bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of Fluorescence in Situ Hybridization (FISH) for EVI1 Rearrangement (3q26) and G-Banding in Patients with Acute Myeloid Leukemia with Inv(3) or t(3;3).
Authors: Choi Qute , Lee Dong Soon , Yoon Sung-Soo , Hwang Sang Mee , Kim Seon Young , Kim Jung Ah , She Cha Ja , Choi Jung Eun , Lee Eun Jung , Kwon Jiseok
Published: October 2019

Abstract 2540

Background:

Acute myeloid leukemia (AML) with inv(3)(q21q26.2) or t(3;3)(q21;q26.2) is a distinct subgroup that may present de novo or arise form a prior MDS, characterized by thrombocytosis and marked dysmegakaryopoiesis. Since the presence of inv(3) or t(3;3) suggests poor prognosis in AML and needs for close monitor in MDS, detection of EVI 1 rearrangement (3q26) is crucial. We performed FISH for inv(3) or t(3;3) and compared those results with those of G-banding in AML, both at initial diagnosis and follow-up.

Methods:

We retrospectively reviewed the results of G-banding in 550 patients with AML (2000 Jan-2012 June). Among them, patients who showed 3q abnormalities by G-banding were subjected to FISH analysis for inv(3) or t(3;3) with frozen bone marrow cells. In patients who showed EVI 1 rearrangement by FISH, we performed EVI 1 rearrangement FISH on serial follow-up bone marrow of those patients, for detection of minimal residual cells. We applied 3 kinds of FISH probes (A company, B company, C company) for the detection of EVI 1 rearrangement.

Results:

By G-banding, the frequency of 3q aberrations involving 3q26 was 1.5% (8/550 patients) in AML and the frequency of AML wit recurrent inv(3) or t(3;3) was 0.2% (1/550 patients) in AML. Among 17 consecutive specimen from 8 patients with 3q26 aberration by G-banding, 11 specimen showed EVI 1 rearrangement by FISH, while 8 specimen showed EVI 1 rearrangement by G-banding. Of note, among 8 patients with 3q26 aberration by G-banding, FISH for EVI 1 rearrangement revealed EVI 1 rearrangement in 5 patients. Furthermore, EVI 1 rearrangement (3q26) FISH detected EVI 1 rearrangement in 1 patients among 30 patients with 3q aberrations other than 3q26 by G-banding. Comparison of FISH results among 3 kinds of FISH probes showed marked discordances. Among 26 consecutive specimens in patients with 3q26 aberrations, probe of each company detected 11 (A company), 4 (B company) and 4 (C company), respectively.

Conclusion:

Incidence of AML with inv(3) or t(3;3) involving EVI 1 rearrangement was rare (0.2%). One third of AML with inv(3) or t(3;3) by G-banding did not harbor EVI 1 rearrangement, suggesting involvement of other gene in the same chromosomal region. Comparison of FISH and G-banding during follow-up in the present study revealed that EVI 1 rearrangement FISH is essential for monitoring for minimal residual disease in AML with inv(3) or t(3;3).

Disclosures:

No relevant conflicts of interest to declare.
EZH2 Mutations Are Associated with Low Blast Percentage in Bone Marrow and −7/Del(7q) Karyotype in De Novo Acute Myeloid Leukemia.
Authors: Wang Xiuli , Dai Haiping , WANG Qian , Wang Qinrong , Xu Yang , Jin Zhengming , Liang Jianying , Qiu Huiying , Sun Aining , Ruan Changgeng , Ruan Jia , Chen Suning , Depei Wu
Published: October 2019

Abstract 2544

Somatic mutation of the EZH2 gene is seen in myelodisplastic syndrome, myelofibrosis, and chronic myelomonocytic leukemia patients. The prevalence and prognostic impact of somatic mutations of EZH2 in patients with acute myelogenous leukemia (AML) remains unknown. In this study, we sought to determine the incidence and clinical implications of somatic EZH2 mutations in 714 patients with de novo AML by PCR amplification of the entire coding region followed by direct bidirectional DNA sequencing. EZH2 mutations were identified in 13/714 (1.8%) of AML patients and occurred almost exclusively in males (11/13, P=0.033). In univariate analysis, the presence of EZH2 mutations was significantly associated with lower blast percentage (21–30%) in bone marrow (P=0.0001) and −7/del(7q) (P=0.025). There was no difference in the incidence of mutations in 13 genes, including ASXL1, CBL, c-KIT, DNMT3A, FLT3, IDH1, IDH2, MLL, NPM1, NRAS, RUNX1, TET2, and WT1, between patients with and without EZH2 mutations. Complete remission, event-free survival or overall survival was similar between AML patients with and without EZH2 mutation (p>0.05). These results demonstrated EZH2 mutation as a recurrent genetic abnormality associated with lower blast percentage in BM and −7/del(7q) in de novo acute myeloid leukemia.

Disclosures:

No relevant conflicts of interest to declare.
Mir-183 Over-Expression: A Potential Biomarker for Juvenile Myelomonocytic Leukemia (JMML).
Authors: Liu Y. Lucy , Yan Yan , Lensing Shelly Y. , Cooper Todd , Emanuel Peter D.
Published: October 2019

Abstract 2558

Juvenile myelomonocytic leukemia (JMML) is a rare disease of early childhood with a predilection for the monocyte/macrophage lineage. The pathogenesis of JMML is linked to dysregulated signal transduction through the NF1/RAS signaling pathway that is partially caused by genetic mutation of Ras, PTPN11, and c-CBL, or loss-of heterozygosity of Nf1. The hallmark of JMML is that JMML cells are selectively hypersensitive to GM-CSF in vitro. We previously reported that protein deficiencies of PTEN, CREB, and Egr-1 were frequently observed in JMML (67–87%). Recent research indicated that CREB was regulated by miR-34b, and Egr-1 was targeted by miR-183. We hypothesized that microRNAs may play an important role in contributing to the deficiency of these proteins. Using relative-quantitative real-time PCR, we evaluated the expression levels of miR-34b and miR-183 in mononuclear cells from 47 JMML patients. We found that the median level of miR-183 was significantly higher in JMML in comparison to normal controls (median=13.8 vs 4.2, p<0.001); but the median level of miR-34b was only slightly higher in JMML subjects, and not significantly so, compared to normal individuals (median=1.4 vs 1.0, p>0.05). This suggests that miR-34b does not play a significant role in JMML. Since extreme monocyte accumulation is one of the critical characteristics of JMML, we analyzed the correlation between the expression level of miR-183 and the monocyte percentage in the peripheral blood. Strikingly, there was a significant correlation between the expression level of miR-183 and the monocyte percentage in the peripheral blood from 34 patients who had available data (p<0.05). Based on a robust regression analysis, for every unit increase in the square root of RQ miR-183, the monocyte percentage significantly increased by 0.73% (SE=0.32%, p=0.023). This is the first evidence suggesting that microRNAs may contribute to the pathogenesis of JMML. miR-183 may also serve as an important biomarker that can be directly and quantitatively linked to significant clinical parameters in JMML. It also may ultimately provide a target for JMML therapy.

Disclosures:

No relevant conflicts of interest to declare.
Cerebrospinal Fluid Flow Cytometry in Acute Lymphoblastic Leukemia/Lymphoma: Is It Beneficial?.
Authors: Rassi Fuad El , Mitri Zahi , Heffner Leonard T , Langston Amelia , Waller Edmund K. , Winton Elliott F. , McLemore Morgan L. , Bernal Leon , Arellano Martha , Khoury H. Jean
Published: October 2019

Abstract 2576

Cerebrospinal fluid (CSF) involvement by leukemic blasts occurs in fewer than 10 % of adult patients with newly diagnosed acute lymphoblastic leukemia/lymphoma (ALL). Leukemic meningitis is diagnosed by microscopic detection of blasts in the CSF. Flow cytometry is a highly sensitive tool for detection of aberrant cells. We sought to analyze the additional benefit flow cytometry might provide for the diagnosis of leukemic meningitis. Between 11/2007 and 8/2011, 80 patients were diagnosed with ALL and treated at Emory University. 800 CSF samples were available for analysis 80 of which were collected from a diagnostic lumbar puncture (LP), 689 from follow-up LPs and 31 from LPs obtained at the time of relapse. As shown in the table, flow cytometry confirmed the presence of leukemic blasts in one, four and five samples diagnosed with leukemic meningitis by cytology at diagnosis, different stages of treatment and relapse, respectively. One and three samples were positive for leukemic blasts by cytology but negative by flow cytometry during different treatment stages and relapse respectively. We conclude that flow cytometry provided no additional benefit to cytology in the diagnosis of leukemic meningitis.

Table: CSF Cytology and Flow Cytometry in 80 Adult ALL patients: CSF samples New Diagnosis N=80 Induction/Consolidation/Intensification/Maintenance/Remission/Post-transplant N = 689 Systemic relapse N = 31 N Cytology N = 80 Flow cytometry N = 66 Cytology N = 689 Flow cytometry N = 188 Cytology N = 31 Flow cytometry N = 13 Negative 79/80 65/66 684/689 184/188 23/31 8/13 Positive 1/80 1/66∼ 5/689 4/188∼* 8/31 5/13∼** ∼ CSF samples positive by flow cytometry were also positive by cytology * One CSF sample was positive by cytology but negative by flow cytometry ** CSF flow cytometry was not done in 3/8 positive CSF samples by cytology

Disclosures:

No relevant conflicts of interest to declare.
Physicians Compliance During Maintenance Therapy in Children with Down Syndrome and Acute Lymphoblastic Leukemia.
Authors: Bohnstedt Cathrine , Levinsen Mette , Rosthøj Susanne , Zeller Bernward , Taskinen Mervi , Hafsteinsdottir Solveig , Björgvinsdóttir Helga , Heyman Mats , Schmiegelow Kjeld
Published: October 2019

Abstract 2577

Children with Down syndrome (DS) and acute lymphoblastic leukemia (ALL) have an inferior prognosis compared to non-DS ALL patients. We reviewed Methotrexate (MTX)/Mercaptopurine (6MP) maintenance therapy data for children with DS treated according to the NOPHO ALL92 or the NOPHO ALL2000 protocols between 1992 and 2007. The five year event-free survival (pEFS5y) for the DS patients was inferior to the non-DS patients (0.50 ± 0.07 vs 0.77 ± 0.01, p<0.001). The 48 DS patients in 1st remission at the beginning of maintenance therapy had pEFS10y below the 522 non-DS control patients (pEFS10y: 0.58, 95%-CI 0.43–0.77 vs. 0.83, 95%-CI 0.80–0.86, p<0.0001). The DS patients received lower median doses of MTX (Median: 11.8 vs 15.4, p<0.0001) and 6MP (median: 45.6 vs. 59.4, p<0.0001). In Cox regression analysis male gender, presence of DS, and high median maintenance therapy white blood cell levels (mWBC) were associated with increased risk for relapse. The mWBC hazard ratio for DS-ALL patients was 2.0, p<0.0005). This supports that DS children with ALL should be treated as vigorously as non-DS ALL patients. Obtaining WBC levels within target range could potentially increase their cure rates.

Figure 1: Kaplan-Meier curves for event free survival for DS and non-DS children. Figure 1:. Kaplan-Meier curves for event free survival for DS and non-DS children.

Non-DS 513 464 443 433 418 DS 47 25 19 12 6

Disclosures:

No relevant conflicts of interest to declare.
Addition of All-Trans Retinoic Acid (ATRA) to the Combination of Fludarabine, Cytarabine, Idarubicin, with or without GCSF in Older, Higher Risk Patients with AML and High-Risk MDS Does Not Improve the Outcome in Those with NPM1 Mutation.
Authors: Nazha Aziz , Bueso-Ramos Carlos E. , Faderl Stefan , Estey Elihu H. , O'Brien Susan M. , Farnandez Michael , Nguyen Martin , Koller Charles , Freireich Emil , Beran Miloslav , Pierce Sherry , Cortes Jorge E. , Kantarjian Hagop M. , Ravandi Farhad
Published: October 2019

Abstract 2616

Background:

Previous studies have been conflicting in their findings regarding whether NPM1 gene mutations may be a marker for response to all-trans retinoic acid (ATRA) given as an adjunct to intensive chemotherapy in patients with acute myeloid leukemia (AML) (Schlenk RF, Haematologica, 2009, 94, 54–60, Burnett AK, Blood, 2010, 115,948, and Schlenk RF, abstract #80, ASH 2011).

Patients and Methods:

we examined the impact of the addition of ATRA among patients with diploid cytogenetics treated on a randomized phase II study of fludarabine + cytarabine + idarubicine +/− GCSF +/− ATRA with available data on their NPM1 mutation status. Between September 1995 and November 1997, 215 patients with newly diagnosed AML (n=153) or high-risk MDS (RAEB or RAEB-T; n=62) were enrolled in the study. They had to have one of the unfavorable features of age > 71 years, antecedent hematological disorder (AHD), therapy- related disease, or high bilirubin (>2.9 mg/ml) or creatinine (>1.5 mg/ml) to be eligible to participate in the study (Estey E, Blood, 1999, 93, 2478). Among them 70 patients had diploid cytogenetic and are the subjects of this analysis.

Results:

The median age of the 70 patients was 66.5 (range, 23–87), 60% had AHD, 15.7% had therapy-related disease, and 40% were older than 71. Bone marrow samples were available for all patients for examination by immunohistochemistry (IHC) for the presence of cytoplasmic NPM (correlating to NPM1 mutation). Twenty (29%) of patients had NPM1 mutation. Among them 7 (35%) did and 13 (65%) did not receive ATRA in combination with chemotherapy. Complete remission (CR) was achieved in 5 (71.4%) of patients treated with ATRA as compared to 9 (69.2%) without ATRA (p=NS). The median event-free survival was 82 weeks vs. 56 weeks for patients receiving ATRA or not [(p=0.5), ranges, (10–173 weeks) and (1–646 weeks), respectively]. The median overall survival was 41 weeks vs. 60 weeks for patients receiving ATRA or not [(p=0.74), ranges, (3–452 weeks) and (0–735 weeks), respectively].

Conclusion:

There was no significant difference in CR rate, EFS, or OS in this higher risk population of patients with NPM1 mutated diploid AML or high-risk MDS receiving ATRA as an adjunct to chemotherapy.

Disclosures:

Kantarjian: Genzyme: Research Funding. Ravandi:genzyme: Research Funding.
Is Interim/Mid-Treatment PET (interim PET, i-PET) Scan Predictive of Outcome in Diffuse Large B-Cell Lymphoma?.
Authors: Verma Dushyant , Takalkar Amol , Shi Runhua , Mills Glenn M. , Paladugu Srikanth , Veillon Diana , Munker Reinhold , Hildebrandt Gerhard , Nair Binu S. , Koshy Nebu
Published: October 2019

Abstract 2675

Background:

Initial treatment of diffuse large B-cell lymphoma (DLBCL) involves 6–8 cycles of chemo-immunotherapy and may be curative in 60–65% of patients. However, in the remaining patients, subsequent therapies appear inadequate for long lasting remission. A strategy to improve patient outcomes could involve early identification of patients who do not respond to treatment as expected and then employing different/aggressive treatment modalities in these patients. PET scan done during mid-treatment (interim PET, i-PET) may help identify these patients early. However, the value of i-PET in DLBCL is not established as there is controversy about its prognostic value and studies are ongoing to evaluate its benefit.

Aims:

To determine predictive value of i-PET on progression-free survival (PFS), overall survival (OS) in DLBCL patients.

Methods:

We performed retrospective analysis of DLBCL patients treated at LSU Health Shreveport, LA, between Jan 2002 – July 2012. All patients were treated with R-CHOP chemotherapy. PET-CT was performed at baseline at time of diagnosis, after 2 to 4 courses (i-PET) and at the end of therapy (final PET, f-PET).

Results:

Forty-four patients were evaluable for analysis. The median age was 55 years (range 21–84), 32 (73%) were males. Ann-arbor staging showed 5 patients each in stage I and II, 11 patients in stage III, 23 in stage IV, and the median IPI score was 3. Median time to i-PET was after 3 cycles of chemotherapy, and median days to i-PET after chemotherapy were 16. The median follow-up duration from start of chemotherapy was 23 months (range 4 – 89). The PET results were as follows: i-PET negative 30 (68%), i-PET positive 14 (32%) patients. Final PET results were: f-PET negative 33 (75%), f-PET positive 11 (25%) patients. The 3-year PFS was 96.3% and 35.7% for i-PET negative versus positive patients respectively (p<0.001), and the 3-year PFS for f-PET negative versus positive patients was 78.9%% versus 30.0% respectively (p<0.001). The 3-year OS was 79.4% and 62.6% for i-PET negative versus positive patients respectively (p=0.3306). The 3-year OS was 79.9% and 58.7% for f-PET negative versus positive patients respectively (p=0.021).

Conclusion:

Interim/mid-treatment PET (i-PET) scan is predictive of progression free survival but not overall survival for DLBCL patients. A final PET (f-PET) scan is predictive of progression free survival as well as overall survival for DLBCL patients. Larger prospective studies are needed to confirm these findings and could also look into the biology of i-PET positive patients by gene expressing profiling (GEP) and evaluate the role of novel agents in modifying the disease course.

Disclosures:

No relevant conflicts of interest to declare.
The Difference in the Incidence and the Trend of Malignant Lymphomas in Japan and the United States.
Authors: Chihara Dai , Ito Hidemi , Matsuda Tomohiro , Shibata Akiko , Sobue Tomotaka , Matsuo Keitaro
Published: October 2019

Abstract 2695

Background:

Malignant lymphomas (ML) are heterogeneous groups that the detailed classification is evolving dramatically. An incidence of malignant disease in certain population reflects cumulative exposure to environment, genetics and their combination overtime. Therefore, a comparison of incidences in various population and their secular trends is very helpful to understand etiology of disease. The aim of this study is to assess the incidence and the trend of each ML subtypes and to evaluate the difference between Japan and US.

Materials and Methods:

We used the data from a population-based cancer registry in Japan and from the Surveillance Epidemiology and End Results (SEER) program 9. Registry data of the US, SEER 9 included 95,155 cases and the data of Japan included 48,658 cases. The period covered in this analyses was 1993 to 2006 in Japan and 1993 to 2008 in the US. Rates of sex-specific, age-standardized incidence with 95% confidence intervals were estimated and standardized by age-adjustment according to the world standard population. We also estimated the annual percent change using joinpoint regression analysis and evaluated the significance of the trend.

Results:

The overall age-standardized incidence rate of all malignant lymphomas per 100,000 in 2006 was 22.4 for males and 16.0 for females in the US, 7.4 for males and 4.9 for females in Japan. The incidence is higher in the US than Japan with most of the subtypes especially for the nodular sclerosis HL, CLL/SLL and FL. In general, B-cell lymphomas showed large difference in incidence while T-cell lymphomas (TCL) showed similar incidence between Japan and the US. The total numbers of ML are constantly increasing in Japan but not in the US as shown in the figure {annual percent change (95%CI), Japan; +2.6% (2.1, 3.1), US; +0.2% (−0.0, 0.4)}. As for details, follicular lymphoma, mantle cell lymphoma, Burkitt lymphoma and the total numbers of TCL are constantly increasing in both countries.

Conclusion:

In conclusion, we showed the marked difference in the incidence and the trend of hematologic malignancies between Japan and the US. The incidence of hematologic malignancies is lower in Japan than the US, but is increasing significantly. The most remarkable difference in the incidence was seen with nodular sclerosis HL, CLL/SLL and FL which was consistent with previous reports. Even with the TCL, the incidence seems to be similar to higher in the US except for the ATLL. The improvement in the quality of cancer registry systems and the organization of these registries between countries enables us to unite the data worldwide that will bring us new insights.

Solid line and circle are the data of the US. Dashed line and hollow circle are the data of Japan.

Disclosures:

No relevant conflicts of interest to declare.
Thrombus Development Processes Are Dependent On Endothelial Injuries: Examined By In Vivo Molecular Imaging
Authors: Nishimura Satoshi , Eto Koji , Nagai Ryozo
Published: October 2019

The thrombotic cellular mechanisms associated with cardiovascular events remains unclear, largely because of an inability to visualize thrombus formation. In addition, the contribution of endothelial cell (EC) injuries to thrombus formation processes are unclear, and we developed in vivo imaging technique based on single- and multi-photon microscopy to revealed the multicellular processes during thrombus development (Figure a,b). We visualized the cell dynamics including single platelet behavior, and assessed dynamic cellular interplay in two thrombosis models. First, we visualized that rapidly developing thrombi composed of discoid platelets without EC disruption was triggered by ROS photochemically induced by moderate power laser irradiation (Figure c). In this model, thrombus consisted by discoid platelet aggregations without leukocyte recruitment. The second model is, thrombus with EC disruption. High power laser induced EC erosion and extravasations of circulating leukocytes with thrombus development. Inflammatory cytokine, adhesion molecules dynamically control these two processes. (Figure d)Figure.Figure.

Using this technique, we elucidated that Lnk (adapter protein) regulates integrin signaling leading to stabilization of developing thrombus without EC disruption. Specifically, adhesion molecules dynamically control these processes. Thrombus formation was initiated by the binding of platelet GPIb-alpha to endothelial von Willebrand Factor in this model, and actin linker talin-dependent activation of alphaIIb-beta3 integrin in platelets was required for late phase thrombus stability.

As for the thrombus formation with EC disruption, chemokine expressions in endothelium and leukocyte (especially neutrophils) recruitment played a significant role in these processes. TLR4 signaling also contributed to these steps.

In sum, using our imaging system can be a powerful tool to analyze thrombus formation and evaluate the therapeutic strategies.

Disclosures:

No relevant conflicts of interest to declare.
The Use Of Platelet Transfusions In The Intensive Care Unit and Impact On Platelet Count: A 30,000 Patient Registry Study
Authors: Ning Shuoyan , Barty, MLT Rebecca , Liu Yang , Heddle Nancy , Arnold Donald
Published: October 2019

Background

Thrombocytopenia is a common complication of critical illness and an independent risk factor for bleeding and death in the intensive care unit (ICU). Platelet transfusions are commonly used to improve platelet counts; however, the expected platelet increment from a transfusion in this setting has not been established. The objective of this study was to describe the frequency of platelet transfusion administration and their effect on platelet count increments in a large cohort of non-oncology critically ill adults.

Methods

We performed an analysis of a registry database, which was developed to capture clinical and laboratory data on all blood transfusions administered in 3 academic hospitals in Hamilton, Ontario, Canada. We included all patients ≥18 years who received one or more platelet transfusion during an ICU admission. Data validation was done by integrity checks with medical records and laboratory information system performed by a biostatistician. Non-transfused ICU patients were used as controls. The absolute increment in platelet count was calculated for each single platelet transfusion using the closest platelet count taken within 24 hours before the transfusion and 4-24 hours after the transfusion.

Results

Between April 2006 and October 2012, 33,222 patients were admitted to ICU, including 29,511 (88.8%) who did not have a diagnosis of cancer. Of those, 4,502 (15.3%) received one or more platelet transfusion during any ICU admission (n=4,690); 31.9% were female and median age at the time of first admission was 69 years (IQR 59-77). Among the 25,009 non-transfused patients admitted to ICU during the same period, 38.1% were female and the median age was 65 years (IQR 52–76). Median pre-transfusion platelet count was 87 x109/L (IQR 59-131) and a single platelet transfusion resulted in a median platelet count increment of 21 x109/L (IQR 6-40) as measured 6.7 hours (IQR 5.1-9.8) after the transfusion. There were 277 (25.4%) transfusions that yielded a platelet count increment of 5 x109/L or less. ICU mortality was 562/4,690 (12.4%) for patients who received a platelet transfusion, compared with 2,251/33,033(6.8%) for patients who were not transfused during their ICU stay.

Summary/Conclusion

Among this large cohort of non-oncology ICU patients, platelet transfusions were commonly administered for thrombocytopenia that was generally mild. In this setting one platelet transfusion resulted in a median platelet count rise of 21 x109/L. Many transfusion episodes yielded no appreciable increase in platelet count. Further studies are needed to determine the clinical effects of platelet transfusion in this setting controlling for confounding.

Disclosures:

Heddle: CIHR: Research Funding; Canadian Blood Services: Membership on an entity’s Board of Directors or advisory committees; Health Canada: Research Funding; Macopharma: Consultancy; ASH: Honoraria.
Procoagulant Activity In Stored Units Of Red Blood Cells
Authors: Butenas Saulius , Aleshnick Maya , Foley Jonathan H , Keating Friederike K.
Published: October 2019

It has been observed that the procoagulant activity of stored units of red blood cells (RBC) increases over time, which is, in part, related to the expression/exposure of tissue factor (TF) on blood cells and microparticles (Keating et al, Transfusion 2011;51:1086). However, in many instances there is a discrepancy between the levels of TF measured in stored units of RBC and changes in procoagulant activity observed over the storage time. We hypothesized that, in addition to TF, an exposure of an acidic phospholipid, phosphatidylserine (PS), on blood cells (Whelihan et al, Blood 2012;120:3837), could be the cause of an elevated procoagulant activity.

Four healthy donors were recruited. RBC units were prepared and stored at 4°C for 30 days. On selected days, aliquots were removed, reconstituted with autologous plasma and recalcified, and were tested in the presence of corn trypsin inhibitor in the thromboelastography assay for procoagulant activity. For all 4 donors, the clotting time decreased from ∼3000-4000 s on day 1 to ∼1000-1600 s on day 30, with the most dramatic changes occurring between days 1 and 10. Addition of an inhibitory anti-TF antibody slightly prolonged clotting times, suggesting the presence of endogenous TF in RBC units. The concentration of TF, quantitated in an activity-based assay, did not change significantly over time for 3 of 4 donors and was within the range of 40-130 fM. For the fourth donor, TF concentration reached a maximum of 310 fM on day 10 and then decreased steadily to 60 fM on day 30.

In an attempt to identify other components in RBC units responsible for the procoagulant activity, the effect of lactadherin, which binds to the phosphatidylserine exposed on cell membranes, was investigated. An addition of 100 nM lactadherin substantially prolonged the clotting time of the reconstituted RBC. Thus, in days 3-5 the clotting time in the presence of lactadherin was by 2.4-3.4-fold longer than in the absence of it. With increasing age of the RBC units, the effect of lactadherin diminished and at day 30, a prolongation of the clotting time did not exceed 1.3-1.8-fold. The addition of anti-TF monoclonal antibody in the presence of lactadherin further enhanced the clotting time of the reconstituted RBC.

In conclusion, our data indicate that in stored units of RBC, the procoagulant activity is dependent upon the presence of active monocyte TF and phosphatidylserine exposed on blood cells and platelets. However, the failure of lactadherin in combination with the anti-TF antibody to completely inhibit the procoagulant activity in RBC units suggests that other components of blood (most likely platelets) support this activity as well.

Disclosures:

No relevant conflicts of interest to declare.
The Transcription Factor IRF8 is a Key Transcription Factor for Basophil Development
Authors: Kurotaki Daisuke , Sasaki Haruka , Osato Naoki , Sasaki Izumi , Kaneda Chika , Sato Hideaki , Nishiyama Akira , Kaisho Tsuneyasu , Aburatani Hiroyuki , Morse Herbert C. , Ozato Keiko , Tamura Tomohiko
Published: October 2019

Basophils are the rarest granulocytes circulating in the peripheral blood. They play critical roles in anti-parasite Th2-type immune responses and chronic allergic disorders. The developmental pathway for basophils has been recently demonstrated; myeloid progenitors pass through common myeloid progenitors, granulocyte-monocyte progenitors, granulocyte-committed progenitors (GPs), and basophil-committed progenitors (BaPs) in the bone marrow. BaPs then give rise to mature basophils. However, our understanding of how this pathway is regulated remains still elusive. Interferon Regulatory Factor-8 (IRF8), a hematopoietic cell-specific IRF transcription factor, is essential for the development of monocytes, dendritic cells, and eosinophils, while it inhibits neutrophil differentiation. Its role in the development of basophils has yet to be analyzed.

In this study, we investigated whether IRF8 has any role in the development of the basophil lineage. We found that Irf8–/– mice displayed a severe reduction of basophil counts in the bone marrow, peripheral blood and spleen compared to wild-type (WT) mice. Irf8–/– mice retained GPs but lacked BaPs. Cell transfer experiments revealed that the defect of basophil development in Irf8–/– mice resides in bone marrow cells. We utilized IRF8-GFP chimera knock-in mice to examine IRF8 protein expression in the basophil lineage at a single cell level. We found that GPs, but not BaPs and mature basophils, expressed IRF8. Furthermore, purified Irf8–/– GPs failed to efficiently give rise to basophils in vitro. These results indicate that IRF8 acts at the stage of GPs in a cell-intrinsic manner. To understand the mechanism by which IRF8 promotes basophil development, we performed transcriptome analysis of purified GPs from WT and Irf8–/– mice by microarray. Because IRF8 is no more expressed in BaPs, we envisaged that IRF8 acts by inducing downstream transcription factors in GPs. The expression of several transcription factor genes such as Gata2 and Spib was reduced in Irf8–/– GPs compared to WT GPs. Analysis of DNA motifs in the promoter regions of genes downregulated in Irf8–/– GPs predicted that GATA transcription factor(s) may act downstream of IRF8. Indeed, retroviral transduction of GATA2, known to be essential for basophil development, into Irf8–/– hematopoietic progenitor cells rescued basophil differentiation in vitro. On the other hand, Spib–/– mice showed no obvious defects in basophil development. Taken together, these results suggest that the IRF8-GATA2 axis in GPs critically regulates basophil development.

Disclosures:

No relevant conflicts of interest to declare.
Cyclosporine A May Affect the Expression of Foxp3 by Regulation of Akt and Stat3 Phosphorylation in CD4+CD25+ Regulatory T Cells from Mouse Model of Severe Aplastic Anemia
Authors: Lin Zenghua , Liu Hong , Wang Xuli , Zhang Yaping , Cai Yifeng , Qian Yu
Published: October 2019

Although Cyclosporine A(CsA) is an effective immunosuppressant in the treatment of aplastic anemia(AA), its affects on CD4+CD25+ regulatory T cells(Tregs) from patients with AA still remains controversial. We have first developed a novel mouse model of severe aplastic anemia(SAA) induced by administration with IFN-γ and busulphan. In this study, the SAA model female mice were intragastrically administered with CsA at daily dose of 5 mg/kg for 10 days(the CsA-treated group, n=60), the SAA group(n=60) and the un-treated group (n=60) were as control. All mice selected to set up the SAA model developed the typical clinical and pathological patterns of SAA from day 10 post dosing. Most of them presented bleedings in association with anemia and infections. At day 15 after treatment, a mortality of 35% was found in the SAA group, while a mortality of 20% was found in the CsA-treated group because of impaired liver function and infections. In order to explore the effect of CsA on CD4+CD25+ Tregs from the novel mouse SAA model, the mononuclear cells of the peripheral blood and spleen were subjected to assess the percentage of CD4+CD25+ Tregs and the intracellular Foxp3 expression in CD4+CD25+ Tregs by flow cytometry(FCM). In the SAA group, the FCM analysis showed decreased ratios of CD4+CD25+ Tregs in CD4+ T cells and down-expression of Foxp3 in CD4+CD25+ Tregs compared with the un-treated group(P<0.05, respectively). These results were in accordance with our previous observations. However, after treatment with CsA, the expression of Foxp3 in CD4+CD25+ Tregs was increased(P<0.05) but not the ratios of CD4+CD25+ Tregs in CD4+ T cells(P>0.05). In addition, after being pured by immunomagnetic beads, the splenic CD4+CD25+ Tregs was subjected to assess the Akt and Stat3 phosphorylation using Western blot. Compared with the un-treated group, CD4+CD25+ Tregs with increased Akt phosphorylation accompanied by increased Stat3 phosphorylation was found in SAA group(P<0.05, respectively). On the contrary, CsA-treated group exhibited CD4+CD25+ Tregs with a reduction in both Akt phosphorylation and Stat3 phosphorylation compared with the SAA group(P<0.05, respectively). These results indicated that CsA may increase the expression of Foxp3 by up-regulation the levels of Akt and Stat3 phosphorylation in CD4+CD25+ Tregs from the novel mice model of severe aplastic anemia.

Disclosures:

No relevant conflicts of interest to declare.
Activation Of Akt Enhances Ribosomal RNA Synthesis In AML Cells Through a Novel Isoform Of TIF-1A and Inhibition Of Filamin A Cleavage
Authors: Truong Nguyen Le Xuan , Huang Min , Chan Steven M. , Majeti Ravi , Mitchell Beverly S.
Published: October 2019

PI3Kinase/Akt signaling is frequently activated in acute myelogenous leukemia (AML) and inhibitors of this pathway have shown some promise in both pre-clinical and early clinical studies. In particular, much attention has been focused on components of the mTOR complex as downstream effectors of Akt activation that promote cell proliferation and survival in AML and other malignancies. We have discovered a direct effect of activated and phosphorylated Akt in increasing ribosomal RNA (rRNA) synthesis in both leukemic cell lines and primary AML cells by a novel pathway that is independent of mTOR. We have defined TIF-90 as a splice variant of TIF-1A, a molecule that tethers RNA Polymerase I to the ribosomal DNA promoter and is required for rRNA synthesis. TIF-90 is localized in the nucleolus and directly co-locates with Pol I in imaging, pull down, and rDNA promoter occupancy studies. Activation and phosphorylation of Akt correlates with high levels of TIF-90 expression and increased TIF-90 occupancy of the rDNA promoter in primary AML cells, while overexpression of constitutively activated Akt (Akt-Myr) increases rRNA synthesis in a TIF-90-dependent fashion in cell lines. Activation of Akt increases the expression of TIF-90 by inhibiting its ubiquitination and proteasomal degradation by the p53 ubiquitin ligase, Mdm2. Furthermore, activated Akt directly inhibits the cleavage of the actin-binding protein, Filamin A, into a 90 kDa fragment that interacts with TIF-90 and prevents it from occupying the rDNA promoter. Expression of the 90 kDa Filamin A cleavage fragment alone is sufficient to inhibit rRNA synthesis by binding to TIF-90. Thus, inhibition of Filamin A cleavage by Akt is associated with increased rRNA synthesis. The Akt inhibitor AZD8055, but not the mTORC1 inhibitor Rapamycin, promotes the cleavage of filamin A, decreasing both TIF-90 occupancy of the rDNA promoter and rRNA synthesis. Examination of 21 primary AML samples reveals an inverse correlation between the phosphorylation of Akt and the cleavage of Filamin A; conversely, AZD8055 inhibits both Akt phosphorylation and rRNA synthesis in these samples while promoting the cleavage of Filamin A. These data demonstrate a novel pathway by which Akt directly enhances rRNA synthesis. This pathway is active in primary AML cells and in cell lines that have high levels of phosphorylated Akt. Inhibition of rRNA synthesis has been shown to result in nucleolar disruption, cell cycle arrest, and ultimately cell death. Our data support an mTOR-independent effect of Akt activation on cell proliferation that would require direct targeting of PI3Kinase or activated Akt to have a major therapeutic effect in AML.

Disclosures:

No relevant conflicts of interest to declare.
Absence Of The E3 Ubiquitin Ligase SKP2 Attenuates Notch-Induced T-Cell Leukemogenesis
Authors: Rodriguez-Rodriguez Sonia , Wang Lin , Zhang Huajia , Zollman Amy , Cardoso Angelo , Carlesso Nadia
Published: October 2019

Acute Lymphoblastic Leukemia (ALL) is the most common malignancy in childhood, accounting for almost 30% of pediatric cancers. In pediatric T-cell acute lymphoblastic leukemia (T-ALL), around 30% of the children still undergo disease relapse, which is associated with poor prognosis. Hence, novel therapeutic strategies for the treatment of T-ALL are necessary, since conventional therapies still fail to cure a significant number of patients. Notch signaling contributes to the regulation of normal T-cells homeostasis, whereas oncogenic, activated form of Notch is present in the majority of the T-ALL cases.

Previous studies in our laboratory demonstrated that Notch1 signaling induces transcriptional activation of SKP2, the F-box protein of the SCF E3-ubiquitin ligase complex. SKP2 targets the major cell cycle inhibitors, the CKIs p21Cip1, p27Kip1, p57Kip2 and p130, for proteasome-mediated degradation. Increased Skp2 expression accelerates cell cycle progression in hematopoietic cells, and its overexpression is frequently found in cancers, in particular lymphomas and leukemias, in which is associated with poor prognosis.

We found that Skp2 expression is dynamically regulated during T-cell differentiation coinciding with the Notch expression pattern. Moreover, primary thymocytes cultured in vitro responded to Notch stimulation by the Delta1 ligand increasing their Skp2 expression and their cell cycle activity. As anticipated, we found that Skp2 expression is increased in T-ALL patient samples. Our hypothesis is that Notch activation promotes T-cell leukemogenesis by altering the cell cycle control through upregulation of SKP2. To better define the role of SKP2 in T-ALL, we analyzed the Skp2 expression in a murine model of Notch-induced T-cell leukemia. Mice transplanted with stem/progenitor cells transduced with the constitutive active form of Notch (ICN), developed T-ALL and died by week 12 after transplant. Analysis of T-ALL cells revealed a 5 fold upregulation of Skp2 expression compared to controls. Next, we tested whether SKP2 was required for T-ALL development. To this end, we evaluated leukemia development in mice transplanted with SKP2 deficient cells overexpressing ICN. Stem/progenitor cells derived from Skp2+/+, Skp2+/- or Skp2-/- mice were transduced with ICN and then transplanted into WT recipients. Loss of SKP2 significantly delayed the development of T-cell leukemia in transplanted mice and increased their survival by 60% at 12 weeks.

Taken together, these results demonstrate a previously unrecognized role for SKP2 in the initiation and progression of T-ALL and its potential role as a therapeutic target.

Disclosures:

No relevant conflicts of interest to declare.
Allelic Forms Of PRDM9 Associated With High Hyperdiploid Childhood Acute Lymphoblastic Leukaemia
Authors: Woodward Eleanor L , Olsson Martin L. , Johansson Bertil , Paulsson Kajsa
Published: October 2019

Whilst a great deal is known about acquired somatic aberrations associated with the diagnosis and prognosis of acute leukaemias, relatively little is known about the effects of germline variation. A recent study reported a link between germline variations in the zinc finger (ZnF) binding domain array of the PR domain-containing 9 (PRDM9) gene, regulator of meiotic recombination, with the development of acute lymphoblastic leukaemia (ALL) in children. An excess of rare PRDM9 alleles that affect meiotic recombination events were seen in parents of children affected with B-cell precursor ALL (pre-B-ALL), and in an independent cohort of children with pre-B-ALL; in particular in aneuploid and infant ALL. In the present study, we carried out Sanger sequencing to investigate variation of PRDM9 alleles in a cohort of parents (n=59) with children diagnosed with high hyperdiploid ALL (HeH; 51-67 chromosomes) (n=31) and a control cohort of individuals (n=66) from the south of Sweden. Whereas similar numbers of rare PRDM9 alleles were observed in both groups, a larger number of rare alleles that affect recombination events were observed in the parents from the family cohort with children diagnosed with HeH ALL compared to individuals of the population control cohort (15.3% vs. 4.5%, P=0.0414). Two-thirds of the parents transmitted the rare allele to their affected children. A previously unreported ZnF repeat was also detected in two individuals of the control cohort but it was not detected in the family cohort. A statistically significant difference in frequency of rare alleles affecting recombination events between the two groups indicates a true association between PRDM9 allelic forms and HeH ALL in an independent cohort. Thus, our results confirm the previous findings that PRDM9 may play a role in the development of childhood ALL.

Disclosures:

No relevant conflicts of interest to declare.
The Polymorphisms Of Base Excision Repair Genes Influence The Cytogenetic Risk Factors In Acute Myeloid Leukemia
Authors: Iwasaki Atsushi , Saitoh Takayuki , Nitta Yasuhiro , Norjimaa Batchimeg , Omiya Chiharu , Kamiya Akira , Hatsumi Nahoko , Shimizu Hiroaki , Ishizaki Takuma , Hoshino Takumi , Takada Satoru , Handa Hiroshi , Sakura Toru , Yokohama Akihiko , Tsukamoto Norifumi , Murakami Hirokazu
Published: October 2019

Background

Base excision repair (BER) systems have important role for repairing oxidative DNA damage, and known to influence the carcinogenesis and the response to anti-cancer treatments. Although few studies have shown that several DNA repair genes are associated with an increased risk of leukemia, the clinical significance of BER polymorphisms in acute myeloid leukemia (AML) patients remains unclassified. The aim of this study was to evaluate the impact of polymorphisms in genes encoding four main proteins of BER system: OGG1 Ser326Cys, MUTYH Gln324His, APE1 Asp148Glu, and XRCC1 Arg399Gln, and on the risk of AML.

Methods

Between December 1991 and May 2013, 99 patients (male/female 55/44, median age 58 years, range 15-86 years) diagnosed as AML and 192 healthy controls were included in this study. Cytogenetic subgroups were classified as good, intermediate, and adverse risk according to NCCN guidelines. Genomic DNA was isolated from peripheral blood using the DNA extraction kit. Genotyping was determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Genotype and allele frequencies were compared between patients group and control group by using χ2-test. Probability values <0.05 were considered statistically significant. All patients and healthy controls received written information about the study. This study was approved by the Institutional Research Board of Gunma University Hospital.

Results

The APE1 Asp/Asp genotype increases the risk of AML (OR 2.30, 95% CI 1.41-3.77, p<0.001), whereas APE1 Glu/Glu genotype reduces the risk of AML (OR 0.34, 95% CI 0.14-0.80, p<0.05). In contrast, there were no significant differences in the genotype frequencies OGG1 Ser326Cys, MUTYH Gln324His, and XRCC1 Arg399Gln between AML patients and control group. Next we compared the frequency of cytogenetic abnormalities according to BER polymorphism. The AML patients with OGG1 Ser/Ser genotype increased the frequencies of (15;17) type (p<0.05) and good risk group. Moreover, the AML patients with MUTYH His/His genotype increased the frequencies of complex type (p<0.02) and reduced the frequencies of t(8;21) type.

Conclusions

According to our data, BER gene polymorphisms may affect the carcinogenesis and the cytogenetic risk of AML.

Disclosures:

No relevant conflicts of interest to declare.
Development Of a Preclinical Human Xenograft Model For Myelodysplastic Syndrome In Immunodeficient Mice Expressing Human Growth Factors
Authors: Scaria George , Bendzick Laura , Shultz Leonard , Kaufman Dan S.
Published: October 2019

Myelodysplastic syndromes (MDS) are a diverse class of hematopoietic disorders, defined by progressive bone marrow failure, myeloid dysplasia, and an increased risk of acute myeloid leukemia (AML). The development of treatments for MDS has been hampered by the lack of an adequate in vivo model of disease. Mouse xenograft models for MDS have been challenging, as samples from patients with MDS have exhibited poor engraftment when transplanted into immunodeficient mice, with little evidence of persistent disease in recipient mice. Here, we tested a novel xenograft mouse model of MDS using an immunodeficient mouse strain that constitutively expresses human hematopoietic growth factors, SCF, GM-SCF and IL-3 (SGM3). These mice demonstrate improved engraftment of human hematopoietic cells from MDS patient samples. We have used bone marrow from patients diagnosed with MDS with blast counts ranging from 2-15% and differing cytogenetic profiles. Unfractionated bone marrow cells we injected into both NOD/LtSz-scid IL2RG-/- (NSG) and NOD/LtSz-scid IL2RG-/- SGM3 (NSGS) mice. These studies demonstrate significantly improved engraftment in the NSGS mice compared to NSG mice. Specifically, 1 out of 4 MDS patient samples demonstrated engraftment in NSG mice, but 3 out of 4 MDS samples demonstrated engraftment in NSGS mice. In NSG mice, we noted approximately 5-6% of human CD45+ cells in peripheral blood at 6 weeks after injection in one MDS sample, but subsequent to that, there was a drop to less than 1 percent of human CD45+ cells detected at 9 and 12 weeks post injection. The NSGS mice demonstrated more robust engraftment up to 15% human CD45+ cells in the peripheral blood at the 6 week time point in some samples, but subsequently fall to 1-2 % at 12 weeks post injection. This demonstrates that the NSGS strain of mice demonstrates improved xenograft efficiency of MDS bone marrow samples. Surprisingly, higher levels of blasts in the patient samples did not necessarily correspond to increased human CD45+ cell engraftment in the NSGS mice. This suggests the NSGS mice do model the MDS patient characteristics rather than overt leukemia. These results support our hypothesis that constitutive expression of human growth factors can promote engraftment of hematopoietic cells from patients with MDS. These studies will allow us to now use of this NSGS mouse model to test novel drug and/or immunotherapy approaches against human MDS in an in vivo mouse model system.

Disclosures:

No relevant conflicts of interest to declare.