bluuebunny/crossref_metadata_2025 huggingface dataset:
High IDH1 Expression Is Associated with a Poor Prognosis in Cytogenetically Normal Acute Myeloid Leukemia
Authors: Ma Qiu-Ling , Wang Jing-Han , Wang Yun-gui , Hu Chao , Mu Qi-Tian , Yu Meng-Xia , Wang Lei , Wang Dong-Mei , Yang Min , Yin Xiu-Feng , Chen Fei-Fei , Lu Sha-Sha , Chen Jian , Zhu Zhi-Juan , Chen Sai-Juan , Jin Jie
Published: October 2019

ABSTRACT

The prognostic value of IDH1 mutations has been systematically evaluated in acute myeloid leukemia (AML) patients recently. However, the role of IDH1 expression in AML is still under exploration. To investigate the clinical significance, we analyzed the IDH1 expression in 320 patients with cytogenetically normal AML (CN-AML) by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). High expression of IDH1 was predominant in patients with FLT3-ITD and DNMT3A mutations, and less prevalent in cases with CEBPA double allele mutations. Strong association was observed between high IDH1 expression and low expression of micro-RNA 181 family. Prognosis was adversely affected by high IDH1 expression with shorter overall survival (OS) and event free survival (EFS) in the context of clinical characteristics including age, WBC, and gene mutations of NPM1, FLT3-ITD, CEBPA, IDH1, IDH2, and DNMT3A in CN-AML. Moreover, the clinical outcome of IDH1 expression in terms of OS, EFS and complete remission rate still remained in multivariate models in CN-AML. Importantly, the prognostic value was validated using the published microarray data from 79 adult patients treated according to the German AMLCG-1999 protocol. Our results demonstrated that high IDH1expression is associated with a poor prognosis of CN-AML.

Disclosures

No relevant conflicts of interest to declare.
Prognostic Factors and Outcomes in Primary Effusion Lymphoma in HIV-Infected Patients: A Single Center Experience
Authors: Sen Shiraj , Marley Eileen , Naina Harris V.
Published: October 2019

Primary effusion lymphoma (PEL) is a human herpesvirus 8-associated non-Hodgkin lymphoma (NHL) that predominantly develops in serous body cavities leading to recurrent lymphomatous effusions. It is poorly understood and very uncommon, accounting for only 3% of all HIV-related NHL. While outcomes have improved in patients with HIV-associated NHL since the widespread use of HAART, the prognosis of PEL remains very poor and median survival is less than 6 months. This study reports our institution’s experience with PEL over the past decade.

Between 2004 and 2014, we treated 8 patients with HIV-associated PEL. All patients were male. The median age was 46.5 years old (range 35-63), three were Caucasian, four were Hispanic, and one was African-American. The primary site of involvement was pericardial in 3 patients, pleural in 3 patients, and extra-cavitary in 2 patients. All patients were Ann Arbor stage IV at the time of diagnosis. The mean LDH and CD4 count at time of diagnosis was 625 U/L and 161 cells/mm3, respectively.

Of the eight patients identified with PEL, four have achieved complete remission (CR). Prognostic factors associated with achieving CR include compliance with HAART therapy prior to PEL diagnosis as well as lower serum lactate dehydrogenase (LDH) levels (202±30 versus 1049±290, p=.03). Patients achieving CR also had a higher average CD4 count (228±93 versus 95±35, p=.22) at time of diagnosis. Median survival is nearly 6 years for those in CR whereas it was 32 days in patients that died (one from an acute stroke, one from septic shock, and two from progressive disease). All patients who achieved CR had previously diagnosed HIV whereas two of the four who died were previously undiagnosed with HIV. Interestingly, pericardial sites of involvement were associated with significantly better outcomes as compared to pleural sites in our case series, as well. There was no association between outcome and HIV viral load, duration of HIV infection prior to PEL diagnosis, or presence of AIDS-defining illnesses. The patients achieving CR were treated with HAART in addition to Hyper-CVAD (1a-3b) or 6 cycles of CHOP or EPOCH. In addition, two patients received bortezomib as initial therapy. Given its rarity, our knowledge of PEL relies heavily on case reports and small case series. Here we report our institution’s experience with PEL, identify LDH and CD4 as possible prognostic factors in PEL, and suggest bortezomib-based chemotherapy as an effective treatment option in HIV-associated PEL.

Disclosures

No relevant conflicts of interest to declare.
Serum Levels of CCL22 and CCL25 Might Predict Skin Rash Induction、the Common Adverse Event By Bendamustine Therapy in Malignant Lymphoma
Authors: Terui Yasuhito , Ueda Kyoko , Kusano Yoshiharu , Gunji Tadahiro , Nishimura Noriko , Hatake Kiyohiko
Published: October 2019

Introduction: Bendamustine has been recently approved for the treatment of low-grade malignant lymphoma. In spite of the better efficacy, skin rash is the commonest adverse event by bendamustine in the treatment of malignant lymphoma. To understand the mechanism of the skin rash, we prospectively examine the relationship between skin rash and chemokines. Methods: After informed consent to the patients, who would received bendamustine (B) or bendmustine+rituximab (BR), the sera were collected before and after the treatment of B or BR. Cytokine/Chemokine array was performed by Bio-Plex system. Results: 20 patients with FL (n=18), MCL (n=1), and low-grade B-cell lymphoma (n=1) were enrolled in this study. All patients wee treated with B or BR regimen. 9 patients showed skin rash after the treatment of bendamustine. In Cytokine/Chemokine array, serum levels of CCL22 and CCL25 before the treatment were significantly higher in skin rash group (p=0.010738476 and p=0.030366028, respectively) (Figure 1 and 2), and serum levels of CXCL10 and CXCL11 after skin rash were higher significantly (p=0.000923527 and p=0.014890795, respectively). Conclusion: Serum levels of CCL22 and CCL25 might predict skin rash inductionAthe commonest adverse event by bendamustine and CXCL10 and CXCL11might contribute to induction of skin rash by bendamustine.

Figure 1 Figure 1. Figure 2 Figure 2.

Disclosures

No relevant conflicts of interest to declare.
Pediatric Hodgkin’s Lymphoma: 5-Year Experience at Single Center in Pakistan
Authors: Riaz Shazia , Khan Safia , Badar Farhana
Published: October 2019

OBJECTIVES:

To review patients’ characteristics and treatment outcome pattern of pediatric hodgkinlympoma in a well funded center in developing country

MATERIALS & METHOD: This was retrospective review of data of 301 pediatric hodgkin lymphoma patients up to 18 years of age treated in Shaukat Khanum Memeorial Cancer Hospital & Research Center, Lahore, Pakistan, during 2009 to 2013.

RESULTS:

Total No. of patients was 301, 81% were male. The median age of studied data was 9 years; ranging from 2 to 18 years. B symptoms in the form of fever, weight loss and night sweats were found in 51%, 29% and 34%, respectively. Median stage was 3 with neck bulky disease in 28% and Medistinal widening in 18% of patients. Trend of histopathology was mixed cellualrity at 56.5%. Most common treatment regime was ChlVPP/ABVD (59.5%); 30.6% of these 301 patient also got radiotherapy. At the end of treatment complete response was reported in 76.5%; 11.3% of these were relapsed afterward.

CONCLUSION:

Our data shows excellent outcome of pediatric hodgkin lympoma and results are comparable to that of developed countries.

Disclosures

No relevant conflicts of interest to declare.
ONC201/TIC10 Is Effective As a Monoagent and Synergizes with Chemotherapy to Induce Cell Death in Non-Hodgkin Lymphoma
Authors: Talekar Mala K. , Allen Josh , Dicker David T. , El-Deiry Wafik S.
Published: October 2019

Outcome for Non-Hodgkin lymphoma (NHL) patients using conventional treatment standards remains unsatisfactory, particularly in advanced stage/ relapsed disease creating an imminent need for investigating novel treatment strategies. ONC201/TIC10 is a small molecule (Allen et al, 2014) that induces p53-independent cell death in tumor cells (sparing normal cells) through inactivation of prosurvival kinases Akt and ERK. ONC201 is scheduled to enter Phase I/II clinical trials as a monoagent for advanced cancers in adults in 2014. We have previously shown that ONC201 induces significant cytotoxicity in preclinical models of human lymphomas (Talekar et al, ASPHO 2014). Here, we show that ONC201 is not only effective as a monoagent across several NHL cell lines, but that it also synergizes with several chemotherapeutic agents to cooperatively induce cell death in vitro.

We found that ONC201 induces significant apoptosis in a diverse panel of seven human NHL cell lines at low micromolar concentrations (1.3 to 5.06 uM). Increased surface TRAIL and surface DR5 expression was noted in a dose-dependent manner across representative cell lines. The increase in surface TRAIL correlated with increase in subG1 DNA content, which suggests TRAIL as a potential biomarker of ONC201 response. ONC201-induced apoptosis was inhibited using a pan-caspase inhibitor and was blocked by an anti-TRAIL antibody RIK-2. Western blot analysis of ONC201-treated representative cell lines suggests ERK inhibition and Foxo3a activation as a potential mechanism of cytotoxicity via TRAIL induction. In agreement with this notion, we observed upregulation of PARP & DR5 and caspase-3 activation in response to ONC201 treatment.

We further found that ONC201 synergizes to potentiate cytotoxicity with several chemotherapeutic agents approved for NHL treatment, particularly anthracyclines (doxorubicin), nitrogen mustard (bendamustine), antimetabolite (cytarabine) and proteasome inhibitor (bortezomib) via CellTiter-Glo cell viability experiments that were corroborated by apoptosis assays.

Together these results suggest that ONC201 is a potent antitumor agent in NHL as monoagent and in combination with approved therapies. The ultimate goal of this project is to provide a preclinical rationale for a phase Ib/II trial of ONC201 as a combination therapy in pediatric lymphoma.

Disclosures

Allen: Oncoceutics, Inc.: Employment, Equity Ownership, Ownership interests (including stock options) in a start-up company, the stock of which is not publicly traded Other, Patents & Royalties. El-Deiry:Oncoceutics, Inc.: Equity Ownership, Founder and Shareholder in Oncoceutics, Inc. The potential conflict of interest has been disclosed to and is being managed by his academic employer. Other.
Clinical Practice Patterns of Anti-Xa Monitoring During Low Molecular Weight Heparin Therapy
Authors: Stutsky Martha E. , Reardon David P. , Lee Alfred I
Published: October 2019

Introduction: Therapeutic low molecular weight heparin (LMWH) regimens were developed empirically based on patient weight and renal function without the need for laboratory monitoring. The role of anti-Xa monitoring for patients receiving LMWH has yet to be fully established and is routinely performed only in high risk patients. At our institution, guidelines for monitoring LMWH recommend drawing anti-Xa levels for patients with chronic kidney disease or those with obesity requiring long-term therapy. The purpose of this analysis was to evaluate compliance with institutional guidelines for monitoring anti-Xa levels for patients receiving LMWH, including indication for anti-Xa monitoring, timing of anti-Xa level in relation to drug administration, and follow up actions of providers in response to abnormal anti-Xa levels.

Methods: We conducted a retrospective chart review of adult patients who received therapeutic LMWH (enoxaparin or dalteparin) and were ordered for an anti-Xa level between September 1, 2014 and July 1, 2015. Patients were excluded if they were ordered for an anti-Xa level while on a prophylactic dose of LMWH or while on a non-LMWH factor Xa inhibitor. Data collected included patient demographics, LMWH dose and indication, ordering service, presence of a Hematology consult, reason for anti-Xa monitoring, timing of the level after the previous LMWH dose, and provider response to the level.

Results: One hundred patients met inclusion criteria, 99 of whom received enoxaparin. Venous thromboembolism was the most common reason for LMWH therapy (85%) followed by atrial fibrillation (11%). The most frequent doses of enoxaparin administered were 1 mg/kg every 12 hours (52%), 1 mg/kg every 24 hours (22%) and 1.5 mg/kg every 24 hours (21%). The most frequently specified indications for anti-Xa monitoring were renal dysfunction and acute kidney injury (30%), followed by abnormal body mass index (13%); no indication was specified by the prescriber or pharmacist in 36% of cases. Of the 100 anti-Xa levels in the study, 65 were drawn at the correct time (3-5 hours after the previous dose of enoxaparin); of the 35 drawn at an incorrect time, 33 had levels measured more than 5 hours after the previous enoxaparin dose. Fifty-four of the initial anti-Xa levels were not within the therapeutic range for the dosing interval of LMWH; of these, follow up action was taken for only 21 patients (39%), most frequently resulting in a dose increase or decrease.

Conclusions: In clinical practice, the use of anti-Xa monitoring is inconsistent and commonly done without a documented need. The majority of anti-Xa levels at our institution are drawn at the appropriate time after the previous dose of LMWH, but action is generally not taken in response to non-therapeutic levels. Additional guidance on the use of LMWH monitoring is needed.

Disclosures

No relevant conflicts of interest to declare.
The Erythroid Intron Retention Program Encompasses Developmentally Stable and Dynamic Networks and Regulates Diverse Gene Classes
Authors: Pimentel Harold , Parra Marilyn , Gee Sherry , Mohandas Narla , Pachter Lior , Conboy John G.
Published: October 2019

Computational analysis of RNA-seq data from highly purified human erythroblasts has been instrumental in revealing changes in pre-mRNA splicing during terminal erythropoiesis. Here we report updated studies of intron retention (IR), a type of alternative splicing in which specific introns are retained in otherwise efficiently-processed transcripts, allowing post-transcriptional modulation of cellular mRNA levels. Differences in differentiation stage-specificity, degree of retention, nuclear/cytoplasmic localization, and sensitivity to nonsense-mediated decay (NMD) suggest the existence of multiple classes of erythroblast IR subject to distinct regulatory controls. Two clusters comprising ~470 "developmentally dynamic" introns in 354 genes exhibit more efficient splicing in proerythroblasts, but elevated intron retention in orthochromatic erythroblasts prior to enucleation. Dynamic regulation of late erythroblast IR parallels previously described splicing switches involving alternative exons. Gene ontology analysis revealed that the dynamic intron group is highly enriched in genes with RNA processing functions. Among these are several spliceosomal factors including SF3B1, a commonly mutated gene in myelodysplasia patients. We also identified several clusters of "developmentally stable" introns whose IR levels are not substantially modulated during erythropoiesis. Among this latter type are two clusters containing 294 introns that are enriched in functions related to metal ion binding. Key genes include mitoferrin-1 (SC25A37; IR~50%) and mitoferrin-2 (SLC25A28; IR~20-30%), mitochondrial iron importers essential for heme biosynthesis. We observed a correlation between splice site strength and percent IR among developmentally stable but not dynamic intron clusters, indicating that splicing regulatory mechanism(s) for the latter must require additional sequence features. A search for such features revealed that IR was significantly higher adjacent to alternative 'PTC' exons containing premature termination codons than it was adjacent to other exons; moreover, by direct RT-PCR analysis we discovered novel (unannotated) PTC exons in additional retained introns. The proposed role of PTC exons in IR is being studied experimentally using an array of minigene splicing reporter constructs. Finally, we noted that while specific IR events are erythroid specific, e.g., in the alpha spectrin gene SPTA1, computational analysis of public RNA-seq data demonstrated that most erythroblast IR events were also observed in granulocytes and in 16 other tissues surveyed by the human BodyMap project. Intron retention is likely to play critical roles in gene regulation in both hematological and non-hematological tissues.

Disclosures

No relevant conflicts of interest to declare.
Identification of Polymorphic Gnpat As a Risk Factor for Porphyria Cutanea Tarda
Authors: Phillips John D , Farrell Colin P , Overbey Jessica , Naik Hetanshi , McLaren Gordon D. , McLaren Christine E , Zhou Luming , Parker Charles J.
Published: October 2019

Symptoms of porphyria cutanea tarda (PCT) resolve when iron stores are depleted by phlebotomy, and a sequence variant of HFE (C282Y) that increases iron absorption by reducing hepcidin expression is a risk factor for PCT. These observations suggest that PCT is an iron dependent disease and that factors that affect iron homeostasis influence the risk of developing the PCT. Recently, a polymorphic variant (D519G) of GNPAT was shown to be enriched in male patients with type I hereditary hemochromatosis (HFE C282Y homozygotes) who presented with a high iron phenotype [McLaren CE, et al. Hepatology Aug;62(2):429-39 2015]. Available evidence suggests that like HFE C282Y, GNPAT D519G increases iron absorption by reducing expression of hepcidin. Therefore, we investigated the prevalence GNPAT D519G in patients with PCT. The study population consisted of 247 patients with PCT. High-resolution DNA melting analysis and Taqman SNP assays were used to identify HFE (C282Y) and GNPAT (D519G) allelic variants, respectively and mutations in uroporphyrinogen decarboxylase (UROD) were identified by nucleotide sequencing. Patients with mutant UROD were categorized as familial PCT (F-PCT) (n=87, 36.0%) and those with wild-type UROD were categorized as sporadic PCT (S-PCT) (n=155, 64.0%). GNPAT D519G was significantly enriched in the patient population (prevalence 22.1%, p<0.001) compared to the general population (prevalence 13.6%) as was HFE C282Y (prevalence of 19.2% in the study population compared to 1.5% in the general population, p<0.001). The relative risk of GNPAT D519G was significantly increased in patients with F-PCT compared to those with S-PCT (odds ratio 1.81, CI 1.06-3.08) whereas the relative risk of HFE C282Y in the F-PCT was lower than in the S-PCT population (odds ratio 0.82, CI 0.46-1.47). Together, these observations suggest that GNPAT D519G, is more likely to require a cofactor (e. g., mutant UROD) to produce PCT whereas HFE C282Y functions as an independent PCT risk factor. This difference may be a consequence of the relative potency of GNPAT D519G (weak) and HFE C282Y (strong) with respect to effects on iron homeostasis. Our studies have identified a new risk factor (polymorphic GNPAT) for PCT that adds support for the involvement of aberrant iron metabolism in the pathobiology of PCT.

Disclosures

No relevant conflicts of interest to declare.
Kindlin-2 Maintains Vascular Barrier Integrity By Stabilizing Endothelial Adherens Junctions
Authors: Pluskota Elzbieta , Szpak Dorota , Bialkowska Katarzyna , Bledzka Kamila , Plow Edward F.
Published: October 2019

Kindlin-2, a widely distributed cytoskeletal protein, has been implicated in integrin activation, angiogenesis, thrombosis and hemostasis. Its absence is embryonically lethal in mice, emphasizing its biological importance. In the present study, we examined the role of Kindlin-2 in regulation of vascular integrity. In vivo, Kindlin-2+/- mice showed enhanced (70%) permeability of tracheal vasculature to fluorescent beads as compared to wild-type (WT) littermates under both basal conditions or when stimulated with Platelet Activating Factor (PAF). Consistent with these in vivo observations, confluent monolayers of aortic endothelial cells (ECs) isolated from Kindlin-2+/- mice had increased (2-3-fold) baseline, PAF- and thrombin-induced permeability for Alexa-488-labeled bovine serum albumin (BSA) or FITC-dextran-10000 as compared to WT cells. Also, reduction of Kindlin-2 expression by 60-70% in human umbilical vein endothelial cell (HUVEC) monolayers with Kindlin-2-specific siRNA resulted in 3-fold increase in their baseline or thrombin/(PAF)-induced permeability of these markers. Mechanistically, Kindlin-2 co-localized with VE-cadherin and actin within adherens junctions in resting, confluent HUVEC monolayers as well as co-immunoprecipitated with these proteins and other components of adherens junctions, including α-, β- and γ-catenin. In contrast, Kindlin-2 did not co-localize or co-immunoprecipitate with any components of GAP or tight junctions. VE-cadherin-based complexes had less associated Kindlin-2 and actin in Kindlin-2+/- ECs and in HUVECs treated with Kindlin-2-specifc siRNA. Also, upon stimulation of HUVECs with PAF or thrombin, Kindlin-2 along with actin dissociated from VE-cadherin-based complexes, suggesting that Kindlin-2 stabilizes adherens junctions. In direct binding studies with recombinant proteins the SPR sensograms revealed direct interaction of Kindlin-2 with β- and γ-catenin, but not with α-catenin nor the VE-cadherin cytoplasmic tail. In addition, using actin spin-down assays we demonstrated that Kindlin-2 directly interacted with actin filaments and linked them to β- or γ-catenin. Using WT and deletion mutants of Kindlin-2 we mapped the β- and γ-catenin binding site to the F1 and F3 domains in Kindlin-2, while actin binding is very dependent on the F0 domain.

Taken together, these data identify a previously unappreciated function of Kindlin-2. It plays a crucial role in maintaining vascular integrity by linking adherens junctions to actin filaments.

Disclosures

No relevant conflicts of interest to declare.
Antibody Engineering of Anti-TFPI Bypass Therapeutic BAY 1093884: Isotype Selection and Sequence Optimization
Authors: Paz Pedro , Xie Jinger , Aswad Fred
Published: October 2019

Background: BAY 1093884 is a fully human monoclonal antibody against tissue factor pathway inhibitor (TFPI) developed as a bypass agent for patients with hemophilia with or without inhibitors. It restores insufficient thrombin burst, leading to stable clot formation in hemophilic conditions in vitro, and effectively stops bleeding in vivo.

Aims: Antibody targeting of TFPI provides the advantages of monoclonal antibodies, such as longer circulation half-life, and the possibility of subcutaneous administration. However, antibody therapeutics can present the challenge of delivering unwanted effects or activities, such as antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) that target healthy tissues in the patient. Additionally, antibody sequence engineering that optimizes binding to the target protein may introduce immunogenic epitopes that can induce antidrug antibodies and compromise efficacy.

Methods: In vitro assays used primary human umbilical vein endothelial cells and EA-hy926 cell lines as target cells. Total peripheral blood mononuclear cells (PBMCs) and purified natural killer (NK) cells isolated from healthy donors were used as effector cells for ADCC. Pooled human serum complement (Quidel®) was used for CDC assays. A proprietary immunogenicity scoring program for T-cell epitopes (Bayer HealthCare) was used to determine immunogenic potential of BAY 1093884 relative to antibodies in the market.

Results: In vitro ADCC assessment of BAY 1093884 using PBMCs and NK cells isolated from healthy donors showed no lytic activity against TFPI-expressing primary and immortalized cell lines. Furthermore, no CDC activity using pooled serum complement was detected with BAY 1093884. In silico scoring for T-cell epitopes using a human leukocyte antigen DR-weighted algorithm compared favorably to therapeutic antibodies that have been reported to have low immunogenicity in the clinic.

Conclusion: In vitro and in silico immunoprofiling has aided in the design of the bypass anti-TFPI immunoglobulin G2 antibody BAY 1093884 with low toxicity potential and immunogenicity in humans.

Disclosures

Paz: Bayer HealthCare: Employment. Xie:Bayer HealthCare: Employment. Aswad:Bayer HealthCare: Employment.
Recombinant FVIII and FVIIa Added to Flowing Hemophiliac Blood Co-Localize with Platelet Aggregates Adherent to a Collagen Surface, Promoting Local Fibrin Deposition in Wake-like Patterns Around the Aggregates
Authors: Grabowski Eric F. , Liu Bohan , Gerace Matthew , Grabowski Paul E. , Gunnarsson Peter , Ezban Mirella
Published: October 2019

Mural platelet thrombi are considered to facilitate hemostasis in hemophilia via rFVIII/ rFVIIa binding to platelet membranes with enhancement of local fibrin deposition. We therefore sought to show that Factors VIII and VIIa co-localize to platelet membranes of adherent platelet aggregates and promote local deposition of fibrin. We collected blood from 9 patients with severe hemophilia A just prior to a prophylactic dose of rFVIII, and from 8 normals, into a plastic tube containing 5 U/ml FC low MW heparin, 1.75 µg/ml of the Tab (anti-CD41) mo antibody, and 1.0 μg/ml of ALEXA 555-conjugated rabbit anti-mouse secondary antibody, plus 0, 11, 33 or 100 ng/ml rFVIII-Oregon green. Similar approaches were used for rFVIIa or fibrin. Fibrin was detected using the monoclonal antibody 59D8 directed against human β fibrin, together with the above Alexa555-conjugated rabbit anti-mouse secondary antibody, and co-localized to platelet aggregate surfaces using 10 µM quinicrine dihydrochloride. Blood at 37° C was withdrawn at 270 sec-1 through a flow chamber, over 150 µm glass cover slips with preadsorbed microfibrillar collagen, using epifluorescence video microscopy and digital image analysis (MetaMorph Premier software). Added rFVIII or rFVIIa co-localized (Fig. 1) with platelet membranes and increased with rFVIII concentration. With hemophiliac blood and 100 ng/ml rFVIII, percent co-localization at 7 min was 52.6 ± 7.7 (N=4). Platelet aggregate growth rate, image pixels/min from time 3 to 9 min, was 26% lower in hemophiliac blood, but increased (p = 0.0001) 2.34 ± 0.62 (6)-fold with rFVIII, and (p = 0.0005) 1.91 ± 0.56 (6)-fold with 30 nM rFVIIa (Fig. 2). These increases essentially restored (or only slightly augmented) platelet deposition to levels observed in normal blood. In contrast, the effect on local fibrin deposition was profound: fibrin deposition was delayed by 3-5 min in normal blood, but by >13 min in hemophiliac blood. Addition of rFVIII or rFVIIa shortened the latter time to 7-9 min. Increases in platelet aggregate growth rate, following a lag phase in growth rate, paralleled the onset of fibrin formation. Fibrin appeared as comet-shaped deposits (comet head 20-25 µm in diameter) centered over each platelet aggregate (3-10 µm), the comet tail trailing 60-75 µm downstream (Fig. 3). In flowing whole blood in real time, these data show that rFVIII and rFVIIa co-localize with platelet membranes of activated, surface-adherent platelet aggregates, and support fibrin deposition in the slow-flow wakes immediately surrounding the platelet aggregates.

Disclosures

Grabowski: CSL Behring: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk, Denmark: Research Funding. Gunnarsson:Novo Nordisk: Employment. Ezban:Novo Nordisk: Employment.
Novel Association of a F11 Variant with Venous Thromboembolism in a Chinese Han Population
Authors: Wu Ying Ying , Tang Liang , Hu Yu , Zeng Wei , Liu Han
Published: October 2019

Background: Coagulation factor XI (FXI) is essential for normal function of the intrinsic pathway of blood coagulation. Nevertheless, high coagulation factor XI (FXI) levels were related to an increase in the risk of venous thromboembosis. The aim of the present study was to investigate whether any genetic variant in the factor XI gene (F11) was associated with susceptibility to venous thromboembosis in the Chinese Han population.

Methods: All exons and the 5'- and 3'-untranslated regions of F11 were initially sequenced to identify informative variants. Potential abnormal variants were analyzed in a population of 1531 venous thrombosis patients and 1343 healthy controls. Finally, functional studies were performed to evaluate the effects of the variant.

Results: A mutation c.539A>G(rs190283743) was identified and harbored a high predisposition to venous thrombosis (adjusted OR = 1.861, 95%CI = 1.221-2.836). The plasma factor XI activity and antigen levels in heterozygotes were increased.

Conclusions: Our results suggested that the novel F11variant is a possible genetic determinant of an increased risk venous thrombosis of and increased coagulant activity of factor XI.

Disclosures

No relevant conflicts of interest to declare.
Increased D-Dimer Levels and Residual Venous Thrombosis Are Associated with Late Recurrence of Deep Venous Thrombosis
Authors: Mazetto Bruna Moraes , Orsi Fernanda Andrade , Flores-Nascimento Mariane , Silveira Sandra , Bittar Luis Fernando , Zapponi Kiara , Colella Marina Pereira , De Paula Erich Vinicius , Annichino-Bizzacchi Joyce Maria
Published: October 2019

Background: Many patients present new thrombotic events even long-time after the first thrombotic episode and risk factors for recurrence in these cases are not fully elucidated.

Aim: To evaluate the risk of late recurrence of thrombosis after a first DVT episode.

Methods: This is a prospective observational study. We evaluated the association of D-dimer (DD) levels and persistent residual venous thrombosis (RVT), by Doppler ultrasound, with the occurrence of new thrombotic events long-time after the acute DVT episode.

Results: Fifty-six patients were enrolled for the study. For all patients, DVT episode occurred more than 12 months apart from the enrollment day. Median follow-up was 28 months. During the follow-up time, 10 patients presented thrombosis recurrence. Patients who suffered DVT recurrence had higher DD levels than those who did not had recurrence (median= 0.99 vs 0.40, respectively). The best cut-off value to discriminate those at risk for recurrence was DD > 0.63mg/L (area= 0.7380 95%CI= 0.5800 to 0.8961, P=0.01). Recurrence occurred in 27.7% of patients with previous DD>0.63mg/L and in 5.9% of patients with previous DD< 0.63mg/L (relative risk = 6.46; 95%CI 1.36- 30.52, P=0.007). New thrombosis events occurred only in patients with previously documented RVT, mainly in patients with hypoechoic RVT. During follow-up, new thrombotic events were diagnosed in 75% of patients with hypoechoic RVT. The relative risk for recurrence according to the presence of RVT was 9.129 (95% CI= 2.60-32.02, P<0.001). The presence of RVT also modified the effect of DD on the recurrence risk. Patients with DD > 0,63mg/L but without RVT had similar risk of recurrence than those with DD< 0.63mg/L, whereas the risk for recurrence increased in 14-15 times in patients with RVT.

Conclusion: These results suggests that the persistence of residual thrombosis combined with high levels of DD, long-time after the acute DVT, are risk factors associated with late thrombosis recurrence.

Disclosures

No relevant conflicts of interest to declare.
Myelodysplastic Syndrome Caused By Activating Kras Mutation in the Non-Hematopoietic Bone Marrow Microenvironment
Authors: Osswald Lena , Miething Cornelius , Duyster Justus , Brummer Tilman , Zeiser Robert
Published: October 2019

Oncogenic Ras mutations occur frequently in myelodysplastic and myeloproliferative syndromes as juvenile myelomonocytic leukemia (JMML) and the myeloproliferative variant of chronic myelomonocytic leukemia (MP-CMML) as well as in acute myeloid leukemia. However in these reports the mutations were in the hematopoietic cells. Here, we show that an activating mutation of Kras in the non-hematopoietic system leads to hematologic disorder resembling human myelodysplastic syndrome (MDS).

Rosa26CreERT2;LSL-KrasG12D mice (CD45.2) were lethally irradiated and transplanted with wild-type bone marrow (CD45.1). After control of engraftment efficiency (above 99.6%), the mice were treated with Tamoxifen to induce the expression of KrasG12D in non-hematopoietic cells.

6-8 weeks after Tamoxifen treatment, the mice developed anemia, leukocytopenia and thrombocytopenia and had a highly increased percentage of myeloid cells in peripheral blood, spleen and bone marrow. FACS-analysis confirmed that these cells were donor-derived and therefore of wild-type origin. The frequency of immature myeloid progenitors (CD11b+ c-kit+) was increased in bone marrow of Rosa26CreERT2;LSL-KrasG12D mice compared to littermate controls suggesting a disturbed differentiation. Morphological analysis of blood smears and bone marrow revealed a high number of dysplastic hypersegmented neutrophils as well as the occurrence of myeloid blasts.

Additionally, a significant decrease of B-lymphocytes was observed in the bone marrow of KrasG12D recipient mice which has also been described in human MDS. Osteoblasts have been shown to contribute to B-cell lymphopoiesis which implicates that decreased B-cell lymphopoiesis in this study may be a result of oncogenic Kras expression in osteoblasts.

All these data indicate that a single mutation in the hematopoietic microenvironment can initiate a severe hematologic disorder. The expression of oncogenic Kras in bone marrow stroma cells leads to a shift to myeloid differentiation, severe anemia and thrombocytopenia as well as reduced B-cell counts recapitulating main signs of human myelodysplastic syndrome.

Disclosures

No relevant conflicts of interest to declare.
Benzene-Induced Leukemogenesis: Irreversible Inhibition of PTPN2 and Subsequent STAT1 Signaling Alteration By the Hematotoxic Metabolite Benzoquinone
Authors: Duval Romain , Bui Linh-Chi , Mathieu Cécile , Petit Emile , Dupret Jean-Marie , Cools Jan , Guidez Fabien , Chomienne Christine , Rodrigues-Lima Fernando
Published: October 2019

Benzene (BZ) is a chemical compound of industrial and toxicological interest classified as a class I human carcinogen. Environmental and occupational exposure to BZ lead to bone marrow malignancies such as leukemia. The leukemogenic effects of BZ relies on its metabolization in bone marrow cells into reactive metabolites, in particular benzoquinone (BQ) that can react with macromolecules (arylation) and/or induce oxidative stress. Although BZ is well recognized as a leukemogenic chemical, most of the key molecular and cellular mechanisms underlying its hematotoxicity are not fully understood.

PTPN2 is a protein tyrosine phosphatase (PTP) mainly expressed in hematopoietic cells and playing a key role in the homeostasis of the hematopoietic system. In particular, this PTP is an important modulator of growth factors and JAK/STAT signaling pathways. Loss of function analyses in patients with mutation/deletion of the PTPN2 gene and knock-out mouse models indicate that PTPN2 acts as a tumor suppressor in haematologic disorders such as leukemia.

We found that BQ, the prime hematotoxic metabolite of BZ, is an irreversible inhibitor of human PTPN2. Kinetic and biochemical analyses using purified PTPN2 indicated that the irreversible inhibition of the enzyme by BQ is mainly due to arylation of its active site cysteine. Exposure of immortalized human hematopoietic cells (Jurkat T and THP-1 lines) to BQ leads to the irreversible inhibition of endogenous PTPN2 activity with a concomitant over activation of JAK/STAT signaling pathway. Irreversible BQ-dependent inhibition of PTPN2 in cells was found to be mainly due to overoxydation of its catalytic cysteine into sulfinic and/or sulfonic forms.

In Vivo experiments conducted in mice confirmed that exposure to BZ leads to irreversible inhibition of PTPN2 in bone marrow and spleen cells.

Our data provide the first mecanistic evidence that irreversible inhibition of PTPN2, a tumor suppressor tyrosine phosphatase, may contribute to benzene-dependent leukemogenesis.

Disclosures

No relevant conflicts of interest to declare.
Leukemia Related Co-Stimulation / Co-Inhibition Predict T-Cell Attack of Acute Lymphoblastic Leukemia Mediated By Blinatumomab
Authors: Feuchtinger Tobias , Feucht Judith , Kayser Simone , Gorodezki David , Döring Michaela , Blaeschke Franziska , Bösmüller Hans , Quintanilla-Fend Leticia , Schlegel Patrick , Ebinger Martin , Lang Peter J. , Handgretinger Rupert
Published: October 2019

Refractory B-precursor acute lymphoblastic leukemia (ALL) remains an unsolved therapeutic challenge. Various T-cell immunotherapies are promising options in relapsed/refractory B-ALL, like the CD19/CD3-bispecific T-cell engaging antibody Blinatumomab. Until now it has not been possible to determine critical factors for T-cell attack against leukemia that decide on in vivo response or non-response to treatment. Immune-checkpoint molecules regulate immune escape of malignant cells and antibody blockade of these inhibitory pathways enhances antitumor immune responses. Therefore, we investigated the role of co-stimulatory and co-inhibitory molecules for effector-target cell interactions and influence on T-cell attack against leukemia. CD19+ lymphoblast lines, primary pediatric B-ALL bone marrow blasts (n=10) and physiologic CD19+ CD10+ pre-B bone marrow precursors from healthy bone marrow were screened for surface expression of 20 different co-signaling molecules. Surface expression of PD-L1, PD-1, LAG3, CD40, CD86, CD27, CD70 and HVEM revealed differences in stimulatory and inhibitory profiles of pediatric ALL blasts as compared to physiologic cells. Pediatric ALL patients refractory to Blinatumomab-treatment (n=4) as well as patients with relapsed leukemia (n=7) showed increased expression of PD-L1 on blasts. Expression of exhaustion markers PD-1 and TIM-3 was significantly higher on patients' T cells as compared to healthy donors and is induced by T-cell attack against blasts. Blinatumomab-mediated T-cell function was examined in healthy donors as compared to pediatric patients with ALL through analysis of proliferation and effector function. Significant differences in Blinatumomab-induced T-cell function were found to be target-cell dependent and correlated to expression of co-signaling molecules on target cells. Blockade of inhibitory PD-1-PD-L and CTLA-4-CD80/CD86 interactions could further enhance effector T-cell function of healthy donors and patients whereas blockade of co-stimulatory CD28-CD80/86 interactions resulted in reduced T-cell effector and proliferation potential. Combined treatment with Blinatumomab and PD-1 blocking antibody Pembrolizumab was feasible and induced an anti-leukemic immune response in a 12 year old patient with refractory ALL. In conclusion, we show that regulation of T-cell activation and inhibition by co-signaling molecules guides the efficacy of T-cell attack against ALL. Inhibitory interactions between leukemia-induced checkpoint molecules on T cells and their counterparts on ALL regulate in vivo resistance to T-cell immunotherapy and will guide future therapeutic interventions.

Disclosures

Off Label Use: Pembrolizumab.
Phase II Trial Daily Pulse Interleukin-2 Therapy with Famotidine during Marrow and Lymphocyte Recovery in Acute Myeloid Leukemia: LJCC 0605
Authors: Friend Reed , Juskevicius Ridas , Salmon Lauren , Murthy Hemant S. , Xenakis James , Asch Adam S. , Walker Paul
Published: October 2019

Background: Therapeutic advances in treating Acute Myelogenous Leukemia (AML) have been limited in the past two decades. The outcome in patients over the age of 60 and those with high risk cytogenetics remains particularly poor with less than 10% of patients in this age group being cured. High dose Interleukin-2 (IL2) alone in relapsed AML has been shown to induce complete remissions. In this study we report the overall survival (OS), event free survival (EFS), responses and tolerability with IL2 therapy administered during marrow/lymphocyte recovery during induction, re-induction or consolidation.

Methods: Eligibility criteria included confirmed hematopathology diagnosis of AML according to WHO classification (not including acute promyelocytic leukemia) receiving marrow suppressive treatment, a total WBC count above 500 mm3, supported platelet count above 20,000, serum creatinine ≤ 2.0 mg/dl, total bilirubin and AST < 3x upper limit of normal and hemodynamic stability. Famotidine 20 mg IV push daily was administered just prior to the IL2. IL2 therapy was infused at 18 million IU/m2 in 50ml 5% D5 or NS IVPB over 15-30 minutes daily for 5 days. Pre-medication prior to each pulse IL2 dose included acetaminophen 650 mg PO, odansetron 8mg IV, and meperidine 25 mg IV.

Results: 18 pts enrolled with 12 receiving IL2 infusion. The median age was 57.5 (range 32-72). 67% women, 33% men. 8 of 12 patients had AML with myelodysplasia-related changes (including complex cytogenetics), 2 AML with inv(16), 1 AML with 11q23 translocation and 1 therapy-related AML. Median bone marrow blast percentage at diagnosis was 62% (range 32-96). 3 pts received IL2 during induction recovery, 1 during re-induction recovery, 8 during consolidation recovery. 2 pts subsequently underwent allogeneic stem cell transplantation in remission. Median follow up was 328 days (range 99-3207). Estimated 18 month overall and event free survival was 42% (CI 0.15-0.67). Of the 4 pts alive past 4 years, 3 had complex cytogenetics, 2 were greater than age 60. IL2 infusion was well tolerated without any grade 4 toxicities or hemodynamic instability requiring ventilator or hemodynamic support.

Conclusion: IL-2 therapy administered during marrow/lymphocyte recovery after standard induction/consolidation chemotherapy in AML treatment achieved a 33% 4 year overall and event free survival.

Disclosures

No relevant conflicts of interest to declare.
Proteomic Analysis of Reprograming of Mesenchymal Stem/Stromal Cells (MSC) Following Interferon Gamma Identifies Pathways That Are Upregulated in Suppression
Authors: Guan Qingdong , Shpiruk Tanner , Ezzati Peyman , Krokhin Oleg , Gilpin Scott , Doerksen James , Spicer Victor , Bhagwat Swaroop , Wilkins John , Wall Donna A.
Published: October 2019

Assessing potency of mesenchymal stem/stromal cells (MSC) used for immunologic applications such as the treatment of GVHD or other inflammatory disorders has been a challenge. Expression of PD-L1 or production of indoleamine-pyrrole 2,3-dioxygenase (IDO-1) has been proposed as potential potency markers. To screen for other pathways involved with suppression we undertook proteomic analysis of IFN-γ stimulated MSC. MSC isolated and expanded from normal healthy donors to 70-80% confluence were treated overnight with human rIFN-γ (30ng/ml). MSC were harvested using TrypLE Select and then immunologic and proteomic studies were performed. IFN-γ exposure increased a) MSC expression of IDO-1 and PD-L1, b) MSC suppression of 3rd party T lymphocyte proliferation, c) MSC inhibition of development of IFN-γ producing T lymphocytes, and d) MSC promotion of Treg expansion. Cellular proteomic changes that occur with IFN-γ exposures were studied in paired samples of control and IFN-γ treated MSC. Samples were prepared using a modified Filter Aided Sample Prep (FASP) and digests were separated using 2D liquid chromatography and analysed by tandem mass spectrometry. Data was processed with the Global Proteome Machine and only proteins with at least two confident peptides were reported. A total of 7621 proteins were identified of which 5575 were seen in all samples and 232 proteins were significantly upregulated in the IFN-γ treated cells relative to their controls. The proteomic analysis identified constitutive proteins seen in MSC. The upregulated proteins were significantly enriched (p<10-17) for GO processes such as "response to IFN-γ" and "cytokine mediated signaling pathway". Known inhibitory mediators (such as IDO-1, PD-L1, PGE2, galectin-9) were upregulated. Interestingly adhesion molecules (ICAM-1, VCAM-1, and CCL9) were increased. Other proteins with increased expression include Bone Marrow Stromal 2 (BST2). Conclusion: Proteomic analysis of response of MSC to IFN-γ has identified a signature of proteins upregulated with the activation of immune suppressive functions of MSC. Once confirmed these findings will support the development of a potency test for immunosuppressive potential of given MSC preparations - something that is sorely needed in the clinical manufacturing of MSC products.

Acknowledgments: Q.D. is holding a postdoctoral fellowship from MS Society of Canada. This research was supported by The Bihlers' Professorship in Stem Cell Research to D.W.

Disclosures

No relevant conflicts of interest to declare.
Number of Circulating t(14;18) Tumor Cells at Diagnosis Is Related to, but Add to the Prognostic Value of Metabolic Tumor Burden in Follicular Lymphoma
Authors: Delfau-Larue Marie-Helene , Nel Isabelle , VAN Der Gucht Axel , Laboure Gaelle , Verret Benjamin , Hamdane Salma , Gaulard Philippe , Meignan Michel , Haioun Corinne , Copie-Bergman Christiane , Dupuis Jehan , Itti Emmanuel
Published: October 2019

Beside the rare true leukemic forms of follicular lymphoma (FL), low levels of circulating tumor cells (CTC) are detected by PCR in the vast majority of FL patients at diagnosis. By a regular recirculating process, those CTC could reflect the total solid tumor mass. Alternatively, they could represent a subpopulation of tumor cells with distinct molecular profile including adhesion molecules and, as such, could add to the prognostic value of solid tumoral mass previously reported in FL (Dupuis, JCO 2013). In order to address these issues, we retrospectively selected FL patients treated in our institution between 2007 and 2014 who were simultaneously evaluated for both t(14;18) cells in peripheral blood (PB) and FDG-PET/CT tumor mass at diagnosis or at relapse. Absolute quantification of t(14;18) positive cells was performed using quantitative droplet digital PCR (ddPCR). Total metabolic tumor volume (TMTV) and total lesion glycolysis (TLG) were calculated from FDG-PET/CT, and baseline clinical characteristics and outcomes were collected. One hundred fourteen patients fulfilled the inclusion criteria. Using a routine 10-4 sensitive biomed 2 t(14;18) PCR assay, 75 had a positive PCR, either in peripheral blood alone (n=37), in peripheral blood and tumor biopsy (n=27) or in tumor biopsy but not PB (n= 11). Absolute quantitative ddPCR was performed for the 56 t(14;18) MBR+ patients. Clinical characteristics are given in Table 1. Median CTC value (number of t(14;18) positive cells out of total peripheral blood nucleated cells) was 1.6 10-3 (range 0-0.96), with only 5 CTC (-) patients and 13 patients with >10% CTC . Median TMTV was 267 cm3 (range 4.61-1900) and median TLG was 1473 (range 10.24-5912). A positive correlation was found between number of CTC and TMTV (R2 = 0.49; P<0.001; Fig.1) and to a lesser extend with TLG (R2 = 0.38; P<0.001). With a median follow-up of 32 months, OS of FLIPI 0-2 patients was 100% vs 87% for FLIPI 3-4 patients (p=0.003). A number of CTC > 6%, TMTV > 432 cm3 and TLG > 2717 tend to be associated with poorer OS (Fig. 2). The combined presence of > 6% CTC and TLG > 2717 allowed to identify a group of patients with 3-year OS of 71%, compared with 100% when both criteria were negative or dissociated (P= 0.01) (Fig. 2). In the subset of 42 patients with an untreated and untransformed FL, incremental prognostic value of circulating mass and metabolic tumor burden remained significant (P=0.03).

Disclosures

Dupuis: ABBVIE: Membership on an entity's Board of Directors or advisory committees; ROCHE: Speakers Bureau.