bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ex Vivo Expansion of SCID Leukemia-Initiating Cells Using NANEX Nanofiber Scaffold
Authors: Fischer Stephen L , Zhao Yukang , Qu Cheng-Kui , Sakthivel Ramasamy 
Published: October 2019

Abstract
Abstract 4994
The concept of the leukemic stem cell (LSC) has gained wide acceptance since it was first definitively established using a NOD-SCID xenotransplantation model nearly 15 years ago. LSCs, which are functionally defined as SCID leukemia-initiating cells (SL-ICs), are believed to possess biological properties that render them resistant to conventional chemotherapy. Although there is still much debate over how to phenotypically define LSCs, there is general agreement that LSCs are rare in acute myeloid leukemia (AML), which has hindered efforts to develop LSC-targeted therapies. In order to provide researchers and pharmaceutical companies with an ample supply of LSCs for testing, methods are needed to generate large numbers of LSCs from patient samples.
Ex vivo expansion of LSCs in culture is one approach that offers tremendous promise for increasing cell numbers for research and drug development. Conditions that enable efficient expansion of normal hematopoietic stem cells (HSCs) can be used as a starting point for developing an optimal culture system for LSCs. The natural bone marrow microenvironment maintains HSCs in close contact with a complex network of stromal cells and extracellular matrix, likely indicating that cell-cell and cell-matrix interactions play an important role in maintaining their stem cell phenotype. With the goal of mimicking the bone marrow stem cell niche, Arteriocyte, Inc. has developed a 3-D NANEX nanofiber based cell culture substrate. The NANEX substrate is designed to provide topographical and substrate-immobilized biochemical cues that act in synergy with media additives to enhance HSC proliferation.
Here, we present our recent work with the NANEX platform towards achieving a high yield ex vivo expansion of LSCs. Using common LSC markers, including CD34, CD38, CD117, and CD123, we quantify and characterize NANEX-expanded leukemic cells using flow cytometry. We compare NANEX to standard tissue culture polystyrene and demonstrate that NANEXÔ significantly improves LSC expansion and reduces clonogenic phenotype loss during ex vivo culture. Additionally, we show that NANEXÔ-expanded cells engraft in NOD-SCID mice and, through limiting dilution analysis, quantify the increase in SL-ICs as a result of culture on NANEXÔ. Our data indicates that NANEX technology provides a robust ex vivo expansion of SL-ICs and, with further development, offers great potential for use in LSC-targeted drug development.

Disclosures:
No relevant conflicts of interest to declare.
Pharmacokinetics of Dasatinib in a Patient with Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia During Treatment with Anti-Tuberculosis Therapy
Authors: Zeller Michelle P. , Beumer Jan H , Christner Susan M , Xenocostas Anargyros 
Published: October 2019

Abstract
Abstract 5006

Objectives:
Dasatinib is a second generation protein-tyrosine kinase inhibitor (TKI) indicated for first line treatment of chronic myeloid leukemia (CML) and second line treatment of Philadelphia chromosome-positive acute lymphoblastic leukemia (ALL) in patients who are imatinib resistant or intolerant.
In this study, we present the case of a patient with Philadelphia chromosome- positive ALL, intolerant to imatinib, who developed reactivated tuberculosis (TB) while receiving dasatinib during maintenance chemotherapy. The purpose of our study was to determine the effects of concomitant anti-TB therapy on dasatinib exposure by measuring plasma dasatinib levels using three different oral doses of the drug in this patient.

Methods:
The patient was treated with isoniazid, rifampin, ethambutol, pyrazinamide and pyridoxine for 2 months, followed by isoniazid, rifampin and pyridoxine for an additional 4 months. Dasatinib therapy was continued at the initiation of TB therapy using 100 mg po od, and titrated by 50 mg increments to 200 mg po od. Dasatinib plasma levels were measured prior to initiating anti-TB treatment and at regular intervals during concomitant treatment using three different dasatinib doses.

Results:
Eight days after initiation of anti-tuberculosis therapy, the apparent clearance of dasatinib had increased from 1410 to 2174 L/h at 100 mg. However, at 150 mg and 200 mg doses, apparent clearance had decreased to levels lower than baseline. As expected, the direction of peak plasma concentration changes was inverse to the changes in apparent clearance: Cmax values were 30 at baseline, and 22, 46, 102 ng/mL for the 3 doses of dasatinib during anti-TB therapy, respectively.

Conclusions:
Our findings suggest dasatinib clearance is increased by approximately 50% shortly after initiating anti-tuberculosis treatment and increasing the dose of dasatinib during anti-TB therapy was able to restore dasatinib plasma levels to those observed before initiation of anti-TB therapy.
(This work was supported by an unrestricted grant from Bristol Myers Squib)

Disclosures:
No relevant conflicts of interest to declare.
Nelfinavir Activates Epstein-Barr Virus and Kaposi's Sarcoma Herpesvirus Lytic Cycle by Inducing ER Stress
Authors: Shirley Courtney , Kalu Nene , Ambinder Richard F 
Published: October 2019

Abstract
Abstract 5011
Epstein-Barr virus (EBV) and Kaposi's Sarcoma herpesvirus (KSHV) are associated with lymphomas and other malignancies. We previously demonstrated that the proteasome inhibitor, bortezomib, leads to ER stress, induction of the unfolded protein response (UPR), and activation of EBV lytic gene expression.1 Here we investigate nelfinavir, an HIV protease inhibitor that has been reported to induce the UPR.2 Nelfinavir treatment of EBV Burkitt's lymphoma (BL) and KSHV primary effusion lymphoma (PEL) cell lines resulted in changes indicative of ER stress: elevated levels of ATF4, XBP1(s), and CHOP10 (Figure 1), as well as the EBV and KSHV immediate early proteins ZTA and RTA (Figure 2), respectively. The appearance of these UPR markers preceded expression of viral lytic RNAs. Regulated knockdown of Bip, an ER-stress sensor and activator of the UPR, by shRNA inhibited viral lytic RNA induction. These effects were observed using drug levels at or just above the levels achieved with standard clinical dosing of nelfinavir. Gantt et al. have reported that nelfinavir inhibits herpes virion production, including KSHV, in vitro.3 The ability to activate viral gene expression in combination with inhibition of virion production may identify nelfinavir as an especially promising agent for virus-targeted cancer therapies.Figure 1Figure 1. Figure 2Figure 2.
Analysis of nelfinavir (NFV) induced ER stress markers by RT-qPCR in Burkitt's lymphoma (Akata) and primary effusion lymphoma (BCBL-1) cells.
Analysis of nelfinavir (NFV) induced lytic activation by RT-qPCR in Burkitt's lymphoma (Akata) and primary effusion lymphoma (BCBL-1) cells.

Disclosures:
No relevant conflicts of interest to declare.
Elevated Chemokine Expression in the Bone Marrow of Patients with Myeloma and MGUS Is Associated with Marked Alterations in the Distribution of CD4+ and CD8+ T Cell Subsets within the Blood and Bone Marrow
Authors: Goodyear Oliver , Essex Sarah , McGettrick Helen , Basu Supratik , Anandram Seetharam , Buckley Christopher D , Filer Andrew , Khanum Rahela , Piper Karen P , Moss Paul AH , Pratt Guy 
Published: October 2019

Abstract
Abstract 5076
Multiple Myeloma (MM) is a malignant disease of bone marrow plasma cells characterised by immune dysfunction and increased chemokine levels. We investigated chemokine receptor expression on T cells, particularly the Th1 and Th2-associated receptors, CXCR3 and CCR4, in patients with paraproteinaemia and compared this to controls and correlated this to the level of related chemokines in blood and bone marrow.
The frequency of CCR4+ T cells within the bone marrow was markedly increased in patients with MM and the normal distribution between blood and marrow was reversed. CXCR3+ Th1 Cells which were rare within normal marrow were increased relative to blood. Th17 CD4+ T cells were markedly increased within marrow with increased Th17 CD4+ CCR4+ and Th17 CD4+ CXCR3 populations and the suppressor CD25highCd127low FoxP3positive subset reduced within blood. The expression of the ligands for CCR4 and CXCR3 was markedly increased within the marrow of patients with MGUS and MM. Mesenchymal stem cells isolated from the bone marrow of myeloma patients supported T cell adhesion and migration with a preferential recruitment of CCR4 and CXCR3-positive cells. Chemokine gradients within the tumour microenvironment alter the distribution of major T cells subsets between blood and marrow. This pattern is established early in the development of paraproteinaemia and may offer a potential target for new forms of treatment.

Disclosures:
No relevant conflicts of interest to declare.
Abrogation of Niche Signals Discovered New Genes Regulating Homing, Expansion and homeostasis of Multiple Myeloma Cells
Authors: Cohen Yossi , Garach-Jehoshua Osnat , Adina Bar-Chaim , Kornberg Abraham 
Published: October 2019

Abstract
Abstract 5080

Introduction:
Multiple myeloma (MM) cells depend on the bone marrow (BM) niche signals for their viability. Thus, tracing the earliest molecular changes in response to the lost of niche signals can disclose new homing related interactions.

Methods:
BM aspirates were collected in heparinized syringes and samples were flesh frozen at different time points. GEP (Affymetrix 1.0 st) was performed for 8 cases with massive BM infiltration with plasma cells. The GEP was also analyzed at different time points following spreading of cultured BM cells from the same patients (maintained in culture for several days).

Results:
Spontaneous induction of genes started within few minutes following aspiration, peaking in hours and persisting for at least 12 hours (Tables 1). RT-PCR results along several time points following BM aspiration from a representative case are presented in Table 2. Interestingly, the induction of genes reverted entirely in cultured conditions imposing cell-cell-matrix contact. However, spreading of the cultured cells re-induced these genes similar to that followed BM aspiration. Furthermore, in comparison of the GEP of cultured cells (frozen immediately after spreading) Vs the GEP from samples taken immediately after BM aspiration, an absolutely different repertoire of genes showed significant changes (Table 3):

Conclusions:
1. BM cells from MM patients respond immediately to changing microenvironment, with prompt upregulation of genes active in tumor expansion (FOS, AREG, JUN, PTGS2). However, such constitutive upregulation plus elevation of tumor suppressor genes (like NR4A2) is intolerable if not reverted by cell-cell-matrix contact. 2. In culture, BM cells from MM patients respond with dramatic upregulation of genes supporting neovascularisation (IL8, PLA27, MMP12, CXCL5), matrix (SPP1, FN1) and osteoclasts. 3. Most interestingly, in culture there is also downregulation of 20 genes critical to mitosis, especially relating to spindle machinery (KIF members, NUF2, TUBB1, ASPM, CENPE, CENPF) suggesting that the failure of long-term culture of primary MM cells results from mitotic crisis. This may also explain the lobulation of primary MM cell nuclei observed. 4. The unannotated gene MOP-1 has a role in MM homing.

Disclosures:
No relevant conflicts of interest to declare.
Anti-Myeloma Activity of LDC-000067, a Novel Selective Inhibitor of Cyclin Dependent Kinase 9: A Targeted Approach
Authors: Niepoth Kathrin , Gustavus Dirk , Wenning Doris , Meisterernst Michael , Kienast Joachim , Berdel Wolfgang E. , Bisping Guido 
Published: October 2019

Abstract
Abstract 5110

Background:
Cyclin-dependent kinase (CDK) inhibitors are reported to induce apoptosis in myeloma cells (Gojo et al., 2002; MacCallum et al., 2005). Here we investigated pro-apoptotic and anti-proliferative effects of LDC-000067, a novel selective CDK9-inhibitor, in three multiple myeloma (MM) cell lines (L-363; RPMI-8226; U-266). In addition, we studied consequences of selective CDK 9-inhibition regarding transcriptional activity (RNA-polymerase II phosphorylation) and related anti-apoptotic pathways (Mcl-1 and NFkB).

Methods:
L-363, RPMI-8226 and U-266 cells were exposed to either DMSO (vehicle control) or serial dilutions of LDC-000067 [0.5–20μM]. Apoptosis was quantified by flow cytometry analyses (FITC Annexin V / propidium iodide staining) after 24 hours in corresponding samples. To further investigate mechanisms of induced apoptosis by LDC-000067 we examined corresponding protein extracts by immunoblot analyses.

Results:
We found that LDC-000067 showed anti-myeloma activity in all myeloma cell lines tested. The proportion of induced apoptosis by exposure to LDC-000067 was equipotent (RPMI-8226) or higher (L-363 and U-266) compared to Seliciclib, a selective CDK 2-, 7- and 9-inhibitor. The IC50 for LDC-000067 was 4.0 μmol/L for L-363, 6.9 μmol/L for U-266 and 11.5 μmol/L for RPMI-8226. LDC-000067 inhibited phosphorylation of RNA polymerase II at Ser2 of the carboxyl-terminal domain in all MM lines tested. In addition, exposure to LDC-000067 resulted in a rapid decline of the protein levels of Mcl-1, known as an anti-apoptotic protein in MM. Furthermore, it is reported that in 40% of MM cell lines and 17% of MM patients the NFkB pathway is constitutionally activated by mutations (Demchenko et al., 2010) and inhibition of the NFkB activity causes induction of apoptosis (Hongyu et al., 2001). In our study we showed that LDC-000067 potently inhibited the NFkB pathway. Immunoblot analyses revealed a decreased phosphorylation and degradation of the NFkB inhibitor IkBa and an inhibition of phosphorylation of the NFkB subunit RelA (p65). Moreover, our data demonstrated that exposure to LDC-000067 caused a significant reduction of the protein levels of cyclin D1 and cyclin D2 strongly correlated to cell cycle arrest.

Conclusion:
In conclusion, our data corroborate that LDC-000067, a novel selective CDK 9-inhibitor, shows anti-myeloma activity by targeting three key pathways in the molecular pathogenesis of MM: firstly, it induces consecutive decline of Mcl-1 protein and inhibition of RNA polymerase II phosphorylation; secondly, it inhibits constitutively activated NFkB pathway; and thirdly, it reduces levels of D-type cyclins.

Disclosures:
No relevant conflicts of interest to declare.
A Phase I Study of Arsenic Trioxide (Trisenox), Ascorbic Acid, and Bortezomib (Velcade) Combination Therapy in Patients with Relapsed/Refractory Mulitple Myeloma
Authors: Held Lauren , Gockerman Jon P. , Diehl Louis F. , de Castro Carlos Manuel , Moore Joseph O. , Rizzieri David A. , Long Gwynn D. , Horwitz Mitchell E. , Chao Nelson J. , Gasparetto Cristina 
Published: October 2019

Abstract
Abstract 5129

Purpose:
Bortezomib is a well recognized standard therapy, however it is not curative for multiple myeloma. New agents and approaches are needed to overcome resistance in multiple myeloma. Arsenic trioxde (ATO) induces apoptosis of plasma cells through a number of mechanisms, including down regulation of gene overexpression, activating cell cycle arrest by inducing p21 cyclin-dependent kinase inhibitor protein, and by triggering apoptosis through caspase-3 in a dose dependent manner. This phase I study assessed the feasibility and tolerability of concomitant administration of arsenic trioxide (ATO), ascorbic acid (AA), and bortezomib (VelcadeÔ) (AAV) in patients with relapsed/refractory multiple myeloma.

Experimental Design:
A standard dose of ATO (0.25 mg/kg IV infused over 1–2 hours) and AA (1g IV infused over 15 minutes after infusion of ATO) were given once weekly × 2 with an escalating dose of bortezomib (cohort 1: 1 mg/m2 or cohort 2: 1.3 mg/m2 IV bolus on days 1, 8) of a 21 day cycle). ATO was given at least 1 hour prior to bortezomib and patients were allowed up to a maximum of 6–8 cycles.

Results:
A total of ten patients (median age, 62 years old) were enrolled with a median follow up of survivors of 25 months. Patients had an average of 4 prior failed therapies. Seven (70%) patients were refractory to bortezomib when previously administered. Despite our patient population being heavily pre-treated, objective responses were observed, with one partial response (Cohort 2), two minimal response (Cohort 1 and 2), and one stable disease (Cohort 2). To date, three of the 10 patients are continuing maintenance therapy 13–43 months from initiating this study.

Conclusion:
Of the patients that completed the treatment, objective responses were observed despite suboptimal dosing and previous bortezomib treatment failure. Tolerability was observed in most patients as discontinuation was not due to treatment toxicities, but due to aggressiveness of disease. Further studies are warranted with a larger patient population to effectively determine the effectiveness of AAV in relapsed/refractory multiple myeloma.

Disclosures:
Rizzieri: Cephalon: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Long:Millennium: Speakers Bureau. Gasparetto:Millennium: Speakers Bureau.
Early Response to Botezomib Combined Chemotherapy Can Be Helpful for Prediction of Progression Free Survival in Patients with Multiple Myeloma Who Were Ineligible for Stem Cell Transplantation
Authors: Lee Ho Sup , Kim Yang Soo , Kim Kihyun , Kim Jin Seok , Kim Hyo Jung , Min Chang-Ki , Suh Cheolwon , Eom Hyeon-Seok , Yoon Sung-Soo , Lee Jae Hoon , Kim Min Kyoung , Kim Sung Hyun , Bae Sung Hwa , Mun Yeung-Chul , Jo Deog Yeon , Chung Joo Seop 
Published: October 2019

Abstract
Abstract 5138

Background:
Response to treatment has been associated with improved survival in multiple myeloma (MM). The use of novel agents changed achievement of CR rates compared with conventional chemotherapy. The purpose of our study is to show influence of early response after treated with bortezomib combined chemotherapy to survival in patients with newly diagnosed MM who are ineligible for stem cell transplantation.

Methods:
We assessed response at least forth cycles before next chemotherapy by international myeloma working group response criteria. The median duration from starting chemotherapy to initial response date was 2.3 month (range; 0.9 – 3.7 months). The enrolled cases were all received bortezomib combined chemotherapy as front line therapy. We divided into good response group (A group) which were included showing more than very good partial response (VGPR) and poor response group (B group) which were partial response (PR) or less than PR. Endpoints were comparision of progression free survival (PFS) and overall survival (OS) between A and B groups.

Results:
We retrospectively analyzed 129 patients registered data for our study from the Korean Multiple Myeloma Working Party (KMMWP) performed a nationwide registration of MM patients. In our results of initial response, 16 patients were in CR (12.4%), 50 were in VGPR (38.8%), 40 were in PR (31.0%), 14 were in SD (10.9%), and 9 were in PD (7.0%). However, in results of best responses, 46 patients were in CR (35.7%), 31 were in VGPR (24.0%), 32 were in PR (24.8%), 11 were in SD (8.5%) and 9 were in PD (7.0%). 3 years PFS of A group and B group were 55.6% and 18.4%, respectively (p-value < 0.001). 3 years OS of A and B group were 65.3% and 52.9%, respectively (p-value 0.078).

Conclusion:
Early response at least forth cycles before next chemotherapy might be helpful for prediction of PFS in patients who were ineligible stem cell transplantation.

Disclosures:
No relevant conflicts of interest to declare.
Selective Accumulation of CD45RO+CCR7- in the Marrow Aspirates in Patients with Essential Thrombocythemia
Authors: Lange Andrzej , Sedzimirska Mariola , Klimczak Aleksandra , Skrzypek Daria Drabczak 
Published: October 2019

Abstract
Abstract 5155
Patients with newly diagnosed essential thrombocythemia (ET) were investigated for selective accumulation/expansion of CD4+ lymphocytes subpopulations in the marrow, to verifie the hypothesis that the neoplastic process associated factor(s) are recognized by the immune system.
Sixteen patients (F/M: 12/4, age 28–72 - median 60 yrs) with ET diagnosed according to the WHO criteria were studied. They had platelets from 496–809 x 10̂3/ul (median 644 × 10̂3 ), (12 heterozygotes, 2 homozygotes). Cytological evaluation revealed normal differentiation of erythroid and myeloid lineages, in contrast an increased number of megakaryocytes often enlarged, hyperlobulated and in clusters were seen in all patients. Trephine biopsy revealed activation of megakaryopoiesis with enlarged numerous megakaryocytes with normal representation of other lineages and a lack of an increase in the reticulin fibers. Marrow cellularity was from 9 – 126 x10̂3/ul (median 42 × 10̂3/ul).
The whole populations of marrow and blood cells were labeled for a purpose of this study with the use CD4, CD25, CD45RO and CCR7 MoAb (Becton Dickinson, San Jose, CA, UmarSA) followed by detection and analysis with the use of flow cytometry (FACS Calibur, Becton Dickinson) equipped with PCLysis programe. Immunostaining of trephine biopsies was used for detection of FOXP3 cells.
It was found:
(i) Lymphocytes in the marrow were in proportions from 4,5%-15,7% (median 7,25%), in numbers from1377/ul to 8245/ul (median 3870/ul) what was significantly higher as compared to blood from 1300/ul to 3900/ul (median 2100/ul) (Wilcoxon Matched Pairs Test, p=0.010).
(ii) CD45RO+CCR7- lymphocytes were higher in numbers in the marrow aspirates as compared to blood (median 1281/ul vs 631,2/ul, p=0,04 Wilcoxon Test for pairs).
(iii) In contrast numbers in the marrow aspirates of CD45 RO-CCR7+ (median 297,4/ul vs median 238,2/ul), as well as CD45 RO+CCR7+ (median 149,6/ul vs 146,4/ul) did not differ from those in blood.
(iv) It was a significant increase of CD4+CD25high+ cells in the marrow aspirates as compared to blood (median 36,2/ul vs 9,1/ul; p=0,02 Wilcoxon Test for pairs). Indeed in 11 out of 14 trephine biopsies investigated FoxP3+ cells were identified.
In conclusion an increase, in the marrow of ET patients, of effector/memory CD4 lymphocytes (CD45RO+CCR7-) but not those of naïve and central memory lymphocytes phenotypes points out on the selective accumulation of these cells in the marrows. Concomitant increase of T regulatory cells (CD4+CD25+high) suggests that an active immunological is taking place at the site. It is possible that effector memory cells are attracted to come to the marrows by ET process associated factors and if so the present observation constitute the primary observation building the rationale behind immunotherapy in ET patients

Disclosures:
No relevant conflicts of interest to declare.