significant in the nilotinib-treated group.
Conclusions: The results showed that imatinib and dasatinib have stronger inhibitory roles than nilotinib when it comes to regulating the number and functions of Tregs. These findings can be used to argue in favor of calls for personalized treatment and follow-up of CML patients during TKIs treatment, particularly for those patients who received combination therapy with allo-transplantation and post-transplant TKIs.

Disclosures
No relevant conflicts of interest to declare.
Flow Cytometry Based Detection of MRD in Bone Marrow of Patients with Multiple Myeloma: A Comparison Between Fluorescent-Based Cytometry Versus Cytof
Authors: Ghosh Arnab , Carreau Nicole , Moscatello Alessandra , Rahman Adeeb , Qi Jingjing , Kim-Schulze Seunghee , Navada Shyamala C. , Chari Ajai , Cho Hearn Jay , Jagannath Sundar , Parekh Samir 
Published: October 2019

Abstract
The introduction of novel agents for the treatment of multiple myeloma (MM) has shifted the emphasis towards achieving a molecular complete remission (CR). Traditional methods to detect malignant plasma cells (PC) in minimal residual disease (MRD) utilize multicolor flow cytometry (MFC) to detect aberrant phenotypes. However fluorescent-based MFC assays are limited by the number of markers, difficulty in standardization of assays and overlap of signal between the fluorescent channels. These limitations can be overcome by cyTOF, a flow cytometry assay based on time-of-flight mass spectrometry using antibodies labeled with heavy-metal ions, permitting simultaneous assessment of large panels of markers. We have developed a novel highly sensitive mass cytometry protocol for the detection of MRD. BM from 5 MM patients and 1 non-myeloma control were RBC-lysed and labeled with MM markers for MFC and cyTOF. cyTOF is a flow cytometry based on mass spectometry where antibodies are labeled with heavy-metal ions, permitting utilization of more markers without concern for spillover. The MFC panel included CD38, CD138, CD45, CD56, CD19, CD117. Labeled cells for MFC were acquired by BD LSR Fortessa.. The panel for cyTOF comprised markers for cells across the hematopoietic spectrum and those specific for MM. Labeled cells acquired with cyTOF were analyzed using SPADE. Limits of detection (LOD) and quantification (LOQ) for MFC are based on prior reports. The results were compared with an independent commercial MFC-based assay (Genoptix). Malignant PC could be detected by MFC and cyTOF based assasys (See Figure). We were able to detect MRD in three subjects that had no detectable disease by an independent commercial MFC based assay (Genoptix). This can be attributed to acquiring at least 1x106 for our assays leading to a lower LOD and LOQ (greater sensitivity). Using cyTOF and SPADE, we could detect MRD in 4/5 patients. Overall our panel with cyTOF has a lower LOD than MFC for detection of MRD. Early detection of MRD in MM patients using a highly sensitive flow cytometry like cyTOF will help in risk stratification, predicting relapse and studying response to therapy.

Disclosures
Chari: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium/Takeda: Consultancy, Research Funding; Onyx: Consultancy, Research Funding; Array Biopharma: Consultancy, Other: Institutional Research Funding, Research Funding; Biotest: Other: Institutional Research Funding; Novartis: Consultancy, Research Funding. Jagannath:Bristol Myers Squibb: Honoraria; Janssen: Honoraria; Merck: Honoraria; Novartis: Honoraria; Celgene: Honoraria.
Factor XI Deficiency Protects Against Atherogenesis
Authors: Shnerb Reut , Harats Dror , Schiby Ginette , Gailiani David , Levkovitz Hanna , Tamarin Ilia , Shaish Aviv , Salomon Ophira 
Published: October 2019

Abstract
In industrialized societies atherosclerosis is still a major cause of morbidity and mortality, in spite of advances in development of cholesterol lowering drugs such as statins and ezetimibe. The detection of procoagulant proteases within atherosclerotic plaques, and the observation that patients with severe factor XI (FXI) deficiency have a reduced tendency to develop ischemic stroke, lead us to investigate whether absence of FXI would affect the process of atherogenesis. For this purpose, we created mice that are homozygous for null alleles for apolipoprotein E (apoE) and FXI (Double knock out [DKO] mice), and compared development of atherosclerosis in these animals to mice lacking apoE alone. At 24 weeks of age, there was a 32% reduction in atherosclerotic plaque area in the aortic sinus in apoE/FXI DKO mice compared to apoEKOmice (p=0.004). At age 42 weeks, apoE/FXI DKO mice had 25% smaller atherosclerotic plaque area in the aortic sinus compared to apoEKO mice (p=0.024), and aortic plaque area was reduced by 49% (p=0.028). Plasma cholesterol and triglycerides levels and lipoprotein profiles were similar in apoE/FXI DKO and apoE KO mice. In some murine infection models, FXI deficiency or FXI inhibition is associated with reduced markers of inflammation and improved survival. Based on this observation, we explored the possibility that reduced atherogenesis in apoE/FXI DKO mice is associated with reduced inflammation. There was a significant reduction in macrophages within atherosclerotic plaque in apoE/FXI DKO mice compared to apoE KO mice as determined by CD68 immunostaining. In conclusion, this work indicates for the first time that factor XI deficiency significantly reduces early, as well as advanced, atherosclerotic burden in an established animal model. We hypothesize that the effect of FXI on atherosclerosis is related to a proinflammatory effect within the atherosclerotic plaque. and propose that therapeutic targeting of FXI may have a role in preventing development of atherosclerosis. As FXI serves a minor role in hemostasis, treatment directed at this protein should not be associated with a high risk of major bleeding.

Disclosures
No relevant conflicts of interest to declare.
The ATP-Competitive mTOR Inhibitor AZD8055 Reduces Cell Proliferation and Tumour Load in Chronic Lymphocytic Leukaemia
Authors: Cosimo Emilio , Tarafdar Anuradha , Holroyd Ailsa , Dunn Karen , Catherwood Mark , Michie Alison M. 
Published: October 2019

Abstract
Chronic lymphocytic leukaemia (CLL), often diagnosed in the elderly, is incurable with current therapeutic regimes. Recent studies demonstrate that the microenvironment within CLL patient lymphoid organs protects leukaemic cells from chemotherapy induced apoptosis. This highlights the need for novel therapies that can overcome such cytoprotective signals. Serine/threonine protein kinase mammalian target for rapamycin (mTOR) is a key regulator of cell survival and proliferation and is commonly deregulated in cancer. mTOR is active in two complexes mTORC1 and mTORC2. We demonstrate that mTORC1 and mTORC2 substrates, including S6 (pS6S235/236) and Akt (pAktS473) respectively, are active although differentially regulated, in CLL cells from distinct prognostic subsets ex vivo, which suggests that mTOR may represent a valid therapeutic target for CLL. Initial studies with everolimus, an analogue of rapamycin, showed only limited antitumor activity in recurrent/refractory CLL patients, possibly because mTORC1 inhibition releases the "brake" that normally active mTORC1 has on mTORC2, thus enabling mTORC2 mediated signalling. To test the impact of inhibiting both mTORC1 and mTORC2 on cell proliferation and survival, we treated primary CLL cells and an aggressive CLL mouse model with the novel ATP-competitive mTOR inhibitor AZD8055 in vitro and in vivo. CLL proliferation and mTORC1/2 activity were significantly reduced upon addition of AZD8055 to NTL-CD154/IL4 co-cultures, which mimic pro-proliferative conditions present in the microenvironment of lymphoid organs. However, in the same conditions use of AZD8055 did not induce apoptosis at clinically-achievable drug concentrations (<150 nM), likely due to ineffective inhibition of Mcl-1 upregulation mediated by the NTL-CD154/IL4 co-cultures. In contrast, B cell receptor (BCR)-mediated survival was inhibited by AZD8055 treatment, inducing significant levels of apoptosis, concomitant with a decreased Mcl-1 expression. These findings suggest that the BCR and CD154/IL4 mediated pathways regulate Mcl-1 expression by distinct mechanisms. In vivo studies using the PKCa-KR-induced CLL mouse model revealed that AZD8055 treatment efficiently reduced tumour burden in the organs (spleen and bone marrow) and blood of mice with established CLL. This was reflected by in vitro studies showing that use of AZD8055 increased apoptosis and reduced proliferation in mouse CLL cells. Overall these data indicate that AZD8055 inhibits CLL proliferation, in vitro and in vivo, and disrupts BCR mediated survival, by downregulating Mcl-1 protein levels. Therefore dual mTOR inhibitors through reduction of tumour burden, show promise as a future therapy for CLL.

Disclosures
No relevant conflicts of interest to declare.
High Expression of AF1q Is an Adverse Prognostic Factor in Multiple Myeloma Patients Treated with Autologous Stem Cell Transplantation
Authors: Hagiwara Shotaro , Schiefer Ana-Iris , MINE Sohtaro , Kenner Lukas , Tse William 
Published: October 2019

Abstract
Introduction: Multiple myeloma is a heterogeneous hematological malignancy. Investigation on the poor prognostic factors contributes the development of the effective stratified treatment.
AF1q is an oncogene which expressed in leukemia cells, located in 1q21. The gene is well known as a one of the partner of 11q23. Recently, the association between the over expression of AF1q and the tumor progression has been reported by several investigators.
We studied the expression of AF1q in patients with multiple myeloma and analyzed the prognostic value.
Method: Newly diagnosed multiple myeloma patients in National Center for Global Health and Medicine hospital from January 2001 to March 2013 were studied. Patients were treated with vincristine-adriamycin-dexamethason or bortezomib-dexamethason induction therapy followed by autologous stem cell transplantation using high dose melphalan.
The expression of AF1q was evaluated using the immunostain of bone marrow clot samples at the diagnosis of multiple myeloma. The expression of AF1q was graded from "-" to "+++". The clinical response, progression free survival, and overall survival were analyzed.
Results: Clinical data and bone marrow clot samples of 61 patients were analyzed. Mean age was 55.5 years old, and 52.5% was male. The grades of AF1q expression were 2 of "-", 18 of "+", 17 of "++", and 24 of "+++". We defined the cases with "-" and "+" as low expression, "++" and "+++" as high expression. Very good partial response or better was obtained after completion of autologous stem cell transplantation in 45% of patients with low AF1q expression and 48.8% of high expression, there was no statistically significant difference.
Survival analysis using Log Rank method showed that high expression of AF1q was associated with significantly shorter progression free survival (p=0.004), but not overall survival.
Conclusion: In this study, we found that the high expression of AF1q was an adverse prognostic factor in multiple myeloma.
Figure 1. Figure 1.

Disclosures
Hagiwara: Celgene Corporation: Membership on an entity's Board of Directors or advisory committees.