bluuebunny/crossref_metadata_2025 huggingface dataset:
Melatonin Protects Against Apoptosis of Megakaryocytes Via Its Receptors and Activation of PI3k/Akt Pathway
Authors: Li Su-yi , Zhou Min , Ye Jie-yu , Lian Qi-zhou , Meng Fan-yi , Yang Mo
Published: October 2019

Melatonin has been shown to be able to lower the frequency of thrombocytopenia in patients with cancer undergoing radiotherapy or chemotherepy in clinical studies (Lissoni et al, J Pineal Res, 1999). Melatonin is an acetylated product of serotonin. Our previous study demonstrated that serotonin stimulates megakaryopoiesis and thrombopoiesis (Yang et al, Stem Cells, 2007; 2014). However, the underlying mechanism of melatonin on platelet formation/survival has not been well investigated. We hypothesized that melatonin has therapeutic effects on thrombocytopenia by reducing apoptosis of megakaryocytes (MKs)/platelets via melatonin receptors and activation of Akt signaling. We investigated the in-vitro effect of melatonin on CFU-MK formation at different doses (0-500 nM). The results showed that melatonin significantly promoted CFU-MK formation at a dose dependant manner. The maximal concentration was at 200 nM (P<0.01, n=6). The size of CFU-MK with melatonin treatment was much larger and each MK cell was more mature looking. Using megakaryocytic cell CHRF-288-11, we identified the expression of melatonin receptors MT2 on these MK cells by RT-PCR and western blotting. We further found that melatonin exerted protective effect on serum-free induced apoptosis of CHRF cells. The cell viability was significantly increased with the treatment of melatonin. Flow cytometric dot-plot analysis demonstrated that the population of apoptotic cells (Annexin V/PI staining) was significantly decreased with melatonin treatment (200nmol/L) for 72 hrs (n=4). It also showed trends of amelioration of caspase-3 expression and the proportion of cells containing JC-1 monomers (indicating a trend in the drop of mitochondrial membrane potential). The population of phospho-Akt were significantly increased after treatment by melatonin and this effect was abolished by melatonin receptor MT2 blocker. The effect of melatonin on in-vivo thrombopoiesis was also investigated in an irradiated mouse model. Eighteen mice were randomly divided into three groups (6 each). Group 1 (normal control) received no irradiation or melatonin; Group 2 (model control) and Group 3 (melatonin treated) both received 4 Gy total body irradiation. After irradiation, melatonin (10mg/kg.d) was injected intraperitoneally into Group 3 consecutively for 21 days. Peripheral blood platelets, white blood cells (WBC), and red blood cells (RBC) were analyzed from the three groups on day 0, 7, 14, and 21.We found that melatonin enhanced the recovery of platelets and WBC production in Group 3. Our results showed that melatonin enhanced platelet formation and survival in radiation induced thrombocytopenia. The underlying mechanisms might involve in direct stimulation of megakaryopoiesis and anti-apoptotic effect on megakaryocytes via it receptors and activation of Akt signaling.

Disclosures

No relevant conflicts of interest to declare.
Thrombin Generation in Zebrafish
Authors: Schurgers Evelien , Moorlag Martijn , Kelchtermans Hilde , Hemker Coenraad , De Laat Bas
Published: October 2019

Introduction Recently, the whole blood calibrated automated thrombogram (CAT) was miniaturized enabling the measurement of thrombin generation (TG) in a limited volume (5 µl) of whole blood. Consequently, this approach may be used to determine TG in small lab animals. Zebrafish are readily available test animals for genetic research. However, their small size has been a hurdle in thrombosis and hemostasis research since most assays require large amounts of plasma. In this study we verified the possibility to measure TG in zebrafish using our newly developed miniaturized whole blood assay.

Methods For TG, 5 µl of whole blood was mixed with 5 µl of buffer containing a rhodamin-based thrombin-sensitive P2Rho substrate (final concentration (fc) 300 µM). 5 µl of this mixture was put on a paper disk and covered with mineral oil to prevent evaporation. Calibration was done as described previously (Ninivaggi et al. Clin Chem 2012) by adding 5 µl of whole blood to 5 µl of a mixture containing P2Rho (fc 300 µM), a2M-thrombin calibrator (fc 100 nM) and citrate (fc 9,8 mM). Fluorescence was detected with a fluorometer (485/538 nm).

Results Due to their limited blood volume, it is impossible to perform both a TG and calibrator measurement on the same fish. Since calibrator measurements performed on blood from different fish demonstrated acceptable variation (CV calibrator slopes < 15%), the average calibrator slope was used for calculations. The calculated TG parameters from 2 independent experiments are depicted in Table 1. TG measured in individual fish showed the same amount of inter-individual variation as in humans. Striking differences with human TG parameters were the short lag time, high peak and high velocity index. Moreover a further analysis of the fibrin network of the clot, by means of scanning electron microscopy (SEM), showed a much denser network composed of thinner fibers compared to humans.

Table 1. Thrombin generation parameters and their coefficient of variance (CV) in zebrafish in two independent experiments. Experiment 1(n=4) Experiment 2(n=9) Mean CV Mean CV Peak ETP (nM.min) 575.68 19.69 689.71 24.91 Peak (nM) 1573.16 29.58 1668.05 14.46 Lag time (min) 0.27 0.00 0.49 10.92 Time to peak (min) 1.08 5.30 1.37 11.49 Velocity (nM/min) 4002.33 38.21 3826.35 17.04

Conclusion These results demonstrate the feasibility of measuring TG in whole blood collected from zebrafish. Consequently, zebrafish may be used as a in vivo model to test the effect of (novel) anticoagulant therapeutics on thrombin generation and serve as a model organism for mechanistical research in thrombosis and haemostasis.

Disclosures

No relevant conflicts of interest to declare.
Small-Scale Mass Spectrometry-Based Phosphoproteomic Analysis of Primary Hematopoietic Stem and Progenitor Cells to Identify Critical Regulators of HSPC Mobilization and Leukemogenesis
Authors: Wang Leo D. , Nguyen Phi , Ficarro Scott B , Hutchinson John , Hofmann Oliver , Marto Jarrod A , Wagers Amy J.
Published: October 2019

Cellular functions are largely effected by proteins, but protein-level analysis of hematopoietic stem and progenitor cell (HSPC) functions has historically been challenged by the difficulty of performing comprehensive and robust proteomic studies of rare cell populations. To confront this challenge, we developed a novel nanoscale multidimensional mass spectrometry-based phosphoproteomic platform that allows, for the first time, comprehensive and unbiased analysis of the activated protein circuits in blood stem cells, as assessed by protein phosphorylation status. We used this platform to interrogate the proteomic features responsible for the growth and maintenance of hematopoietic progenitors. Our analysis pipeline is capable of returning 12,000 unique phosphopeptide sequences (corresponding to several thousand proteins) from an input of 400,000 FACS-sorted primary mouse HSPCs. Among these phosphorylated proteins, the novel Rac-GAP Arhgap25 emerged as an important regulator of mobilization in HSPCs. Arhgap25 is phosphorylated upon treatment of HSPCs with a standard cyclophosphamide-GCSF mobilization protocol. Germline deletion of Arhgap25 in mice impairs HSPC egress from the bone marrow, both at rest and after mobilizing stimuli. These findings validate the use of this platform in the discovery of new therapeutic targets in hematopoiesis, and present a clear pathway for identifying novel targets in other rare subsets of human progenitor cells, including leukemia stem cells.

Disclosures

No relevant conflicts of interest to declare.
PAX5 Tyrosine Phosphorylation By Syk Co-Operatively Works with Its Serine Phosphorylation By ERK1/2 and Cancels PAX5-Dependent Repression of BLIMP1: A Mechanism of Antigen-Triggering Plasma Cell Differentiation through B Cell Receptor Signal
Authors: Inagaki Yuichiro , Hayakawa Fumihiko , Kojima Yuki , Morishita Takanobu , Imoto Naoto , Naoe Tomoki , Kiyoi Hitoshi
Published: October 2019

Pax5 is an essential transcription factor to maintain B cell identity. Pax5 is expressed in stages from pro-B to mature B cells and promotes the B cell differentiation program by transcriptional activation of many B cell receptor (BCR)-related genes such as CD19, CD79a, and BLNK. On the contrary, it inhibits plasma cell differentiation by suppressing the expression of BLIMP1 and XBP-1, transcription factors essential for plasma cell differentiation. After BCR stimulation by antigen, upregulation of BLIMP1 and XBP-1 and subsequent suppression of PAX5 by BLIMP1 were observed and thought to be the trigger of plasma cell differentiation. We previously demonstrated that serine phosphorylation of PAX5 by ERK1/2, a main component of BCR signal, attenuated the BLIMP1 suppression by PAX5 and that the PAX5 phosphorylation might be the initial event for plasma cell differentiation (Yasuda T et al, J Immunol. 2012; 188: 6127-34). Here, we investigated additional PAX5 phosphorylation by BCR signal and found that another BCR signal component, Syk, caused PAX5 phosphorylation in vitro (Figure A). We identified the tyrosines that were phosphorylated by Syk in vitro by making phosphorylation-defective mutants, and confirmed that Syk phosphorylated PAX5 at the same sites in vivo (Figure B). In the luciferase reporter assays, PAX5 tyrosine phosphorylation by Syk attenuated the BLIMP1 suppression by PAX5, similarly to its serine phosphorylation by ERK1/2, and both phosphorylations co-operatively worked for it (Figure C). Furthermore, we demonstrated that B cell receptor stimulation with anti-IgM antibody induced Syk and ERK1/2 activation, tyrosine and serine phosphorylation of endogenous Pax5, and upregulation of Blimp1 mRNA. These results suggested that PAX5 phosphorylations by Syk and ERK1/2 co-operatively work for the cancelation of transcriptional repression of Blimp1 by PAX5 after BCR activation by antigen. This might be a trigger of plasma cell differentiation. Our findings give a new insight into the regulation of the terminal differentiation of B cells.

Figure 1 Figure 1.

Disclosures

Naoe: Zenyaku Kogyo: Research Funding; Dainippon Sumitomo Pharma: Research Funding; Kyowa Hakko Kirin Co. LTD: Research Funding; Chugai Pharmaceutical Co. LTD: Research Funding; Novartis Pharma,: Research Funding; Bristol-Myers Squibb: Research Funding; Otsuka Pharmaceutical Co. LTD: Research Funding; FUJIFILM Corporation: Research Funding. Kiyoi:Zenyaku Kogyo: Research Funding; Dainippon Sumitomo Pharma: Research Funding; Kyowa Hakko Kirin Co. LTD.: Research Funding; Chugai Pharmaceutical Co. LTD: Research Funding; Bristol-Myers Squibb: Research Funding; FUJIFILM Corporation: Research Funding.
Function of Growth Factor Independence 1 (GFI1) in the Polarization AML -Associated Stroma
Authors: Botezatu Lacramioara , Al-Matary Yahya S. A. , Opalka Bertram , Dürig Jan , Schroeder Thomas , Haas Rainer , Dührsen Ulrich , Khandanpour Cyrus
Published: October 2019

The growth of various solid tumors, lymphoma and leukemias is not only the result of cell-specific changes at the genetic and epigenetic level, but is also affected by the surrounding stroma and the cells therein. It has been shown that tumor cells induce surrounding immune cells to express various cytokines and other factors, which promote further growth and spread of tumor cells. Most studies have been conducted with solid tumors, however, not much is known about the role of stroma cells with regard to haematopoietic tumors. In addition, our knowledge is limited with regard to the cell intrinsic factors governing the polarization of stroma cells. We intended to study the role of stroma cells in acute myeloid leukemia (AML), a malignant disease of the myeloid lineage and in myelodysplastic syndrome (MDS), a disease which is characterized by disturbed function of the bone marrow, which can often progress to AML.

We focused our studies on two different components of the stroma: mesenchymal stroma cells (MSC) and monocytes.

Bone marrow derived MSCs and monocytes originating from AML patients better supported the growth of human leukemia cells in vitrothan MSCs and monocytes from control healthy persons. Interestingly, after achieving a remission, MSCs and monocytes from the cured patients did not any more support the growth of leukemia cells to the same extent as monocytes and MSCs from leukemic patients.

To better understand the mechanism behind this observation, we used different murine MDS and AML models. Similar to the finding in human patients, bone marrow derived MSCs and monocytes from MDS and AML mice better supported the growth of leukemic cells in vitro than monocytes and MDSc from healthy mice. In addition, we observed a higher number of non-malignant MSCs and monocytes in the bone marrow of leukemic mice than in the bone marrow of healthy mice. Furthermore, the higher the number of monocytes was in the bone marrow of the mice, the more aggressive was the course of the leukemia. On a molecular level we found that the transcription factor Gfi1 is 3-4 fold upregulated in the MSCs and monocytes of leukemic mice. To verify whether Gfi1 is indeed required for the polarization of MSCs and monocytes we used leukemic Gfi1-deficient mice. In these mice the accumulation of monocytes was less pronounced than in the bone marrow of Gfi1 wildtype mice. In addition MSCs and monocytes from Gfi1-deficient mice did not support to the same extent the growth of leukemic cell lines in vitro, as did the monocytes and MSCs of Gfi1 wildtype leukemic mice. Thus, we have first indications that MSCs and monocytes play an important role in the support of leukemic cells and that Gfi1 is involved in the polarization of these cells and thus Gfi1 could serve as an additional approach to treat leukemia and MDS.

Disclosures

No relevant conflicts of interest to declare.
Bone Marrow Memory CD8+ T Cells Play a Supporting Role in Hematopoiesis
Authors: Geerman Sulima , Pascutti Fernanda M , Bhushal Sudeep , Nolte Martijn A.
Published: October 2019

The bone marrow (BM) has an important function as primary lymphoid organ through the process of hematopoiesis. This process is sustained by hematopoietic stem cells (HSCs) and their life-long production of new blood cells, which is made possible by their unique ability to self-renew. Next to this, BM also functions as a secondary lymphoid organ, as it can mediate primary T cell responses against invading pathogens. We and others have shown that activated T cells can influence the hematopoietic process through the production of pro-inflammatory cytokines. This indicates that adaptive immune responses and hematopoiesis are intertwined in the BM, though most of the cellular and molecular interactions that occur during this crosstalk are yet unknown.

Based on previous observations that T cell deficient mice have altered hematopoiesis and that depletion of T cells from allogeneic BM grafts compromises HSC engraftment, we questioned to what extent BM T cells can directly affect the function of HSCs. To test this, we sorted and co-cultured murine BM T cells with HSCs (Lin-Sca-1+c-Kit+CD48-CD150+). We found that particularly BM CD8+ central memory (CD44+CD62L+) T cells (Tcm) enhance the capacity of HSCs to self-renew. Furthermore, we found that TCR-transgenic mice, which do not have memory T cells, have lower numbers of HSCs, which could subsequently be increased by transferring BM CD8+ Tcm. Remarkably, an increase in HSC numbers was also observed when HSCs were cultured with only supernatant derived from BM CD8+ Tcm. Moreover, HSCs cultured with supernatant from BM CD8+ Tcm and later transplanted in myeloablated hosts displayed a strongly enhanced ability to restore hematopoiesis. Importantly, the strong impact of BM T cells on HSCs was not only apparent in the steady state situation, but also following a viral infection with either acute or chronic lymphocytic choriomeningitis virus (LCMV). We could establish that both acute and chronic LCMV-specific CD8+ T cells or supernatant from these cells could increase the HSC self-renewal capacity.

In conclusion, our findings demonstrate that BM memory CD8+ T cells can positively influence the function of HSC through soluble mediators. We postulate that this process is particularly relevant after immune activation, in order to protect and/or restore the HSC pool and the subsequent hematopoietic recovery. We are currently using a proteomics approach to identify these soluble mediator produced by CD8+ T cells.

Disclosures

No relevant conflicts of interest to declare.
Diagnostic Efficacy of Whole-Exome Sequencing in 250 Patients with Congenital Bone Marrow Failure
Authors: Muramatsu Hideki , Okuno Yusuke , Yoshida Kenichi , Doisaki Sayoko , Hama Asahito , Wang Xinan , Narita Atsushi , Kawashima Nozomu , Xu Yinyan , Sakaguchi Hirotoshi , Takahashi Yoshiyuki , Sanada Masashi , Shiraishi Yuichi , Chiba Kenichi , Tanaka Hiroko , Miyano Satoru , Ogawa Seishi , Kojima Seiji
Published: October 2019

Introduction: Congenital bone marrow failure syndromes (CBMFSs) are a heterogeneous class of diseases with overlapping phenotypes. Therefore, a precise and comprehensive genetic diagnostic system is strongly warranted to arrive at appropriate clinical decisions to avoid ineffective therapies and/or lethal complications of allogeneic hematopoietic stem cell transplantation. However, a large panel of newly identified causative genes of CBMFSs have been identified in recent years; therefore, it is virtually impossible to establish a routine genetic diagnostic test using conventional Sanger sequencing. Whole-exome sequencing (WES) is a promising solution for the diagnosis of inherited diseases because it tests virtually all genes simultaneously. For the introduction of WES into clinical practice, it is necessary to clarify whether this technique has superior diagnostic efficacy to conventional clinical genetic tests.

Methods: We performed WES in 250 patients with CBMFSs lacking genetic diagnoses. Exome capture was performed using the SureSelect® Human All Exon V3–5 kit (Agilent Technologies, Santa Clara, CA, USA), which covers all known coding exons, followed by massively parallel sequencing using the HiSeq 2000 Sequencing System (Illumina, San Diego, CA, USA). Our established pipeline for WES (genomon: http://genomon.hgc.jp/exome/) detected >20,000 candidate variants per patient. Diagnoses were based on variants of 130 genes with pathogenicities confirmed by published studies.

Results: Genetic diagnoses were possible in 68 patients (27%). The best efficacy was achieved in patients with Fanconi anemia [35/73, 48%; FANCG (n = 17), FANCA (n = 14), FANCB (n = 1), FANCF (n = 1), SLX4 (n = 1), and BRCA2 (n = 1)], although Sanger sequencing was not applied because of the large sizes of its causative genes. Encouraging results were obtained in patients with Diamond–Blackfan anemia [11/ 61, 18%; RPS26 (n = 3), RPS7 (n = 2), RPS19 (n = 2), RPL5 (n = 2), RPL35A (n = 1), and RPL11 (n = 1)] and dyskeratosis congenita [7/29, 24%; TERT (n = 3), TINF2 (n = 2), and DKC1 (n = 2)]. Five genetic diagnoses (7%) were inconsistent with clinical diagnoses, possibly because of overlapping disease phenotypes.

Conclusion: Relative to conventional genetic testing, WES was found to be effective for the diagnoses of CBMFSs. Furthermore, the efficacy of WES will increase as our knowledge of gene mutations expands. In conclusion, the use of WES in clinical practice is warranted.

Disclosures

No relevant conflicts of interest to declare.
Expression of c-MYC and MMP9 in Alternatively Activated Macrophages (M2) of Classical Hodgkin Lymphoma
Authors: Yang Long , Harris Jonathan A. , Ibrahim Sherif , Liu Cynthia
Published: October 2019

Classical Hodgkin Lymphoma (CHL) is a monoclonal lymphoid neoplasm that accounts for approximately 30% of all lymphomas in the United States. Although multiagent chemotherapy and radiation therapy have made CHL a highly curable disease, approximately 15-20% of patients are refractory to the treatment or relapsed after remission. The rich inflammatory cellular microenvironment plays an important role in the pathogenesis of CHL. Gene-expression profiling has identified a gene signature of tumor-associated macrophages (TAMs) that was significantly associated with primary treatment failure. The potential of macrophage-specific markers, CD68 and CD163, as prognostic markers for resistance to therapy in CHL patients has been investigated; however, these studies generated inconsistent results. Roughly TAMs could be characterized into two distinct polarization states; the classically activated type 1 macrophages (M1) associated with acute inflammation and T-cell immunity and the alternatively activated type 2 macrophages (M2) associated with the promotion of the tumor growth. In the present study, we hypothesize that the specific type of TAMs, M2, may predict poor prognosis in CHL. To test this hypothesis, we examined the correlation between expressions of two M2-specific markers, c-MYC and matrix metalloproteinase 9 (MMP-9), with disease recurrence in a total of 42 cases of CHL, mostly nodular sclerosis subtype. Both c-MYC and MMP-9 are good candidate markers because c-MYC has been shown to be a specific M2 marker and plays a central role in M2 differentiation. In addition, matrix metalloproteinase 9 (MMP-9) is a M2 specific marker that directly related to its protumoral function. Immunohistochemical staining showed that both c-MYC and MMP9 are expressed in a subset of macrophages, but not in Hodgkin Reed-Sternberg (HRS) cells. By comparing the percentage of positive stains of these two markers within HRS rich areas in the primary tumor samples, we found that there was no significant difference in either c-MYC or MMP9 staining between patients who later recurred (n=6) or did not recur (n=36). The role of TAMs or an unidentified subtype of TAMs in predicting resistance to therapy in CHL patients remains to be determined.

Disclosures

No relevant conflicts of interest to declare.
Clinical Presentation and Outcome of ABVD-Treated Elderly Versus Younger Patients with Classical Hodgkin´s Lymphoma
Authors: Acosta-Maldonado Brenda Lizeth , Ramirez-Ibarguen Ana , Grimaldo-Gomez Flavio A , Rivas-Vera Silvia
Published: October 2019

Background

Approximately 20% of classical Hodgkin’s Lymphoma (cHL) patients are over 60 years of age. There is no standard of care for this age group. Although most patients receive ABVD (Doxorubicin, bleomycin, vinblastine and dacarbazine), a standard regimen in younger patients, little is known regarding the clinical presentation of cHL, and the long term efficacy of ABVD in young and elderly Latin American Mestizo patients.. Defining whether individuals in all age groups and social circumstances benefit from a common treatment approach requires investigation..

Thus we sought to evaluate and compare the clinical presentation and the efficacy of ABVD in the elderly (60 years of age and above) versus the younger population of individuals with cHD.

Methods

We retrospectively analyzed 44 consecutive cHL patients treated with ABVD at the National Cancer Institute in Mexico between 2006 and 2013, as compared to 218 patients under 60 years of age.

Results

Median age for the elderly was 65 (60-89y), without a difference in gender (21m/2 f). According to Ann Arbor staging 6-8-13-14 were stage I, II, III, or IV , respectively. The International Prognostic Score (IPS) was > 3 in all patients. B symptoms were present in 34 patients (77.3%). Mixed cellularity subtype was present in 52.3% of cases and nodular sclerosis subtype (34.1%). The great majority of patients (93%) received at least 1 ABVD (1-6), 27 patients received chemotherapy alone, 14 patients received combined modality treatment and 3 patients only received radiotherapy. Twenty four patients achieved a CR (54.5%), 5 partial responses, 5 had refractory /relapsed disease and 10 couldn’t be evaluated. Unlike younger patients a better response was not demonstrated with combined treatment modality (Table 1). Overall survival for the whole group at 7.2 years was estimated at 75.4% (95% CI 57.1-79.4) The OS (7 y) in elderly group was 75% versus 92% in younger group, however patients with age >75 had a lower survival, 50%. The median follow-up was of 8 years.

Summary

Our study showed no clinical differences between elderly vs younger population and confirms the efficacy of ABVD in elderly patients. The lower OS compared to other series may be related to the advanced stage of disease and poor IPS in our series Table 1.Clinical characteristics of elderly versus younger patientsFeature< 60 years (%)>60 years (%)P valueGenderMale/female61.8/38.247.5/52.30.10B Symptoms77.077.30.86Ann Arbor stage I II III IV7.424.931.835.913.618.224.538.60.45IPS >348.865.90.07Bulky disease48.838.60.26Histology Mixed cellularity Nodular Sclerosis43.848.852.334.10.60Treatment ABVD ABVD + RT RT Other42.455.32.32.361.431.86.84.50.37Response CR PR Relapse/progression NV60.89.718.910.654.511.411.422.70.37Overall survival (8 years)92%75%0.000

Disclosures

No relevant conflicts of interest to declare.
Peripheral Absolute Neutrophil, Mmonocyte and Lymphocyte Counts and Clinical Ooutcome in Diffuse Large B-Cell Lymphoma
Authors: Chen Yiming , Rodriguez Maria Alma , Feng Lei , Bi Weiqi , Zhang Liang , Wang Michael
Published: October 2019

Background

Baseline levels of peripheral absolute neutrophil (ANC), lymphocyte (ALC) and monocyte (AMC) counts have prognostic value in solid tumors. There are limited data on their impact on outcomes in patients with diffuse large B-cell lymphoma (DLBCL). The present study is to confirm whether the baseline levels of peripheral ANC, ALC and AMC can predict outcomes in patients with newly diagnosed DLBCL.

Patients and Methods

Data from lymphoma database at the University of Texas MD Anderson Cancer Center (UTMDACC) were used. The correlation of baseline ANC, AMC and ALC with progression-free survival (PFS) and overall survival (OS) were analyzed in 817 newly diagnosed previously untreated patients with DLBCL who received frontline treatments at UTMDACC between 2000-2011. Kaplan-Meier method was used for time-to event analysis including PFS and OS. Multi Cox proportional hazards model were fitted to evaluate important patient characteristics on the time-to event endpoints.

Results

Median ages at diagnoses were 58 years (range, 18 to 91 years). Seventy-nine percent of patients received conventional immunochemotherapy(CHOP-R) while 21 % of patients had intensive immunochemotherapy (HyperCVAD-R or R-EPOCH) or intensive chemotherapy(n=5; HyperCVAD or R-EPOCH) due to CD 20 negative; two patients received radiation therapy only. IPI low, intermediate and high were 38%, 46% and 16%, respectively. Overall response rate was 98% with a complete remission rate of 73%. Patients with abnormal ANC (> 7.3 X109/L) had an inferior PFS (P<0.001) and OS (P<0.001) compared to patients with ANC ≤ 7.3 X109/L; patients with abnormal ALC (< 1.0 X109/L) had significant short PFS (P<0.001) and OS (P<0.001) compared to patients with ALC ≥ 1.0 X109/L; similarly, patients with abnormal AMC (> 0.7X109/L) had inferior PFS (P< 0.001) and OS (P< 0.001) compared to patients with AMC≤ 0.7 X109/L. Multivariate analysis identified abnormal ALC and AMC as an independent adverse prognostic factors for PFS (hazard ratio [HR] 1.39 and 1.49, respectively; P<0.016 and P=0.004, respectively) and abnormal ANC and AMC as an independent adverse prognostic factors for OS (P=0.017 and P=0.024, respectively; HR 1.56 and 1.42, respectively).

Conclusions

An abnormal Neutrophil, lymphocyte and monocyte counts in peripheral blood are independent, poor prognostic factor for PFS and OS in patients with DLBCL. Incorporation of these immunological markers into IPI prognostic model may improve outcome prediction.

Disclosures

No relevant conflicts of interest to declare.
Phase 2 Trial of Entospletinib (GS-9973), a Selective SYK Inhibitor, in Follicular Lymphoma (FL)
Authors: Sharman Jeff P. , Klein Leonard M. , Boxer Michael , Kolibaba Kathryn S. , Hawkins Michael J. , Abella Steve , Wu Meihua , Yasenchak Christopher A.
Published: October 2019

Background: Spleen tyrosine kinase (Syk) is a mediator of B-cell receptor signaling in normal and transformed B-cells. Entospletinib is an orally bioavailable, selective inhibitor of Syk (Kd 7.6 nM, no other kinase < 100 nM).

Methods: This Phase 2 trial is evaluating Entospletinib 800 mg BID in a 41 subject cohort with previously treated FL in a study of 165 subjects with lymphoid malignancies. Tumor imaging was planned at weeks 8, 16, 24 and then every 12. Tumor response was assessed per Cheson 2007 criteria.

Results: For the subjects with FL included in this analysis, median age was 67 years (range 41 – 89), 49% were male. The median number of prior Rx regimens was 2 (range 1-8). Prior treatments (Rxs) included anti-CD20 antibodies (rituximab 100%, ofatumumab 5%), alkylating agents (95%; bendamustine 51%) and anthracyclines (51%). Baseline risk factors: Ann Arbor Stg III-IV (66%), Gr 3a FL (27%), FLIPI ≥3 (34%). At the time of this analysis, 41 subjects with follicular lymphoma were enrolled and 38 subjects have been treated through first response assessment (1 subject ongoing prior to first response assessment, 1subject discontinued due to AE and 1 subject withdrew consent). Entospletinib was generally well tolerated. The most common TEAEs (any Grade/≥Gr 3, independent of causality) were fatigue (46%/12%), nausea (42%/2%), diarrhea(27%/0%), decreased appetite (22%/0%), vomiting (22%/0%) and common laboratory abnormalities were increased AST (37%/17%), increased ALT (34%/20%), increased total bilirubin (27%/5%), anemia (34%/10%) and neutropenia (22%/10%). Reversible Grade 3 or 4 ALT/AST elevations occurred in 8 (19.5%) FL subjects. 3 subjects died while on study: 2 from progressive disease and 1 from acute renal failure investigator reported as unrelated to study drug. Investigator response assessments are available for 29 subjects, 16/29 (55%) subjects experienced reduced tumor bulk measured by SPD; 3 (10%) achieved a decrease of ≥ 50%. CR has not been observed at this early evaluation. 14/41 subjects continue on Rx. Median duration of Rx for all patients was 15 weeks. Among all patients who experienced reduction in tumor volume, median duration of Rx was 25 weeks (range 4-51).

Conclusions: Entospletinib monotherapy given with this dose and schedule was generally well tolerated and demonstrated moderate activity in subjects with advanced relapsed FL, including those with poor prognostic features. Updated data with longer follow-up duration will be presented at the meeting.

Disclosures

Sharman: Gilead Sciences: Research Funding. Klein:Gilead Sciences: Research Funding. Boxer:Gilead Sciences: Research Funding. Kolibaba:Gilead Sciences: Research Funding. Hawkins:Gilead Sciences: Research Funding. Abella:Gilead Sciences: Employment, Equity Ownership. Wu:Gilead Sciences: Employment, Equity Ownership. Yasenchak:Gilead Sciences: Research Funding.
GADD45a Is a Tumor Suppressor in BCR-ABL-Driven Leukemogenesis
Authors: Mukherjee Kaushiki , Sha Xiaojin , Skorski Tomasz , Bhatia Ravi , Hoffman Barbara , Liebermann Dan
Published: October 2019

The bcr-abl oncogene causes chronic myelogenous leukemia (CML). Growth arrest DNA damage 45a (Gadd45a) gene, a member in the gadd45 family of genes including Gadd45b & Gadd45g, is upregulated during myeloid lineage terminal differentiation. It is involved in G2/M cell cycle arrest and apoptosis in response to multiple stressors, including genotoxic and oncogenic stress . To investigate the effect of GADD45A in the development of CML, syngeneic wild type lethally irradiated mice were reconstituted with either wild type or gadd45a null myeloid progenitors transduced with a retrovirally expressed 210-kD BCR-ABL fusion oncoprotein. It was observed that loss of gadd45a accelerates BCR-ABL driven CML resulting in the development of a more aggressive AML like disease. Loss of Gadd45a resulted in increase of BCR-ABL expressing leukemic Stem/progenitor cells (GFP+Lin- cKit+Sca+). BCR-ABL transformed GADD45A deficient progenitors exhibit increased proliferation and decreased apoptosis, associated with enhanced PI3K-AKT-mTOR-4E-BP1 signaling and upregulation of p30C/EBPα and MCL-1 expression. Moreover, loss of Gadd45a was observed to facilitate transformation of BCR-ABL expressing BM cells to factor independence. These results provide novel evidence that gadd45a functions as a suppressor of BCR/ABL driven myeloid leukemogenesis. Since Gadd45a functions as a tumor suppressor in murine BCR-Abl driven leukemia, the status of Gadd45a mRNA expression levels was also investigated in human CML samples by utilizing real time PCR analysis. It was found that Gadd45a transcript levels were significantly up-regulated in chronic phase I CML samples. However in chronic phase-II, accelerated phase and blast crisis phase samples, expression was significantly down-regulated relative to normal controls. Thus, Gadd45a expression was observed to be altered in human CML samples correlating with disease progression. These results provide novel evidence that gadd45a functions as a suppressor of BCR/ABL driven myeloid leukemogenesis. These data also provide the impetus to further elucidate the role Gadd45a plays in suppressing the development of CML, and explore how its loss contributes to the progression of CML to a more aggressive leukemic phenotype.

Disclosures

No relevant conflicts of interest to declare.
Index to Predict MMR at 12 Months in Chronic Phase Chronic Myeloid Leukemia Patients Based on the Area Under the Lymphocyte Curve
Authors: Murai Kazunori , Ito Shigeki , Kowata Shugo , Oyake Tatsuo , Ishida Yoji
Published: October 2019

BACKGROUND: Dasatinib is a second-generation BCR-ABL inhibitor that has a 325-fold higher potency than imatinib and a 16-fold higher potency than nilotinib in vitro. Recent reports suggested that absolute lymphocytosis in response to dasatinib for chronic phase (CP) –chronic myeloid leukemia (CML) might be associated with favorable response. However, as the fluctuation of lymphocytosis was often observed, it is sometimes difficult to define lymphocytosis. To evaluate how much increase and how long continuation of lymphocytosis affects the better clinical efficacy, we developed an index (DL index; Dasatinib induced Lymphocytosis index) that combines duration and increase of lymphocytosis. The aim of this study was to test the DL index as a predictor of Major Molecular Response (MMR) at 12 month.

PATIENTS and METHODS: The DL area was based on a graph plotting the absolute lymphocyte counts and was the area under the lymphocyte curve using trapezoidal method. DL index was DL area divided by weeks. For example, DL index12W was defined as (DL area from 4 to 12 weeks) divided by (12-4) weeks. We tested the DL index12W in 30 patients who developed MMR at 12 months (MMR group) and 9 patients without MMR (non-MMR group).

RESULTS and DISCUSSION: DL score12w in MMR group and non-MMR group were 2714.0 (95% CI; 2286.0-3142.2, n=30) and 1559.9 (95% CI; 1006.9- 2113.0, n=9) respectively. There was a significant difference between MMR group and non-MMR group in DL index12W by t-test(Figure 1, p=0.0016). A significant linear relationship was obtained between DL index12w and the peaked lymphocyte counts within 12 weeks by Spearman's correlation coefficient rank test (Figure 2, p=1.97E-07, correlation coefficient=0.91).

The DL index12w, 2,113, which was the upper limit of non-MMR group 95% CI, is equivalent to 2,909/µl in lymphocyte count, and the DL index12w, 2,286, which is the lower limit of MMR group 95% CI, equivalent to 3,123/µl in lymphocyte count. Therefore, we divided all patients (n=39) into two groups, equal or more than 3,123/µl and less than 3,123/µl in the peaked lymphocyte counts. The patients with the peaked lymphocyte counts ≥ 3,123/µl reached MMR at 12 months (19/20=95.0%) to compare those less than 3,123/µl (11/19=57.9%) (p=0.0075).

CONCLUSION: The peaked lymphocyte number, induced by the DL index12w, which calculation was extremely easy, was associated with molecular response. These data suggests that this tool can be useful in interpreting the results of molecular response at 12 months.

Figure 1 Figure 1.

Figure 2 Figure 2.

Disclosures

No relevant conflicts of interest to declare.
Exosomes Derived from AML/MDS Cells Is Involved in Stromal Dysfunction and Bone Marrow Failure
Authors: Horiguchi Hiroto , Kobune Masayoshi , Kikuchi Shohei , Jomen Wataru , Murase Kazuyuki , Ibata Soushi , Iyama Satoshi , Sato Tsutomu , Kamihara Yusuke , Miyanishi Koji , Sato Yasushi , Hayashi Tsuyoshi , Takimoto Rishu , Kato Junji
Published: October 2019

The failure of normal hematopoiesis in myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) could be induced by a variety of mechanism such as the alteration of property of hematopoietic stem cells and stem cell niche. However, it has not yet been clarified precise mechanism how MDS stem/progenitor cells could replace normal hematopoietic stem/progenitor cells especially regarding involvement of mesenchymal stromal cells (MSCs). To gain insight into the mechanism of stromal dysfunction, comparative analyses of transcriptomes were conducted between normal and MDS/AML-derived MSCs. Further, we attempted to identify certain effectors originated from MDS/AML cells could alter the function of bone marrow (BM) MSCs.

The MSCs derived from healthy volunteer (HV)-derived (normal) and MDS/AML-derived stromal cells were established and analyzed mRNA expression by quantitive PCR (qPCR) array. Additionally, the supporting activity of MSCs for BM CD34+ progenitor/stem cells was examined using serum free coculture system. The interaction between MDS/AML cells and MSCs were evaluated by using Boyden Chamber and the changes of mRNA expression were analyzed.

The results of qPCR array revealed that the expression of hematopoietic factors was drastically altered in MDS/AML-derived MSCs as compared with normal MSCs. Among these factors, the expression of SCF and JAG1 mRNA were significantly and consistently reduced in all MDS/AML patients examined. Functional assay of these MSCs demonstrated that the number of colony-forming units (CFU) mixed cells (MIXs) and cobblestone area-forming cells (CAFCs) derived from CD34+ cells was significantly reduced after coculture with MDS/AML-derived MSCs as compared with normal MSCs. Even non-contact culture using Boyden Chamber between leukemic cells and MSCs induced the reduction of SCF and JAG1 mRNA, indicating that certain inducers could be soluble factors. Interestingly, this effect of transcriptomes alteration was negated by nSMase2 inhibitor (GW4869). Exosome transfer assay using Boyden Chamber revealed that GFP and PKH26 in leukemic cells transmit onto MSCs in non-contact coculture system and this transfer of exosome was significantly inhibited by GW4869 or nSMase siRNA. The multiple type of microRNA in exosome derived from MDS/AML cells was transferred into MSCs, suggesting that exosome could contribute to the alteration of mRNA expression in stromal cells.

Collectively, these results indicated that exosome derived from MDS/AML cells could be involved in the reduction of SCF/JAG mRNA and the stromal supporting activity of normal hematopoietic stem/progenitor cells.

Disclosures

No relevant conflicts of interest to declare.
Gene Editing of CCR5 in Hematopoietic Stem Cells in a Nonhuman Primate Model of HIV/AIDS
Authors: Peterson Christopher W. , Wang Jianbin , Polacino Patricia , Holmes Michael C. , Hu Shiu-Lok , Gregory Philip D , Kiem Hans-Peter
Published: October 2019

Background: Hematopoietic stem cell (HSC) transplantation remains the only clinically observed path to functional cure of HIV infection. To better understand the mechanism of HSC-driven HIV control, and apply this therapy to a greater number of patients, we have developed a model of combination antiretroviral therapy (cART)-suppressed HIV infection in the pigtailed macaque, applicable to both gene therapy- and allogeneic transplant-based cure strategies. Following transplantation of HIV-resistant, autologous cells into conditioned animals, we are evaluating the extent to which protected cell progeny impede infection by SIV/HIV (SHIV) chimeric virus in vivo.

Methods: Animals are challenged with SHIV virus containing an HIV envelope, after which a 3-drug cART regimen is initiated. Autologous HSCs are engineered to resist infection through targeted disruption of the CCR5 genetic locus using Zinc Finger Nucleases (ZFNs). Engraftment, persistence, and SHIV response of these autologous stem cells, and stem cell-derived lymphoid and myeloid cells, are measured in vivo.

Results: SHIV infection in the pigtailed macaque model results in sustained viremia with consequent reduction in CD4+ T cells. Moreover, administration of three-drug cART leads to rapid and durable suppression of plasma viremia to <30 copies/mL plasma - suggesting that this model recapitulates key features of HIV infection and treatment in humans. CCR5 targeting experiments yield up to 60% gene disruption in CD34+ cells ex vivo, translating to approximately 5% disruption in vivo following transplant. Importantly, up to 10% of transplanted cells carry two disrupted alleles of CCR5; these cells should preferentially reconstitute CD4+ T-cell pools and other susceptible subsets following SHIV challenge. Consistent with this prediction, our preliminary data suggest that CCR5-deleted cells undergo positive selection following SHIV challenge in vivo.

Conclusions: Our pigtailed macaque model of HIV infection and cART represents a promising platform for modeling functional cure strategies. Here we show that CCR5 deletion does not impair HSC engraftment or differentiation, and that CCR5-deleted cells can undergo SHIV-dependent positive selection even when present at low levels. Our model enables the evaluation of novel therapeutic approaches in the clinically relevant context of cART controlled SHIV infection - a setting of particular importance to approaches aimed at addressing the viral reservoir.

Disclosures

Wang: Sangamo Biosciences: Employment. Holmes:Sangamo Biosciences: Employment. Gregory:Sangamo Biosciences: Employment.
Glycopeptide-Specific Chimeric Antigen Receptor Targeting of T Cell Leukemia
Authors: Posey Avery D. , Schwab Robert D. , Boestaneau Alina , Johnson Laura A. , June Carl H.
Published: October 2019

Background

Adoptive transfer of T cells redirected with chimeric antigen receptors (CARs) has shown tremendous efficacy in targeting CD19-expressing leukemic cells; however, these CAR T cells also deplete normal CD19-expressing B cells. Although B cell lymphopenia can be overcome by gamma-globulin supplementation, other forms of on-target, off-site toxicity emphasizes the need for cancer cell-specific epitopes in CAR T cell therapy. To date, T cell leukemia has not been targeted due to possible fratricide because of shared antigens expressed on leukemic T cells and CAR T cells. Cellular transformation often produces altered glycosylation patterns, making cancer-specific glycoantigens attractive targets for CAR development. Here we demonstrate the successful eradication of the Jurkat T cell leukemia with Tn-specific CAR T cells.

Methods

The Jurkat E6.1 T cell leukemia line contains a frameshift mutation in the T synthase chaperone protein Cosmc that prevents core 1 structure formation on O-linked protein glycosylation, resulting in the presentation of Tn antigen on many glycoproteins. Here we describe the development of a CAR using a scFv sequence derived from the 5E5 mAb, which was developed against the Tn antigen of Mucin-1 (Muc1). A CAR was constructed by linking the 5E5 scFv to CD8alpha hinge and transmembrane region to the signaling endodomain comprised of 4-1BB and CD3zeta. The resultant Tn specific CAR was expressed in a lentiviral vector.

Results

In preliminary experiments Tn-CAR T cells had cytotoxicity to a variety of primary ovarian, breast and pancreatic tumor cells. Specific killing of Jurkat T leukemic cells was demonstrated by lysis of wild type Jurkat while the CAR T cells failed to kill Jurakat after overexpression of Cosmc. In specific, expression of full-length Cosmc expression in the Jurkat E6.1 cell line abolished staining by the 5E5 mAb, restoredT synthase activity, and protected the cells from lysis by 5E5-CAR T cells. Human T cells expressing the 5E5-CAR demonstrate rapid clearance of established Jurkat tumors in NSG mouse xenograft models, as assessed by serial bioluminescence imaging and prolonged survival compared to mice expressing a control CD19-specific CAR T cells (P<0.005).

Conclusions

These results demonstrate that the 5E5-CAR is specific for the Tn antigen and provides a potent cancer-specific immunotherapy for the targeting of non-dispensable tissues such as T cell leukemias.

Disclosures

June: Novartis: IP licensed by University of Pennsylvania to Novartis. Author entitled to royalties from the University. Patents & Royalties, Research Funding.
Pulmonary Embolism Begets Pulmonary Embolism?: A Meta-Analysis Depicting the Type of Recurrence Following an Index Pulmonary Embolism
Authors: Coleman Craig I , Kohn Christine G , Patel Dhaval
Published: October 2019

INTRODUCTION: Conflicting evidence suggests a person who suffers an index pulmonary embolism (PE) is more likely to develop a recurrent PE than a deep vein thrombosis (DVT). We performed a meta-analysis of randomized controlled trials (RCTs) to determine the proportion of recurrent events that will be PE in such people.

METHODS: A literature search of MEDLINE, EMBASE and CENTRAL (through 7/2014) was performed to identify RCTs of acute treatment and prevention of recurrent VTE in persons suffering an index PE, enrolling ≥50 subjects, requiring anticoagulation for ≥3 months and reporting the proportion of recurrent events that were DVT or PE (±DVT). RCTs not in English, enrolling only cancer, surgical or thrombophilia patients, primary prevention and extended treatment trials were excluded. We extracted recurrent VTE event data; and used a random-effects approach to pool the proportion [along with 95% confidence intervals (CIs)] of recurrent events that were PE and fatal PE, as well as, the proportion of recurrent PEs that were fatal.

RESULTS: Nine RCTs (n=13,606 subjects; 413 recurrent events) were included. In persons presenting with an index PE, 67%; 95%CI, 62-71% of their recurrent events were PE and 27%; 95%CI, 22-33%) were fatal PE. Of all recurrent PEs observed, 41%; 95%CI, 33-48% resulted in death. No statistical heterogeneity was observed in any analysis (I2<32% for all).

CONCLUSION: Our meta-analysis suggests a majority of recurrent events in those experiencing an index PE will be PEs. Moreover, recurrent PEs are associated with a high mortality rate.

Disclosures

No relevant conflicts of interest to declare.