bluuebunny/crossref_metadata_2025_split huggingface dataset:
IGH-FR1 and Leader Analysis By Next Generation Sequencing for the Detection of Clonality and Somatic Hypermutation in Lymphoid Malignancies
Authors: Piccaluga Pier Paolo , Gazzola Anna , Huang Ying , Panganiban Jeff , Hutt Kasey , Klass Michael 
Published: October 2019

Abstract
The standard method to analyze clonality and somatic hypermutation of immunoglobulin genes in B-cell non Hodgkin lymphomas is a multiplex PCR followed by capillary electrophoresis and/or Sanger sequencing. Recently, Next Generation Sequencing (NGS) of immune receptor genes was demonstrated to be highly effective, with increased sensitivity for detecting sequences of interest. Based on that, it was proposed for the diagnosis and monitoring of lymphoid neoplasms. A phase 3 diagnostic accuracy study was designed to compare the value of the first commercially available kit for NGS of IGH/FR1 with the gold standard analysis. One hundred twenty samples were evaluated for IGH rearrangements with both traditional capillary electrophoresis (IdentiClone™ IGH Gene Clonality Assay by Invivoscribe Technologies) and NGS (LymphoTrackTM IGH Assay - MiSeq and LymphoTrack® IGH Somatic Hypermutation Assay - MiSeq by Invivoscribe Technologies on an Illumina MiSeq).
The NGS clonality assay compared to conventional analysis had an overall diagnostic accuracy of 96% (63/66 cases), sensitivity (ST), specificity (SP), positive predictive value (PPV), and negative predictive value (NPV) of 95%, 100%, 100%, and 75%, respectively.
Regarding mutational analysis, conventional detection and NGS gave comparable results. Notably, NGS was capable of analyzing all cases, including those failed by Sanger sequencing. In discrepant cases NGS results were confirmed by a different set of primers that provided coverage of the IGH Leader sequence.
In conclusion NGS was effective for IGH analysis in lymphoid disease (even on FFPE samples). In order to apply them for routine diagnostics, NGS based approaches should be evaluated prospectively and cost-effectiveness assessments should be performed.

Disclosures
Huang: Invivoscribe: Employment. Panganiban:Invivoscribe: Employment. Hutt:Invivoscribe: Employment. Klass:Invivoscribe: Employment.
Preclinical Development of Bispecific Chimeric Antigen Receptor Targeting Both CD19 and CD22
Authors: Qin Haiying , Haso Waleed , Nguyen Sang Minh , Fry Terry J 
Published: October 2019

Abstract
Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy. Despite advances in upfront therapy, relapsed and refractory ALL remains a primary cause of cancer-related mortality in children. Immunotherapy using genetically engineered T cells expressing chimeric antigen receptors (CARs) targeting CD19 has demonstrated dramatic clinical results in children and young adults with pre-B cell ALL with a 70-90% complete remission rate in multiple clinical trials. However, not all patients respond and at least 10% of patients will relapse due to loss of the targeted CD19 epitope. CD22 is another pan-B cell surface antigen that is broadly expressed on ALL blasts and has been targeted successfully using immunoconjugates. We have recently developed a highly active CD22 CAR currently being tested in a clinical trial. Conceptually, dual targeting both CD19 and CD22 on Pre-B ALL with a single bispecific CAR may result in enhanced avidity and increased T cell activation which may result in additive or synergistic responses compared to targeting a single antigen. In addition, since there are heterogeneous expression levels of CD19 and CD22 on leukemic blasts, dual targeting may also provide a more broadly active therapy. Finally, simultaneously targeting of both CD19 and CD22 on the surface of pre-B cell ALL may reduce the likelihood of antigen escape due to downregulation or deletion. We describe the preclinical development of two novel CD19 and CD22 bispecific CARs assembled with CD19 and CD22 single chain fragment variable regions (scFv) binding domains on a single construct. T cells transduced the CD19/CD22 bispecific CARs have comparable CAR surface expression when compared to T cells expressing either the CD19 or CD22 CAR. T cells expressing the bispecific CAR are active in vitro against ALL cell lines expressing both CD19 and CD22 as measured by interferon gamma production and chromium release. In vitro activity is maintained against K562 cells expressing either CD19 or CD22 alone. Interestingly, the order and the linker between the CD19 and CD22 scFvs in the CAR construct has a major impact on the efficacy of the dual CAR with a membrane-distal CD22 binding scFv demonstrating improved efficacy. Finally, in xenograft models both bi-specific CARs efficiently eradicate ALL cell lines and patient derived xenografts. In vivo treatment of a Crisper-knockdown-CD19-negative leukemia line and a CD19-negative patient-derived xenograft is ongoing. In summary, CD19/CD22 bispecific CARs demonstrate robust pre-clinical activity against pre-B cell ALL and provides a strategy to improve the clinical efficacy of CAR therapy.

Disclosures
No relevant conflicts of interest to declare.
Novel Bromodomain Inhibitors Suppress Proliferation of Multiple Myeloma Cells
Authors: Ashihara Eishi , Oki Ryoko , Imayoshi Natsuki , Yoshioka Makoto , Strovel Jeffrey , Honjo Ayako , Sakai Yumi , Takada Tetsuya , Chauhan Jay , Raje Mithun , Fletcher Steven , Takata Kazuyuki 
Published: October 2019

Abstract
New molecular targeting agents against multiple myeloma (MM) have been developed and the prognosis of patients with MM has been improved. However, MM still remains an incurable disease. It is important to continue to investigate new therapeutic agents based on the biology of MM cells. Bromodomain (BRD4) inhibitors such as JQ-1 and I-BET151 have been shown as candidates for a molecular targeting agent against MM. In this study, we searched for chemical compounds inhibiting BRD4 binding ability and we investigated their effects on MM cell proliferation. We identified novel compounds CA1 and CA2 as a BRD4 inhibitor using an alpha screening assay. The IC50 values of the binding ability of recombinant human BRD4-BD1-BD2 of CA1 and CA2 are 0.44 and 1.1 mM, respectively (Figure 1). We next investigated the effects of CA1 and CA2 on MM cell lines (MM.1s, AMO-1, NCIH929, and OPM-2 cells). Analyses with MTT assay showed that these compounds inhibited the proliferation of MM cells in a dose-dependent manner. c-myc and cyclinD1 mRNA transcripts were decreased by the treatment of CA1 and CA2, while, p21 mRNA transcripts were increased (Figure 2). In flow cytometric analysis, cell cycle arrest occurred in a G1 phase and apoptosis was induced. Western blotting analysis showed the decrease of c-myc protein levels as well as the cleavage of caspase-3. In conclusion, novel BRD4 inhibitors CA1 and CA2 inhibited binding of BRD4 to its ligands, resulting in the suppression of MM cell proliferation and the induction of apoptosis via the caspase activation.
Figure 1. Figure 1. Figure 2. Figure 2.

Disclosures
Yoshioka: ConverGene LLC: Employment. Strovel:ConverGene LLC: Employment.
Survival Outcomes in Blastic Plasmacytoid Dendritic Cell Neoplasm (2001-2012)
Authors: Mosalpuria Kailash , Bhatt Vijaya R. , Nawal Lata 
Published: October 2019

Abstract
Background: BPDCN is a rare subtype of aggressive acute leukemia that usually has an indolent clinical presentation such as isolated solitary or multiple cutaneous lesions. Despite initial clinical responses to systemic chemotherapy, BPDCN follows an aggressive course with a reported median OS of approximately 8-14 months based on single-center studies. We aimed to determine the OS of BPDCN and its predictors utilizing a US population-based database.
Methods: Using International Classification of Disease for Oncology, 3rd edition (ICD-O-3) code 9727/3, SEER 18 database was analyzed to identify adult patients with BPDCN diagnosed from 2001-2012. We divided the entire cohort into 3 groups based on time periods of 4 years each (2001-2004, 2005-2008, 2008-2012) to represent early (E), middle (M) and late (L) cohorts. Age, sex, race, marital status, radiation use, and OS were compared across the three time periods. Multivariate analyses of OS were performed using Cox regression to adjust for significant covariates. A p-value of <0.05 was considered statistically significant.
Results: Of 417 patients, the majority were males (67%), patients aged ≤ 60 years (75%) and whites (82%). A higher proportion of patients were diagnosed in the early time period (E=43% vs. M=29% vs. L = 29%). About a sixth of the cohort (17%) received radiation therapy. Median OS was not reached for the cohort. Five-year OS was higher for patients ≤ 60 years, as compared to patients >60 years (70% vs. 50%, p<0.01). On a multivariate analysis, age >60 years (hazard ratio, HR 2.05; 95% confidence interval, CI 1.28-3.27, p <0.008) and African American ethnicity (HR 1.79; 95% CI 1.10-2.89, p=0.018) were associated with a higher hazard for death whereas OS did not differ based on sex, marital status, time period, or the use of radiation therapy. Of the 106 patients >60 years, the majority were males (68%), whites (93%), married (74%), and did not receive radiation (92%). On a multivariate analysis after adjusting for age, sex, race, time of diagnosis, and radiation therapy, being single (marital status) was associated with a worse OS (HR2.24; 95% CI 1.05-4.77, p=0.034) among patients >60 years.
Conclusions: To our knowledge, this is the largest and most recent population-based study in BPDCN. Compared to prior single-center studies, our results demonstrate much better OS. Older age and African American ethnicity are determined to be negative prognostic factors for OS. Single older patients are at even a higher risk of mortality. The study findings may help in patient counseling and informed decision-making. Racial disparities in OS require further investigation.

Disclosures
No relevant conflicts of interest to declare.
Low Density Granulocytes in Patients after Allogeneic Hematopoietic Stem Cells Transplantation (allo-HSCT): A Distinct Class of Neutrophils in Systemic Alloimmunity
Authors: Mikhaltsova Ekaterina , Savchenko Valeri G. , Kuzmina Larisa A. , Drokov Mikhail , Vasilyeva Vera , Koroleva Olga , Dubnyak Daria , Parovichnikova Elena N. 
Published: October 2019

Abstract
Introduction
It's generally considered that all alloimmune process such as acute graft-versus host disease (aGVHD) after allo-HSCT are mostly controlled by lymphocytes. The role of neutrophils in systemic alloimmunity after allo-HSCT is still illusive. In 1987 a distinct subset of proinflammatory, low-density granulocytes (LDGs) isolated from the peripheral blood mononuclear cell fractions of patients with system lupus erythematosus has been described. There is no LDG's in healthy donors. While the origin and role of LDGs still needs to be fully characterized, we try to describe this population in patients with hematological malignancies after allo-HSCT
Patients and methods.
Peripheral blood samples were collected in EDTA-tubes before allo-HSCT, on day +30,+60,+90 after allo-HSCT and at day of aGVHD from 47 patients with hematological malignancies (AML=22, ALL n=17, LPD=3, MDS =2; CML=2; 17 with active disease, 30 - in CR) after allo-HSCT (from matched unrelated donor n=34, from matched related donor n=13; MAC = 13, RIC=34). Isolation of mononuclear cells from human peripheral blood was made by standard protocol using Lympholyte®-M Cell Separation Media (Cedarlane Labs). The anti-CD66b-PE (Biolegend, USA) antibodies and FSC/SSC were used to determine LDGs cells as FSChigh \SSChigh \CD66b+. 100000 of cells were analyzed on a BD FACSCanto II (Becton Dickinson, USA).
Results. Results of blood evaluation of 47 patients with hematological malignancies, whose blood was examined after allo-HSCT presented in table 1.
Conclusion Despite the fact that we don't get significant differences. LDG's detection in allo-HSCT patients need further investigation.
Table 1. Incidence of LDG after allo-HSCT in patients with and without aGVHD Table 1. Incidence of LDG after allo-HSCT in patients with and without aGVHD

Disclosures
No relevant conflicts of interest to declare.
Thrombophilic Factors in Retinal Vessel Thrombosis: A Single Center Experience
Authors: Maxey Nicole , Mohammadi Zeinab , Dominguez-Perez Pamela , Cortes Erasto , Antony Nishaal , Landeros Mark M , Arango Carmen , Campos-Gines Angel , Vazquez Carlos W , Herrada Juan 
Published: October 2019

Abstract
Background: Retinal Vessel Thrombosis (RVT) is an ophthalmologic emergency that can result in permanent vision loss. The retinal vessels are considered a rare site for thrombosis, obligating physicians to evaluate the underlying cause. Classic risk factors include hypertension, cardiovascular disease, diabetes, and glaucoma. In addition, there is a variable degree of association with inherited and acquired thrombophilia. Our aim is to describe our experience with eleven patients (pts) evaluated for inherited and acquired thrombophilia who presented with RVT.
Methods: Retrospective analysis.
Results: We reviewed the medical records of 11 consecutive pts seen in our institution between 2001 and 2015 who were referred with the diagnosis of RVT. Eight out of eleven were females and three were males; median age was 47 years, with an age range from 30 to 78. Clinical presentation was transient visual loss in the affected eye in 10 pts and persistent conjunctivitis in 1 pt. Laboratory evaluation of thrombophilia in blood samples showed the following: 1 pt had both factor V Leiden mutation (heterozygous) along with prothrombin-20210A mutation (heterozygous); 3 pts had MTHFR gene mutations (two were heterozygous and one was homozygous); 1 pt had MTHFR gene mutation and positive lupus anticoagulant; 1 pt had antiprothrombin antibodies and homocysteinemia; 1 pt had factor V Leiden (heterozygous); 1 pt had factor V Leiden (heterozygous) and elevated factor VIII; 1 pt had protein S deficiency and elevated factor VIII; 1 pt had anticardiolipin antibodies; and lastly 1 pt had no laboratory abnormalities found.
Conclusion: In our limited pt sample, we have found that retinal vessel thrombosis can be associated with inherited and acquired thrombophilia. The clinical implication of these findings remains incompletely understood and merits further research.

Disclosures
No relevant conflicts of interest to declare.
Developing a Model of Human Pluripotent to Hematopoietic Stem Cell Development in Mistrg Mice
Authors: Astle John , Xiang Yangfei , Rongvaux Anthony , Weibel Carla , Elizabeth Henchey , Halene Stephanie , Park In-Hyun , Flavell Richard 
Published: October 2019

Abstract
De novo generation of HSCs has been described as a "holy grail" of stem cell biology, however the factors required for converting human pluripotent stem cells (PSCs) to true hematopoietic stem cells (HSCs) capable of robust long-term engraftment have yet to be fully characterized. Two groups have shown that injection of PSCs into immunodeficient mice leads to teratomas containing niches producing hematopoietic progenitors capable of long-term engraftment. Once these hematopoietic progenitors and their microenvironments are better characterized, this system could be used as a model to help direct in vitro differentiation of PSCs to HSCs. Toward this end, we have injected human PSCs into immunodeficient mice expressing human rather than mouse M-CSF, IL-3, GM-CSF, and thrombopoietin, as well as both human and mouse versions of the "don't eat me signal" Sirpa (collectively termed MISTRG mice). These cytokines are known to support different aspects of hematopoiesis, and thrombopoietin in particular has been shown to support HSC maintenance, suggesting these mice may provide a better environment for human PSC-derived HSCs than the more traditional mice used for human HSC engraftment. The majority of teratomas developed so far in MISTRG contain human hematopoietic cells, and the CD34+ population isolated from over half of the teratomas contained hematopoietic colony forming cells by colony forming assay. These findings further corroborate this approach as a viable method for studying human PSC to HSC differentiation.

Disclosures
No relevant conflicts of interest to declare.
Decreased Tim-3 Expression Level of Peripheral Blood Natural Killer Cells in Severe Aplastic Anemia
Authors: Fu Rong , Yuan Xin , Liu Chunyan , Liu Hui , Wang Yihao , Chen Tong , Li Lijuan , Song Jia , Wang Guojin , Shao Zonghong 
Published: October 2019

Abstract
Severe aplastic anemia (SAA) is a life threatening disease characterized by severe pancytopenia and bone marrow failure. T-cell immunoglobulin- and mucin domain-containing (Tim)-3 has been initially used to identify dysfunctional T cells, but recent studies have demonstrated that Tim-3 is widely detected on nature killer cells (NK cell) and may serves as a marker for activation and maturation of NK cells. In our study, Tim-3, expressed on peripheral blood of NK cells in SAA patients, was quantitatively analyzed by Flow cytometry before and after immunosuppressive therapy (IST). Results showed that the expression of Tim-3 in patients before IST [(61.11±15.46)%] was significantly lower than that in patients after IST[(74.30±12.63)%, P<0.05] and normal controls[(70.39±12.73)%, p<0.05]. However, no difference was observed between patients before and after IST. Therefore, we concluded that low expression of Tim-3 in NK cells may play a crucial role in early stage of SAA.

Disclosures
No relevant conflicts of interest to declare.
Very Severe Aplastic Anemia Due to Doxycycline Therapy
Authors: Singh Prabhsimranjot , Sinha Ankur , Kamath Anisha , Malhotra Sonali , Chandra Abhinav B 
Published: October 2019

Abstract
Very severe aplastic anemia due to doxycycline therapy
A 42 y/o male presented to the emergency department with 3 weeks history of fatigue and epistaxis. On initial blood work he was found to have pancytopenia with hemoglobin of 4.7g/dl, platelet count of 4,000 and absolute neutrophil count of 160. He has no significant family history. His past medical history was only significant for a skin rash, which was biopsied 3 months ago and reported to be follicular dermatitis. The only home medication he has taking was doxycycline 100mg twice a day for the past few weeks before the current hospital admission. He was transfused with packed red blood cells and platelets to maintain hemodynamic stability. His bone marrow biopsy was reported to be hypocellular (<5% cellularity) with normal morphological features (see figure 1). Paroxysmal nocturnal hemoglobinuria was ruled out by flow cytometry. Workup including HIV, EBV, Hepatitis B and C and parvovirus serology was reported to be negative. His history of long-term Doxycycline use as well as no family history of any similar disorder, it is plausible that doxycycline could have caused his aplastic anemia. In congruence with the above mentioned findings, he is diagnosed with very severe aplastic anemia and is planned to be started on immunosuppressive therapy with equine anti-thymocyte globulin and cyclosporine along with consultation for allogeneic transplant.
Aplastic anemia is a remarkably rare but serious adverse effect of drug therapy like antimicrobials. We report this as the first case, to the best of our knowledge based on the literature review, of possible doxycycline induced severe aplastic anemia. Our patient had history of skin rash for which he was taking doxycycline for few weeks before he presented with pancytopenia. Acquired aplastic anemia is a rare hematological disorder presenting with pancytopenia and a predominantly empty marrow. It is a fatal disease and irrespective of the etiology, without treatment, patients usually succumb due to infection and bleeding. Cases have been reported with tetracycline as a causative agent of aplastic anemia. We hypothesize doxycycline to be the possible etiology of severe aplastic anemia in this patient. Practitioners need to be aware of this rare but fatal complication of this widely used antibiotic.
Figure 1. Hypo-cellular Bone Marrow Figure 1. Hypo-cellular Bone Marrow

Disclosures
No relevant conflicts of interest to declare.
De Novo Nethyltransferases, DNMT3a and DNMT3b Are Underexpressed in Multiple Myeloma
Authors: Latalova Pavla , Minarik Jiri , Smesny Trtkova Katerina 
Published: October 2019

Abstract
Background and aims: Presently, there is growing evidence that along with the important role of genetic abnormalities, epigenetic aberrations are relevant factors in multiple myeloma (MM). As was recently found, genome-wide analysis of DNA methylation reveals epigenetic alterations in plasma cells from patients with MM and individuals with monoclonal gammopathy of undetermined significance (MGUS). MGUS is characterized by predominant hypomethylation. Transformation into MM is accompanied by progressive hypermethylation with maximum methylation seen in relapsed disease. DNA methyltransferases (DNMTs) catalyze DNA methylation through transfer of methyl group to cytosine of the CpG dinucleotides, resulting in 5-methylcytostine. DNMT1 maintains patterns of methylated cytosine residues in human genome. DNMT3A and DNMT3B are de novo DNA methyltransferases, whose role is to maintain new methylation pattern that forms due to formation of the cancer.
Methods: 30 bone-marrow aspirates from individuals with MGUS or MM patients before the treatment initiation were used. The cDNA was synthesized using 100 ng of total RNA in a 20 µl reaction volume (Roche, Diagnostics, Basel, Switzerland). Quantification of DNMT1, DNMT3a and DNMT3b levels by TaqMan® probes (Life Technologies, Grand Island, NY) with Xceed qPCR Master Mix (IAB, BioTech-Europe, Czech Republic) was performed. For normalization, the GAPDH was used.
Results: Although MM is characterized by widespread alterations in DNA methylation, we observed that DNMT3a and DNMT3b de novo methyltransferases were underexpressed in both, MGUS individuals and MM patients when compared to DNMT1 expression level (Figure 1). The transcribed genes have increased levels of 5-hydroxymethylcytosine, then the DNMTs activities might compensate for active hydroxymethylation - demethylation.
Conclusions: Our results confirm that the expression of de novo DNA methyltransferases is deregulated in MM cell lines. The presented analysis is first of its kind that was performed on human myeloma cell lines, especially with the focus on the residual expression of Dnmt3a.
With support of the grant NT14393.
Figure 1. Quantitative RT-PCR for DNMT1, DNMT3a and DNMT3b in MGUS individuals and MM patients. Figure 1. Quantitative RT-PCR for DNMT1, DNMT3a and DNMT3b in MGUS individuals and MM patients.

Disclosures
No relevant conflicts of interest to declare.
Oroxylin a Heightens Tumor Necrosis Factor a-Induced Differentiation Effects on Acute Myeloid Leukemia Cells Via Modulation on AKT and NF-Kb Pathways Involving RXRa
Authors: Xu Jingyan , Ouyang Jian , Hui Hui 
Published: October 2019

Abstract
Tumor necrosis factor alpha (TNFα) is a complicated cytokine which could induce differentiation of acute myeloid leukemia (AML) cells. We have found that Oroxylin A, a natural compound isolated from Scutellariae radix, markedly enhanced TNFα-induced NBT reduction and CD11b/CD14 expression of AML cells. Besides, Giemsa staining also displayed that the combination group induced U937-MDR cells to differentiate into monocyte-like cells yet NB4 and HL-60-resistant cells to granulocytic-like cells. Further study showed that TNFα induced PI3K subunit p85α binding to N-terminal truncated nuclear receptor RXRα (tRXRα) proteins and activating AKT. On the contrary, these could be inhibited by Oroxylin A through inhibiting interaction between tRXRα and p85α in NB4 and HL-60-resistant cells. Moreover, Oroxylin A inhibited the activation of NF-κB signaling and the DNA binding activity by TNFα proved by EMSA in these two AML cells. All these suggested that Oroxylin A was able to inhibit NF-κB signaling and RXRα-dependent AKT signaling, the negative effects of TNFα for AML therapy, suggesting that combination of Oroxylin A and TNFα might be a novel candidate for differentiation therapy for AML cells and required further investigation.

Disclosures
No relevant conflicts of interest to declare.