bluuebunny/crossref_metadata_2025 huggingface dataset:
Mir-223 Is Dispensable for the Onset of Acute Myeloid Leukemia
Authors: Kuchenbauer Florian , Berg Tobias , Mah Sarah M , mirkovic-Hosle Milijana , Salmi Anisa , Ruschmann Jens , Muranyi Andrew , Agiropoulos Bob , Rouhi Arefeh , Starczynowski Daniel T , Heuser Michael , Hogge Donna E. , Camargo Fernando D. , Dohner Hartmut , Buske Christian , Humphries R. Keith
Published: October 2019

Abstract 501

The functional roles of microRNAs in the development of acute myeloid leukemia (AML) are not yet clear. Due to its myeloid-specific expression, miR-223 has been one of the most-investigated miRNAs in normal and malignant hematopoiesis. However, the role of miR-223 in myeloid differentiation is not completely understood, as contradicting data exists. Genetic depletion of miR-223 led to a significant increase of myeloid progenitor cells as well as circulating hyperreactive neutrophils. Here, we investigate the role of miR-223 in the development of AML in vivo, using retroviral overexpression models of Hoxa9 with Meis1 or MN1 as two potent models of leukemic transformation in a miR-223+/+ or miR-223−/− background. In contrast to the observed high level expression of miR-223 in human CD34- bulk AML cells (p=0.0106), we could show that miR-223 was dispensable for the development of AML and did not impact on either the leukemic stem cell frequency nor the AML cell phenotype in Hoxa9-Meis1 AML cells. While these findings reveal that miR-223 is not necessary for leukemic transformation in highly aggressive AML models, we became interested if miR-223 functions rather as modulator of disease progression, especially at the early development of AML. Therefore, we investigated the role of miR-223 with regards to differentiation and self-renewal in two preleukemic model systems by retrovirally infecting miR-223−/− and miR-223+/+ BM cells with AML1-ETO and Hoxa9 respectively. Characterization of these models demonstrated that miR-223 expression is a determinant of differentiation, as miR-223−/− preleukemic cells exhibit a significant lower Mac-1 expression (p=0.0003). However, in contrast to normal miR-223−/− BM cells, which show a significantly higher colony forming capacity in methylcellulose compared to miR-233+/+ BM cells, the colony forming capacity of miR-223−/− or miR-223+/+ preleukemic cells did not significantly change.

These findings demonstrate that miRNA miR-223 is hierarchically expressed in AML cells, and functionally link miR-223 to impaired differentiation rather than increased self-renewal in the initiation of AML. This indicates that miR-223 is more likely a fine tuner of leukemic development than a potent tumor suppressor or oncogenes as suggested in the literature. However, it still remains to be shown if the presence of miR-223 influences the susceptibility of preleukemic cells to convert into leukemia initiating cells.

Disclosures:

No relevant conflicts of interest to declare.
Membrane Associated TGF-β1 on Leukemia Blast-Derived Microvesicles In Sera of Acute Myeloid Leukemia Patients Suppresses NK Cell Function
Authors: Boyiadzis Michael , Szczepanski Miroslaw , Whiteside Theresa
Published: October 2019

Abstract 502

Natural killer (NK) cell cytotoxicity in patients with acute myeloid leukemia (AML) is significantly decreased relative to that in normal controls (NC). TGF-β1 is a potent inhibitor of NK cell cytotoxicity. We considered the possibility that suppression of NK cell activity in AML patients is mediated by leukemia blast-derived microvesicles (MV) via TGF-β1. Sera of 19 patients newly diagnosed with AML prior to any treatment were used to isolate MV by ultracentrifugation. MV from AML patients were positive for the blast-associated markers (CD33, CD34, CD117) by flow cytometry and for membrane-associated TGFb-1 in Western blots, whereas neither these markers nor TGFb-1 were detected in MV from NC. The protein content of blast-derived MV was higher (p<0.001) than that of MV isolated from sera of 25 NC (75μ g±12/mL vs 1.2μ g±0.4/mL). To evaluate MV-mediated NK cell suppression, NK cells obtained from NC were co-incubated with MV isolated from patients' sera. A significant decrease (p<0.002) in NK cell cytotoxicity (2412 LU pre- vs 1640 LU post co-incubation) was accompanied by a concomitant decrease (p<0.004) in the NK-cell activating receptor, NKG2DR, expression levels. To determine whether MV-associated TGF-β1 was responsible for these adverse effects of MV on NK cells, neutralizing anti-TGF-β1 mAb was added to the co-cultures. This mAb significantly abrogated MV-mediated inhibition of NK cell cytotoxicity and down-regulation of the NKG2DR expression level. We next measured the plasma concentrations of TGF-β1 in AML patients and NC. The mean ± SD plasma concentration of TGF-β1 was 2,600 pg/mL ± 2,500 in patients vs. 101 pg/mL ± 55 in NC (p<0.0001). The use of urea, which disassociated membrane-bound TGF-β1 in MV, resulted in a significant increase of the measured TGF-β1 levels (mean 13,700 pg/mL ± 9,800) only in the sera of AML patients but not in the sera of NC. To determine whether the SMAD pathway used by the TGF-β family members is involved in the MV-NK cell interactions, we co-incubated NK cells with MV isolated from sera of AML patients. These MV induced SMAD phosphorylation in NK cells. The addition of anti-TGF-β1 mAbs to the co-culture inhibited SMAD phosphorylation but did not alter expression of the SMAD protein itself. These data provide evidence for a novel mechanism responsible for down-regulation of NK cell activity and NKG2DR expression levels by TGF-β1 associated with leukemia blast-derived MV in AML patients.

Disclosures:

No relevant conflicts of interest to declare.
Investigation of the Molecular Action of Bortezomib Treatment In Multiple Myeloma
Authors: Ballabio Erica , Chen Xiao-He , Tramonti Daniela , Wainscoat James S. , Boultwood Jacqueline , Hatton Chris S.R , Lawrie Charles H.
Published: October 2019

Abstract 5038

Bortezomib (Velcade®) is a 26S proteasome inhibitor currently used for the treatment of relapsed multiple myeloma (MM) and more recently mantle cell lymphoma patients. Clinical trials are under way to evaluate its efficacy in other hematological malignancies diseases including diffuse large B cell lymphoma (DLBCL). Although it was widely accepted that bortezomib acts as a NF-κB inhibitor, this view has recently been challenged by work by Hideshima et al. (Blood 2009) who found that treated myeloma cells actually displayed down-regulation of NF-κB inhibitors (IκBα), increased activity of pro-NF-κB molecules (RIP2 and IKKβ) as well as increasing levels of NF-κB DNA binding. Additionally, although bortezomib has a potent anti-tumor activity, not all MM patients are responsive. The identification and understanding of the molecular mechanisms leading to bortezomib-resistance will help with the development of new approaches and will improve the treatment of these patients. In order to investigate the direct effect of bortezomib on lymphomas, we treated different cell lines with this drug and levels of cell proliferation and apoptosis were measured. Bortezomib was shown to significantly inhibit proliferation and promote apoptosis in MM cell lines (RPMI-8226, NCI-H929, JJN-3 and Thiel) and DLBCL cell lines (SU-DHL-4 and SU-DHL-10, HLY-1, HLB-1 and RIVA). Since MM cell lines were shown to be the most sensitive to the bortezomib, we selected them to carry out further studies. We next employed whole genome expression arrays (Affymetrix U113plus2.0) and microRNA arrays to identify molecules associated with treatment in these cell lines. Using unsupervised cluster analysis, we observed that the cells treated with bortezomib have a distinct microRNA and gene expression profile from their counterpart controls. Additionally, we used a shRNA whole-genome library to carry out a loss-of-function screen for cells resistant to bortezomib-induced apoptosis. The RPMI-8226 cell line was transduced with the shRNA library. One half of the cells was then treated with bortezomib. Relative gene expression levels were measured by hybridizing samples to custom-made arrays. Unsupervised cluster analysis identified a number of shRNAs which might be involved in mechanisms of resistance to bortezomib. Further functional studies will be necessary to investigate the role of these microRNAs in the mechanism of action of bortezomib.

Disclosures:

No relevant conflicts of interest to declare.
New Cases of Acute Promyelocytic Leukemia Treatment by Arsenic Trioxide Alone and Their Long Term Follow up
Authors: Ghavamzadeh Ardeshir , Alimoghaddam Kamran , Rostami Shahrbano , Ghaffari Seyed Hamidolah , Jahani Mohamad , Jalili Mahdi , Mousavi Seyed Asadollah , Iravani Masood , Baybordi Eisa , Bahar Babak , Alizadeh Nassrin
Published: October 2019

Abstract 506

Long term follow-up of APL patients treated by ATRA and chemotherapy is available and shows promising results of DFS and OS. Although several studies show acceptable efficacy of Arsenic Trioxide in new cases of APL, its long term result needs better clarification.

Materials and methods:

one hundred ninety seven, new cases of APL treated by Arsenic trioxide (0.15 mg/kg daily i.v infusion till complete remission). After achieving to CR patients received 4 more courses of Arsenic trioxide as consolidation. Then patients followed by CBC and RT-PCR on peripheral blood for detection of MRD every three months or till relapse or death.

Results:

Morphologic complete remission rate was 85.8% and most common cause of remission failure was early death due to hemorrhage of APL differentiation syndrome.(14.7%) Most important prognostic factor for early mortality is high WBC count at presentation. Disease free survival was 90%+/−2%, 72.7%+/−3% and 66.7%+/−4% for 2, 3 and 5 years respectively. Relapse for patients who remained in CR after 5 years was very uncommon. Overall survival for patients after diagnosis by intention to treat analysis was 80.2%+/−3%, 75.9%+/−3% and 64.4%+/− 4% for 2, 3 and 5 years. Also OS and DFS were the same between patients with high and low WBC count.

Conclusion:

Long term follow up of newly diagnosed cases of APL, treated with single agent Arsenic Trioxide shows promising result. Arsenic trioxide potentially eliminates the adverse effect of prognostic factors of APL treatment such as high WBC count. We suggest that it is time to integrate Arsenic trioxide in treatment of new cases of APL.

Disclosures:

No relevant conflicts of interest to declare.
Decitabine and Gemtuzumab Ozogamicin In Patients with Advanced Myelofibrosis
Authors: Al-Ameri Ali Al-Ameri , Kantarjian Hagop , Malik Asifa , Badoux Xavier , Andreeff Michael , Quintas-Cardama Alfonso , Borthakur Gautam
Published: October 2019

Abstract 5061

Background:

While hypomethylating agents, azacitidine and decitabine, are approved for use in treatment of myelodysplastic syndromes (MDS), their role in treatment of myeloproliferative disorders is evolving. The modified dosing schedule of decitabine (20 mg/m2 IV for 5 days) in MDS has shown overall response rates of approximately 40%. A clinical study of azacitidine in myelofibrosis (MF) has shown a response rate of 32%. We investigated the activity of the combination of decitabine and gemtuzumab ozogamicin (GO) (anti-CD33 antibody), a combination with activity in patients with MDS and acute myelogenous leukemia (AML), in patients with advanced myelofibrosis.

Patients and Method:

We reviewed the records of patients with MF treated with the combination of decitabine and GO.

Result:

Seven patients were treated till the decisions by FDA to withdraw GO from the market. Age ranged from 60–69 years (median 65 years), 5 patients were male and no of prior therapies ranged 0–4 (median2). Three patients had MF progressed to acute myeloid leukemia. Prior treatments included, hydroxyura, thalidomide, prednisone, pomolidamide etc. All patients were positive for JAK 2 V617F mutation. Cytogenetic abnormalities were seen in 4 patients (hyperdiploid, complex or -5/-7) and 3 were with diploid cytogenetics. Five patients received 3 cycles of Decitabine plus myelotarg while 2 patients had only one cycle. Four patients had stable disease, one had clearance of marrow blasts (40%>0%), two had no response. Three patients showed decreases in splenic size. Clinically significant infectious complications were encountered in 4 patients.

Conclusions:

The combination of decitabine and GO showed early signs of activity in patients with MF but future investigation of this combination will be limited due to lack of access to GO.

Disclosures:

Borthakur: eisai: Research Funding.
Statins and Venous Thromboembolism In Lung Cancer
Authors: Khushman Moh'd , Abu-samra Abdel-ghanie , Farhan Shatha , Jacobsen Gordon , Hanbali Amr , Wang Ding
Published: October 2019

Abstract 5122

Background:

Dyslipidemia plays a major role in the pathophysiology of atherosclerosis which may also contributed to an increased risk of and thromboembolism from the injury of the vascular endothelium and its activation of platelets, thereafter, the coagulation cascade. It has been well-established that the use of statins in patients with dyslipidemia reduces cardiovascular events and mortality, therefore, improves survival. In addition to a cholesterol-lowering effect, statins exhibit antithrombotic and anti-inflammatory properties that may confer a protective benefit to an increased risk of thromboembolism in cancer patients. Several case-controlled studies reported that cancer patients receiving long-term statins, but not other non-statin lipid lowering treatment, had a lower incidence of venous thromboembolism.

Methods:

To investigate any protective benefit from statin use in lung cancer patients, we have conducted this retrospective, case-controlled study. Total of 1548 patients with lung cancer were included from the tumor registery at Henry Ford Health System, Detroit, Michigan, between January 1999 and December 2004. The data were extracted from available electronic medical records of these patients. Statistical analyses were performed and stratified for statin users versus non statin users.

Results:

Out of 1,548 patients, 315 patients (average age was 68, range 46–90) were statin users at the time of their lung cancer diagnosis. The remaining 1233 patients (average age was 65.9, range 29–94) were non- statin users. After stratifying for statin use, 25 of the 315 statin users developed DVT/PE (7.9%), while 100 of the 1233 non-statin users developed DVT/PE (8.1%). This potential benefit from statin use can also be better visualized from comparing the development of DVT/PE between the statin and non-statin groups in a Kaplan-Meier curve for the probability of freedom from DVT/PE across time. Though statistical significance was not reached (log-rank p-value = 0.377), a trend towards a slightly decreased incidence of DVT/PE onset in the patients who receiving statin as the lipid-lowering agent has been observed.

Conclusion:

Based on our preliminary data, statin use in patients with lung cancer has demonstrated a weak but favorable protective effect on the development of a venous thromboembolism. The nature as a retrospective study and not well-balanced two group of patients (e.g., the average age of statin users were 2 years older than the non-statin users) may limit our results from reaching statistical significance. In addition, some of the patients who had no clear documentation of their home medications had also been categorized as non-statin users in our study. The results from our final data analysis will be presented.

Disclosures:

No relevant conflicts of interest to declare.
A Biotin-Avidin-EL ISA for Studying on Platelet Membrane Surface P-Selectin and Its Clinical Application
Authors: Zhao Yiming , Zhang Youtao , Lu Shiqi , Zhu Mingqing , He Yang , Ruan Changgeng
Published: October 2019

Abstract 5132

Introduction

To define the abnormalities in surface GPs expression on circulating platelets and provide better biomarker of bleeding and thrombotic disorders, we develop a high-throughput assay using biotin-avidin enzyme-linked immunosorbent assay (BA-ELISA) to test platelet responses to key platelet agonists and antagonists.

Methods

SZ51(anti-P-selectin mAb) coated-plate was added into 100μ l PRP with 10μ l ADP (27μ mol/L ADP)or 10μ l PBS without ADP and incubated for 1 h. Activated platelets captured were reacted with biotin-SZ-2 for 1 h. Then, the plate was re-incubated with avidin-HRP coiugated for 1h. Binding avidin was detected with OPD. The levels of P-selectin were detected in AMI group, ICH group, DM group or control group using the BA-ELISA method. This BA-ELISA was compared with flow cytometer (FCM). Inhibition of platelet GPs function was evaluated with inhibitory mAb, SZ21 and aspirin respectively.

Results

The BA-ELISA can detect platelet count as low as 6.25 × 109/L in PRP. Both of the inter-assay and intra-assay coefficient variation (C V) were less than 10%. The OD492nm values of the ADP-induced P-selectin in 30 AMI, 30 ICH and 30 DM platelets in PRP were 1.76± 0.56, 1.95±0.53 and 1.51±0.14 which was significantly higher than that in controls (0.68±0.21, P<0.01). The OD 492nm values of non ADP-induced P-selectin in 30 AMI, 30 ICH or 30 DM platelets in PRP were 0.72±0.25, 1.53±0.61 and 0.61±0.16 significantly higher than that in 30 controls (0.18±0.09, P<0.01). The correlation between the BA-ELISA and FCM was concordant (R=0.86). SZ21 or aspirin could inhibit the expression of P-selectin induced by ADP.

Conclusion

BA-ELISA is suitable for measuring platelet GP function and is also useful for diagnostic tests and for screening of inhibitors/activators of platelet activation.

Disclosures:

No relevant conflicts of interest to declare.
Anaemia In Nonagenarians: Incidence, Severity and Aetiology
Authors: Brady Justine S , van Bilsen Nenna , Han Ping , Mulligan Stephen P
Published: October 2019

Abstract 5138

INTRODUCTION:

The population of Australians aged 90–99 years has increased by 204% (42,234 to 128,654) [0.25% to 0.59%] between 1990 and 2010 compared to a 30% increase in the general population (17.06 million to 21.99 million) highlighting the trend to aging of the population. At this age bracket the M:F ratio is ~1:3. One third of the Australian population lives in the state of New South Wales (NSW) (www.abs.gov.au). The Australian heathcare system of federally funded pathology has resulted in marked consolidation of pathology providers servicing all non-hospital healthcare settings. Symbion Pathology account for ~35% of all pathology services in NSW and hence closely reflects the community incidence for laboratory diagnosed disorders. Community-based pathology laboratories now see large numbers of patients in this 90–99 year old age range.

METHODS:

We assessed all individual patients aged between 90 and 99 years with anaemia over the 5 year period 2005–2009. Patients were included in the analysis if they had at least one full blood count (FBC/CBC) showing a hemoglobin below the laboratory reference range (RR) of 130g/L for males and 115g/L for females. All duplicate individuals and full blood counts (FBC) were excluded.

RESULTS:

There were 4,369 (33.8%) of 12,921 individuals with anaemia. Classification by red cell size showed the majority were normochromic (80.6%), while 5.8% were microcytic and 13.6% were macrocytic.

In microcytic anaemias, 136 (53%) had a ferritin level performed and 63 (46% of this 136 patients) were iron deficient. In macrocytic anaemias, only 149 (25%) had B12 assays and 13 were deficient. In normocytic anaemias, 2910 (82.7%) had urea performed with 1413 patients had urea >10 mmol/L (48%) and 976 patients had urea >12 mmol/L (33%) (RR 3.5–9.0 mmol/L). Pancytopenia was present in 44 (1%) patients. In most patients, anaemia was mild but 25% of females and 50% of males had Hb >15g/L below the lower limit of the RR.

CONCLUSION:

Anaemia is very common in individuals aged 90–99 years occurring in one-third of the population at this age. Half the males and a quarter of females have a Hb >15g/L below the RR making this a significant comorbidity in the very elderly. As might be expected the aetiology is heterogeneous but appears to be frequently under investigated. In particular, simple haematinic assays appear to be under-utilised to better define the aetiology despite being inexpensive and readily available. The study highlights the substantial social, economic and healthcare implications in this era of significant ageing of the population.

Disclosures:

No relevant conflicts of interest to declare.
Population Screening for Thalassemia In West Bengal with a Goal to Make Zero Thalassmia Growth
Authors: Basak Jayasri , Majumdar Deboshree , Mukhopadhyay Soma , Dasgupta Swati , Das Priyabrata , Pal Nabamita , Dey Subhasish , Mukhopadhyay Ashis
Published: October 2019

Abstract 5175

Background:

India is in the Thalassemia belt of the world. Presently prevalence of Thalassemia carrier in India is 3.7% of its total population. Annual increment of Thalassemia carrier is 50,000. Both Alpha and Beta Thalassemia are found in West Bengal, a state in Eastern part of India. Beta Thalassemia is most common in West Bengal. Annual Increment of beta carrier is 2000–2500. With a motivation to make zero Thalassemia growth in West Bengal we started this program in 2005 taking Cyprus as an example. We regularly arranged camps in different districts and rural areas of West Bengal. To aware the general people seminars on Thalassemia were organized. Usually we took help from Principals of colleges & schools, Presidents of club, Gram Pradhans, Priests, Fathers & Celebrities etc.

Materials & Methods:

Duration of this screening study is from January 2005 to July 2010. Interested people came for thalassemia screening irrespective of cast & creed. Screening procedure consisted of the following steps. NESTROF test for all individuals was done on the spot which was followed by CBC and HPLC, done in the Laboratory. Finally to reveal different mutations in b globin gene and deletions in alpha globin gene, ARMS-PCR and gap PCR were performed respectively with the DNA of the suspected individuals.

Result:

For thalassemia carrier screening we had organized 122 camps in different rural as well as urban areas of West Bengal from January 2005 to July 2010. Total number of individuals screened is 29,350 (Male: 18197, Female: 11153). 24% individuals were found positive from NESTROF test and 28% has doubtful CBC. NESTROF positive and doubtful CBC samples were undergone HPLC test. Finally, 13% individuals were found beta carrier. In two camps at north Bengal we have collected about 278 samples having positive NESTROFT value and having abnormal CBC values (MCV < 80 fl, MCH< 27 pg, MCHC > 30 g/dl, RDW > 13%) were tested for HPLC. HbA2 values were found in the normal range. DNA analysis by gap PCR revealed that approximately 47% of them are alpha Thalassemia carrier, 32% having 3.7 kb deletion, 11% having 4.2 kb deletion and 4% other deletions.

Conclusion:

As the percentage of thalassemia carrier in West Bengal is very high we have to take concrete steps to control the disease from the Government level. From our part we are screening the boys and girls at marriageable age and advising them not to marry between two carriers.

Disclosures:

No relevant conflicts of interest to declare.
Clinical Scale Central Memory T Cell Enrichment for Adoptive T Cell Therapy
Authors: Casati Anna , Schmitz J\)rgen , Abken Hinrich , Chmielewski Markus , Nahvi Azam , Dudley Mark , Assenmacher Mario , Scheffold Alexander
Published: October 2019

Abstract 5180

The adoptive transfer of genetically engineered lymphocytes is a promising strategy to treat cancer. However, the efficacy of adoptive immunotherapy in humans is often limited and the critical parameters for the in vivo efficacy remain poorly defined. One aspect may be the limited survival time and self renewal capacity of transferred T cells in vivo.

Antigen-experienced memory T cells (TM) can be divided further into central memory (TCM) and effector memory (TEM) subsets. CD8+ TEM lack CCR7/CD62L, migrate preferentially to peripheral tissues, and exhibit immediate effector function, whereas CD8+ TCM express CD62L and CCR7, which promote migration into LNs. TCM proliferate rapidly if re-exposed to antigen and there is evidence that T cell lines derived from TCM have increased persistence and functionality in vivo. Thus TCM might be a superior starting population for T cell therapy using transduced T cells.

We have developed a GMP compatible sorting strategy for TCM allowing their selective transduction and large-scale expansion. TCM cell were isolated by a sequential depletion of CD45RA+ and CD4+ and subsequent enrichment of CD62L+ cells using the CliniMACS® Plus Instrument. CD8+ TCM from peripheral blood of cancer patients were obtained in large numbers from leukapheresis products and genetically engineered with tumor-specific TCRs. After expansion in vitro to numbers sufficient for patient treatment the TCM population retained high CD62L expression. This protocol can be the basis for clinical studies on the efficiency of TCM derived T cell lines in T cell therapy against tumors.

Disclosures:

Casati: Miltenyi Biotec: Employment. Schmitz:Miltenyi Biotec: Employment. Assenmacher:m: Employment. Scheffold:Miltenyi Biotec: Employment.
Upregulation of Inflammatory Mediators In End-Stage Renal Disease as Measured Via Biochip Array Technology
Authors: Bansal Vinod , Davis Rachael , Litinas Evangelos , Hoppensteadt Debra , Thethi Indermohan , Fareed Jawed
Published: October 2019

Abstract 5186

End-Stage Renal Disease (ESRD) is a complex syndrome in which systemic vascular pathophysiologic changes contribute to adverse cardiovascular and cerebrovascular manifestations. Cardiovascular disease alone is present in over 60% of patients with ESRD and contributes heavily to mortality among this population. Given that inflammatory and hemostatic aberrations contribute to the overall pathogenesis of the syndrome, the purpose of this study is to profile several inflammatory mediators in order to better understand their role in the underlying mechanism of vascular changes in ESRD. Plasma samples from 49 patients with ESRD were collected prior to maintenance hemodialysis sessions. A group of 56 normal individuals, both male and female, was included as control. Cerebral Array II chips were used in the Randox® system to simultaneously measure Neuron Specific Enolase (NSE), Neutrophil Gelatinase-associated Lipocalin (NGAL), Soluble Tumor Necrosis Factor Receptor I (TNFRI), D-Dimer (DD), Thrombomodulin (TM), and C-reactive protein (CRP). The Randox® Evidence Investigator™ is a new biochip array technology that utilizes multiple discrete test regions of immobilized antibody to simultaneously quantify multiple markers from a single patient plasma sample based on the light signal generated from each test region. The data was statistically analyzed using the Mann-Whitney U test (two-tailed with Gaussian approximation). As compared to the normal individual, all of the markers studied showed an upregulation in patients with ESRD. Most notably, TNFRI showed a 19.8 fold increase in patients with ESRD (mean 7.8 ± 2.8 ng/ml, range 0.8 to 13.7) compared to the control (mean 0.4 ± 0.2, range 0.1 to 1.0). TM was increased 5.2 fold (mean 6.5 ± 2.6, range 0.7 to 14.1) compared to control (mean 1.2 ± 0.4, range 0.6 to 2.3). Also, NGAL showed a 4.6 fold increase (mean 1390 ± 257, range 406 to 1729), compared to control (mean 299 ± 99, range 115 to 603), and CRP a 4.2 fold increase (mean 5.7 ± 4.2 ug/ml, range 0.6 to 13.2) compared to control (mean 1.4 ± 1.7, range 0.2 to 11.4). DD and NSE were also increased 3.0 and 1.8 fold respectively. These studies show that some newer markers such as TNFRI, NGAL and NSE are upregulated in ESRD. The marked increase in TM is highly suggestive of endothelial damage. Similarly, the increase in TNFRI supports a state of increased cellular damage. The elevations in NGAL and CRP imply a state of increased inflammation and indicate a polypathologic process, which may predispose ESRD patients to both cardiovascular and cerebrovascular thromboembolic events. Finally, this study further validates the role of endothelial damage and endogenous thrombotic processes in ESRD as evidenced by the increased levels of TM and DD. However, the clinical significance of these markers still needs to be further explored.

Disclosures:

No relevant conflicts of interest to declare.
A Novel Synthetic GMCSF and MCP3 Fusion Cytokine Induces IL10-Producing B Cells with Immune Modulatory Properties on Antigen Presentation and Th17 Development
Authors: Hsieh Hsiang-Chuan (Jeremy) , Williams Patrick , Rafei Moutih , Birman Elena , Young Yoon Now , Galipeau Jacques
Published: October 2019

Abstract 587

Therapeutic strategies based on the modulation of immune system can be exploited to treat a variety of pathologies including autoimmune diseases and cancer. We previously described the immune suppressive and receptor-specific apoptotic effect of a fusion cytokine GMME1, generated by the linkage of granulocyte macrophage-colony stimulating factor (GM-CSF) at the N-terminal of MCP1 (6-76). To better understand the immunobiology of N-terminal-modified CC chemokine, we described the novel function of GMCSF-MCP3 (5-76) (GMME3) in its induction of IL10+, regulatory-competent B cells. Ex vivo cultured B lymphocyte with GMME3 leads to a significant increase in S and G2/M phases, IL10 production and the expression of CD1.d and CD5. Treatment of experimental autoimmune encephalomyelitis (EAE) mice with GMME3-activated wild type B cells (BGMME3), but not IL10 null B cells, significantly inhibits the disease progression and immune infiltrates into the CNS. We demonstrated that the mechanism by which BGMME3 exerts their effect is through IL10-mediated inhibition of class II antigen presentation of macrophages; moreover, BGMME3 inhibits the reactivation of EAE-derived Th17 cells by augmenting Th1 pathway. Hence, N-terminal-GMCSF-fused CC chemokine, GMME3 modulates the immunological phenotype of B cells in a manner permissive for personalized cell therapy of autoimmune ailments.

Disclosures:

No relevant conflicts of interest to declare.
Dissecting the Mechanism of Apoptosis and Leukemic Transformation In Myelodysplasia
Authors: Slape Christopher I , Saw Jesslyn , Failla Laura M , Aplan Peter D , Jane Stephen M , Curtis David J
Published: October 2019

Abstract 610

A central question in the pathogenesis of the myelodysplastic syndromes (MDS) is the mechanism of apoptosis in early disease, and how this is overcome during disease progression to acute myeloid leukemia (AML). In humans, increased serum and bone marrow levels of TNF and/or FASL in early MDS implicate the cell-extrinsic apoptotic pathway, whereas increased expression of anti-apoptotic BCL2-related proteins (the cell-intrinsic pathway) has been linked to leukemic transformation. Here, we have employed the Vav-NUP98-HOXD13 (NHD13) transgenic mouse model to genetically dissect the molecular mechanisms underpinning apoptosis and leukemic progression in MDS. Three month old NHD13 mice recapitulate ‘low-risk’ human MDS with peripheral blood cytopenias, impaired in vitro progenitor cell growth, dysplasia of myeloid cells and megakaryocytes, and prominent apoptosis. Leukemic transformation in NHD13 mice occurs between 6 and 14 months of age. Using gene targeted mouse lines crossed with NHD13 mice, we show that neither TNF nor FasL are required for apoptosis. In contrast, enforced expression of Bcl2 rescues the early MDS phenotype including the macrocytic anemia, dysplasia of megakaryocytes, apoptosis and impaired erythroid and myeloid progenitor cell growth. Remarkably, enforced expression of Bcl2 completely prevented transformation to AML. Our results challenge many of the concepts regarding apoptosis in MDS. First, apoptosis is the predominant mechanism of peripheral cytopenias including erythroid-megakaryocyte dysplasia. Second, apoptosis is mediated by the BCL2-related cell intrinsic pathway. Third, overcoming apoptosis is unlikely to be an important mechanism of leukemic transformation. Indeed, our results extend recent evidence that apoptosis may be a ‘driver’ of leukemic transformation rather than a protective mechanism. These findings raise the therapeutic potential of BCL2 mimetic drugs for treating cytopenias and preventing leukemic transformation of MDS.

Disclosures:

No relevant conflicts of interest to declare.
Correction of the Fanconi Anemia Phenotype at the Cellular Level by S Phase Delay: Implications for Etiopathogenesis and Treatment
Authors: Lambert W. Clark , Centurion Santiago , Brown Monique
Published: October 2019

Abstract 879

Fanconi Anemia (FA) is a rare, complex, recessively inherited disease, with 13 known complementation groups, in which many patients develop bone marrow failure and/or leukemia, especially myelogenous leukemia, at an early age. Although bone marrow transplantation (BMT) has been beneficial, a high proportion of these patients go on to develop head and neck cancers and other solid malignancies, regardless of whether BMT has been carried out. Our laboratory, and others, have shown that FA is associated with a cell cycle defect in which FA cells fail to slow or arrest their rate of replicative, S-phase DNA synthesis as do normal cells following treatment with a DNA interstrand cross-linking (ICL) agent. FA cells are also markedly hypersensitive to the clastogenic and cytotoxic effects of such ICL agents, characteristics used to define the disease in the classic diepoxybutane, or DEB test. We wished to test whether, if the cell cycle defect were corrected by treating the cells with an inhibitor of DNA synthesis, the cytoclastic and cytotoxic effects of an ICL agent, psoralen plus ultraviolet A light (PUVA), would be affected in FA cells. Among DNA synthesis inhibitors, we were particularly interested in examining hydroxyurea and 5-fluorouracil (HU and 5-FU), because they have been used successfully to treat other blood diseases and cancers, respectively, such as sickle cell anemia and colon adenocarcinomas. Following treatment with PUVA, normal lymphoblastoid cells (two cell lines), FA lymphoblastoid cells (two FA-A, one FA-C and one FA-G lines), and two genetically corrected FA lymphoblastoid cell lines (FA-A and FA-C) were either mock treated or treated with an inhibitor of DNA synthesis (high dose thymidine, methotrexate, or HU) for 24 hours. Chromosome breaks and both short term (trypan blue exclusion) and long term cell viability (colony forming ability) were then measured. Except for one FA-A cell line that had low thymidine kinase activity, and therefore did not respond to thymidine, all uncorrected FA cell lines, but not corrected FA cell lines, showed dramatic reductions in clastogenicity and increases in viability following PUVA and treatment with any of the DNA synthesis inhibitors. Normal cells and corrected FA cells failed to show a comparable response.

These results indicate that the S-phase cell cycle defect in FA is important in its etiopathogenesis. The stalled DNA replication forks documented in FA cells containing DNA ICLs may actually be due, in part or entirely, to a prior defect in cell cycle regulation when damaged FA cells enter S phase. These results also suggest at least two possible modes of therapeutic intervention, treatment of FA patients with HU or 5-FU to prevent or delay onset of complications including bone marrow failure, leukemia, or, particularly in patients who have undergone BMT, head, neck, and other tumors.

Disclosures:

No relevant conflicts of interest to declare.
Noninvasive Prediction of Graft-Verus-Host Disease Following Allogeneic Hematopoietic Cell Transplantation by Gene Expression Profiling
Authors: Kohrt Holbrook E , Li Li , Alizadeh Ash A , Goldstein Matthew J , Strober Samuel , Sarwal Minnie , Lowsky Robert
Published: October 2019

Abstract 896

Background:

Chronic graft-versus-host disease (cGvHD) and immunosuppressive medications for its treatment result in significant morbidity and mortality, limiting the clinical benefit of allogeneic hematopoietic cell transplantation (HCT). A noninvasive, diagnostic and prognostic test is needed to determine which patients have active cGvHD at present and which subset will have active cGvHD in the future.

Methods:

We performed peripheral blood gene expression profiling to identify an array of transcriptional biomarkers that discriminate presence of active cGvHD requiring immunosuppression following allogeneic HCT. Peripheral blood mononuclear cells were prepared by Ficoll gradient and cryopreserved from 63 patients (median age 50 yrs; range 19–64) following allogeneic HCT (median 475 days post-HCT) on an IRB approved protocol. Samples were randomly selected into a training set (23 cGvHD and 19 without cGvHD) and test set (12 cGvHD and 9 without cGvHD) and processed on the Agilent whole human genome 44k microarray platform. Bioinformatics programs including AILUN, GeneSpring, SAM, and PAM were used to select a minimum gene-set (false discovery rate, FDR<5%) to predict cGvHD in the training set. Patient characteristics in the two groups were collected for confounder/interaction analysis across the following parameters: age, gender, disease histology, disease status at HCT, graft source, conditioning regimen, white blood count (WBC) at sample, and follow-up status including relapse and survival. Type, grade, and activity of acute GvHD and cGvHD at time of sample and at most recent follow-up were determined by two independent investigators by standard clinical criteria.

Results:

There is considerable heterogeneity among cGvHD patients, not attributable to multiple confounding factors, suggesting molecular heterogeneity in the development and progression of cGvHD. A minimum of 10 unique genes selected from SAM and PAM predict active cGvHD at the time of the peripheral blood sample with 95% sensitivity and 78% specificity in the training set, and 75% sensitivity and 78% specificity in the test set. Eight and four of ten genes are enriched in NK and CD4 T cells, respectively, and involved in the IL-10 signaling pathway (p<0.01). The 10 gene-set predicted the presence of active cGvHD at last follow-up (median 849 days post-HCT) with 85% sensitivity and 83% specificity.

Conclusion:

Peripheral blood gene expression across a 10 gene-set shows strong correlation of the predictive probability of cGvHD and the clinical phenotype. Given reasonable diagnostic sensitivity and specicifity for prediction of future cGvHD, additional longitudinal studies of this cohort and a prospective cohort are underway to further improve the accuracy of this approach and predictive power of this gene-set.

Disclosures:

No relevant conflicts of interest to declare.
MEF2C as Novel Oncogene for Early T-Cell Precursor (ETP) Leukemia
Authors: Homminga Irene , Pieters Rob , Langerak Anton , de Rooi Johan , Stubbs Andrew , Verstegen Monique , Vuerhard Maartje , Buijs-Gladdines Jessica , Kooi Clarissa , Klous Petra , Van lierberghe Pieter , Ferrando Adolfo , Cayuela Jean-Michel , Verhaaf Brenda , Beverloo Berna , Horstmann Martin A. , de Haas Valerie , Wiekmeijer Anna-Sophia , Pike-Overzet Karin , Staal Frank , de Laat Wouter , Soulier Jean , Sigaux Francois , Meijerink Jules
Published: October 2019

Abstract 9

To identify novel oncogenic pathways in T-cell acute lymphoblastic leukemia (T-ALL), we combined expression profiling of 117 pediatric patient samples and detailed molecular cytogenetic analyses. Using unsupervised and supervised analyses, we identified a T-ALL cluster that was associated with an immature immunophenotype (CD1−, CD4−, CD8−), frequent expression of CD34 and co-expression of the myeloid markers CD13/CD33. Patients in this cluster lacked any of the known oncogenic rearrangements, but ectopically expressed MEF2C, which was recently demonstrated as an important transcription factor for T-cell development1. Molecular-cytogenetic analyses including the Chromatine Conformation Capture on Chip (4C) method revealed novel rearrangements of the MEF2C locus at 5q14, rearrangement of transcription factors that target MEF2C (PU.1, NKX2-5, RUNX1) or MEF2C-associated cofactors (NCOA2/GRIP1) in about half of the patients in this cluster. Four out of the 6 rearrangements identified have never been observed before in human cancer. Nearly all of these patients in this cluster could be predicted by the early T-cell precursor (ETP) signature2 using PAM statistics. This indicates that MEF2C may represent the oncogene for ETP T-ALL, an entity that has been associated with poor outcome2. Inhibition of MEF2C in a cell line model system provoked relieve of developmental arrest, indicating that ectopic MEF2C expression blocks T-cell development at an early stage. We demonstrated that MEF2C is a transcriptional regulator for many differentially expressed genes that were associated with the immature cluster including LYL1 and LMO2. Although LYL1 has been suggested as potential oncogene for immature T-ALL cases3, oncogenic rearrangements were never identified in T-ALL cases with immature immunophenotype. Our data therefore imply that high expression of LYL1 (and LMO2) is part of a pathogenic pathway for immature T-ALL that is regulated by the MEF2C oncogene.

1 Stehling-Sun, S., Dade, J., Nutt, S. L., DeKoter, R. P. & Camargo, F. D. Regulation of lymphoid versus myeloid fate ’choice’ by the transcription factor Mef2c. Nat Immunol 10, 289–296, (2009).

2 Coustan-Smith, E. et al. Early T-cell precursor leukaemia: a subtype of very high-risk acute lymphoblastic leukaemia. Lancet Oncol 10, 147–156, (2009).

3 Ferrando, A. A. et al. Gene expression signatures define novel oncogenic pathways in T cell acute lymphoblastic leukemia. Cancer Cell 1, 75–87 (2002).

Disclosures:

No relevant conflicts of interest to declare.
In Vivo Roles for Factor XII
Authors: Renné Thomas
Published: October 2019

Abstract SCI-19

The coagulation system is critical for limiting blood loss at a site of injury (hemostasis), but may also contribute to thrombotic disease. The model of a coagulation balance represents these two sides of the same coin. Thrombosis may occur in the venous or arterial circulation, causing pulmonary embolism or myocardial infarction and stroke, collectively the most common causes of death in the developed world.

Recent data from genetically altered mouse models have challenged the dogma of a coagulation balance. Deficiency of coagulation factor XII (Hageman factor), a serine protease, which initiates the intrinsic pathway of coagulation, severely impairs thrombus formation but is not associated with any excessive bleedings in humans or in mice. Targeting factor XII protects from occlusive disease in experimental animal models. Individuals with hereditary deficiency in the factor XII substrate factor XI are largely protected form ischemic stroke and deep-vein thrombosis. These findings indicate that fibrin-forming mechanisms, which operate during pathologic thrombus formation, involve pathways distinct from those proceeding during normal hemostasis. As the factor XII-driven contact system selectively contributes to thrombosis, but not to hemostasis, inhibition of the system offers novel anticoagulation strategies associated with minimal or no bleeding risk. In contrast to factor XII deficiency states, a single missense mutation (Thr328Lys) in the coagulation protein is associated with a life-threatening swelling disease, hereditary angioedema type III. Pharmacological factor XII inhibitors interfered both with pathological thrombosis and edema formation suggesting broad medical implications.

Factor XII is activated in vivo by platelet-released inorganic polyphosphate, a linear polymer of 60–100 phosphate residues that directly bound to the coagulation factor. Polyphosphates-driven factor XII activation triggered release of the inflammatory mediator bradykinin. Polyphosphates increased vascular permeability and induced skin edema formation in mice and animals deficient in factor XII or bradykinin receptors were resistant to polyphosphates-induced leakage. Polyphosphates were procoagulant via the intrinsic pathway of coagulation. Ablation of intrinsic coagulation pathway proteases factors XII and XI protected mice from polyphosphate-triggered lethal pulmonary embolism. Targeting polyphosphates with phosphatases interfered with procoagulant activity of activated platelets and blocked platelet-induced thrombosis in mice. Addition of polyphosphates restored defective plasma clotting of Hermansky-Pudlak Syndrome patients, who platelet lack storage organelles for polyphosphates. The data identify polyphosphates as the endogenous activator of factor XII having fundamental roles in platelet-driven proinflammatory and procoagulant disorders.

Disclosures:

No relevant conflicts of interest to declare.
Gemtuzumab Ozogamicin as Post-Consolidation Therapy Does Not Prevent Relapse In Children with AML. Results of the NOPHO-AML 2004 Study
Authors: Hasle Henrik , Abrahamsson Jonas , Forestier Erik , Ha Shau-Yin , Heldrup Jesper , Jahnukainen Kirsi , Jonsson Olafur G , Lausen Birgitte , Palle Josefine , Zeller Bernward
Published: October 2019

Abstract 1534

There are no data on the role of post-consolidation therapy with gemtuzumab ozogamicin, (GO, Mylotarg) in children with AML. The NOPHO-AML 2004 protocol included two induction courses and four consolidation courses followed by a post-consolidation randomization to GO or no further therapy (ClinicalTrials.gov identifier NCT00476541).

GO was administered as 5 mg/m2 four weeks after last consolidation and repeated once after three weeks. We randomized 120 patients; 59 to receive GO, 61 to no further therapy. The children in the two groups had similar distribution of sex, age, WBC, and cytogenetics. Treatment was given as randomized in 113 (94% of the patients). Survival was analyzed on an intention to treat basis. The median follow-up for patients alive was 3.2 years (range 0.9 – 7.0).

Toxicity data from the children who received GO showed modest elevation of transaminase and bilirubin but without signs of VOD. No significant decrease in hemoglobin was observed whereas severe neutropenia occurred in 96% of the patients. Recovery to neutrophils > 0.5 lasted a median of 15 days. Febrile neutropenia followed 39% of the GO courses but none were life-threatening. A moderate decline in platelet count was noted with platelets < 50 × 109/L in 27% and transfusion was given following 11% of the GO courses.

Relapse occurred in 22 and 23 of those randomized to GO or no further therapy, respectively. The median time to relapse was 16 months in GO treated vs. 10 months in those receiving no GO (n.s.) (figure). Therapy-related MDS developed in two patients treated with GO. The 3-year EFS and OS in those randomized to receive GO vs. no further therapy was 54% vs. 63% (n.s.) and 75% vs. 84% (n.s.), respectively. The results were similar in all subgroups analyzed.

In conclusion GO therapy post consolidation was well tolerated with severe neutropenia, modest thrombocytopenia and no clinical liver toxicity. GO did not change the frequency of relapses but there was a non-significant delay in the time to relapse from 10 to 16 months for patients randomized to GO.

Disclosures:

Hasle: Pfizer: Research Funding. Off Label Use: A randomized study of gemtuzumab ozagamicin in childhood AML is presented.