bluuebunny/crossref_metadata_2025 huggingface dataset:
Cyclin A2 Plays a Critical Role Not Only in Self-Renewal of Hematopoietic Stem Cells, but Also in Non-Self-Renewing Proliferation of Lymphoid Progenitors.
Authors: Kohno Kentaro , Iino Tadafumi , Ito Kyoko , Mizuno Shin-ichi , Sicinski Piotr , Akashi Koichi
Published: October 2019

Abstract 381

Cyclins are regulatory subunits of cyclin-dependent kinase, and are important components of cell cycle engine. The A-type cyclin is generally the S-phase cyclin. Mammalian cells express two A-type cyclins, including cyclin A1 that is exclusively expressed in the testis, and cyclin A2 whose expression is ubiquitous. We have recently reported that cyclin A2 is not required for fibroblast proliferation but it is indispensable in maintenance of self-renewal of stem cells, including embryonic stem cells and hematopoietic stem cells (HSCs) (Cell 138 2009). The question is whether cyclin A2 plays a role in proliferation of hematopoietic progenitors downstream of the HSC. Here we further assessed the requirement of A-type cyclin in non-self-renewing hematopoietic progenitors. Quantitative RT-PCR analysis showed that cyclin A2 was expressed in hematopoietic stem and progenitor cells, but its expression level is highest in lymphoid-committed progenitor stages of both T and B cell lineages. Thus, in order to test the role of cylin A2 in early lymphopoiesis, we crossed cyclin A2 floxed mice with Rag1-Cre knock-in mice. Rag1 expression is initiated at the preproB to the proB stages, and the DN1-DN3 stages in the thymus, while their proliferation is dependent at least upon pre-BCR or pre-TCR signal at these stages. Interestingly, the Rag1-Cre cyclin A2 floxed/floxed mice were viable, and have normal numbers of HSCs and myeloid progenitors in the bone marrow. They, however, displayed severe reduction of T and B cell numbers that were only 1/100 - 1/10 of wild-type controls; the number of common lymphoid progenitor was unchanged, but there were almost complete loss of proB and preB cells. Similarly, all thymic T cell progenitor compartments such as CD4-CD8- double negative, and CD4+CD8+ double positive populations were severely reduced. These findings clearly demonstrate that cyclin A2 is indispensable not only for self-renewal of HSCs, but also for proliferation of T and B cell progenitors.

Disclosures:

No relevant conflicts of interest to declare.
Modulation of ER Stress/Unfolded Protein Response (UPR) Pathways in Multiple Myeloma Cells by Inhibition of Hsp90 and Serine-Threonine Kinase CK2.
Authors: Piazza Francesco , Manni Sabrina , Gurrieri Carmela , Colpo Anna , Tubi Laura Quotti , Cabrelle Anna , Trentin Livio , Semenzato Gianpietro
Published: October 2019

Abstract 3840

Poster Board III-776

Hsp90 is an essential chaperone molecule that helps in the maturation and folding of a number of cellular client proteins. Hsp90 function is essential for malignant plasma cell survival, since its inhibition in multiple myeloma (MM) cells results in cell death and activation of apoptosis. Clinical trials using Hsp90 inhibitors are currently ongoing in MM patients.

Hsp90 inactivation in MM cells causes perturbation of the endoplasmic reticulum (ER) stress/unfolded protein response (UPR), eventually triggering the apoptotic cascades. Protein kinase CK2 critically regulates the activity of the chaperone complex formed by the Cdc37 and Hsp90 proteins. We already described that CK2 is over-expressed in a fraction of MM patients and is an essential MM pro-survival molecule. We have here investigated its role in the ER stress/UPR pathways and in Hsp90 inhibition-induced apoptosis in MM cells.

Down-regulation of the catalytic CK2 alpha subunit with selective chemical inhibitors or RNA interference resulted in significant modifications of the main UPR regulating signaling cascades: 1) a marked reduction of IRE1alpha protein levels; 2) a reduction of BiP/GRP78 and Hsp70 chaperone protein levels; 3) an increase of PERK activity and phospho eIF2alpha levels. When UPR was triggered by thapsigargin in CK2-inactivated cells, we observed that the IRE1alpha-dependent axis of the UPR was greatly impaired, as XBP-1short isoform generation and the levels of some induced chaperones were reduced. Interestingly, thapsigargin was able to induce CK2 kinase activity. Remarkably, treatment of CK2-silenced MM cells with Hsp90 inhibitors geldanamycin or its derivative 17-AAG (17-(demethoxy)-17-allylamino geldanamycin) resulted in 1) an even more pronounced reduction of IRE1 alpha protein levels; 2) a marked inhibition of GA or 17-AAG-triggered BiP/GRP78 protein level raise; 3) a more evident increase of eIF2 alpha phosphorylation. Of note, CK2 plus Hsp90 inhibition was followed by apoptotic cell death to a much greater extent than that obtained with the single inhibition of the two molecules. Noteworthy, these effects were also reproduced upon modelling the MM bone marrow (BM) microenvironment by co-culturing MM cells with BM stromal cells.

These data suggest that CK2-mediated signaling regulates the ER stress/UPR pathways and modulates the threshold to apoptosis of ER stressed MM cells. CK2 interacts with Hsp90, since its inhibition synergizes with GA or 17-AAG treatments in terms of induction of apoptosis and shift of the ER stress/UPR pathways towards the terminal phase. These results might be useful to set the groundwork in designing novel combination treatments for MM patients.

Disclosures:

No relevant conflicts of interest to declare.
Pomalidomide Monotherapy for Relapsed Myeloma Is Associated with Excellent Responses and Prolonged Progression Free and Overall Survival.
Authors: Streetly Matthew , Stewart Orla , Gyertson Kylie , Kazmi Majid A. , Schey Steve
Published: October 2019

Abstract 3878

Poster Board III-814

Introduction

Despite recent therapeutic improvements in the management of myeloma it remains an incurable disease. New therapies are therefore required. Pomalidomide (POM, Celgene) is a thalidomide derived immunomodulatory agent with similar preclinical spectrum of activity as lenalidomide. In Phase 1 studies we have previously demonstrated that POM monotherapy is well tolerated by patients with relapsed myeloma determining a maximum tolerated dose of 2mg od or 5mg on alternate days (Schey et al. JCO 2004; Streetly et al. BJHaem 2008). The toxicity profile was acceptable and overall response rates of 54 and 50% were observed with daily and alternate day dosing respectively. The current study examines long term responses, progression and survival outcomes. Patients were entered into the POM daily dosing or alternate day dosing Phase 1 studies between March 2001 and September 2003 and continued to receive treatment with POM until progressive disease (PD) or Grade 3 or greater non-haematological toxicity. Patients with PD were eligible to receive dexamethasone in addition to POM. POM became unavailable in May 2005 and patients still receiving drug were switched to receive lenalidomide. All patients gave informed consent.

Results

44 patients received treatment with POM at a dose of 1mg alternate days – 10mg od. A median of 3 (range 1 – 8) prior lines of therapy had been received. POM was received for a median of 9.3 months (1 – 53). Following POM withdrawal 8/44 patients who had not developed PD subsequently received lenalidomide from a median of 30 months after starting POM. Overall responses by IMWG criteria to POM monotherapy were: CR 13.6%, VGPR 4.5%, PR 34%, MR 9%, SD 29.5% and PD 7% giving an overall response rate (>PR) of 52%. Dexamethasone was introduced for PD for 10 patients and prolonged SD for 1 patient. 5/10 of these patients had >MR response to the addition of dexamethasone. With a median follow-up of 28 months the median PFS was 13.7 months and median OS was 28 months. Patients who had a PR response or better received POM for a median of 17.5 months and had improved PFS (median 19.8 months) and OS (median 42 months). 8/44 patients subsequently received lenalidomide. 7/8 of these patients have now developed PD at a median 26 months from commencing lenalidomide and 74 months from starting POM.

Conclusions

POM is a very well tolerated drug with excellent long term responses observed in this Phase 1/2 setting predominantly as monotherapy. Phase 2 studies are ongoing and results of these are awaited with interest.

Disclosures:

Streetly: Celgene: Honoraria. Schey:Celgene: Honoraria.
AML-1/RUNX-1 Is Overexpressed in Patients with Myeloproliferative Neoplasms and Mediates JAK2 V617F-Independent Overexpression of NF-E2.
Authors: Wang Wei , Schwemmers Sven , Pahl Heike L
Published: October 2019

Abstract 3893

Poster Board III-829

The transcription factor nuclear factor erythroid-2 (NF-E2) plays an essential role in both erythropoiesis and megakaryopoiesis. In addition to erythroid, megakaryocytic and granulocytic progenitors, NF-E2 is expressed in hematopoietic stem cells and in mature granulocytes. We have previously shown that NF-E2 is overexpressed in the vast majority of patients with polycythemia vera (PV). Concomitantly, 90 – 95% of these patients carry the JAK2V617F point mutation. Even though NF-E2 levels correlated with JAK2V671F allele burden in some PV cohorts, the molecular mechanism causing aberrant NF-E2 expression in PV patients has not been described. Here we show that NF-E2 expression is also increased in patients with Essential Thrombocythemia (ET) and that this is independent of the presence of the JAK2V617F mutation. Characterization of the NF-E2 promoter revealed multiple binding sites for Acute Myeloid Leukemia-1 (AML-1/RUNX-1), which were shown to be functional by deletion analysis and site directed mutagenesis. Chromatin Immunoprecipitation (ChIP) demonstrated AML-1 binding to the NF-E2 promoter in vivo. Moreover, AML-1 binding to the NF-E2 promoter in vivo is significantly increased in primary granulocytes from PV patients compared to healthy controls. Subsequently, we demonstrated that mRNA and protein expression of AML-1 is elevated in PV and ET patients, both in the presence and absence of JAK2V617F. In addition, AML-1 and NF-E2 expression are highly correlated. RNAi-mediated suppression of AML-1 levels significantly decreased NF-E2 expression. In summary, our data identify NF-E2 as a novel AML-1 target gene and point to a role of aberrant AML-1 expression in mediating elevated NF-E2 expression in MPN patients.

Disclosures:

No relevant conflicts of interest to declare.
Generation and Characterization of Transgenic Mice Expressing MplW515L.
Authors: Zhao Wanming , Xing Shu , Gao Rufei , Al-Kali Aref , Ho Wanting Tina , Zhao Zhizhuang Joe
Published: October 2019

Abstract 3901

Poster Board III-837

Myeloproliferative neoplasias (MPNs) are a group of conditions characterized by chronic increases in some or all of the blood cells (platelets, white blood cells, and red blood cells). JAK2V617F, a gain-of-function mutation of tyrosine kinase JAK2, is found in over 90% of patients with polycythemia vera (PV) and about 50% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). Attempt to identify other signaling components involving the JAK2 signaling pathways has led to discovery of acquired mutations of Mpl, the receptor of thrombopoietin, in 5-10% patients with PMF and ET. To prove the pathogenesis of Mpl mutants, we have generated transgenic mice expressing the most frequently occurred Mpl mutant designated MplW515L by using the vav gene promoter which drives expression of transgenes in the hematopoietic system. We obtained three lines of MplW515L transgenic mice which all displayed similar hematological abnormalities. As expected, the mice developed ET- and PMF-like phenotypes with much elevated platelet counts, severe splenomegaly/hepatomegaly, and bone marrow/spleen myelofibrosis. Interestingly, these mice also had markedly increased white blood cells in the peripheral blood, majority of which are IgD-positive mature B-cells. Histochemical staining and flow cytometric analyses revealed infiltrations of megkaryocytes and B cells into the spleen, the presence of megkaryocytes and erythroid blast cells in the liver, and infiltrations of the bone marrow with B-cells. Reticulin staining revealed that MplW515L transgenic mice developed profound myelofibrosis in the bone marrow and spleen. In vitro hematopoietic colony assays demonstrated increased numbers of hematopoietic progenitor cells including BFU-E, CFU-GM, CFU-Mk, and CFU-Pre-B in the bone marrow, mobilization of these stem/progenitor cells to peripheral blood and spleen, and their autonomous growth in the absence of growth factors and cytokines. Finally, transplantation of bone marrow cells from MplW515L mice into irradiated normal mice installed the aforementioned phenotypes into the recipient mice, indicating that expression of MplW515L altered the activity of hematopoietic stem cells. Together, our data demonstrated that transgenic expression of MplW515L not only causes PMF- and ET-like phenotypes but also lymphoproliferative disorders. Considering that Mpl is expressed in hematopoietic stem cells and that oncogenic gene mutations are often associated with alteration of gene expression, we believe that MplW515L may be involved in a wider spectrum of human hematological diseases than MPNs.

Disclosures:

No relevant conflicts of interest to declare.
Weight Loss, Splenomegaly, and Hypocholesterolemia in Myeloproliferative Neoplasms: Patterns and Relevance from the Pre JAK2 Inhibitor Era.
Authors: Mesa Ruben A. , Schwager Susan , Huang Jocelin , Pardanani Animesh D. , Hussein Kebede , Camoriano John , Tefferi Ayalew
Published: October 2019

Abstract 3918

Poster Board III-854

BACK GROUND

We have previously demonstrated that the myeloproliferative neoplasms (MPNs) of primary myelofibrosis (PMF), polycythemia vera (PV), and essential thrombocythemia (ET) can lead to weight loss, splenomegaly and constitutional symptoms (Cancer 2007;109:68–76). Additionally we have demonstrated that hypocholesterolemia in MPN patients is associated with decreased survival (Blood 2007;110:a2548). Given that current JAK2 inhibitor trials are demonstrating the ability to reverse MPN associated splenomegaly (Haematologica 2009;94(Suppl 2)439 a1088) and cachexia (Blood 2008;112(11):a1760) we sought to determine the baseline natural history for these variables in patients treated prior to the JAK2 inhibitor era.

METHODS

We analyzed the Mayo MPN database for patients (not treated with JAK2 inhibitors) with information on disease prognosis, presentation, therapies, height and weight at diagnosis, and outcomes. Additionally, when available, we analyzed additional weights during the clinical course, the body mass index (BMI- (weight/(height*height)), spleen size, and peripheral blood studies including lipids.

Results: Patients

783 patients with MPNs (followed for a median of 51 months (range 1-871 months); 60% having expired) were identified for the analysis (PV=158, ET=255, PMF=370) with 541 (69%) having a weight at the time of diagnosis, the remainder had a weight obtained a median of 7.8 months after diagnosis. Additionally, 508 patients (65%) had a weight value available from 1–3 additional time points during the course of their disease. Corresponding measurements of splenomegaly, or absence thereof, were noted in 766 cases (98%). Lipid panels (obtained within 18 months of diagnosis) were available in 264 patients. Results by MPN disease type are listed in the Table.

Impact on prognosis

Univariate analysis of variables discussed which negatively impacted survival included the subtype of MPN (not surprisingly worse for PMF p<0.001), weight loss of greater than 10% during the course of follow-up (P<0.001), or development of splenomegaly of >10 cm below the left costal margin (p=0.004) whereas hypocholesterolemia was significant only for the subset of PMF patients (P=0.03). The IWG-MRT International Prognostic Score (IPSS - Cervantes et. al. Blood 2009) was the only variable prognostically relevant in multivariate analysis (P<0.001).

Conclusions

Progressive splenomegaly, weight loss, and hypocholesterolemia are common across all MPNs but are most prognostically detrimental in PMF. Ongoing and future trials of JAK2 inhibitors will answer whether reversal of these latter hypercatabolic and proliferative manifestations of disease will improve outcomes for MPN patients.

Disclosures:

No relevant conflicts of interest to declare.
High Incidence of Hepatitis B or C Virus Infection in Lymphoma.
Authors: Tajima Katsushi
Published: October 2019

Abstract 3934

Poster Board III-870

Introduction

In lymphoma, several reports from different countries have shown that patients with lymphoma have a higher prevalence of HCV or HBV infection. The infection rates were very different even in virus endemic areas as Asia-Pacific countries including Japan. However, an association between virus infection and lymphoma should be evaluated and discussed with the background of virus prevalence in the community. This study aimed to investigate the prevalence of HBV or HCV infection status in lymphoma patients in Tohoku distinct in Japan.

Patients and Methods

The study, designed by the Tohoku Hematology Forum Group, was conducted in 9 clinical centers in Tohoku Distinct in Japan. This study included 1331 newly diagnosed consecutive series of lymphoma patients admitted in Tohoku Hematology Forum Group Hospital (Japan) and 848 non-lymphoma control patients enrolled from January 2005 to October 2008. We evaluated the prevalence of HBV (as indicated by hepatitis B surface antigen) or HCV infection in both group patients and compared the clinic-pathological characteristics of HBV- or HCV-positive and its negative lymphoma cases.

Results

The carrier rate of HBsAg was higher in lymphoma patients (57 of 1331, 4.3%) than control group (1.7%, p <0.001) The higher prevalence was observed in both sexes and especially diffuse large B cell lymphoma. Compared with HBV negative lymphoma group, the positive group displayed more liver or spleen involvement (p=0.002). The rate of HCV infection was higher than those in control group In HCV-infected case, more liver involvement was indicated (p<0.004). No difference in HBV or HCV prevalence was found between B-cell and T/NK-cell lymphoma.

Conclusions

The present study, so far the largest trial of incidence of HBV or HCV infection in lymphoma patients, demonstrated that patients with lymphoma in Japan had higher prevalence of HBV and HCV infection. These appears to be a possible role for HBV or HCV in the induction of malignant transformation, resulting in the development of lymphoma. However, the precise role of HBV or HCV in the pathogenesis of lymphoma remains to be elucidated and warrants further research.

Disclosures:

No relevant conflicts of interest to declare.
Divergent Effects of Notch Signaling On Leukemia Stem Cells and Hematopoietic Stem Cells.
Authors: Chiang Mark Y , Shestova Olga , Childs M. Eden , Romany Candice , Aster Jon , Pear Warren S.
Published: October 2019

Abstract 395

The Leukemia Stem Cell (LSC) hypothesis proposes that a subset of the cells in the bulk tumor cell population is responsible for leukemia maintenance and propagation. We tested the LSC hypothesis in murine T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) induced by retroviruses expressing strong gain-of-function Notch1 alleles. This model is relevant to human disease, as Notch1 is activated in ∼70% of human T-ALLs. By transferring sorted leukemia cells to syngeneic secondary recipients at limiting dilution, we found that LSC activity was enriched in the CD8+CD4-HSAhi (Immature Single Positive or “ISP”) T-cell subset. The LSC frequency was ∼1 in 1000 cells, two orders of magnitude higher than in the CD8+CD4+ (Double positive or “DP”) T-cell subset. We found similar results in a KrasG12D-driven T-ALL mouse model where activating Notch mutations occur spontaneously in ∼80% cases. Surprisingly, we were unable to isolate Notch-activated hematopoietic stem and progenitor cells (HSPCs) to test for LSC activity. Upon further analysis, we observed that activation of Notch in HSPCs caused loss of stem cell quiescence and progressive, complete loss of long-term hematopoietic stem cells (LT-HSCs) over several weeks. Notch signals had no significant effects on stem cell homing, apoptosis, or senescence. Similar results were obtained in both noncompetitive and competitive secondary transplants as well as in a mouse model in which Notch activation is conditionally induced in HSCs by Cre recombinase. We conclude that while Notch signaling promotes LSC activity, it extinguishes HSCs. These results provide a rationale for therapeutic targeting of the ISP-like T-cell subset in Notch-activated T-ALL while underscoring the potential difficulty of manipulating Notch signaling to expand normal, long-term stem cell populations for clinical applications.

Disclosures:

No relevant conflicts of interest to declare.
Epigenetic Signaling Is Required for HoxA9-Based Leukemic Transformation.
Authors: Chaubey Aditya , Velu Chinavenmeni S. , Horman Shane , Jegga Anil , Guzman Monica L. , Risner Laurie , Zeleznik-Le Nancy J. , Jordan Craig T. , Carroll Martin , Gebelein Brian , Grimes H. Leighton
Published: October 2019

Abstract 3966

Poster Board III-902

Since oncogenic activation of HoxA9 is induced by multiple chromosomal translocations affecting MLL1 (11q23)(e.g. MLL-Af9) or Nup98 (11p15)(e.g. Nup98-HoxA9 or Nup98-Nsd1), the function of the HoxA9 transcription factor is of critical interest in human acute myeloid leukemia (AML). HoxA9 forms heterodimeric DNA binding complexes with members of the Pbx and/or Meis family of homeodomain proteins. Importantly, the direct transcriptional targets of endogenous HoxA9 that mediate transformation remain largely unknown. The Growth factor independent-1 (Gfi1) transcriptional repressor is known to induce granulopoiesis and inhibit myeloid progenitor proliferation. GFI1 is mutated in patients with severe congenital neutropenia (SCN). SCN patients are at increased risk for AML. In a transcriptional circuit conserved to Drosophila, we have recently shown that Gfi1 represses HoxA9, Meis1 and Pbx1 expression, that Gfi1 and HoxA9 demonstrate dramatic epistatic relationships, and that Gfi1 loss of function is potently preleukemic. Our new bioinformatic, biochemical and expression data reveal microRNA genes to be targets of endogenous HoxA9 versus Gfi1 antagonism. Moreover, these miR are activated by Hox-signaling leukemia oncoproteins. Next, in both murine leukemia models and primary human AML samples, antagomir-mediated inhibition of microRNA function specifically disrupts oncogenic signaling by HoxA9, Nup98-HoxA9 and MLL-Af9 (but not AML-ETO which does not signal through HoxA9). In vivo, antagomir treatment blocked MLL-Af9-initiated leukemia lethality. These data establish microRNA genes as functional downstream targets of endogenous HoxA9, and implicate epigenetic signaling as critical client/mediators of Hox-based leukemia oncoproteins.

Disclosures:

No relevant conflicts of interest to declare.
Connecticut Attorney General Blumenthal's Citizen Petition Regarding Thalidomide-Associated VTE Redux: Patient Safety Concerns Still Persist.
Authors: McKoy June M. , Boen Monica , Deleon Dianne , Feliciano Josephine , Courtney D. Mark , Bennett Charles L.
Published: October 2019

Abstract 3990

Poster Board III-926

Background

In 2005, the first ever Citizen Petition filed by a State Attorneys General, Attorney General Richard Blumenthal, requested that the FDA adopt 6 measures to improve VTE prophylaxis and patient safety of thalidomide (thal). While 4 recommendations were accepted, the FDA refused to mandate a Phase IV randomized, controlled trial designed to identify the best VTE prophylaxis stating that substantial data already existed, but no specific prophylaxis was identified. We review the progress of this safety concern pre- and post-FDA approval of thal in 2006, specifically for the use of multiple myeloma patients. Similar safety concerns apply to lenalidomide.

Methods

A literature search was performed from 2006 to 2009 through Pubmed and Ovid using the key words “thalidomide,” “lenalidomide,” “thrombosis,” and “multiple myeloma.” High dose thal was defined as higher than 200mg/d, low dose thal as 200mg/d or lower, and high dose dexamethasone (dex) as higher than 20mg/d and low dose dex as 20mg/d and lower.

Results

See table

Conclusion

Since 2006, we have found information on 2657 patients with no prophylaxis, 539 patients with thal/dex with prophylaxis, and 2210 patients with len/dex with prophylaxis. VTE rates remain high, except for the low dose thal and/or low dose dex group. Overall, optimal VTE prophylaxis remains an enigma. Current recommendations for VTE prophylaxis are not supported by empirical data. Against this backdrop, Blumenthal's team should consider resubmitting their safety request.

Disclosures:

No relevant conflicts of interest to declare.
Residual Vein Thrombosis Improves Screening for Occult Cancer: Results On 537 Patients with Idiopathic Deep Vein Thrombosis.
Authors: Siragusa Sergio , Malato Alessandra , Fulfaro Fabio , Saccullo Giorgia , Caramazza Domenica , Pizzo Giusy , lo Coco Lucio , Gebbia Nicola
Published: October 2019

Abstract 3999

Poster Board III-935

Background

Clinical advantage of extensive screening for occult cancer in patients with idiopathic Deep Vein Thrombosis (DVT) is unknown. We have demonstrated that the Residual Vein Thrombosis (RVT)-based screening for occult cancer improves early detection as well as cancer-related mortality (Siragusa S et al. Blood 2007;110(699):OC). Here we report on final analysis of 537 patients over a period of 8 years.

Objective of the study

We conducted a prospective study evaluating whether a RVT-based screening for cancer is sensitive and influences cancer-related mortality.

Study design

Prospective with two cohorts of DVT patients: the first cohort was monitored for clinical overt cancer only (Group A), while the second (Group B) received complete screening for occult neoplasm and subsequent surveillance.

Materials and methods

Consecutive patients with a first episode of DVT who presented RVT after 3 month of anticoagulation and without signs and/or symptoms for overt cancer. Screening for occult cancer was based on: ultrasound and/or CT scan of the abdomen and pelvis, gastroscopy, colonoscopy or sigmoidoscopy, hemoccult, sputum cytology and tumor markers. These tests were extended with mammography and Pap smear for women and ultrasound of the prostate and total specific prostatic antigen (PSA) for men. All investigations had to be completed within four-weeks from the assessment of RVT. All patients were followed-up for at least 2 years. Incidence and cancer-related mortality was compared between the two groups by survival curves (Kaplan-Mayer) and related Breslow test for statistics.

Results

Over a period of 8 years, 537 patients were included in the analysis: first cohort included 346 patients (Group A), second cohort 191 (Group B). Clinical characteristics between groups were homogenous. During the follow-up, 8.3% of patients developed overt cancer in group A; in group B, 7.8% of patients had diagnosed cancer at the moment of extensive screening while 2 new cases (0.7%) occurred during the follow-up (Table). The sensitivity of this approach was 92.1% (95% confidence intervals 75.2-104.2). Cancer-related mortality was 7.5% in group A and 3.6% in group B (p< 0.001).

Conclusions

The RVT-based screening for occult cancer is highly effective for improving early detection as well as cancer-related mortality in a cohort of 537 patient with DVT of the lower limbs.

Disclosures:

Off Label Use: Hydroxyurea use in myelofibrosis.
Thrombospondin-1 and ADAMTS13 Competitive Binding with VWF Mainly in Both A2 and A3 Domain.
Authors: Wang Anyou , Dong Ningzheng , Ma Zhenni , Zhang Jingyu , Su Jian , Ruan Changgeng
Published: October 2019

Abstract 4008

Poster Board III-944

Introduction

Thrombospondin-1 (TSP1) is one multifunctional glycoprotein belonging to the thrombospondins family. It is mainly synthesized by megakaryocytes, stored in the intermediate zone of platelet -granules, and secreted by platelets upon thrombin activation. TSP1 has been postulated to play an important role in hemostasis for a long time. Recently, in a TSP1 knockout mouse, study showed that TSP1 protected unfolded endothelium-bound and subendothelial VWF from degradation by its plasma protease ADAMTS13, however, concrete mechanism that how to inhitbit ADAMTS13 activity by TSP1 was unclear.

Materials and Methods

we constructed and expressed different domains of human VWF, mainly including D'D3, A1, A2, A3 domain, full-length VWF and ADAMTS13, then investigated the binding effect of TSP1 or ADAMTS13 with these different domains and full-length VWF by enzyme linked immunosorbent assay. We also observed the direct inhibition effect of different concentrations TSP1 on ADAMTS13 activity by Western blot method and residual-collagen binding assay (R-CBA) in denaturing condition and high shear stress condition

Results

We found that ADAMTS13 could interact with the A1, A2, A3 domain and full-length VWF, and TSP1 could bind with the A3 domain. Meanwhile, we observed that TSP1 also had significant interaction with the A2 domain. And TSP1 competed with ADAMTS13 mainly existing in both A2 and A3 domain. The results of Western blot and R-CBA also showed that TSP1 dramatically restrained ADAMTS13 activity, especially at high concentrations, and the inhibitory effect could add up to 50%.

Conclusions

Our study showed that TSP1 played competitive inhibitor role during ADAMTS13 binding and cleaving VWF, and the competitive process maybe mainly exist in both A2 and A3 domain.

Disclosures:

No relevant conflicts of interest to declare.
Shear-Induced Von Willebrand Factor-Mediated Platelet Glycoprotein Ibα Shedding.
Authors: Cheng Hong , Yan Rong , Li Suping , Yuan Yanhong , Liu Jun , Ruan Changgeng , Dai Kesheng
Published: October 2019

Abstract 4024

Poster Board III-960

Introduction

Shear-induced platelet adhesion through the interaction of glycoprotein (GP) Ibα with von Willebrand factor (VWF) exposed at the injured vessel wall or atherosclerotic plaque rupture is the first step for the physiological hematosis or pathologic thrombus formation in stenosed arteries. However, the role of shear stress in the regulation of platelet function remains poorly defined. Methods: Washed platelets were exposed to VWF-coated surface at different shear conditions, provided by syringe pump connecting with glass capillary or cone-and-plate viscometer. GPIbα shedding was investigated by flow cytometry and western blot.

Results

GPIbα ectodomain was shed from platelets, while a small mass of GPIbα C-terminal peptide around 17 kDa was increased correspondingly. Although GPIbα ectodomain shedding was found under all shear conditions, the extent of GPIbα shedding was maximum at 250/s and dramatically reduced with increased shear rate, which was consistent with the number of stable adhesive platelets. There was a correlation between the levels of platelet adhesion and the extent of GPIbα shedding. Furthermore, GPIbα shedding increased with the concentration of immobilized VWF and time duration (within 1 minute) of constant shear stress. The potential protease(s) and signaling pathway involved in this process were investigated. Pretreatment of platelets with membrane-permeable calpain inhibitors (MDL28170, calpain inhibitor I and II) and metalloproteinase inhibitor (GM6001) abolished shear-induced GPIbα shedding. Though platelets showed partial activation detected by PAC-1 binding, GPIbα shedding was not impacted by apyrase and PGE1. However, integrin αIIbβ3 antibody (SZ-21) and phosphoinositide 3-kinase inhibitors (wortmanine) obviously inhibited GPIbα shedding. Conclusions: These results indicate that shear-induced platelet-VWF interaction results in calpain and metalloproteinase-dependent GPIbα ectodomain shedding. Fluid shear stress and VWF always exist in both the normal circulation of blood and pathological medical conditions, particularly, the interaction of GPIbα with VWF under flow initiates platelet adhesion and thrombus formation. Thus, the current finding that shear-induced the interaction of GPIbα with VWF incurs GPIbα ectodomain shedding not only has physiological implication in understanding the presence of glycocalicin in normal circulation, but also suggests a novel mechanism for the negative regulation of platelet function and the limitation of platelet thrombus growth under pathophysiological flow conditions.

Disclosures:

No relevant conflicts of interest to declare.
Role of Upstream cAMP Response Element (CRE) in γ-Globin Gene Regulation.
Authors: Ramakrishnan Valya , Bhattacharya Kajari , Pace Betty
Published: October 2019

Abstract 4076

Poster Board III-1011

The reactivation of fetal hemoglobin has been shown to ameliorate symptoms of sickle cell disease. Previous work from our laboratory demonstrated that histone deacetylase inhibitors such as Trichostatin A and sodium butyrate activate γ-globin gene expression via p38 MAPK signaling. Using a transient luciferase genetic reporter system, the trans-factors cAMP response element binding protein 1 (CREB1) and activating transcription factor-2 (ATF-2) were shown to trans-activate the Gγ-globin CRE, located upstream at nucleotide -1222. To study this regulatory element in a native chromatin conformation, we cloned the Gγ-globin promoter (-1500 to +36) into pGL4.17 Luc2/neo to produce the pGγLuc2 reporter construct. In addition, three mutant plasmids were created by site directed mutagenesis including the -1225 G/A mutation (pGγLuc2m1), -1227 AC/TG mutations (pGγLuc2m2) and a scrambled G-CRE, 5'-TCTATGTA-3' (pGγLuc2m3S). Each reporter along with the empty vector control (pGL4.17 Luc2/neo) was transfected into K562 cells and five stable lines including KLuc2, KGγLuc2, KGγLuc2(m1), KGγLuc2(m2) and KGγLuc2(m3s) were established using the G418 selectable marker. Luciferase activity in KGγLuc2 was 700-fold higher than the promoterless Kluc2 stable line. When the G-CRE element was mutated, we observed a 70 to 80 % decrease (p<0.05) in luciferase activity in the three mutant lines. Furthermore, a loss of Gγ-promoter inducibility was observed in the mutant lines. Treatment with butyrate (2mM) or Trichostatin A (0.5μM) resulted in a 3-fold increase of luciferase activity for the wild-type KGγLuc2 line while a 5-fold loss of inducibility was observed in the mutant stable lines. These studies support a functional role for the G-CRE in γ-globin transcription. To expand our understanding of p38 MAPK signaling in G-CRE function, siRNA studies were completed. In wild-type K562 cells, p38 siRNA molecules produced a 95% decrease in target p38 MAPK mRNA levels and a concomitant 65% loss of γ-globin expression was observed. This demonstrated that p38 MAPK is required for steady-state γ-globin gene transcription. When p38 MAPK siRNA knockdown was performed in the KGγLuc2 stable line we observed 30% reduction in luciferase activity, while this effect was lost in the mutant stable lines. Additional siRNA studies will be completed in the stable lines to determine if the target trans-factors CREB1 and ATF-2 mediate the effect of p38 MAPK in the context of chromatin. Chromatin immunoprecipitation assay will also be performed to determine in vivo binding to the G-CRE. Elucidating regulatory elements involved in γ-globin gene regulation will provide novel approaches for fetal hemoglobin induction as a treatment for sickle cell disease.

Disclosures:

No relevant conflicts of interest to declare.
Indirubin-3-Monoxime Induces Apoptosis and Autophagy in Human Lymphocytic Leukemia Cells and Human Chronic Myelogenous Leukemia Cells.
Authors: Lee Ming-Yang , Chuang Jing-Jing , Liu Yi-Wen
Published: October 2019

Abstract 4106

Introduction

Indirubin-3-monoxime (IO) is the active ingredient of Danggui Longhui Wan, a mixture of plants that is used in traditional Chinese medicine. It is a potent inhibitor of cyclin-dependent kinases (CDKs), especially CDK2. In clinical studies demonstrated that indirubin did not cause major side effects in patients with chronic myelogenous leukemia. However, the functional action of indirubin on acute lymphoblastic leukemia (ALL) is still unclear. In the present study, we investigated that cytotoxic effect and mechanism study of IO in human acute lymphocytic leukemia cell line JM1 and human chronic myeloid leukemia cell line K562. We also analyzed the viability influence of IO in normal human granulocytes and lymphocytes.

Materials & Methods

After the treatment of IO, cell viability of JM1 and K562 cells was determined by WST-1 assay. The influence of IO on cell-cycle distribution was analyzed by FACScan. To identify which cell death types were induced during IO treatment we measured the caspase-3 activity for analysis the induction of apoptosis; evaluated the LDH release in the necrotic analysis and the expression level of LC3-II was determined by Western blotting analysis for autophagic cell death. Furthermore, we also analyzed the toxicity of IO in normal human granulocytes and lymphocytes by the treatment dose of IO with value of IC50.

Results

IO significantly affected the cell viability on JM1 but also on K562 cells in a dose dependent manner. The G2/M phase of cell cycle was arrested and sub-G1 proportion was relative increased. In addition, the finding showed that IO initiated caspase-3-dependent apoptosis in JM1 and K562 cells. The expression of autophagosome-incorporated LC3-II protein was also clearly increased once cells treated with IO. However, the necrotic phenomenon through measuring LDH release from K562 and JM1 cells could not be observed. Excitingly, by the treatment dose of IO with value of IC50, the cell viability of lymphocytes was marginally affected, whereas the cell cytotoxitity of granulocytes was not induced.

Conclusions

IO has the ability to induce potent cytotoxic effect on K562 and JM1 cells. The cell death after IO treatment is mainly caused by apoptosis and autophagy, not by necrosis. This effect is also associated with interrupted cell cycle. Importantly, IO has few or little cytotoxic effect on human lymphocytes and granulocytes. The results suggest that IO might be useful for clinical anti- ALL treatment.

Disclosures:

No relevant conflicts of interest to declare.
Surgery Treatment and Pathological Results of Patients of Malignant Hematological Disorders Complicated with Lung Diseases.
Authors: Tang Xiaowen , Huang Haoyue , Zhan Shenghua , Sun Xingwei , Shi Xiaolan , Sun Aining , Shen Zhenya , Kang Suya , Depei Wu , Jin Zhengming , Qiu Huiying , Miao Miao , Fu Chengcheng , Han Yue , Chen Suning , Xue Shengli , Ma Xiao
Published: October 2019

Abstract 4107

Objectives

To determine the pulmonary pathological changes in patients of hematological malignancies with pulmonary complications using surgical or thoracoscopic technologie.

Methods

17 hematological malignant patients who underwent surgical treatment were evaluated retrospectively in our study. Pulmonary infection was presented in 14 cases following chemotherapy, and lesions can not be completely absorbed after a broad-spectrum anti-bacterial and anti-fungal treatment. Furthermore, computerized tomographic scanning showed that there remained several kinds of localized lesions. Subsequently, all the 17 patients underwent open lung or thoracoscopic biopsies (lobar, partial, or wedge resection). The pathological changes of all the surgical specimens were examined postoperatively by standard hematoxylin and eosin staining.

Results

Pathological examination confirmed: Aspergillus infection in 9 patients, sub-acute inflammation (fibrosis and hematoma formation) in 3 patients, pulmonary infarction with granulomatous tissue in the periphery in 1 patient, granulomatous inflammation with calcified tubercle in 1 patient, alveolar dilation and hemorrhage, interstitial fibrosis and focal vasculitis in 1 patient, intercostal neurilemmoma in 1 patient, and moderate-differentiated adenocarcinoma accompanied by intrapulmonary metastasis in 1 patient. And several operative complications (1 case of fungal implantation, 3 cases of pleural effusion and adhesions and 2 cases of pulmonary hematoma) were occurred. The coincidence rate of pre- and post-operative diagnosis was 9/14 (64.3%). After surgery, 8 patients were received hematopoietic stem cell transplantation (HSCT, allo-gene or autologous), in which 7 cases were succeeded. Following the effective secondary antifungal prophylaxis,4 of 5 patients of aspergillosis were succeeded in transplantation free from mycotic relapse,just one patient was dead from fungal relapse.

Conclusion

Hematological malignancies with certain pulmonary complications, that is, persistent and/or medical-management-resistant pulmonary infection, hemoptysis, or lung diseases of diagnosis unknown, should be treated in time by surgical resection to effectively eliminate the residual disease and to achieve definitive diagnosis, so as to create a prerequisite condition for the following treatments. Moreover, the secondary antifungal prophylaxis could provide positive roles in protecting patients scheduled for chemotherapy and/or HSCT.

Keywords

hematological malignancies; immunocompromise; pulmonary aspergillosis; pulmonary resection; histopathology ; secondary antifungal prophylaxis

Disclosures:

No relevant conflicts of interest to declare.
CD22 Expression in Pediatric B-Lineage Acute Lymphoblastic Leukemia.
Authors: Jegalian Armin G. , Wayne Alan S. , Kreitman Robert J. , Mussai Francis J. , Pastan Ira , Yuan Constance M. , Stetler-Stevenson Maryalice
Published: October 2019

Abstract 4119

While the majority of pediatric patients with newly diagnosed B-lineage acute lymphoblastic leukemia (ALL) are cured with standard chemotherapy regimens, treatment is associated with multiple toxicities, and ALL remains the most frequent cause of cancer mortality in childhood. CD22, a B-lineage surface glycoprotein involved in B cell signaling and adhesion, is expressed in most cases of B-lineage ALL. We are conducting clinical trials of anti-CD22 immunotoxins [RFB4(dsFv)-PE38] for pediatric ALL. To assess eligibility for such targeted therapy, CD22 expression by ALL cells was studied in peripheral blood and/or bone marrow aspirate samples from 50 patients with relapsed ALL. The level of CD22 expression by ALL cells was quantitated by measuring mean anti-CD22 antibody binding per ALL cell (ABC) under saturating conditions using flow cytometry and the BD Biosciences QuantiBRITE system for fluorescence quantitation. Patients ranged in age from 3 to 22 years (median 10 years) and included 27 males and 23 females. CD22 expression was detected in all samples, and the vast majority of cases demonstrated expression of CD22 in 100% of leukemic blasts. CD22 antigen density in ALL cells varied widely among patients at baseline (range 451 - 14,519; mean 4276; median 3824; standard deviation 2976; see graph). CD22-directed immunotoxin therapy was initiated in 29 of the 50 patients, 19 of whom had samples quantitated for CD22 expression levels both before and after immunotoxin therapy. Most patients exhibited limited variation in the mean number of anti-CD22 molecules bound per ALL cell when comparing multiple specimens. In conclusion, CD22 expression varies widely in pediatric B-lineage ALL and persists despite repeated exposure to CD22-directed therapy.

(MedImmune, LLC, sponsored the clinical studies of anti-CD22 immunotoxin CAT-8015.)

Disclosures:

No relevant conflicts of interest to declare.
The Analysis of FLT3 and NPM1 Gene Mutations in Chinese Patients with Acute Myeloid Leukemia.
Authors: Zhang Su-Jiang , Li Jianyong , Han Yangli
Published: October 2019

Abstract 4136

Objective

To investigate the frequency of nucleophosmin(NPM1) gene mutations and FLT3 mutations in Chinese patients with acute myeloid leukemia (AML) and its correlation with clinical feature and prognosis.

Methods

123 first diagnosed AML patients were involved in our study. Polymerase chain reaction (PCR) combined with electrophoresis was directly used to detect FLT3-ITD mutations, PCR combined with EcoRV digestion was used to detect FLT3-TKD mutation, and PCR combined with directly sequencing was used to detect NPM1 mutations.

Results

(1) In the 123 first diagnosed AML patients, 16 patients (13.0%) were found harboring FLT3-ITD mutation, 6 patients (4.9%) were FLT3-TKD positive, and one patient was found harboring co-existing FLT3-ITD and FLT3-TKD mutation. In addition, 24 patients (19.8%) were NPM1 positive, including 22 patients with A-type mutation, one patient with B-type mutation and one patient with D-type mutation. 5 patients were found harboring co-existing FLT3-ITD and NPM1 mutation. (2) FLT3-ITD and NPM1 mutations were identified as 17.5% (10/57) and 31.6% (18/57) individually in 57 AML patients with normal karyotype, which were significantly higher than patients with abnormal karyotype. All of patients with double mutation had normal karyotype. (3) White blood cell (WBC) count and leukemia cell of bone marrow in FLT3-ITD or FLT3-ITD+/NPM1+ patients were both higher than those patients with FLT3-ITD-/NPM1-. The CR rate of FLT3-ITD+/NPM1-, FLT3-ITD+/NPM1+, FLT3-ITD-/NPM1+, FLT3-ITD-/NPM1- were 50% (4/8), 20% (1/5), 87.5% (7/8), 79.5% (35/44) individually.

Conclusions

FLT3 gene and NPM1 gene mutations are common in AML patients, and early detection of FLT3 gene and NPM1 gene mutations will be beneficial for treatment and prognosis analysis of AML patients.

Disclosures:

No relevant conflicts of interest to declare.
HL-60 Cell Death Induced by Lycorine Is Due to p21-Mediated Cell Cycle Arrest and TNF-α Signaling Stimulation.
Authors: Liu Jing , Hu Ji-liang , He Yang , Shi Bi-Wei , Hu Wei-Xin
Published: October 2019

Abstract 4158

Lycorine displays various biological functions including remarkable anti-tumor effect. We previously reported that lycorine induced anti-leukemia effect via arresting cell cycle and inducing apoptosis on human acute promyelocytic leukemia (APL) cell line HL-60. To explore the molecular mechanism how lycorine induced HL-60 cell apoptosis, cDNA microarray was used to investigate the expression profile of 494 apoptosis-associated genes. Real-time RT-PCR, Western blotting and immunocytochemistry were used to analyze the expression of related genes, as well as the modification and distribution of related proteins in the presence of lycorine. The results showed that 89 differential genes were expressed significantly (Cy3:Cy5 > 2 or < 0.5) among all the 494 apoptosis-related genes. 78 genes were up-regulated and 11 genes were down-regulated. We are particularly interested in the expression increase of p21 (9.271 folds) and TNF-α (8.242 folds). Furthermore, we found that lycorine could down-regulate p21-related gene expression including Cdc2, Cyclin B, Cdk2 and Cyclin E, promote the truncation of Bid protein and the release of cytochrome c from mitochondria, decrease the phosphorylation of IκB, block the nuclear import of NF-κB and down-regulate expression of survivin. This study revealed that lycorine decreased HL-60 cells survival through p21-mediated cell cycle arrest and stimulation of TNF-α signaling pathway which induced apoptosis.

Disclosures:

No relevant conflicts of interest to declare.