bluuebunny/crossref_metadata_2025_split huggingface dataset:
Erythroid Lineage-Restricted Expression of Jak2V617F Is Sufficient to Induce a Myeloproliferative Disease in Mice,
Authors: Akada Hajime , Hamada Saeko , Mohi Golam 
Published: October 2019

Abstract
Abstract 3861
A somatic point mutation (V617F) in the JAK2 tyrosine kinase was found in most cases of Ph-negative myeloproliferative neoplasms (MPNs) including ∼95% patients with polycythemia vera (PV) and 50–60% patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). To investigate the contribution of JAK2V617F in MPNs, we generated a conditional Jak2V617F knock-in mouse (Akada et al., Blood 2010; 115: 3589–3597). Expression of Jak2V617F in all hematopoietic compartments including the hematopoietic stem cells (HSC) resulted in a PV-like disease associated with a marked expansion of erythroid progenitors in the bone marrow and spleen. Since Jak2 is essential for normal erythropoiesis and expression of Jak2V617F mutant enhances erythropoiesis, so we asked if erythroid progenitors are actual target cells for Jak2V617F mutation. To address this question, we have specifically expressed Jak2V617F in erythroid progenitors using the EpoR-Cre mice. Expression of heterozygous Jak2V617F in erythroid progenitors resulted in a polycythemia-like phenotype characterized by increase in hematocrit and hemoglobin, increased red blood cells, Epo-independent erythroid colonies, and splenomegaly. Erythroid lineage-specific expression of homozygous Jak2V617F resulted in significantly greater increase in hematocrit, hemoglobin, red blood cells, Epo-independent erythroid colonies, and splenomegaly compared to heterozygous Jak2V617F expression. These results suggest that erythroid lineage-restricted expression of Jak2V617F is sufficient to induce a polycythemia-like disease in a gene-dose dependent manner. However, transplantation of Jak2V617F-expressing erythroid progenitors (c-kithighTer119lowCD71high or c-kitlowTer119highCD71high) from the diseased mice into lethally irradiated recipients could not transfer the disease suggesting that Jak2V617F mutation does not confer self-renewal capacity to erythroid progenitors. We also observed that only Jak2V617F-expressing HSC has the unique capacity to serially transplant the myeloproliferative disease in mice. Taken together, our results suggest that HSCs are the disease-initiating cancer stem cells and erythroid progenitors are the target cells in Jak2V617F-evoked MPN.

Disclosures:
No relevant conflicts of interest to declare.
Frequent Involvement of PVT1 in Multiple Myeloma Carrying 8q24 Rearrangement and Identification of Novel PVT1-NBEA Chimeric Gene,
Authors: Nagoshi Hisao , Taki Tomohiko , Hanamura Ichiro , Nitta Masakazu , Otsuki Takemi , Nishida Kazuhiro , Okuda Keiko , Sakamoto Natsumi , Kobayashi Satoru , Yamamoto Mio , Tsutsumi Yasuhiko , Kobayashi Tsutomu , Matsumoto Yosuke , Horiike Shigeo , Kuroda Junya , Taniwaki Masafumi 
Published: October 2019

Abstract
Abstract 3917
The 8q24 rearrangement has been identified in 3.5–5.0 % of multiple myeloma (MM) patients with conventional cytogenetic analyses, such as G-banding, and in 9.5–20% of MM patients with fluorescence in situ hybridization (FISH) and spectral karyotyping (SKY) techniques. Of note, 8q24 rearrangements accompany with advanced MM patients and MM cell lines more frequently. Chromosomal translocation of the immunoglobulin gene (Ig), such as t(8;14)(q24;q32) and t(8;22)(q24;q11), occur in approximately 25% in MM with 8q24 rearrangements, while non-Ig chromosomal loci, including 1p13, 1p21–22, 6p21, 6q12–15, 13q14, and 16q22, have been also identified as translocation partners where no candidate genes have been delineated so far. In this study, we precisely investigate molecular features of chromosomal 8q24 rearrangements to access pathophysiology of MM. FISH, SKY, and RT-PCR analyses were performed as described previously. DNA gain and loss assay based on oligonucleotide array (GeneChip Human Mapping 50K, 250K, or 6.0 array, Affymetrix) was performed on the genomic DNA extracted from MM cells. Oligonucleotide array data was analyzed using the CNAG3.0 or 3.3 programs. Various types of 8q24 rearrangements were detected in 12 (22.2 %) of 54 MM patients and 8 (72.7 %) of 11 MM cell lines by means of FISH procedure. A breakpoint cluster of approximately 360 kb region containing myelocytomatosis oncogene (MYC) and plasmacytoma variant translocation 1 (PVT1) genes at 8q24 was divided into three subclusters according to rearranged region, that is, PVT1 region, region which is centromerically adjacent to PVT1 (including MYC), and region which is 120kb centromeric to MYC. Seven of 12 patient-derived cells and 5 of 8 cell lines with 8q24 abnormalities showed PVT1 rearrangements with various partner loci, such as 4p16, 4q13, 13q13, 14q32, or 16q23–24 in SKY combined with FISH analyses (SKY-FISH). Oligonucleotide array combined with SKY analysis delineated the candidate genes within partner loci of 8q24 rearrangements by mapping boundary breakpoints of copy number gains and losses, identifying MMSET, EPHA5, NBEA, and WWOX as candidate genes at 4p16, 4q13, 13q13, and 16q23–24, respectively. We identified the novel chimeric gene PVT1-NBEA in AMU-MM1 cell line with t(8;13)(q24;q13). The PVT1-NBEA fusion transcript in which PVT1 exon 1 was fused to NBEA exon 3 was accompanied with overexpression of abnormal NBEA lacking its part of N-terminus. In conclusion, PVT1 rearrangement may play pivotal roles in pathophysiology of MM harboring 8q24 abnormalities.

Disclosures:
No relevant conflicts of interest to declare.
Inhibition of mTOR with Everolimus (RAD001) and Silencing by Vascular Endothelial Cell Growth Factor Specific siRNA Induces Additive Antitumor Activity in Multiple Myeloma Cells,
Authors: Koldehoff Michael , Elmaagacli Ahmet , Beelen Dietrich W. 
Published: October 2019

Abstract
Abstract 3984

Background:
Angiogenesis plays an important role in the pathogenesis and progression in multiple myeloma (MM). MM cells secrete vascular endothelial growth factor (VEGF), which further promotes proliferation of the tumor cells. Several RNA interference (RNAi) methodologies are rapidly being established and hold promise to specifically inhibit gene expression in mammals. RNAi is the sequence-specific, posttranscription gene silencing methods initiated by double-stranded RNAs, which are homologous to the gene being suppressed. Mammalian target of rapamycin (mTOR) is an essential part of tumour growth being capable of integrating proliferative, antiapoptotic and angiogenic signalling by connecting VEGF, hypoxia-inducible factor 1 (HIF-1) and HER family receptors. Several reports have demonstrated the anti-tumor activity of everolimus (RAD001), an mTOR inhibitor, in a variety of cancers, including various leukemias and lymphomas.

Aims:
Therefore, we evaluated the anti-myeloma effect of VEGF siRNA silencing in MM cells and whether it can be augmented by the additional application of everolimus.
Methods and Results: After transfection with VEGF siRNA we observed a reduction of VEGF expression in all studied cell lines: OPM-2, RPMI-8226, INA-6, Jurkat, Raji and Karpas-299, as well as in cells of MM- and lymphoma patients. Next, using the MM cell line OPM-2 we studied the time courses of VEGF siRNA transfection in order to investigate the knock-down efficiency of VEGF expression. The efficiency of VEGF siRNA transfection in treated OPM-2 cells showed a reduction of 75.5% VEGF protein levels after 24 h compared to the untreated OPM-2 cells (p<0.001). Further, VEGF siRNA both significantly induced apoptosis and inhibited proliferation in OPM-2 cells (p<0.001), RPMI-8226 (p<0.001), and in INA-6 (p<0.01) versus controls. To assess whether everolimus treatment of OPM-2 and RPMI-8226 affects the viability of these cells, cells were treated with various doses (1–20 nM) of everolimus for 24 hours, harvested, and analyzed for cell viability by MTT assay. Everolimus significantly decreased the viability of OPM-2 cells (IC50 = 5.9 nM) and of RPMI-8226 cells (IC50 = 0.6 nM), respectively. Everolimus and siRNA both together might to reduce the VEGF gene expression up to 33% (p<0.001) compare to siRNA VEGF (61%) alone or everolimus alone (39%), that demonstrated additive effects of everolimus and siRNA.

Conclusions:
These findings suggest that mTOR inhibition and silencing by VEGF specific siRNA may be associated with an additive antitumor activity and might be a suitable target for new therapeutic strategies using RNA interference in MM.

Disclosures:
No relevant conflicts of interest to declare.
Cell Autonomous Role of TGFβ and IL-2 Receptor in the In Vivo Generation of CD4 and CD8 Inducible Regulatory T Cells During Graft-Versus-Host Disease,
Authors: Satake Atsushi , Sawamukai Norifumi , Kambayashi Taku 
Published: October 2019

Abstract
Abstract 4015
FoxP3+ regulatory T cells (Tregs) suppress graft-versus-host disease (GVHD) while preserving graft-versus-tumor effects, making them an attractive target for GVHD therapy. The donor-derived Treg pool can potentially be derived from expansion of pre-existing natural Tregs (nTregs) or from de novo generation of inducible Tregs (iTregs) from donor conventional T cells (Tconvs) in the transplant recipient. Although the co-adoptive transfer of nTregs or in vitro -derived iTregs has been shown to prevent the development of GVHD, the relative contribution of these two Treg subsets in protection against GVHD has been unclear. To investigate the contribution of the different FoxP3+ Treg subsets, we used a MHC-mismatched mouse model of acute GVHD. Lethally irradiated (500cGy × 2) B6D2F1.SJL (H-2bxd) host mice were injected with T cell-depleted bone marrow cells and FACS-sorted Tconvs (WT or Foxp3-deficient) with or without FACS-sorted Tregs of C57BL/6 (H-2b) mouse origin. Weight loss in mice receiving Foxp3-deficient Tconvs alone was significantly more pronounced compared to other groups. The presence of either donor-derived nTregs or iTregs alone protected against GVHD-induced weight loss but was suboptimal compared to the presence of both donor-derived nTregs and iTregs.
Next, we sought to determine how the donor-derived Treg pool was established during acute GVHD and tracked the appearance of Tregs in the secondary lymphoid organs at different time points post transplant. On Day 8 post GVHD induction, ∼5% of the donor-derived CD4+ T cells in the spleen were FoxP3+. We found that the Treg pool was comprised equally of donor-derived nTregs and iTregs. Unexpectedly, we found a significant fraction of CD8+FoxP3+ T cells (1–3% of all CD8+ T cells) in the spleen and in the lymph nodes. These CD8+FoxP3+ T cells representing ∼70% of the iTreg pool on Day 8 post GVHD induction. These CD8+FoxP3+ T cells shared phenotypic markers with their CD4+ counterparts and displayed suppressive activity, suggesting that they were bona fide iTregs. Both CD4+ and CD8+ Tregs expanded in vivo with IL-2 treatment and required IL-2 and TGFβ receptor expression for their generation.
In summary, we found that donor derived-iTregs are generated during GVHD and contribute to suppression of acute GVHD induced-weight loss. Surprisingly, CD8+Foxp3+T cells were a major contributor to the donor derived-iTreg pool after transplantation. The generation of CD8+ and CD4+ iTregs occurred at least in part by a cell autonomous IL-2 and TGFβ receptor-dependent mechanism. Thus, our data suggest that in addition to increasing nTregs, concomitant strategies aimed at enhancing the conversion of donor-derived Tconvs to iTregs for example by engaging the IL-2 and TGFβ signaling pathways might be beneficial for the treatment of GVHD.

Disclosures:
No relevant conflicts of interest to declare.
Evaluation of the Toxicity of Total Body Irradation As a Separate Treatment in Preparation for Hematopoietic Cell Transplantation
Authors: Giebel Sebastian , Kruzel Tomasz , Sadus-Wojciechowska Maria , Najda Jacek , Czerw Tomasz , Miszczyk Leszek , Holowiecki Jerzy 
Published: October 2019

Abstract
Abstract 4579

BACKGROUND:
Myeloablative regimens used for allogeneic or autologous hematopoietic cell transplantation (HCT) may be based on either chemotherapy alone or total body irradiation (TBI). TBI is generally considered highly toxic, however, it is usually combined with high-doses of cyclophosphamide or other cytostatics, which does not allow reliable assessment of its tolerance. In Maria Sklodowska-Curie Cancer Center and Institute of Oncology in Gliwice TBI is used a single treatment in the preparation for the first autologous HCT in patients with multiple myeloma (MM). Second HCT in a tandem procedure is performed after 4 months interval with high-dose melphalan as conditioning. The protocol gives opportunity to separately assess early toxicity of TBI, which is a goal of this study.

PATIENTS AND METHODS:
The clinical course of 30 patients with MM, aged 58 years (range, 50–66), including 14 men and 16 women was evaluated. The disease status prior to autoHCT was CR (n=9), VGPR (n=7), or PR (n=14). 47% patients were pre-treated with 2 or more lines of chemotherapy and the median number of preceding cycles was 8. TBI was administered at the dose of 12 Gy divided in 3 fractions on days -3, -2, -1. 6 MV X-ray beam was used with lung shielding after 6 Gy and additional irradiation of chest wall using electrons 9–15 MeV to the planned total dose. Peripheral blood was used as a source of stem cells. The incidence of adverse events was assessed using ‘Common Toxicity Criteria v4.03’.

RESULTS:
Within 100 days follow-up all patients were alive. Median time to neutrophil >0.5 ×10e9/L and platelet >50 ×10e9/L recovery was 12 days (11–21) and 14 days (9–18), respectively. Patients required the median of 1 (0–3) platelet and 0 (0–6) red blood cell transfusions. The median hospital stay since HCT was 16 days (14–21). The most frequent grade 3 or 4 adverse events were infections (43%), including neutropenic fever (23%). Other severe toxicities were rare. In particular no patient experienced hepatic or renal complications (Table 1).

CONCLUSION:
TBI as a separate treatment is well tolerated and may be safely used for conditioning in a population of patients up to 66 years old. Our observation may potentially be applied to both autologous and allogeneic transplantations.

Disclosures:
No relevant conflicts of interest to declare.
Aberrant Expression of Golgin-84 in Non-Hodgkin Lymphomas and Plasma Cell Myeloma
Authors: Chen Ling , Yang Yaling , Yin C. Cameron , Lu Gary , Chen Su , Huang Xiaoyan , Chen Jiayu , Chang Felix , Rahman Effie , Calin Steliana , Medeiros L. Jeffrey , Miranda Roberto N. , You M. James 
Published: October 2019

Abstract
Abstract 4633

Background:
Golgins are proteins of the Golgi complex. Several Golgins have been implicated in apoptosis. Expression of Golgin-84, a Golgin protein, is altered in apoptotic WEHI-231, a B-cell lymphoma line, suggesting that Golgin-84 may play a role in lymphoid tumorigenesis. Here, we aimed to determine the expression levels of Golgin-84 in human primary non-Hodgkin lymphomas and plasma cell myeloma.

Design:
Golgin-84 expression was investigated in non-Hodgkin lymphoma cell lines by using Western blot analysis and polyclonal antibodies. Using immunohistochemical stains, Western blotting analysis and Q-PCR, Golgin-84 expression was assessed in 5 reactive lymph nodes, 149 cases of primary non-Hodgkin lymphoma and 28 cases of primary plasma cell myeloma.

Results:
Immunohistochemical stains, Western blotting analysis and Q-PCR on 5 reactive lymph nodes demonstrated that Golgin-84 was expressed at low levels in lymphoid cells of germinal centers, mantle cells, marginal zones, and interfollicular areas. Golgin-84 was variably expressed in non-Hodgkin lymphoma cell lines tested, with the highest levels in cells from high-grade tumors (e.g. anaplastic large cell lymphoma; ALCL, Diffuse large B-cell lymphoma (DLBCL), ALCL and peripheral T-cell lymphoma unspecified (PTCL)) and the lowest levels in mantle cell lymphoma (MCL) cells. DLBCL, ALCL and PTCL frequently showed high expression of Golgin-84. Most lymphoplasmacytic lymphomas (LPL) and plasma cell myeloma (PCM) expressed high levels of Golgin-84. Expression levels of Golgin-84 were lower in MCL and low-grade B-cell non-Hodgkin lymphomas, including chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), follicular lymphoma (FL), and marginal zone lymphoma (MZL).

Conclusions:
Golgin-84 expression levels are low in lymphoid cells of normal lymph nodes. Most (>90%) cases of LPL and PCM, and at least half of cases of DLBCL, ALCL and PTCL express high levels of Golgin-84. These findings suggest that Golgin-84 may be involved in tumorigenesis or lymphoma progression, particularly in neoplasms with plasmacytic differentiation.

Disclosures:
No relevant conflicts of interest to declare.
Enforced E47 Expression Has Differential Effects on Lmo2-Induced T-Cell Leukemia
Authors: Goodings Charnise , Dave Utpal P. 
Published: October 2019

Abstract
Abstract 4637
Lmo2 is one of the most commonly deregulated oncogenes in human T-cell acute lymphoblastic leukemia (T-ALL). In mouse models, Lmo2 overexpression causes a differentiation block before the onset of T-ALL at a developmental stage that is similar to the block seen in E47 knockout mice. Furthermore, Lmo2 and E47 are part of an oligomeric protein complex in hematopoietic stem and progenitor cells. Since E47 knockout mice also develop T-ALL, it has been hypothesized that Lmo2 may induce T-ALL by redirecting E47 activity away from its normal target genes. We noted downregulation of many E2A targets in Lmo2-induced T-ALL. To directly test whether E47 is required in Lmo2-induced T-ALLs, we transduced four stable T-ALL lines established from Lmo2 transgenic mice with retrovirus expressing E47 fused with estrogen receptor. All 4 lines tolerated stable high- level protein expression of E47-ER with no change in from their baseline growth rates. The E47-ER fusion protein allowed forced dimerization upon treatment with 4-hydroxytamoxifen. Tamoxifen treatment increased expression CD4 and other described E2A targets in all 4 T-ALL lines; but two lines underwent G0/G1 cell cycle arrest. Our data suggest that E47 deficiency is not a universal feature of Lmo2- induced T-ALL and E47 forced expression has differential effects on T-ALL.

Disclosures:
No relevant conflicts of interest to declare.
A Comparison of INNOVANCE® PFA P2Y and VerifyNow P2Y12 Assay for the Assessment of Clopidogrel Resistance in Patients Undergoing Percutaneous Coronary Intervention
Authors: Shin Soyoung , Kim Yonggoo , Lim Jihyang , Jang Jiyoung , Kim Jayoung 
Published: October 2019

Abstract
Abstract 4671

Introduction:
VerifyNow P2Y12 is commonly used to measure responsiveness to clopidogrel. We sought to compare the results obtained from novel INNOVANCE® PFA P2Y and VerifyNow P2Y12 assay for the assessment of clopidogrel resistance in patients with undergoing percutaneous coronary intervention.

Methods:
255 patients undergoing percutaneous coronary intervention, preliminarily treated with 100 mg/day of aspirin followed co- administration of clopidogrel(loading dose 600 mg, maintenance dose 75 mg/day), were enrolled in this study. Platelet aggregation was measured by INNOVANCE® PFA P2Y and VerifyNow P2Y12.

Results:
INNOVANCE® PFA P2Y and VerifyNow P2Y12 assay showed moderate correlations with INNOVANCE® PFA P2Y Vs VerifyNow %inhibition: r = 0.412, p < 0.0001; INNOVANCE® PFA P2Y Vs VerifyNow platelet reactivity units(PRU): r = −0.402, p < 0.0001. The agreement between INNOVANCE® PFA P2Y and VerifyNow %inhibition was 85 % and that of INNOVANCE® PFA P2Y and VerifyNow platelet reactivity units(PRU) was 79%: The k statistics between INNOVANCE® PFA P2Y and VerifyNow %inhibition and platelet reactivity units(PRU) were 0.52 and 0.44, respectively.

Conclusions:
INNOVANCE® PFA P2Y’s sensitivity in detecting clopidogrel resistance is comparable to VerifyNow P2Y12 assay. As the PFA-100® system was already widely used, the new test cartilage may be useful tool for the assessment of clopidogrel effects. Additional Clinical correlation studies were required to validate the effectiveness of INNOVANCE® PFA P2Y for predicting long term clinical outcomes.

Disclosures:
No relevant conflicts of interest to declare.
A Novel Mutation of ADAMTS13 Gene in Congenital Thrombotic Thrombocytopenic Purpura
Authors: Bai Xia , Su Jian , Jiang Minghua , Wang Zhaoyue , Ruan Changgeng 
Published: October 2019

Abstract
Abstract 4674
Congenital thrombotic thrombocytopenic purura (TTP) is caused by gene mutations of von Willebrand factor-cleaving protease (a disintegrin and metalloprotease with thrombospondin type I domains 13, ADAMTS13). In this study, one novel mutation in the ADAMTS13 gene was found in a woman whose presents are first cousins. Thrombocytopenia occurred during the second trimesters in her first pregnancy, and she died of recurrent attacks after diagnosis of TTP. The ADAMTS13 activity measured using the recombinant FRET-VWF73 during her acute episode was less than 5%. ADAMTS13 inhibitor was negative measured by 9:1 mixture of patient and pooled normal plasma followed by ADAMTS13 activity assay using the VWF multimer electrophoresis. The 29 exons and exon-intron boundary sites of ADAMTS13 gene was analyzed using the human genomic DNA extracted from peripheral leukocytes of the patient. The results demonstrated she was homozygous for R498C. This novel mutant was constructed using the expression plasmid pSectag containing ADAMTS13 cDNA, and the vector was introduced by linpofectamine 2000 to Hela cells. Western Blot revealed that rADAMTS13-wide type (WT) was synthesized as a single band with molecular mass close to 190 Kda in the conditioned media, however, no detectable ADAMTS13 of this mutant existed. The lysates of cells expressing the mutant showed the same protein amounts compared to the rADAMTS13-WT. The immunofluorescence study demonstrated that mutant had the same localization pattern at Endoplasmic Reticulum(ER)and Golgi-compartments compared to the rADAMTS13-WT. The results imply that this mutant may be retained in the cellular ER and Golgi-comparments, but rapidly degraded or insufficiently secreted.

Disclosures:
No relevant conflicts of interest to declare.