bluuebunny/crossref_metadata_2025_split huggingface dataset:
Anaplastic Lymphoma Kinase (ALK)-Positive Diffuse Large B-Cell Lymphoma Presenting with CNS Involvement: A Case Report
Authors: Kute Blakely D , Spalding Aaron , Martin Alvin W. , Sun David 
Published: October 2019

Abstract
Abstract 5225

Introduction
Anaplastic lymphoma kinase (ALK)- positive large B-cell lymphoma is a rare form of large B-cell lymphoma originally described in 19971. Since its initial description, approximately fifty cases have been reported in the literature, none of which describe extranodal involvement of the CNS as we will present below.

Case
The patient is a 30 year old previously healthy male who presented with visual changes described as decreased acuity and headaches. Papilledema was present on exam, but exam was otherwise negative. Imaging showed an enhancing mass posterior to the Torcula Herophili. With the working diagnosis of meningioma, the lesion was embolized and then debulked. Final pathology, however, revealed an ALK-positive large B-cell lymphoma with cytoplasmic granular ALK staining. Immunohistochemistry was positive for CD 138, CD 45, CD 38, CD 4, MUM1, epithelial membrane antigen (EMA) and monoclonal IgA lambda, and negative for CD 20, CD30, CD 3, and CD 8. FISH revealed ALK rearrangement (likely with clathrin, but pending at time of abstract) and c-MYC amplification, but not translocation. Serum immunoglobulins were normal. PET/CT showed skeletal metastasis but no additional visceral disease.

Discussion
ALK-positive large cell lymphomas described previously show immunoblastic/plasmablastic-like phenotypes (CD138+, CD 45+, CD 20-, and cytoplasmic IgA expression), but are CD30-negative and have uniform EMA expression and ALK translocations, as did our patient. The majority of ALK-positive DLBCL exhibit an ALK translocation with the long arm of the clathrin gene to form t(2;17)(p23;q23)2.
ALK-positive large cell tumors typically present nodally, but cases have been reported extranodally in the nasopharynx, tongue, bone, soft tissue, and stomach. There is a 3:1 male predominance and a poor overall prognosis with a median survival of 12.2 months, with isolated cases having longer survival. Most patients were treated with CHOP-based regimens with or without radiation therapy2,3. The need for more intense or biologically directed therapy in these cases, including early intensification with or without autologous stem cell rescue is recognized. Gambacorti-Passerini and Poglinani recently described lymphoma sensitivity with crizotinib, an anaplastic lymphoma tyrosine kinase inhibitor, in relapsed anaplastic large cell lymphoma after standard treatment with promising results. Investigation into the use of crizotinib in ALK-positive large cell lymphomas as salvage therapy is potentially warranted4.
This case presents an interesting treatment dilemma given the ALK mutation, c-myc amplification, and CNS disease. Crizotinib is likely not a salvage option for this patient given unclear CNS availability and the possibility of this tumor being motivated by c-myc as well as ALK.

Disclosures:
No relevant conflicts of interest to declare.
NF-κB Independent Protection Against Apoptosis by Overexpression of A20 in B-Cells
Authors: Alamome Isabelle , Feuillard Jean , Faumont Nathalie 
Published: October 2019

Abstract
Abstract 5233

Introduction
In Activated B-Cell like Diffuse Large B-Cell Lymphomas (ABC DLBCLs) the most commonly affected gene is TNFAIP3, which encodes A20, an ubiquitin modifying enzyme involved in the negative regulation of NF-κB signaling. A20 is itself a target gene of NF-κB and needs to be induced in order to exert its negative feedback effect on NF-κB. Mutations resulting in the inactivation of A20 have been found in a significant proportion of ABC DLBCL cases (Compagno M et al, Nature 2009). The tumor suppressor role of A20 was established by the reintroduction of wild-type A20 in some mutant A20 lymphoma B-cell lines promoting apoptosis, supposedly due to the loss of the ability to inactivate NF-κB. However, a protective effect of A20 against apoptotic stimuli was demonstrated in some cell types. In endothelial cells, A20 protects from TNF- and Fas-mediated apoptosis by inhibiting caspase-8 activation (Daniel S et al, Blood 2004). We thus wanted to understand the relationship between A20 expression, NF-κB activation and protection against apoptosis in B-cells.

Methods
We have cloned cDNAs coding for wild-type A20 (A20wt), for IκBαS32,36A (constitutively active form of IκBα), and for luciferase (as a control) into a doxycycline double-inducible episomal vector pRT-1, and stably transfected these vectors into an EBV positive lymphoblastoid B-cells line. Induction of p53 mediated apoptosis was performed by treatment of cells with Fludarabine*, whereas induction of caspase-8 dependant was performed with agonist antibodies against Fas (CD95).

Results
Both A20 and IκBαS32,36A overexpression suppressed NF-κB activity with downregulation of NF-κB target genes. Loss of NF-κB activation by induction of IκBαS32,36A led to increase of spontaneous apoptosis. Oppositely, loss of NF-κB activation by induction of A20 led to decrease of spontaneous apoptosis. Compared with luciferase transfected cells, no effect of A20 overexpression was seen after treatment of cells with anti-CD95 antibodies. Caspase-9 cleavage was significantly decreased and increase of TP53 protein levels was lower in A20 overexpressing cells after Fludarabine* treatment. By contrast, Fludarabine* treatment of IκBαS32,36A expressing cells led to an increase of both TP53 protein levels and caspase-9 cleavage. Therefore, A20 negatively modulated mitochondrial apoptosis pathway despite NF-κB downregulation.

Conclusions
These results suggest that A20 may exert an anti-apoptotic effect on its own independently of NF-κB activation. We are now looking after the functional properties of A20 natural mutants. Indeed, we can hypothesize that A20 mutants described in ABC DLBCLs have lost their negative feedback effect on NF-κB, but not their protective effect against apoptosis. So, A20 mutants may actively contribute to oncogenesis through their own protective effect on tumor cells.

Disclosures:
No relevant conflicts of interest to declare.
Evaluation of a-Catenin(CTNNA1) As a Tumor Suppressor Gene in Acute Myeloid Leukemis
Authors: Zeng Xianwei , Liu Ya , Lv Cui , Hu Zhenbo 
Published: October 2019

Abstract
Abstract 5240
Human a-catenin (CTNNA1) is a 102-kDa cadherin-binding protein that has been recognized as a tumor suppressor gene in solid tumors. Recent studies suggested that a-catenin functional as a tumor suppressor gene in leukemia with 5q deletion as well. In the present study, we speculate that a-catenin may play an important role as a tumor suppressor gene in AML and MDS cases with del(5q), since it is located on chromosome 5, band q31, and within the interval that is consistently deleted in these malignancies. Here, we report the evaluation of a-catenin as a tumor suppressor in myeloid disorders by mutational and functional analysis. Northern blot analysis revealed a very low level of expression of a-catenin mRNA in a panel of leukemia cell lines, whereas it was slightly higher in breast, colon, and prostate cancer lines. Protein truncation analysis in 11 myeloid leukemia cell lines revealed no mutations. By genomic sequencing selected exons in 23 clinical samples of AML/MDS patients with del(5q), we found that 1 of the samples carries a mismatch mutation on exon 5 of the gene. Using pcDNA-catenin expression vector for gene transfection analysis, we found that constitutive expression of a-catenin in the del(5q) leukemia cell line MUTZ-8 inhibited colony formation by 38.7%±6.3%(mea±sd) in the catenin-expressing cells when compared to those with empty vector and caused G1 arrest in the catenin-expressing cells (85.2%±7.3% vs 65.1%±4.6% in control cells) by cell cycle analysis. We conclude that a-catenin causes growth suppression in myeloid leukemia cells, which is consistent with a tumor suppressor gene, and endogenous expression of a-catenin is decreased in most of these cells; However, the finding a-catenin mutation in 1 out of 23 leukemia samples provides limited evidence that it is mutationally inactivated in human leukemias.

Disclosures:
No relevant conflicts of interest to declare.
Characterization of Single Platelet Mass, Volume, and Density in Response to Agonist Stimulation
Authors: Haley Kristina M. , Loren Cassandra P. , Phillips Kevin G. , McCarty Owen J.T. 
Published: October 2019

Abstract
Abstract 5261
An increase in mean platelet volume (MPV) is correlated with platelet activation and subsequent shape change. Pathologic processes marked by increased platelet activity such as myocardial infarction, cerebral vascular accidents, diabetes mellitus, and hypertension are associated with an increased MPV. Elevated MPV in these conditions reflect both a higher level of platelet activation as well as increased platelet turnover secondary to platelet consumption within thrombus formation. Assessment of MPV can be used to risk stratify patients as well as assign them to prognostic categories. However, MPV does not assess platelet heterogeneity or the specific change in single platelet mass, volume, or density. Current methods provide little insight into changes in physical parameters at the single platelet level. In order to overcome this limitation, we developed a quantitative tomographic differential interference contrast (QTDIC) microscopy technique to measure dry mass, volume, and density of platelets at the single-cell level. This technique is based on determining the axially resolved refractive index from a series of through-focus DIC images. Single cell platelet mass was observed to reduce from 1.84 ± 0.14 pg to 1.60 ± 0.13 pg in response to stimulation with thrombin-receptor agonist peptide (TRAP), while single cell platelet volume reduced from 7.28 ± 0.56 fL to 6.03 ± 0.48 fL (mean ± SEM). Single cell platelet density increased from 0.25 ± 0.001 pg/fL to 0.26 ± 0.002 pg/fL (mean ± SEM). Taken together, we have characterized the physical parameters of platelets in response to agonist stimulation. Our data suggest that platelet activation may correlate with decreased mass and volume, perhaps as a consequence of platelet degranulation. Further elucidation of the morphological changes of activated platelets at the single platelet level may allow for better understanding of platelet function and dysfunction in patients affected by platelet granule deficiencies, giant platelet syndromes, and disorders associated with membrane receptorsFigure 1.Characterization of physical parameters of platelets. (a) DIC image of human platelets, (b) refractive index map, (c) dry mass density map determined from refractive index using the Barer calibration, (d) Histogram of platelet dry mass, (e) Histogram of platelet volume, (f) Histogram of platelet density.Figure 1. Characterization of physical parameters of platelets. (a) DIC image of human platelets, (b) refractive index map, (c) dry mass density map determined from refractive index using the Barer calibration, (d) Histogram of platelet dry mass, (e) Histogram of platelet volume, (f) Histogram of platelet density.

Disclosures:
No relevant conflicts of interest to declare.
Successful Therapy of Cold Agglutinin Disease Utilizing Rituximab
Authors: Cholankeril Michelle , Bradley Thomas P. , Devoe Craig , Ghiuzeli Cristina Maria , Kolitz Jonathan E. , Allen Steven L. 
Published: October 2019

Abstract 5271
Cold agglutinin disease is an autoimmune hemolytic anemia mediated by cold reactive autoantibodies triggering a complement mediated hemolysis. This condition, when not associated with infection, is characterized by clonal proliferation of CD20+ B cells that produce monoclonal IgM cold agglutinins. Conventional therapies for primary cold agglutinin disease (CAD) are ineffective, but case reports suggest that rituximab, an anti-CD20 monoclonal antibody, may be effective. In this retrospective single institution study, we evaluated the use of rituximab therapy in 6 patients (pts) [1 male, 5 female; median age 70 years (range 62 – 89)]. Three pts had primary CAD, 2 pts had mixed CAD and warm AIHA, and 2 pts had CAD in the setting of CLL. Five pts had received steroid therapy, 2 pts IVIG, 1 pt azathioprine, and 5 pts PRBC transfusion (2–10 units). Five received induction therapy with rituximab 375 mg/m2 IV weekly for four weeks and 1 for seven weeks. Four patients received maintenance rituximab 375 mg/m2 IV every two months (4+ to 12 cycles). All responded to therapy with a median rise in hemoglobin of 1.8 g/dl at 2 months from initiation of induction with further improvement over time (figure 1). Re-induction was performed in 2 pts; both had an initial one year duration of response and both responded following re-treatment. Median duration of response is 3+ yrs (range 1–8+ yrs); the 8+ yr response was in pt 2 who received only induction therapy. Two pts have completed two years of maintenance therapy and remain in remission at 4 months and 2 years post, respectively. Cold agglutinin titers decreased by 4 fold in 2 patients, 1 fold in 1 pt and remained stable in 1. Despite the improvement in hgb in all pts, laboratory evidence of low grade hemolysis persisted in 4 pts. All pts were able to be tapered off steroids and all remain transfusion free. No unexpected adverse events were noted. Rituximab appears to be a well tolerated and effective therapy for cold agglutinin disease. The need for and length of maintenance therapy remains to be determined.

Disclosures:
No relevant conflicts of interest to declare.
Von Willebrand Factor, ADAMTS13 Activity, TNF-Alpha, and Their Relationships in Patients with Chronic Kidney Disease
Authors: Shen Lei , Lu Guoyuan , Dong Ningzheng , Jiang Liqiong , Ma Zhenni , Ruan Changgeng 
Published: October 2019

Abstract
Abstract 5312
Patients with chronic kidney disease (CKD) have increased risks of endothelial dysfunction and thrombosis. Systemic inflammation may contribute to the endothelial dysfunction and accelerated thrombosis observed in CKD patients. von Willebrand factor (VWF), a well-known index of endothelial damage, has been reported to increase both in CKD and inflammatory states. ADAMTS13 is a specific VWF-cleaving protease, and some reports suggested that the ratio of VWF and ADAMTS13 maybe more useful in the diagnosis and treatment evaluation of patients in the prothrombotic state. So we assessed the relationships among endothelial dysfunction, ADAMTS13 activity, and levels of inflammatory cytokines in CKD patients. CKD patients were classified into 3 groups: chronic glomerulonephritis group (CGN, n = 31), idiopathic nephritic syndrome group (NS, n = 32), and lupus nephritis group (LN, n = 41). We measured the plasma levels of TNF-α, VWF antigen (VWF:Ag), and ADAMTS13 activity by using an ELISA-based method in CKD patients (n = 104) and normal controls (n = 32). The ratio of the VWF:Ag level to ADAMTS13 activity was calculated. The VWF:Ag level was significantly higher and the ADAMTS13 activity was significantly lower in all the disease groups than in the controls (P < 0.01). ADAMTS13 activity was lower in the NS group than in the CGN and LN groups (P < 0.05) (Table 1). The TNF-α (pg/mL) level was higher in the CKD group than in the control group (CGN: 3.19 ± 1.01; NS: 3.26 ± 1.18; LN: 3.24 ± 1.24; Control: 2.27 ± 1.23; P < 0.01; P < 0.01). TNF-α was positively correlated with the VWF:Ag level (r = 0.242, P = 0.013) and negatively correlated with the glomerular filtration rate (GFR) (r = −0.193, P = 0.049). ADAMTS13 activity was negatively correlated with the cholesterol level in CKD patients (r = −0.2, P = 0.042). TNF-α level in CKD was positively correlated with the VWF:Ag level and negatively correlated with GFR, which suggests that inflammation might be a major cause of endothelial dysfunction and an index for renal function. The VWF:Ag level increased and ADAMTS13 activity decreased in CKD patients, which indicates that CKD leads to a prothrombotic state.
Table 1. The plasma VWF:Ag level, ADAMTS13 activity, and the VWF/ADAMTS13 ratio in the CGN, NS, LN, and control groups N VWF (%) ADAMTS13 (%) VWF/ADAMTS13 CGN 31 198.25 ± 140.20** 61.93 ± 22.47**# 3.83 ± 3.29** NS 32 149.94 ± 74.50** 48.87 ± 18.63** 3.42 ± 1.59** LN 41 234.75 ± 134.91**## 67.81 ± 22.30**## 3.79 ± 2.52** CONTROL 32 99.55 ± 21.37 94.37 ± 23.66 1.12 ± 0.37 vs. CONTROL ** P < 0.01; vs. NS ## p < 0.01, # P < 0.05

Disclosures:
No relevant conflicts of interest to declare.
Urokinase-Type Plasminogen Activator Receptor (uPAR, CD87) Regulates Integrin Redistribution in Endothelial Cells Via Interaction with Low Density Lipoprotein Receptor- (LDLR-) Like Proteins
Authors: Prager Gerald , Novotny Rene , Unseld Matthias , Poettler Marina , Kalinowska Waclawa , Breuss Johannes , Zielinski Christoph 
Published: October 2019

Abstract
Abstract 5315
angiogenesis by degradation of extracellular matrix proteins as well as induction of intracellular signal transduction. We recently could demonstrate that in VEGF-stimulated endothelial cells pro-uPA becomes activated, which leads to uPAR-complex formation, it's internalization and redistribution of uPAR to newly formed focal adhesions ad the leading edge of migrating endothelial cells. Thereby, uPAR surface expression is tightly transcriptional regulated via the Density Enhanced Phosphatase-1 (DEP-1), but also via the LDLR-family members, which regulate subcellular uPAR distribution.
Here, we describe a mechanisms by which uPAR-internalization regulates integrin redistribution. We have characterized a novel binding motif on uPAR domain 3 for LDLR-protein interaction by using affinity chromatography as well as co-immunoprecipitation experiments. To proof a functional relevance of a direct uPAR/LDLR protein interaction, we reconstituted either uPAR mutants (mutL3/uPAR), lacking the binding site for LDLR-proteins, or wild type uPAR into endothelial cells derived from uPAR−/− mice. Reconstitution of mutL3/uPAR was incapable to redistribute uPAR as well as integrins during VEGF-induced endothelial cell migration when compared to wild type uPAR reconstitutes. The functional importance of uPAR / LDLR interaction was further reflected by the use of an inhibitory peptide (P1) interfering with uPAR/LDLR-protein interaction, which functionally reverted full length uPAR reconstitution, or the chaperon Receptor Associated Protein (RAP), a high affinity ligand for LDLR-proteins, which prevents uPAR/LDLR interactions. Thus, interfering with uPAR/LDLR-protein interaction at different levels led to an impaired endothelial cell spreading behavior on integrin-adhesive matrix proteins as well as a reduced pY576 FAK phosphorylation upon endothelial cell adhesion, leading to an reduced migratory response towards VEGF.
These data suggest a central role of uPAR/LDLR-protein interaction in VEGF-induced endothelial cell migration via induction of integrin redistribution. Thus, uPAR/LDLR interaction might represent a novel therapeutic target in angiogenesis-related diseases.

Disclosures:
No relevant conflicts of interest to declare.
Disruption of MYD88 Pathway Signaling Leads to Loss of Constitutive IRAK1, NF-κβ and JAK/STAT Signaling and Induces Apoptosis of Cells Expressing the MYD88 L265P Mutation in Waldenstrom's Macroglobulinemia
Authors: Yang Guang , Zhou Yangsheng , Liu Xia , Cao Yang , Hunter Zachary , Treon Steven P 
Published: October 2019

Abstract
Abstract 597

Introduction:
Through whole genome sequencing of bone marrow lymphoplasmacytic cells (LPCs) from WM patients, we recently identified a broadly expressed somatic variant (MYD88 L265P) which was present in 90% of WM patients, as well as in BCWM.1 and MWCL-1 WM cells. The presence of this variant has been reported to confer oncogenic activity in cell lines derived from patients with DLBCL ABC subtype lymphomas by induction of IRAK 1/4 kinase activity, and downstream signaling via NF-κβ (Ngo et al, Nature 2011, 470:115-119). We therefore sought to delineate the activity of agents which either disrupt MYD88 homodimerization, a prerequisite to IRAK 1/4 auto-phosphorylation, or by direct inhibition of IRAK 1/4 kinase activity in WM.

Patients and Methods:
Primary WM LPCs were isolated from bone marrow biopsy specimens from WM patients, and CD19+ B-cells were isolated from the peripheral blood of healthy donors. The mutational status of MYD88 was identified by Sanger sequencing, which confirmed the presence of the L265P mutation in BCWM.1 and MWCL-1 cells, and absent in Ramos and MM1S cells. Western blot and phospho-flow studies using phospho-specific antibodies were performed for signaling proteins downstream of MYD88, cell killing evaluated by Annexin V/PI staining and AlamarBlue, and IgM and IL-6 release determined after treatment with and without inhibitors of MYD88 homodimerization (IMG-2005-5, IMGENEX, San Diego CA), and IRAK 1/4 kinase function (407601, EMD, Gibbstown, USA).

Results:
Phosphorylation of the MYD88 downstream signaling proteins IRAK1, IκBα, NFκβ-p65 and STAT3 was greater in MYD88 L265P expressing BCWM.1 and MWCL-1 cells, and primary WM patient LPC, versus MYD88 wild type expressing Ramos, MM1S, and healthy donor CD19+ B-cells. Inhibition of MYD88 homodimerization and IRAK 1/4 kinase activity led to decreased phosphorylation of IRAK1, IκBα, NFκβ-p65 and STAT3 in BCWM.1 and MWCL-1 cells. Importantly, both inhibitors induced robust apoptosis of BCWM.1 and MWCL-1 cells, and primary WM LPCs bearing the MYD88 L265P mutation in comparison to Ramos, MM1S, or healthy donor CD19+ B-cells. Activation of caspase-3 and PARP, decreased BCL-2 expression, and decreased release of IL-6 and IgM accompanied treatment of WM cells following treatment with inhibitors of MYD88 homodimerization and/or IRAK 1/4 kinase activity.

Conclusion:
Disruption of MYD88 pathway signaling leads to loss of constitutive IRAK1, NF-κβ and JAK/STAT activation and induces apoptosis of cells expressing the MYD88 L265P mutation in WM. The results of this study provide novel insights into the pathogenesis of WM, and provide a framework for the clinical investigation of inhibitors of the MYD88 pathway in WM.

Disclosures:
No relevant conflicts of interest to declare.