bluuebunny/crossref_metadata_2025 huggingface dataset:
Surface IgM of Chronic Lymphocytic Leukemia Cells Displays Unusual Glycans Indicative of Antigen Engagement In Vivo.
Authors: Packham Graham , Krysov Serge , Mockridge Christopher Ian , Potter Kathy N , Stevenson Freda K
Published: October 2019

Abstract 55

Several lines of evidence support the idea that surface immunoglobulin M (sIgM) plays a key role in determining the clinical behavior of chronic lymphocytic leukemia (CLL). For example, the presence of somatic mutations in immunoglobulin variable region genes is a strong prognostic marker with unmutated CLL (U-CLL) associated with a poor outcome relative to mutated CLL (M-CLL). U-CLL also generally express higher levels of sIgM and retain the ability to signal via this receptor. In this study, we used surface biotinylation to analyse sIgM in CLL and discovered that it exists in two forms with differing mobility on SDS-PAGE. Treatment with glycosidases revealed that these forms were due to different N-glycosylation patterns in the μ constant region. One form is similar to that of normal B cells in bearing mature complex glycans common to most cell surface glycoproteins. The other is an immature mannosylated form more characteristic of endoplasmic reticulum (ER)-located μ chains. CLL cells expressed variable proportions of the immature mannosylated form and quantitative analysis demonstrated that, on average, the proportion of mannosylated sIgM was approximately 2-fold higher (p=0.006) in U-CLL compared to M-CLL. Although normal B cells isolated from blood expressed only the mature form of sIgM, in vitro treatment with anti-μ resulted in upregulation of the immature form, suggesting that glycan modification is a consequence of antigen exposure. Consistent with this, in vitro incubation of CLL cells was associated with increased expression of the mature form of sIgM. Phosphotyrosine analysis demonstrated that both forms of sIgM were able to signal following sIgM engagement in vitro. Taken together, these findings support the concept that CLL cells are continuously exposed to antigen in vivo. This process leads to a change in the N-glycosylation pattern of the re-expressed sIgM so that a mannosylated form predominates, especially in U-CLL. Strikingly, expression of mannosylated sIgM is also characteristic of follicular lymphoma, where it is constitutively displayed via N-glycosylation sites in the Ig variable region (Radcliffe et al. J Biol Chem. 2007; 282, 7405-15). Persistent mannosylation of sIgM appears to be a feature common to several B-cell malignancies, suggesting a role in pathogenesis.

Disclosures:

No relevant conflicts of interest to declare.
Accuracy of Reporting of Venous Thromboembolism in Hospitalized Patients.
Authors: Perez Alejandro , Hubman Margie S. , Martin Craig , Michaels Jeannie , Bartholomew John R.
Published: October 2019

Abstract 557

Introduction:

Venous thromboembolism (VTE), which includes deep vein thrombosis (DVT) and pulmonary embolism (PE), is an important clinical event that needs to be accurately reported. The Agency for Healthcare Research and Quality (AHRQ) has categorized VTE as a reportable patient safety indicator (PSI) and has distributed a software package for recording events such as VTE based upon diagnosis codes. At our institution, there was concern that VTEs identified by this software method may be over estimating the actual number.

Objective:

To verify the cases of VTE as identified by AHRQ methodology through a review of the medical record for each event reported.

Methods:

Through a review of the electronic medical record (EMR), the events recorded as a VTE by PSI software over a 7 month period from November 2008 to May 2009 were further characterized into categories of acute, remote or age indeterminate DVTs and were distinguished from events that were either isolated superficial vein thrombosis (SVT) or that were not VTE at all. Discharge summaries, hospital notes, operative reports and imaging reports were used to verify identified cases of VTE. VTE occurrences were grouped by the month of discharge of the patient. The PSI software utilized diagnosis codes that have been abstracted for a patient's hospitalization based upon physician documentation.

Results:

437 events were recorded as VTE over a 7 month period. 59 cases (13.5%) were incorrectly coded as VTE. 35 cases (8.0%) were superficial vein thrombosis mistakenly classified as DVT. 12 cases (2.7%) had remote or age indeterminate DVTs. 12 cases (2.7%) cases had no VTE and were considered further abstraction errors. After interim reporting of the first 4 months of VTE data, vascular laboratory study reports were amended so that superficial vein thrombosis would no longer be identified by this terminology and would subsequently be classified as superficial vein thrombophlebitis. The last three months of data collection had 9 cases (4.7%) incorrectly identified as DVTs that were actually SVTs versus 26 cases (10.6%) prior to implementation of the nomenclature change.

Conclusion:

Hospitals must be aware of reporting practices in their own institution as national protocols may not completely depict individual hospital occurrences. Careful attention to the manner in which the medical record reflects events can make substantial difference in accounting of VTE occurrence. Because of the lack of specificity of current diagnosis coding guidelines, an appropriate medical term was not sufficient for medical record abstractors to categorize an event correctly in this study. As hospitals become monitored for quality of care with these methodologies, the accuracy of reporting becomes paramount.

Disclosures:

No relevant conflicts of interest to declare.
Regulation of ABCB1 (p-glycoprotein) by the FOXO1 Transcription Factor in Acute Myeloid Leukemia.
Authors: Seedhouse Claire , Ahluwalia Sophie , Mills Ken I , Grundy Martin , Shang Shili , Burnett Alan K. , Russell Nigel H. , Pallis Monica
Published: October 2019

Abstract 589

FOXO1 mediates a cellular stress response by inhibiting cell growth and up-regulating expression of molecules involved in apoptosis and antioxidant defences. The membrane pump p-glycoprotein (pgp, expressed by the ABCB1 gene) is responsible for the efflux of chemotherapeutic drugs and the redistribution of components of the ceramide signalling pathway. In AML, pgp over-expression is associated with failure to respond to remission-induction chemotherapy, but attempts to modulate pgp function have enjoyed little success and new approaches are needed. We studied transcriptional control of the ABCB1 gene in AML cells. The Pgp protein was measured using MRK-16; ABCB1 mRNA was measured using realtime PCR in patients entered into UK AML 14 and AML 15 clinical trials. 141 of these trial samples were also studied using the Affymetrix U133A (22,283 probesets) or the U133Plus2 (∼54,000 probesets) gene chips to establish correlations between pgp and factors which might influence expression. FOXO1 was one of the most closely correlated genes with pgp (P<0.001). FOXO3 and 4 did not correlate. To confirm these results FOXO1 mRNA levels were measured by real time PCR in a separate cohort of 178 trial samples: FOXO1 and ABCB1 were also strongly correlated in this cohort (P<0.001). Protein levels of FOXO1 were measured by flow cytometry, and corresponded to mRNA levels in patient samples. Using the TF1 cell line, which normally expresses high levels of FOXO1 and ABCB1 we studied the regulation of pgp by FOXO1 using siRNA. FOXO1 siRNA was found to decrease both FOXO1 and ABCB1 mRNA expression by 70% and 60% respectively. High pgp expression rarely occurs in a sample with a FLT3-ITD mutation. In this context we established that the presence of a FLT3-ITD in AML samples is associated with low FOXO1 expression (P<0.001, median FOXO1/B2M ratio = 0.51 in wildtype samples (n=268) and 0.18 in ITD samples (n=101)) and that siRNA to FLT3 in MV4.11 (FLT3-ITD) cells upregulates FOXO1 more than 2-fold. As the ABCB1 promoter is known to have a FOXO1 binding site, we conclude that FOXO1 is likely to be a major regulator of ABCB1 expression in AML and may therefore be a therapeutic target to modulate drug resistance. We further conclude that FOXO1 may be at least partially responsible for the distinction between two major poor risk phenotypes in AML–namely FLT3-ITD (causing low FOXO1, predictive for relapse) and pgp overexpression (induced by high FOXO1, predictive for chemoresistant disease).

Disclosures:

No relevant conflicts of interest to declare.
Supportive Care Only Versus Allogeneic Hematopoietic Cell Transplantation in Patients ≥ 60 Years with De Novo High-Risk MDS.
Authors: Platzbecker Uwe , Finke Jürgen , Trenschel Rudolf , Kerstin Schaefer-Eckart , Bornhauser Martin , Kobbe Guido , Scott Bart L , Ehninger Gerhard , Beelen Dietrich , Deeg H. J. , Germing Ulrich
Published: October 2019

Abstract 596

Myelodysplastic syndromes (MDS) occur primarily in older patients, and allogeneic hematopoietic cell transplantation (HCT) only infrequently is considered a therapeutic option. Until recently best supportive care (BSC), only including transfusion support, has been the standard of care for the majority of these patients (pts). Novel treatment modalities and innovations in transplant protocols have changed that approach and have led to increased frequency of allogeneic HCT in pts more than 60 or 65 years of age. However, no randomized trial comparing outcomes with BSC and allogeneic HCT has been conducted.

Therefore, we performed a matched pair analysis including 126 patients 60-77 (median 65) years old with de novo high-risk MDS by FAB classification (RAEB n=88, RAEB-t n=20, CMML n=18) who underwent allogeneic HCT at a median of 8.6 months from initial MDS diagnosis. Cytogenetics were availabel in 108 pts (good: n=63, intermediate: n=18, poor: n=27), allowing for IPSS scoring in 107 pts: 28 were INT-1, 45 INT-2, and 34 HIGH risk. Before HCT most pts had progressed to more advanced disease (RAEB n=45, RAEB-t n=10, CMML n=10, AML n=61). At the time of HCT the median blast count in the marrow was 12%. While 51 pts had received supportive care only prior to HCT the remaining had undergone chemotherapy of various intensities including methyltransferase-inhibitors (n=10). Patients were prepared for HCT with one of several reduced intensity (RIC, n=79) or more conventional intensity (CI, n=47) conditioning regimens and transplanted from related (n=50) or unrelated (n=76) donors. The outcome after HCT was compared to outcome with BSC only in a matched pair group from the Düsseldorf registry. Matching criteria were age, gender, marrow blast count, FAB and IPSS category. One prerequisite was that controls were still under supportive care at the time of HCT of the corresponding matched patient.

With a median follow-up of 60 months from MDS diagnosis the 5-year overall survival (OS) rate was 45% for the HCT and 25% for the BSC cohort (p=0.008). These retrospective data suggest, therefore, that allogeneic HCT from related or unrelated donors, using various conditioning approaches, compared to BSC only, offers a meaningful survival advantage and the potential of cure for patients with high-risk MDS, even in the 7th decade of life. There is a need for prospective clinical trials in order to determine the place of allogeneic HCT within the growing therapeutic opportunities for pts with MDS.

Disclosures:

No relevant conflicts of interest to declare.
Molecular Response <1% BCR-ABL IS at 12 Months Is Associated with Improved Overall and Progression-Free Survival. the Randomized German CML-Study IV
Authors: Müller Martin C. , Hochhaus Andreas , Lauseker Michael , Jung-Munkwitz Susanne , Pfirrmann Markus , Leitner Armin , Krause Stefan W. , Pletsch Nadine , Hanfstein Benjamin , Schnittger Susanne , Gratwohl Alois , Hasford Jörg , Saussele Susanne , Hehlmann Rüdiger ,   for The German CML-Study Group
Published: October 2019

Abstract 669

Introduction:

The prognostic relevance of major molecular remission (MMR, <0.1% BCR-ABL according International Scale, IS) for survival has remained uncertain. Gold standard for the evaluation of treatment response is the achievement of complete cytogenetic remission in spite of its limited sensitivity and the requirement of bone marrow puncture. The standardization of PCR methods and the introduction of conversion factors to account for differences among European laboratories, has resulted in a uniform reporting system allowing comparable BCR-ABL expression levels derived from peripheral blood samples. We sought to evaluate an association of the degree of molecular response and survival.

Patients and Methods:

We have analyzed 848 patients within the CML-Study IV (randomized comparison of imatinib 800 mg vs 400 mg vs 400 mg + IFN). BCR-ABL (IS) was determined by quantitative RT-PCR. Patients with atypical BCR-ABL transcripts were excluded from the analysis. Median observation time was 40 months (minimum 12). Landmark analyses have been performed at 12 months for overall and progression-free survival using 3 groups of response (<0.1%, 0.1%-1%, >1% BCR-ABL IS).

Results:

341 patients achieved a BCR-ABL expression <0.1% (MMR), 240 patients between 0.1% and 1% and 267 patients >1% by 12 months. Independent of treatment approach, the groups of patients achieving MMR and 0.1%-<1% at 12 months showed significantly higher progression free survival (PFS) (p=0.0023; 99% [95% CI: 97–100%] vs 97% [95% CI: 94–99%] vs 94% [95% CI: 90–97%] at 3 years) and better overall survival (p=0.0011; 99% [95% CI: 97–100%] vs 98% [95% CI: 95–100%] vs 93% [95% CI: 90–96%] at 3 years) compared to the group with >1% BCR-ABL by 12 months (Figure).

Conclusion:

Faster and deeper response to imatinib-based treatment by 12 months revealed to be associated with improved PFS and overall survival. The critical cutoff level seems to be 1% BCR-ABL IS which has been shown to closely correlate with complete cytogenetic remission.

Disclosures:

Müller: Novartis: Honoraria, Research Funding. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. German CML-Study Group:Deutsche Krebshilfe: Research Funding; Novartis: Research Funding; Roche: Research Funding; BMBF: Research Funding; Essex: Research Funding.
Methylation Profiling of Mediastinal Gray Zone Lymphoma Reveals a Distinctive Signature with Elements Shared by Classical Hodgkin's Lymphoma and Mediastinal Large B-Cell Lymphoma
Authors: Eberle Franziska C. , Rodriguez-Canales Jaime , Wei Lai , Hanson Jeffrey C. , Killian J. Keith , Sun Hong-Wei , Adams Lisa G. , Hewitt Stephen M. , Wilson Wyndham H. , Meltzer Paul S. , Staudt Louis M. , Emmert-Buck Michael R. , Pittaluga Stefania , Jaffe Elaine S.
Published: October 2019

Abstract 747

Background:

Mediastinal gray zone lymphoma (MGZL) is a newly recognized entity demonstrating transitional morphologic and immunophenotypic features between classical Hodgkin's lymphoma, nodular sclerosis subtype (CHLNS) and primary mediastinal large B-cell lymphoma (PMLBCL). CHLNS and PMLBCL differ in morphology, immunophenotype, and therapeutic consequences. MGZLs present a challenge both to the pathologist and clinician, as the criteria to distinguish MGZL from CHLNS and PMLBCL are still imprecise, and the optimal treatment approach is as yet undetermined.

Purpose of Study:

Epigenetic changes have been implicated in the loss of the B-cell program in CHL, and might provide a basis for the immunophenotypic alterations seen in MGZL. Thus, we performed a large scale DNA methylation array of MGZL, CHLNS, and PMLBCL as well as diffuse large B-cell lymphoma (DLBCL) to investigate the biological underpinnings of MGZL and how it corresponds to the two related entities CHLNS and PMLBCL and the less related entity DLBCL. Microdissection of tumor cells was performed to identify changes in the tumor cell population, and allow comparison with the background inflammatory and stromal milieu.

Results:

Principal component analysis (PCA) demonstrated that MGZLs have a distinct epigenetic profile intermediate between CHLNS and PMLBCL but clearly different from that of DLBCL. Analysis of common hypo- and hypermethylated CpG targets in MGZL, CHLNS, PMLBCL and DLBCL was performed. MGZL showed great overlap with CHLNS (49 common targets) and PMLBCL (50 common targets). In contrast, MGZL shared only two common targets with DLBCL. However, using the epigenetic profiles we were able to establish class prediction models that could distinguish between MGZL, CHLNS and PMLBCL with a final combined prediction of 100%. Pyrosequencing for selected CpG sites from different genes were performed and confirmed the high accuracy of methylation array results.

Conclusions:

MGZLs share several clinical and pathological features with CHLNS and PMLBCL. Our findings further underscore the close biological relationship between MGZL, CHLNS and PMLBCL, and ready distinction from DLBCL. However, MGZL has a distinct epigenetic identity that shares elements of both parent disorders. As the first biological study on MGZL, our results provide novel insights into MGZL pathogenesis, and its relationship to CHLNS and PMLBCL.

Disclosures:

Emmert-Buck: NIH: Michael Emmert-Buck is an inventor on all NIH patents covering laser capture microdissection technology and receives royalty payments through the NIH technology transfer program., Patents & Royalties.
Reinforcement of Cancer Immunotherapy by Adoptive Transfer of Cblb-Deficient Cytotoxic T Lymphocytes Combined with a Dendritic Cell Vaccine
Authors: Lutz-Nicoladoni Christina , Stoizner Patrizia , Pircher Magdalena , Wallner Stephanie , Wolf Anna Maria , Gastl Guenther , Penninger Josef , Baier Gottfried , Wolf Dominik
Published: October 2019

Abstract 957

Introduction:

Various approaches to induce immunological rejection of tumors including transfer of autologous tumor infiltrating lymhocytes (TIL) after ex vivo clonal expansion or application of ex vivo transduced antigen specific T cell (TCR) transgenic T cells have been elaborated. In general, adoptive T cell transfer (ATC) has been combined with lympho-depleting agents (e.g. cyclophosphamide). However, the therapeutic efficacy of these cancer immunotherapy approaches is limited due to insufficient in vivo activation, expansion and survival of transferred effector immune cells, which is mainly due to suppressive mileu signals and immune evasion mechanisms induced by TGF-β. The E3 ubiquitin ligase Cbl-b is a key regulator of T cell activation and is assumed to confer TGF-β resistance. Thus we performed a proof-of-concept study evaluating Cbl-b targeting as “intracellular adjuvant” strategy to improve ATC for cancer immunotherapy.

Material and Methods:

We first tested the in vitro sensitivity of CTL towards TGF-β mediated immuno-suppressive cues and then in vivo evaluated the anti-tumor reactivity of cblb-deficient cytotoxic T lymphocytes (CTL) in murine tumor models alone or in combination with a dendritic cell (DC) vaccine.

Results:

Cblb-deficient CTL are hyper-responsive to TCR/CD28-stimulation in vitro and protected from the negative cues induced by TGF-β as determined by quantification fo IFN-g secretion and quantification of their proliferative capacity. Unexpectedly, adoptive transfer of polyclonal, non TCR-transgenic cblb-deficient CD8+ CTL, however, is not sufficient to reject B16ova or EG7 tumors in vivo, which is in clear contrast to previous reports using lymphopenic animals receiving adoptively transferred TCR-transgenic T cells. Thus, we next evaluated in vivo re-activation of adoptively transferred cblb-deficient T cells by a DC vaccine (i.e. SIINFEKL-pulsed DC). In strict contrast to ATC monotherapy, this approach now markedly delays tumor outgrowth and significantly increase survival rates, which is paralleled by an increased CTL infiltration rate to the tumor site and an enrichment of ova-specific and IFN-g-secreting CTL in the draining lymph nodes. Moreover, compared to wild-type CTL, cblb-deficient mice vaccinated with the DC vaccine show an increased cytolytic activity in vivo.

Conclusions:

In summary, we provide experimental evidence that genetic inactivation of cblb in polyclonal, non-TCR transgenic adoptively transferred CTL might serve as a novel “adjuvant approach”, suitable to augment the effectiveness of anti-cancer immunotherapies using ATC in immune-competent recipients.

Disclosures:

No relevant conflicts of interest to declare.
A Commitment to Lineage
Authors: Glimcher Laurie H. , Lazarevic Vanja , Ermann Joerg , Garrett Wendy
Published: October 2019

Abstract SCI-22

The transcription factor T-bet, isolated in our laboratory a decade ago, is a master regulator of Type 1 immunity in cells of both the adaptive and innate immune system. In adaptive immunity, T-bet instigates genetic programs in T helper 1 (Th1) cells and is required for production of the hallmark Th1 cytokine IFNg. It simultaneously represses the differentiation of T helper 2 cells and the profibrotic cytokines IL-13 and TGFb. We have recently determined that T-bet is also a repressor of the Th17 genetic program and have established the molecular mechanisms that underpin that function. T-bet also controls the optimal differentiation and function of the cytolytic CD8 cell, and is required for the development of the natural killer T cell. T-bet deficient animals are largely protected from autoimmune/inflammatory diseases such as multiple sclerosis, systemic lupus, type 1 diabetes and inflammatory arthritis, but are susceptible to type 2 driven diseases such as asthma and scleroderma. An exception to this overall rule is our recent discovery that in the absence of an adaptive immune system, the majority of mice lacking T-bet develop a spontaneous ulcerative colitis that progresses to colonic dysplasia and rectal adenocarcinoma. This colitis and inflammation associated colorectal cancer are MyD88 independent, driven by colitogenic flora and ameliorated by treatment with TNF blockade, antibiotics, and transfer of T regulatory cells. This phenotype maps to the T-bet deficient dendritic cell that drives this pro-inflammatory program; selective over-expression of T-bet in DCs was sufficient to reduce colonic inflammation and prevent the progression to neoplasia. The molecular pathogenesis of TRUC colitis and colitis-associated colorectal (caCRC) shares several key features with human caCRC. This model of colitis and colitis-associated colorectal cancer provides opportunities to further understand host-microbial relationships in inflammation and neoplasia and test preventative and therapeutic strategies pre-clinically. The function and mechanism of action of T-bet in the pathogenesis of immune system driven diseases will be discussed.

Disclosures:

Glimcher: Merck: Consultancy, Patents & Royalties, Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.
Th17/Treg Imbalance in Sickle Cell Disease and Hydroxyurea Therapy
Authors: Gilli Simone , Pericole Fernando V , Fernandes Luis Gustavo , Ribeiro Karina A. R. , Castilho Lilian , Costa Fernando Ferreira , Saad Sara T Olalla
Published: October 2019

Abstract 1050

Background:

Tregs and proinflammatory Th17 cells have been described as two distinct subsets from Th1 and Th2 cells and have opposite effects in autoimmunity and inflammation. Th17/Treg balance controls inflammation and is important in the pathogenesis of autoimmune, infectious and chronic diseases. In view of growing evidence linking sickle cell disease (SS) and chronic inflammation and the potential anti-inflammatory role of Tregs cells, we hypothesized that SS is associated with decreased Treg subpopulation and consequent T cell dysfunction. Furthermore, the effects of hydroxyurea (HU) in this balance are not understood. To assess whether the Th17/Treg balance is impaired in patients with sickle cell disease, we investigated the peripheral blood Th17 and Treg frequencies and the expression of TGFβ1 and RORγT, a master regulator of Th17 differentiation, in sickle cell patients under HU therapy (SS-HU) and without HU (SSw/oHU). Methods. Thirty-eight patients with steady-state sickle cell disease (mean age 43 ± 10.5 yo; range 19–57 yo; 21 female/17 male), 19 SS-HU, 19 SSw/oHU and 20 healthy blood donors were evaluated. Tregs and Th17 quantification was performed by flow cytometry analysis and the expression of TGFβ1 and RORγT by q-PCR. Results. We found significant decrease in the Treg frequency (Foxp3+CD25+CD4+ population) comparing total SS patients and healthy controls (p=0.001) and this difference was more expressive between SS-HU and healthy controls (P=0.0006). Moreover, we found statistically increase of Th17 in SS-HU (p=0.02) and in SSw/oHU (p=0.01), comparing both with healthy controls. The TGFβ1 expression was increased in SS-HU (p=0.04) and SSw/oHU patients (p=0.01) when compared to control group. Interestingly, RORγT expression was significantly elevated only in SSw/oHU vs controls (p=0.03). Conclusion: Our results demonstrated a numerical imbalance of Th17/Treg in the peripheral blood of patients with SS disease. HU has no role mediating Th17/Treg plasticity and even SS patients under HU therapy remains Treg depleted. However, HU is able to modulated RORγT, corroborating the importance of other regulating factors during Th17 differentiation. Moreover, the higher expression of TGFβ1 may be an important positive instructive signal for Th17 differentiation. Th17/Treg imbalance might impact the disease presentation and phenotype and HU therapy may be involved in this issue.

Disclosures:

No relevant conflicts of interest to declare.
CD8+ CD28null T Cell Expansion Impairs Lenalidomide Immunomodulatory Function in Myelodysplastic Syndrome
Authors: McDaniel Jessica M. , Zou JianXiang , List Alan F. , Epling-Burnette P.K.
Published: October 2019

Abstract 1117

Lenalidomide, a thalidomide analog, is known to induce high rates of transfusion independence in Myelodysplastic Syndrome (MDS) patients harboring a commonly deleted region on chromosome 5q (del(5q)). Erythroid response is also seen in 20–30% of low-risk, non-del(5q) patients, although the mechanism of response is incompletely understood. Lenalidomide is a potent immunomodulating agent and hematopoietic response has been linked to increased lymphocyte infiltration in the bone marrow and improved T cell and NK cell proliferation/function. Lenalidomide's effect on the erythroid compartment is well documented, but the effect of lenalidomide on the immune compartment, and the relationship between the T cell compartment and erythroid response in vivo in MDS is not known. We therefore examined 23 T cell parameters before lenalidomide treatment and correlated them to hematologic response. We found that patients who fail lenalidomide therapy had higher CD8+ Terminal Effector Memory (TEM) T cells than did responders (p=0.02). TEM cells express CD45RA, have variable expression of the CD45RO memory marker and loss of L-selectin (CD62L) lymph node homing receptor. Interestingly, T cells within the TEM compartment are uniformly CD28 deficient (CD28null). Lenalidomide has been shown by us and others to increase the proliferation and function of T cells by providing co-stimulation (LeBlanc et al. 2004, Blood) through CD28, so CD28 expression may be important for immunomodulatory response. We found that MDS patients non-responsive to lenalidomide had an overall increase in CD4 (p=0.0195) and CD8 (p=0.0317) CD28null T cells, as well as an increase in CD28null cells (p=0.0159) within the TEM compartment compared to hematologic responders. To determine if CD28 expression is necessary for lenalidomide action in T cells, we sorted healthy donor T cells into CD8+CD28+ and CD8+CD28− populations, and found that lenalidomide-induced proliferation and interleukin-2 (IL-2) production were completely ablated within the CD28null subset. Because CD28null T cells displayed less proliferation (p<0.001) and produced less IL-2 (p<0.001) after stimulation, possibly related to excessive proliferative history in vivo, we therefore used shRNA to knockdown CD28 in healthy (CD28+) T cells and determined the functional response of these cells to lenalidomide. With CD28 knockdown, lenalidomide produced significantly less IL-2 compared to CD28+ controls (p<0.001), indicating the necessity of the immunotyrosine-based activation motifs (ITAMs) on the intracellular domain of the CD28 receptor for lenalidomide action. These results indicate that lenalidomide-mediated immune modulation requires CD28 expression on T cells and expansion of CD8+ CD28 deficient T cells may represent a predictor of erythroid non-response in non-del(5q) low-risk MDS patients.

Disclosures:

List: Celgene: Consultancy. Epling-Burnette:Celgene: Research Funding.
The Disulfide Isomerase Erp57 Mediates Platelet Aggregation and Hemostasis
Authors: Wu Yi , Ahmad Syed S. , Zhou Junsong , Wang Lu , Cully Matthew P , Essex David W
Published: October 2019

Abstract 1129

A close homologue to protein disulfide isomerase (PDI) called ERp57 forms disulfide bonds in glycoproteins in the endoplasmic reticulum and is expressed on the platelet surface. We generated two rabbit antibodies to ERp57. One antibody strongly inhibited ERp57 in a functional assay and strongly inhibited platelet aggregation. There was minimal cross reactivity of this antibody with PDI by Western blot or in the functional assay. Using flow cytometry this antibody substantially inhibited activation of the alphaIIbbeta3 fibrinogen receptor. Furthermore, adding ERp57 to platelets potentiated aggregation. In contrast, adding a catalytically inactive ERp57 inhibited platelet aggregation. When the inactive ERp57 was infused into mice the tail bleeding time was prolonged. We generated two IgG2a monoclonal antibodies that reacted with ERp57 by immunoblot. One of these antibodies inhibited both ERp57 activity and platelet aggregation. The other antibody did not inhibit ERp57 activity or platelet aggregation. When the antibodies were infused into mice the tail bleeding time was prolonged by the inhibitory antibody, but not by the non-inhibitory antibody. Finally, we found that a commonly used monoclonal antibody to PDI also inhibited ERp57 activity. We conclude that a glycoprotein specific member of the PDI family, ERp57, is required for platelet aggregation and hemostasis.

Disclosures:

No relevant conflicts of interest to declare.
Markers of Thrombogenesis Activation and Endothelial Dysfunction in End Stage Renal Disease
Authors: Bansal Vinod , Sharain Korosh , Hoppensteadt Debra , Litinas Evangelos , Lewis Bruce E , Fareed Jawed
Published: October 2019

Abstract 1151

INTRODUCTION

The pathogenesis of ESRD is complex involving inflammatory, thrombotic, and calcification processes. The leading cause of morbidity and mortality in ESRD is cardiovascular disease (CVD), accounting for 50% of all deaths. Current studies suggest that chronic inflammation is a major risk factor in ESRD. This investigation profiles markers of inflammation, thrombogenesis, and vascular dysfunction in patients with ESRD. METHODS Samples from ESRD patients (n=117) over the age of 18 on maintenance hemodialysis for at least 3 months and aged matched control samples (n=50) obtained from healthy volunteers were included in the study. The Randox Evidence Investigator (Antrim, United Kingdom) was used to measure IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, Vascular Endothelial Growth Factor (VEGF), Interferon-γ (INF-γ), Tumor Necrosis Factor-α (TNF-α), Monocyte Chemotactic Protein-1 (MCP-1), Epidermal Growth Factor (EGF), C-Reactive Protein (CRP), D-Dimer, and Thrombomodulin (TM). Plasminogen Activator Inhibitor -1 (PAI-1) and Asymmetric Dimethylarginine (ADMA) were measured using ELISA kits from Diagnostica Stago (Paris, France) and DLD Diagnostika GmbH (Hamburg, Germany), respectively. RESULTS IL-6 levels in ESRD were 4.3 times the controls. VEGF and INF-γ were both elevated at 2.6 times the controls. MCP-1, IL-8, IL-10, EGF, TNF-α, and IL-1α were elevated at 2.0, 1.6, 1.4, 1.4, 1.3, 1.2 times the controls, respectively. IL-4, IL-2, and IL-1 β levels were reduced at 0.9, 0.9, and 0.5 times the controls. TM, CRP, DDMER, PAI-1, and ADMA were increased at 5.9, 4.4, 3.0, 1.4, and 1.6 times the controls, respectively, each demonstrating statistically significant p-values. DISCUSSION The elevation of the interleukins, INF-γ, TNF-α and MCP-1 reveals intense inflammatory activation of leukocytes. The increase in these cytokines also demonstrates the vascular and tissue damage that occurs in ESRD. VEGF and EGF are both elevated in the ESRD samples indicating a state of cellular proliferation, possibly in an attempt to replace damaged tissue caused by the heightened inflammatory state. These results also demonstrate that ESRD patients have a significant upregulation in markers of thrombogenesis and endothelial dysfunction. The upregulation of the nitric oxide inhibitor ADMA points to disturbances in vascular lumen size regulation while CRP and TM suggest significant endothelial damage. Additionally, the upregulation of PAI-1 and D-Dimer indicates an activation of coagulation in ESRD. The combined disruption of endothelial regulation and endothelial damage along with the upregulation of thrombogenic and inflammatory markers helps to explain the significant prevalence of DVT, PE and CVD in ESRD. These markers should be evaluated for their roles in kidney disease risk stratification and prognosis.

Disclosures:

No relevant conflicts of interest to declare.
NEW FINDINGS From the FRENCH COHORT of MYH9-RELATED DISORDERS: PHENOTYPE PECULIARITIES and GENOTYPE NOVELTIES on Behalf of the National French Reference Center on Inherited Platelet Disorders (CRPP)
Authors: Saposnik Béatrice , Binard Sylvie , Nurden Alan T. , Nurden Paquita , Schlegel Nicole
Published: October 2019

Abstract 1155

MYH9-Related Diseases (RD) are inherited platelet disorders combining thrombocytopenia and giant platelets with various associations of leukocyte inclusions, deafness, nephritis and cataracts. Mutations in the MYH9 gene, that encodes the nonmuscle myosin heavy chain IIA (NMMHC-IIA), are the hallmark of these disorders. Our cohort includes a total of 161 subjects (90 women, 71 men): 76 propositi (37 isolated, 39 family index cases) with MYH9 mutations and 85 family members (45 women, 40 men) belonging to the 39 unrelated families. The cases with an identified mutation were classified according to the location of the mutation either in the motor domain (MD patients (P): “MDP”) or in the tail domain (TD patients (P): “TDP”) of NMMHC-IIA. MYH9 mutations were found in a total of 109 subjects: the 76 propositi (45 women, 31 men) and 33/85 (38.8%) family members (19 women, 14 men, from 22 families). Among the 41 different mutations detected, the majority were missense mutations (33 = 80.5%) and new (25 = 61%). Two substitutions in intron 39 were discovered for the first time: 1 in the donor splice site (corresponding to a classical MYH9-RD phenotype) and the other in position minus 3 of the acceptor splice site (moderate thrombocytopenia only without giant platelets). A total of 15 different exons were identified as having mutations, 4 exons remained unaffected. Mutations were more frequently found in exons 1, 16, 26, 30, 38 and 40, as already published. The majority of patients (102 = 93.6%) were heterozygous for one mutation but 7 patients (6.4%) were compound heterozygous for 2 mutations. Significant differences between MDP and TDP were found for bleeding symptoms (65% of MDP, 33% of TDP, p=0.0037), nephropathy (31.5% of MDP, 11.6% of TDP, p=0.015), age (years, mean) at onset for nephropathy (21 for MDP, 48 for TDP, p=0.0003), deafness (54% of MDP, 23.7% of TDP, p=0.0025), and association bleeding/extra-hematological symptoms (68.2% of MDP, 27.7% of TDP, p=0.01). Patients with bleeding had a lower platelet count than non-bleeders (mean= 42G/L versus 62G/L, p=0.0024) but there were no significant differences in the percentage of giant platelets. ITP (splenectomy for 7) was the initial misdiagnosis for 29/69 (42%), (51.7% of MDP, 35% of TDP, p= 0.165). Cataracts occurred in 4 patients only. Altogether these results showed that the clinical phenotype of MYH9-RD was more severe for MDP than TDP. New mutations were discovered and were widespread along the exons and in some cases two mutations were identified in the same patient. The sequencing of the full gene and all exons-introns boundaries should be mandatory for each patient whatever the initial result.

Disclosures:

No relevant conflicts of interest to declare.
Thrombin Generation Inhibition by Newer Anti Xa and Anti IIa Drugs in Prothrombin Complex Concentrates: Relevance to Endogenous Modulation of Thrombogenesis
Authors: Gray Angel , Litinas Evangelos , Hoppensteadt Debra , Cunanan Josephine , Lewis Bruce E , Iqbal Omer , Fareed Jawed
Published: October 2019

Abstract 1196

Several anti-Xa agents (apixaban, otamixaban, rivaroxaban and edoxaban) and anti-IIa agents (dabigatran and melagatran) have been developed for clinical use. These agents are strong inhibitors of factor Xa and IIa and their potency is usually expressed in terms of the inhibitory parameters of these enzymes. These synthetic agents can also modulate thrombin and factor Xa generation in complex systems such as the platelet rich and platelet poor plasma and whole blood. The order of potency differs depending upon the type of matrix used due to cellular and protein binding and interaction with generated proteases and protein fragments. In order to compare these agents for their relative thrombin generation inhibitory profile, prothrombin complex based assay systems are developed utilizing 5 units/mL of non activated prothrombin complex such as Konyne® and Preconativ. Thrombin generation is triggered by tissue factor and monitored using a thrombin specific florometric substrate based assay (Technoclone, Vienna, Austria). All agents were tested in a concentration range of 0–1000 ng/mL. The relative inhibition of thrombin generation was calculated in terms of IC50. The factor Xa inhibitors were found to be relatively potent inhibitors of thrombin generation exhibiting an IC50 value of 25–96 ng/mL with the following order of potency- Rivaroxaban>Otamixaban>Apixaban. The anti IIa agents exhibited potency in the range of 125–225 ng/mL. Melagatran was more potent than dabigatran. In the plasma based anticoagulant assays such as PT, aPTT, heptest, the anti IIa agents were more potent than factor Xa drugs. Assay based differences were noted in all agents which did not follow the same patterns as thrombin generation inhibition in the prothrombin complex systems. These observations suggest that although considered to be single target, the newer anticoagulant drugs produce variable inhibitory effects on the protease network in complex systems such as plasma and prothrombin complexes. These effects may be related to the relative safety and efficacy profile of these agents and should be included in the preclinical profiling of these agents.

Disclosures:

No relevant conflicts of interest to declare.
Curcumin Prevents Cytokine-Mediated Tissue Factor Pathway Inhibitor Down-Regulation in Human Endothelial Cells
Authors: Maruyama Keiko , Morishita Eriko , Goto Yukie , Sekiya Akiko , Asakura Hidesaku , Ohtake Shigeki , Nakao Shinji
Published: October 2019

Abstract 1202

Objective:

Curcumin (diferuloyl methane), an active component of the spice turmeric, has been shown to exhibit anti-inflammatory and antioxidant activities in addition to an anticartinogenic activity in vitro and in vivo. Furthermore, we reported that curcumin inhibited the induction of tissue factor (TF) expression in human umbilical vein endothelial cells (HUVECs) at 52nd ASH 2010. Therefore, curcumin may ameliorate hyper-coagulable state associated with inflammation or oxidative stress. On the other hand, tissue factor pathway inhibitor (TFPI) which is expressed by endothelial cells plays a crucial role in hemostasis by regulating TF-induced initiation of coagulation. This study examined whether curcumin modulates the expression of TFPI in HUVECs. Methods: HUVECs were pretreated with curcumin at the concentration of 20 μM for 3h, washed and stimulated with tumor necrosis factor-alpha (TNF-alpha, 10 ng/ml) for additional 12 or 24h. The mRNA and protein levels of TFPI in the cultured HUVECs were determined by reverse transcriptase polymerase chain reaction (RT-PCR) and western blotting, respectively. To determine whether curcumin affects the MAPK signaling pathways, the phosphorylation of p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) in the HUVECs were analyzed with western blotting. Additionally, to determine whether curcumin affects nuclear factor-kappa B (NF-kB) pathway, nuclear and cytoplasmic fractions were extracted and protein levels were determined by western blotting for NF-kB (p65), p-IkB and IkB. Results: After TNF-alpha stimulation, TFPI mRNA levels were approximately decreased by 40% compared to the control (p<0.05; Figure 1B). Similarly to the mRNA expression, TFPI protein levels were decreased (Figure 1A). On the other hand, pretreatment of HUVECs with curcumin significantly suppresses TNF-alpha-induced TFPI mRNA and protein down-regulation (p<0.05; Figure 1A, B). Curcumin inhibited TNF-alpha-induced activation of p38MAPK, ERK1/2, and JNK. Moreover, curcumin inhibits TNF-alpha-induced IkB activation in HUVECs. And, translocation of NF-kB from the cytosol into the nucleus by TNF-alpha was inhibited by curcumin. Conclusions: These results indicate that curcumin may suppress the TNF-alpha-induced TFPI down-regulation via NF-kB pathways. Thus, curcumin may offer a novel antithrombotic option for treatment of the hypercoagulable state associated with inflammation.

Disclosures:

No relevant conflicts of interest to declare.
Identification of Unique Biomarkers in Sepsis Associated Coagulopathy
Authors: Sharma Gautam , Bijlani Rahul , Litinas Evangelos , Hoppensteadt Debra , Cunanan Josephine , Khan Hussein , Sharma Gautum , Kaul Inder , Fareed Jawed
Published: October 2019

Abstract 1237

Introduction:

Disseminated Intravascular Coagulation represents a complex multi-factorial pathophysiological process in which protease disregulation along with other pathologic processes play an important role. The uncontrolled protease activities result in generation of several mediators which are not fully characterized. Biomarker profiling using protein chip array technology has been used in the identification of unique mediators in various diseases. Surface Enhanced Laser Desorption Ionisation(SELDI) is an ionization method in mass spectrometry that is used for the analysis of protein mixtures and correspondingly in the profiling of biomarkers in various diseases. Purpose: The purpose of this study was to profile and identify unique biomarkers in hospitalized patients with sepsis associated coagulopathy. Study design: There were 385 plasma samples included in this study. Baseline protein chip array profile in Molecular weight range of 0–200 kDa was carried out using SELDI technique employing gold chips. Plasma samples from normal humans (n=100) and pooled preparation from both the normal (NHP) and suspect liver disease/coagulopathy were utilized as controls. Results: Of the 385 baseline samples analyzed 371 patients (96.86%) exhibited the presence of a unique biomarker at 11.6 kDa. In addition down regulation of a biomarker at 56.4 kDa was also noted in 218 of patients (56.74%) In the normal human pooled plasma the 11.6 kDa peak was nearly absent. However in Pathological pooled plasma a distinct biomarker at 11.6 kDa with a much lower intensity was noted. Additional markers in baseline samples in 1–10 kDa range were also noted. Conclusions: These observations suggest that sepsis associated coagulopathy results in the generation of specific unique biomarker at 11.6kDa which requires further characterization. The downregulation of biomarker at 56.4 kDa and the apparent reduction levels during hospitalization require further investigations.

Disclosures:

No relevant conflicts of interest to declare.
FANCL Ubiquitinates Beta-Catenin and Enhances Its Nuclear Function
Authors: Dao Kim-Hien T. , Rotelli Michael D. , Petersen Curtis L. , Brown Brie R. , Nelson Whitney D. , Yates Jane E. , Newell Amy Hanlon , Olson Susan B. , Druker Brian J. , Bagby Grover C.
Published: October 2019

Abstract 1335

Fanconi anemia (FA) is associated with a hereditary predisposition to bone marrow failure. The proteins encoded by the FANC genes are primarily involved in DNA repair responses through the formation of a large, multisubunit complex that has E3 ubiquitin ligase activity (Annual Review of Genetics 2009;43:223). FA hematopoietic stem cells display defective stem cell properties and limited replicative potential. However, the molecular basis for how a FA genetic background contributes to those defects remains poorly understood. Here we provide evidence that FANCL, which has E3 ubiquitin ligase activity, enhances beta-catenin activity (Figure A) and protein expression. Beta-catenin is a nuclear effector of canonical Wnt signaling. The Wnt/beta-catenin pathway is active in normal hematopoietic stem cells in the native bone marrow environment and disruption of this signaling pathway results in defective hematopoietic stem cells (Nature 2003;423:409). To test whether FANCL positively regulates beta-catenin through its ubiquitination activity, we performed cell-based ubiquitination assays. We show that FANCL functionally ubiquitinates beta-catenin (Figure B) and that ubiquitin chain extension can occur via non-lysine-48 ubiquitin linkages. Accumulating evidence reveal diverse, non-proteolytic biological roles for proteins modified by atypical ubiquitin chains (EMBO Reports 2008;9:536). Our data suggests that FANCL may enhance the protein function of beta-catenin via ubiquitination with atypical ubiquitin chains. Importantly, we demonstrate that suppression of FANCL expression in human CD34+ cord blood stem cells reduces beta-catenin expression (Figure C) and multilineage progenitor expansion. These results demonstrate a role for the FA pathway in regulating Wnt/beta-catenin signaling. Therefore, diminished Wnt/beta-catenin signaling may be an important underlying molecular defect in FA hematopoietic stem cells leading to their accelerated loss.

A, LEF-TCF-luciferase reporter assay showing increasing beta-catenin activity in 293FT cells with increasing FANCL expression compared with vector-control (VC) (n=4). B, Immunoprecipitation of beta-catenin in cells transfected with vector-control or FANCL and probed for hemagglutinin (HA)-tagged ubiquitin shows increased ubiquitinated forms of beta-catenin with FANCL expression (n=4). C, shRNA suppression of FANCL expression in CD34+ cord blood stem cells results in decreased beta-catenin expression compared with a scramble control (Scr) by immunofluorescence analysis (three different shRNA constructs, n=3 for each construct).

Disclosures:

No relevant conflicts of interest to declare.