bluuebunny/crossref_metadata_2025 huggingface dataset:
CAL-101, a Potent Selective Inhibitor of the p110δ Isoform of Phosphatidylinositol 3-kinase, Attenuates Pathway Signaling, Induces Apoptosis, and Overcomes Signals From the Microenvironment In Cellular Models of Hodgkin Lymphoma
Authors: Meadows Sarah A , Kashishian Adam , Johnson Dave , Diehl Volker , Lannutti Brian
Published: October 2019

Abstract 3926

Phosphatidylinositide 3-kinases (PI3Ks) are a family of lipid kinases that are involved in signaling events which control a diverse number of cellular processes. The class I kinases contain 4 isoforms designated p110α, β, δ, γ, and are activated by cell surface receptors. Aberrant regulation of the PI3K signaling pathway is frequently observed in human malignancies including those of hematological origin. CAL-101 is an oral p110δ-specific inhibitor which has shown preclinical and clinical activity in non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL). This compound is a potent p110δ inhibitor (EC50 of 65 nM in a whole-blood assay) with >200-fold selectivity over the other class I PI3K isoforms and no activity against Class II and III PI3K family members or other PI3K-related proteins, including mTOR and DNA-PK. Prior in vitro NHL studies revealed that CAL-101 induces caspase-dependent apoptosis, and inhibits CD40L-, BAFF-, CXCL12- and CXCL13-derived survival signals in cellular models (Lannutti BJ, et al., Blood 2010).

To investigate the potential role of p110δ in Hodgkin lymphoma (HL) we screened a number of HL cell lines for p110δ isoform expression and constitutive PI3K pathway activation. We report high levels of p110δ protein and activated Akt in 5 of 5 HL cell lines evaluated (L428, L540, L591, L1236, KM-H2). Inhibition of p110δ with CAL-101 treatment of cell lines resulted in a reduction of Akt phosphorylation and a decrease in cellular viability. Because previous studies have established the importance of signals from the microenvironment for the survival and proliferation of malignant cells as well as for their resistance to standard therapies, we investigated the effect of p110δ inhibition by CAL-101 in HL cell line-stroma cocultures. In this setting, CAL-101 overcame tumor cell growth induced by coculture of HL cells with bone marrow stromal cells. In addition, CAL-101 induced dose-dependent apoptosis of HL cells at 48 hours. Furthermore, stromal cell coculture resulted in increased CCL5, CCL17, and CCL22 levels; productions of these chemokines by HL cells cultured in the presence of stromal cells were reduced by CAL-101 in a dose-dependent manner. These results indicate that specific inhibition of p110δ may disrupt signals between HL cells and their microenvironment, thereby providing the preclinical rationale for clinical evaluation of CAL-101 as a novel therapeutic approach in patients with Hodgkin lymphoma.

Disclosures:

Meadows: Calistoga Pharmaceuticals: Employment. Kashishian:Calistoga Pharmaceuticals: Employment. Johnson:Calistoga Pharmaceuticals: Employment. Lannutti:Calistoga Pharmaceutical Inc.: Employment.
Remarkable Efficacy of IL-1 Receptor Antagonist In Schnitzler's Syndrome: a Series of 6 Cases
Authors: Lachmann Helen J , Harding Stephen , Wechalekar Ashutosh , Gillmore Julian D , Amft E. Nicole , Hawkins Philip N
Published: October 2019

Abstract 3958

Schnitzler syndrome is a rare acquired autoinflammatory disease characterized by relapsing urticarial rashes, periodic fevers, arthralgias, lymphadenopathy and IgM paraproteinaemia, which can be very low level. Fewer than 100 patients have been reported and in general chemotherapy directed at the B cell clone has not been effective in controlling the inflammatory symptoms.

We describe our series of six patients, their phenotype and response to treatment. The median age at symptom onset was 55.6 years (range 52.8–78.9 years) and 4 of the patients were male. All patients presented with rash, recurrent fever, constitutional upset with appetite loss, fatigue and myalgia. The rash was urticarial in appearance but non itchy and on biopsy demonstrated a marked neutrophil infiltrate. Three patients had intermittent lymphadenopathy and one disabling bone pain. An IgM paraprotein was detected in all cases, median 7g/L (range: detected on immunofixation only – 8g/L). The IgM paraprotein was kappa in 5 cases and only one patient had abnormal serum free light chains. The Hevylite™ assay was abnormal in all 4 cases in whom the test was performed. Of note the patient with an IgM lambda paraprotein detected only on immunofixation has a clear IgM lambda excess on Hevylite™ assay. Baseline bone marrow demonstrated significant abnormalities in three patients: one has a 10% infiltrate of clonal cells consistent with lymphoplasmacytic lymphoma, one demonstrated a myeloproliferative picture with dysplastic features but no obvious plasma or lymphoid infiltrate and one was reported as reactive. Three patients had been treated with broad spectrum immunosupression without benefit and all had had corticosteroids with only partial benefit. In all cases treatment with anakinra (recombinant IL-1 receptor antagonist) as monotherapy produced a complete clinical response with normalisation of inflammatory markers. The median duration of treatment has been 9 months (range 0.1–2.7 years) and no patients have lost their response to daily ankinra injections. Median follow up from presentation has been 7.1 years with no evidence of evolution of overt lymphoproliferative disease nor change in the paraprotein concentration. In conclusion Schnitzler's syndrome is a rare acquired autoinflammatory disease which can be very effectively treated by anakinra suggesting that IL-1 is central to its pathogenesis. Treatment does not appear to have had any direct effect on clonal markers and the role of the IgM paraprotein in this disease remains unclear.

Disclosures:

Off Label Use: Anakinra - IL-1 receptor blockade, used off licence in Schniztlers syndrome.
CD40 Stimulation Sensitizes CLL Cells to CD20-Triggered Cell Death by Rituximab and GA101 Via a Different Mechanism
Authors: Jak Margot , van Bochove Gregor , Klein Christian , Umana Pablo , Eldering Eric , Van Oers Marinus H.J.
Published: October 2019

Abstract 3979

Although treatment results for Chronic Lymphocytic Leukemia (CLL) have improved considerably over the last decade, unfortunately a curative treatment is still not available. In part this might be due to the interaction of CLL cells with their micro-environment in lymph nodes, spleen and bone marrow. Micro-environment derived signals are not only capable of driving proliferation of CLL cells, but can also induce resistance of CLL to cytotoxic drugs. Previously we have shown that in vitro CD40 stimulation of peripheral blood derived CLL cells can to a certain extent mimic the lymph node microenvironment and result in resistance to cytotoxic drugs (Kater AP et al. Br J Haematol 2004;127:404; Smit LA et al. Blood 2007;109:1660.; Hallaert DY et al. Blood 2008;112:5141). At present it is not known whether sensitivity of CLL cells to CD20 monoclonal antibodies (mAbs) is modulated by micro environmental stimuli. Therefore in the present study we investigated anti-CD20 mediated cell death of CD40-stimulated CLL cells from 17 CLL patients. We observed that in sharp contrast with the response towards cytotoxic drugs, CD40 stimulation sensitizes CLL cells to cell death mediated by anti-CD20 mAbs. In CD40-stimulated CLL cells, cell death induced by both Rituximab (a type I anti-CD20 mAb) and GA101 (a novel type II anti-CD20 mAb) (Moessner E et al. Blood, 2010;127:404, 115(22):4393-402) is increased. Both anti-CD20 mAbs induce a non-apoptotic, caspase- and p53-independent rapid cell death, but interestingly the mechanism of Rituximab and GA101-induced cell death appears to be different. Rituximab-induced cell death is dependent on extracellular Ca2+ and ROS production and CD40 stimulation sensitizes CLL cells by increasing basal ROS production. In contrast, GA101 induces cell death via a lysosome-dependent mechanism and CD40 stimulation sensitizes CLL cells by increasing the lysosomal volume of the cell. Moreover, in contrast to Rituximab, GA101 induces cell death in the absence of a secondary crosslinking mAb. Combination of GA101 with fludarabine, chlorambucil, bortezomib or bendamustine shows additive effects and results in strong cell death of CD40-stimulated CLL cells, even in p53 dysfunctional CLL cells. Our findings not only provide a rationale for combining cytotoxic drugs and anti-CD20 monoclonal antibodies, but also show that GA101 is a potent promising new anti-CD20 mAb for the treatment of CLL.

Disclosures:

Klein: Roche: Employment, Equity Ownership, Patents & Royalties.
Combined Low-Dose Homoharringtonine, Cytarabine and Granulocyte Colony-Stimulating Factor for Patients with Advanced Myelodysplastic Syndrome (MDS) or Acute Myeloid Leukemia Transformed From MDS
Authors: Wu Lingyun , Li Xiao , Chang Chunkang , Shu Jiying , Xu Li , Zhang Zheng
Published: October 2019

Abstract 4004

A pilot study of the efficacy and toxicity of the low-dose cytarabine and homoharringtonine in combination with granulocyte colony-stimulating factor (G-CSF) (CHG protocol) in patients with advanced myelodysplastic syndromes (MDS) or MDS-transformed acute myeloid leukaemia (t-AML) was conducted. A total of 50 patients (31, advanced MDS; 19, t-AML) were enrolled in this study. All patients were administered the CHG regimen comprising low-dose cytarabine (25 mg/d, intravenous continuous infusion, days 1 C14), homoharringtonine (1 mg/d, intravenous continuous infusion, days 1 C14) and G-CSF (300 μg/d, subcutaneous injection, days 0 C14, intermitted when the peripheral WBC count reached >20 °Á 109/L). This regimen was followed by conventional chemotherapy as post-remission therapy when complete remission (CR) was achieved. The overall response rate was 68.0% after one course of the CHG regimen was applied. Of the total 50 patients, 24 (48.0%) achieved CR and 9 (18.0%) achieved partial remission (PR). The median overall survival (OS) was 14.1 months. Among the patients aged ≥ 70 years, the response rate was 81.0% (71.4% CR and 9.6% PR), while in patients aged < 70 years, the response rate was 55.2% (31.0% CR and 24.1% PR) (P < 0.01). There were no statistically significant differences for CR, PR and OS when the patients were grouped by blast percentage in bone marrow and karyotypes, respectively. 8 of the 24 CR patients who just received post-remission regimens of HA (homoharringtonine and cytarabine) and DA (daunorubicin and cytarabine) relapsed rapidly and just kept a mean CR of 4.3 months. Otherwise, the other 14 CR patients alternatively received succeeded chemotherapy, which combined mitoxantrone, idarubicin, pirarubicin or aclarubicin with cytarabine or decitabine. The mean CR duration of these 14 patients reached 15.5 months with 4 still maintaining continuous CR. No treatment-related deaths were observed. Myelosuppression was mild to moderate, and no severe non-haematological toxicity was observed. Thus, CHG priming regimen as an induction therapy is well tolerated and effective in advanced MDS or t-AML patients.

Disclosures:

No relevant conflicts of interest to declare.
MiR-15a and Mir-16-1 Affects the Angiogenesis of Multiple Myeloma by Targeting VEGF-A
Authors: Sun Chunyan , Hu Yu , She Xiaomei , Chu Zhangbo , Fang Jun , Chen Lei
Published: October 2019

Abstract 4039

MicroRNAs (miRNAs) are non-coding small RNAs that negatively modulate protein expression at a post-transcriptional level and are deeply involved in the pathogenesis of a variety of cancers. Two miRNAs, miR-15a and miR-16, which act as putative tumor suppressor by targeting the oncogene BCL2, have been implicated in cell cycle, apoptosis and proliferation. Here we investigated the possible role of miR-15a/miR-16 in the angiogenesis of multiple myeloma (MM). Using stem-loop quantitative reverse transcription-PCR analysis, we showed that miR-15a/miR-16 are significantly underexpressed in primary MM cells as well as MM cell lines (RPMI 8226, ARH-77, OPM-2, KM3, U266 and NIH929). The aberrant expression of miR-15a/miR-16 were detected especially in advanced stage multiple myeloma. The expression of miR-15a and miR-16-1 were remarkably lower in stage III MM patients (n=23), whereas it were significantly higher in healthy individuals (n=18) and stage 2, II MM patients (n=14) (P<0.01 and P<0.001, respectively). In human MM cell lines and normal plasma cell, expression of miR-15a/miR-16 inversely correlated with the expression of vascular endothelial growth factor (VEGF). Consistently with the proposed role of miRNAs as key regulators of angiogenesis, here we identified VEGF-A as a target for miR-15a and -16. Enforced miR-15a or miR-16 expression reduced VEGF-A protein level and the luciferase reporter assays demonstrated that miR-15a and -16 specifically suppress expression of VEGF-A by directly interacting with its 3′-untranslated region. Moreover, Ectopic overexpression of miR-15a and -16 led to decreased pro-angiogenic activity of MM cells. Conditioned medium of pri-miR-15a- and pri-miR-16- transfected RPMI 8226 cells inhibited human bone marrow microvascular endothelial cell (BMEC-1) proliferation, chemotactic motility and capillary formation in vitro as compared with conditioned medium of scramble probe-transfected RPMI 8226 cells (P<0.05). Finally, miR-15a/miR-16 was shown to greatly inhibit the process of tumor formation in an animal model. Infection of lentivirus-miR-15a or lentivirus-miR-16 can significantly inhibit the xenograft tumor growth in nude mice. The average tumor volume after 4 weeks for GFP-transfected cells was 372.5 mm3. While the average tumor volume for miR-15a-transfected cells was 47 mm3 (P<0.05) and for miR-16-transfected cells was 93.6 mm3 (P<0.05). Of interest, neoangiogenesis analysis by immunohistochemistry staining of anti-CD31 showed that there were significant reductions in microvessel density present in the miR-15a group and miR-16 group as compared to control (P<0.05). Take together, our findings suggested that miR-15a and miR-16 could play a role in the tumorigenesis of MM at least in part by modulation of angiogenesis through targeting VEGF-A. These findings have therapeutic implications and may be exploited for future treatment of MM.

Disclosures:

No relevant conflicts of interest to declare.
Expression Profile and up-Regulation of Telomere-Associated Proteins In Multiple Myeloma
Authors: de la Guardia Rafael Díaz , Elosua Carolina , Catalina Purificación , Walker Brian A , Johnson David C , Gonzalez David , Davies Faith E. , Morgan Gareth J , Leone Paola
Published: October 2019

Abstract 4050

The role of the telomeres in the mechanisms of ageing and carcinogenesis has generated a considerable interest as a novel approach to the treatment of many cancers. Telomeres are nucleoproteins structures that protect the ends of eukaryotic chromosomes, which are particularly vulnerable due to progressive shortening in almost all dividing cells. The telomere length was observed as a critical factor in the initiation and progression of human cancers, and it is associated to chromosomal instability.

Most immortal cells possess enzymatic activity of telomerase. This suggests that telomerase activity and telomere length maintenance may be required for unlimited cell proliferation, tumorigenesis, and protection, allowing the evasion of apoptosis in cancer development. The telomerase activity could also be regulated positively or negatively by post-trancriptional and/or post-translational modification of the enzyme without transcriptional up-regulation of human telomerase reverse transcriptase (hTERT) mRNA. In this work, we analyze the expression data of all genes involved in telomerase activity. Patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), multiple myeloma (MM) and plasma cell leukemia (PLC) were studied through gene expression profiling analysis (Human Genome U133 Plus 2.0 arrays, Affymetrix). We identify 21 deregulated genes, implicated directly in telomere length maintenance activity in clonal plasma cells compared with normal cells (20 up-regulated and 1 down-regulated). These genes are MYC, KRAS, HSPA9, RB1 and members of the families: Small nucleolar ribonucleoproteins (H/ACA snoRNPs), A/B subfamily of ubiquitously expressed heterogeneous nuclear ribonucleoproteins (hnRNPs), and 14-3 -3 family. In conclusion, the myeloma cells acquire the telomere maintenance capability without deregulation of the human telomerase RNA gene (hTERC) and hTERT gene expression. It is an alternative lengthening of telomeres mechanism that has effect in the regulation of the BAD activity in apoptosis. The mechanism is based on preventing the partially-denatured proteins from aggregating, telomere maintenance through the correct processing and intranuclear trafficking of hTERC, telomerase reactivation and telomere stabilization, and efficient accumulation of hTERT in the nucleus. Thus, the findings of this study may help to improve telomerase-based therapy for multiple myeloma.

Disclosures:

No relevant conflicts of interest to declare.
Activity of Dasatinib Against Novel KIT-N822I Mutation In Familial Cutaneous Mastocytosis
Authors: Wasag Bartosz , Niedoszytko Marek , Piskorz Anna , Lange Magdalena , Renke Joanna , Jassem Ewa , Debiec-Rychter Maria , Limon Janusz
Published: October 2019

Abstract 4088

Background:

In contrast to mastocytosis associated with somatic KIT mutations, the accounts of familial forms of mastocytosis with KIT germline mutations are extremely rare. We report the family that met the WHO criteria for cutaneous mastocytosis, which was diagnosed in the father and two children.

Patients and Methods:

The clinical follow-up of mastocytosis in the father included bone marrow histopatological/cytological examinations and flow cytometry, and histopatological examination of the skin. In children tryptase measurement and skin histopatological examination was performed. The father and children presented with urticaria pigmentosa as the only manifestation of the disease. Blood, urine and buccal swabs specimens were collected from the family members. Molecular analysis of the KIT coding sequence revealed a novel missense mutation (p.N822I) in the affected members of the family.

Ba/F3 cell lines expressing KIT-N822I, KIT-D816V and KIT-V559D mutants were treated with different concentrations of imatinib and dasatinib. The effect of treatment on proliferation, survival, and signaling was determined. In addition, Cos-7 cells were transiently transfected with plasmids expressing KIT-WT and KIT-N882I.

Results:

By in vitro assays, both imatinib and dasatinib exhibited a high efficacy toward the control, imatinib-sensitive KIT-V559D mutant. In contrast, only dasatinib potently inhibited the KIT-N822I and control, imatinib-resistant KIT-D816V isoform with an IC50 value of 68 nmol/L and 77nmol/L, respectively. Finally, experiments with Cos-7 cells showed that on the contrary to KIT-WT the KIT-N822I mutation constitutively activated KIT tyrosine phosphorylation.

Conclusion:

In summary, we proved that novel germline KIT mutation (p.N822I) has transforming potential and cause a constitutive activation of KIT. Moreover, we demonstrated that treatment with dasatinib may provide a therapeutic alternative for patients with mastocytosis whose carry imatinib-resistant KIT mutant isoforms.

Disclosures:

No relevant conflicts of interest to declare.
In Vivo Imaging of Cerebral Circulation In Mouse Models of Polycythemia Vera
Authors: Santisakultarm Thom P. , Nishimura Nozomi , Paduano Claire , Skoda Radek C. , Olbricht William L. , Schafer Andrew I. , Schaffer Chris B. , Silver Richard T.
Published: October 2019

Abstract 4091

Polycythemia vera (PV) is a myeloproliferative disease which is often associated with compromised cerebral microcirculation due to thrombotic and other rheological complications; this, in turn, may contribute to cognitive decline. Elevated blood viscosity may also lead to leukocyte adhesion, which further impacts cortical perfusion. An activating mutation, JAK2V617F, in the gene for JAK2, a cytoplasmic tyrosine kinase, is associated with PV in humans and transgenic mice carrying the same mutation have been developed. Our study aims to quantify the alterations in cerebral microcirculation in polycythemic mice and to determine the role of leukocyte adherence in disrupting blood flow, raising the possibility of a possible clinical therapeutic target for patients with PV. We used two models of PV: erythropoietin (EPO) injection (10-100 IU daily subcutaneous injections for 5 days) and a JAK2V617F transgenic mouse. Vascular topology and blood flow was imaged in anesthetized mice, through a craniotomy, using in vivo two-photon excited fluorescence microscopy. Texas-red dextran (0.05 mL of 2.5% w/v) and rhodamine 6G (0.05 mL of 0.1% w/v) were intravenously injected to label blood plasma and leukocytes, respectively. RBC flow speed was measured in arterioles, capillaries, and venules. Brain capillaries were classified as flowing or stalled by evaluating the motion of RBCs within individual vessels. We found a significant decrease in average capillary flow speed in EPO-injected mice, with an average hematocrit (Hct) of 60% (0.70±0.533 mm/s. 10 mice, 105 vessels, p<0.01) and JAK2V617F transgenic mice with Hct of 67% (0.55±0.362 mm/s. 3 mice, 47 vessels, p<0.01) compared to wild type controls with Hct of 48% (1.24±0.986 mm/s. 9 mice, 92 vessels), but not in surface arterioles or venules. In EPO-injected mice, 20% of the capillaries were stalled (12 mice, 6594 vessels, p<0.01), compared to only 3% in control mice (5 mice, 2431 vessels). In JAK2V617F transgenic mice, we found 25% of the capillaries were stalled (4 mice, 4574 vessels, p<0.01). Further, we observed firm leukocyte adherence in a large fraction of the stalled capillaries in the EPO-injected and JAK2V617F transgenic mice (see Figure). Our findings suggest that high Hct creates flow conditions which lead to leukocyte adherence, and may result in leukocyte activation. This work suggests that targeting leukocyte adherence and reducing the hematocrit may be clinically important in patients with PV and other diseases with high blood viscosity to ameliorate abnormal cerebral blood flow.

Disclosures:

No relevant conflicts of interest to declare.
Infliximab Drives Gamma-Delta T Cell Expansion In Crohn's Disease – a Predictor of Lymphoma Risk?
Authors: Kelsen Jens , Schwindt Heinrich , Dige Anders , d'Amore Francesco , Pedersen Finn Skou , Agnholt J∅rgen , Christensen Lisbet Ambrosius , Dahlerup Jens Frederik , Hvas Christian Lodberg
Published: October 2019

Abstract 4131

Background:

Due to the widespread use of combined immunosuppressive therapy in the management of Crohn's disease (CD), the risk of malignant lymphoproliferation, including the fatal hepato-splenic T cell lymphoma (HSTCL), has become a major concern. We investigated dynamic changes of peripheral gamma-delta (γδ)-T cells during CD treatment with the anti-TNF-α-antibodies infliximab (Remicade®) and adalimumab (Humira®).

Methods:

Forty-six patients with active CD and nine healthy volunteers were analysed. Patients delivered blood samples before and 1, 7, and 42 days after infliximab 5 mg/kg (20 patients) or adalimumab given as induction with 160 mg and 80 mg after 2 weeks and subsequently 40 mg every other week (26 patients). The γδ-T cells were analysed using FACS analysis. Patients with high percentages of peripheral γδ-T cells were characterized by PCR-assessment of γδ-T cell clonality.

Results:

Of 46 patients included in the analysis, 35 (76%) had γδ-T cell levels comparable to those of healthy individuals (mean: 1.6%; 95% CI: 1.3–2.0%). Higher γδ-T cell levels from 5% up to 15% occurred in 11 patients (24% of the total cohort). A high γδ-T cell level was associated with non-smoker status and young age. In 18 patients receiving thiopurines or methotrexate, the mean baseline γδ-T cell percentage was 4.4% (95% CI: 2.1–6.7%). In three male CD patients with high baseline values, the γδ-T cell percentage doubled within 24 hours following infliximab therapy. Another male patient on infliximab monotherapy presented with a predominantly clonal baseline γδ-T cell population as high as 20%, further increasing to 25% shortly after infliximab treatment.

Conclusion:

One fourth of the CD patients treated with immunomodulators had constitutive high levels of circulating γδ-T cells, and infliximab aggravates this γδ-T cell expansion. We raise the hypothesis that such patients may be at increased risk of developing a malignant γδ-T cell lymphoproliferation.

Disclosures:

No relevant conflicts of interest to declare.
The BCL11B Tumor Suppressor Is Mutated In Human T-Cell Acute Lymphoblastic Leukemia
Authors: Gutierrez Alejandro , Kentsis Alex , Sanda Takaomi , Etchin Julia , Protopopov Alexei , Zhang Jianhua , Chin Lynda , Silverman Lewis B. , Winter Stuart , Hunger Stephen , Sallan Stephen E. , Zha Shan , Alt Frederick , Look A. Thomas
Published: October 2019

Abstract 4177

The BCL11B transcription factor, previously identified as a central player in normal α/β T-cell development, has recently been found to play critical roles in the maintenance of T-cell identity, with biallelic Bcl11b inactivation in T-cell precursors severely impairing their continued development into the T-cell lineage. Previous work has demonstrated that Bcl11b haploinsufficiency accelerates the onset of thymic lymphomas in p53-mutant mice, as well as T-lymphoid blast crisis in a mouse model of chronic myeloid leukemia induced by BCR-ABL. Furthermore, we have recently identified recurrent monoallelic Bcl11b deletions in 91% of T-cell acute lymphoblastic leukemias (T-ALL) arising in Atm-deficient mice, further supporting a role for Bcl11b haploinsufficiency in T-cell leukemogenesis. In order to determine whether BCL11B inactivation plays a role in the molecular pathogenesis of human T-ALL, we analyzed BCL11B status in primary T-ALL patient samples by array CGH and sequencing analysis. Monoallelic BCL11B deletions were identified in 6% of cases (n = 3 of 47) analyzed by array CGH, including one microdeletion within the BCL11B locus, one small deletion involving BCL11B and 6 additional genes, and one large 26 Mbp deletion of the distal arm of chromosome 14. BCL11B sequencing revealed heterozygous missense mutations in an additional 9% (n = 4 of 43) of primary T-ALL patient samples and in 19% (n = 3 of 16) of T-ALL cell lines. Structural homology modeling revealed that many of the mutations identified, including 3 of the 4 in the primary patient samples, disrupted key amino acids within BCL11B zinc finger domains that are involved in DNA recognition or structural stabilization required for zinc finger domain-mediated transcriptional activity. Analysis of TCRγ rearrangement status and gene expression data revealed that most cases with BCL11B inactivation were characterized by biallelic TCRγ rearrangements together with an early thymocyte precursor (ETP) gene expression signature, indicating developmental arrest at a prethymocyte stage of T-cell development, which we and others have shown confers an increased risk of treatment failure (Coustan-Smith et al. Lancet Oncol 2009; Gutierrez et al. J Clin Oncol 2010). Given that BCL11B expression during normal T-cell development increases markedly at the prethymocyte stage, our findings suggest that BCL11B inactivation may be directly responsible for developmental arrest at a prethymocyte stage during thymocyte transformation. Our findings provide compelling evidence that BCL11B is a tumor suppressor in a subset of human T-ALLs with a high risk of treatment failure.

Disclosures:

No relevant conflicts of interest to declare.
Endogenous Oncogenic Nras Mutation Initiates T-Cell Leukemia/Lymphoma In a Dose-Dependent Manner
Authors: Wang Jinyong , Li Zeyang , Wang Zhongde , Liu Yangang , Ryu Myung-Jeom , Meline Benjamin , Du Juan , Young Ken H , Chang Qiang , Zhang Jing
Published: October 2019

Abstract 4183

The oncogenic NRAS mutations are frequently identified in myeloid diseases but rare in lymphoid diseases. They occur in 4% of acute T-cell lymphoblastic leukemia/lymphoma (T-ALL) patients and 22% of human T-ALL cell lines. Its differential roles in myeloid versus lymphoid disease development remain unclear. Here we examine the tumorigenic potential of oncogenic Nras in T-cells using two conditional Nras G12D murine knock-in models that either hypomorphically (NrasG12D Hypo) or normally (NrasG12D Norm) expresses oncogenic Nras G12D from its endogenous locus. Mice expressing monoallelic or biallelic NrasG12D Hypo develop normally and are tumor free. However, NrasG12D Norm leads to acute T-cell leukemia/lymphoma (TAL/L) in a bone marrow transplantation model, with a low incidence (∼8%) when expressing one allele (TAL/L-het) and a complete penetrance when expressing two alleles (TAL/L-homo). TAL/L-het tumors are associated with spontaneous up-regulation of oncogenic Nras in ∼67% of animals, and tumor cells are TdT positive, suggesting that they are transformed at an immature stage. In contrast, TAL/L-homo tumors express comparable levels of Nras to control thymocytes, and tumor cells are TdT negative, suggesting that they are transformed at a more mature stage. Both TAL/L-het and TAL/L-homo tumors are oligoclonal or polyclonal. Above 70% of these tumors contain clonal Notch1 mutations and are sensitive to gamma-secretase inhibitor. These data indicate that Notch1 mutations are acquired at an early stage and play an important role in the development of TAL/L-het and TAL/L-homo tumors. Together, our results show that engdogenous oncogenic Nras mutation leads to TAL/L in a dose-dependent manner, and thus explain the low incidence of oncogenic NRAS mutations in human T-cell diseases.

Disclosures:

No relevant conflicts of interest to declare.
JAK2 Germline Genetic Variation In Budd-Chiari Syndrome and Portal Vein Thrombosis
Authors: Smalberg Jasper , Koehler Edith , Murad Sarwa Darwish , Plessier Aurelie , Garcia-Pagan Juan-Carlos , Seijó Susana , Langlet Philippe , Trebicka Jonel , Primignani Massimo , Maat Moniek De , Valla Dominique , Janssen Harry , Leebeek Frank
Published: October 2019

Abstract 4212

Primary Budd-Chiari syndrome (BCS) and non-malignant, non-cirrhotic portal vein thrombosis (PVT) are rare disorders with a considerable overlap in etiology. Myeloproliferative neoplasms (MPN) are the most frequent underlying prothrombotic factor in both entities. The JAK2 V617F mutation (VF) has been identified in over half of the individuals with MPN. Recently, a JAK2 haplotype, designated ‘46/1’, has been described. Previous studies suggest that the JAK2 46/1 haplotype represents a disease susceptibility to MPN, independent of VF status. The aim of this study was to determine the role of the JAK2 46/1 haplotype in the etiology of BCS and PVT. Patients were recruited from the EN-Vie cohort, consisting of 163 BCS and 138 PVT patients, consecutively enrolled in nine European countries between October 2003 and October 2005. DNA was available from 116 BCS patients (50 males and 66 females; median age 38.1), 97 PVT patients (47 males and 50 females; median age 49.8) and 104 healthy controls (43 males and 61 females; median age 36.8). The JAK2 46/1 haplotype was tagged by the rs12343867 single nucleotide polymorphism. Frequency of the JAK2 haplotype 46/1 was higher in BCS (36%, p=0.06) compared to controls (27%), while similar in PVT patients (28%, p=0.89). When stratified for VF status, haplotype 46/1 frequency was higher in VF positive BCS (44%, p=0.01) and VF positive PVT patients (40%, p=0.06) compared to controls. Haplotype 46/1 frequency was similar in VF negative BCS (33%, p=0.29) and PVT patients (24%, p=0.47) compared to controls. VF negative BCS patients with a proven MPN also showed increased frequency of the 46/1 haplotype (56%, p=0.07). Logistic regression, adjusted for age and sex, showed an association between the 46/1 haplotype and risk of VF positive BCS (OR: 2.10; 1.16–3.80), VF positive PVT (OR 2.07; 0.95–4.52) and VF negative BCS patients with a proven MPN (OR 3.04; 1.02–9.06). We conclude that the JAK2 46/1 haplotype may be associated with BCS and that this was limited to patients with a proven MPN, independent of VF status. In PVT, the 46/1 haplotype was only associated with patients who were VF positive.

This study was carried out on behalf of the European Network for Vascular Disorders of the Liver (EN-Vie).

Disclosures:

No relevant conflicts of interest to declare.
Clearance of Platelet Microvesicles by Endothelium. the Role of Developmental Endothelial Locus–1 (Del-1)
Authors: Dasgupta Swapan Kumar , Le Anhquyen , Thiagarajan Perumal
Published: October 2019

Abstract 4303

Activation of platelets with certain agonists induces release of small submicron size membrane-enclosed compartments of platelet content called microvesicles. During microvesiculation, phosphatidylserine, an anionic phospholipid, moves from the inner to the outer leaflet of membrane bilayer accounting for the procoagulant effect of microvesicles. Microvesicles circulate in the blood with a short half-life of less than 5–10 minutes and the exposure of phosphatidylserine may mediate its rapid clearance. Developmental endothelial locus–1 (del-1) is a 52kDa glycoprotein secreted by endothelial cells. It contains three epidermal growth factor (EGF)-like repeats in amino terminus and two discoidin I-like domains in carboxy terminus, similar to the macrophage opsonin lactadherin. The second EGF like repeat contains the canonical Arg-Gly-Asp (RGD) motif that enables Del-1 to bind integrin αVβ3 and αVβ5. Here, we tested the role of Del-1 in endothelial uptake of platelet-derived microvesicles.

In vitro, resting human umbilical vein endothelial cells engulf BODIPY-labeled phosphatidylserine expressing platelets microvesicles avidly. Evidence of uptake was obtained by confocal microscopy and by flow cytometry. Following uptake, the microvesicles were distributed throughout the cytoplasm of the endothelial cells. We quantified the internalization of fluorescent microvesicles by measuring the intracytoplasmic fluorescence associated with endothelial cells in a flow cytometer after quenching cell surface fluorescence with Trypan Blue. A monoclonal antibody to Del-1 inhibits (p=0.03) the uptake while exogenous Del-1 promotes the uptake by endothelial cells. Abciximab inhibited -dependent phagocytosis to about ∼40 % while under similar conditions an irrelevant control antibody had no effect (p=0.016). In addition, annexin A5 also inhibited the uptake of platelet-derived microvesicles by endothelial cells (p=0.002). These results show Del-1 mediates phosphatidylserine and integrin-dependent uptake of microvesicles by endothelial cells. To assess the in vivo significance, we infused BODIPY labeled platelet-derived microvesicles into the inferior vena cava of the mouse and harvested endothelial cells from the pulmonary circulation. Microvesicles were detected within the endothelial cells. In Del-1 deficient mice has impaired uptake of microvesicles compared wild type littermate controls (P=0.02). We also measured the levels of microvesicles six after administration lipopolysaccharide. Del-1 deficient mice have increased microvesicles compared to their littermate controls (p=0.02).

These studies show a physiological role for Del-1 in the clearance of microvesicles by endothelium.

Disclosures:

No relevant conflicts of interest to declare.
Genetic Determinants of Plasma Von Willebrand Factor Antigen Levels: A Target Gene SNP and Haplotype Analysis of the ARIC Cohort
Authors: Campos Marco , Sun Wei , Yu Fuli , Barbalic Maja , Chambless Lloyd , Wu Kenneth , Ballantyne Christie , Folsom Aaron , Boerwinkle Eric , Dong Jing-fei
Published: October 2019

Abstract 4310

Background and Objectives:

Von Willebrand factor (VWF) is an essential component of hemostasis. It is known that multimer size and the amount of circulating VWF impact its hemostatic function. Genetic factors play a major role in regulating VWF synthesis and clearance and are reported to contribute to 66% of the variation in plasma VWF antigen level. We have analyzed 78 SNPs distributed throughout the VWF gene and haplotypes constructed from those SNPs for an association with VWF antigen level in 7,856 subjects of European descent in the Atherosclerosis Risk in Communities cohort. Methods and Results: Blood was drawn after an 8 hour fasting period and VWF antigen level was determined by a commercial ELISA kit. All subjects underwent analysis for 78 SNPs available from Affymetrix 6.0 chip in the region encompassing the VWF gene. We used the fastPHASE 1.2 program to resolve haplotypes from the unphased SNP genotype data. Using Haploview we determined SNPs in strong linkage disequilibrium (LD), their co-segregation rates and underlying haplotypes. Linear models were used to evaluate the association of actual or log VWF antigen levels with each SNP and haplotypes derived from those SNPs. Eighteen (16 are intronic) of the 78 SNPs were found to significantly associate with levels of VWF antigen (Table 1). All are clustered in a 50 kb region of the VWF gene in spite of the fact that the 78 SNPs studied are distributed throughout the VWF gene without apparent clusters. All 78 SNPs are co-segregated in 9 LD haplotype blocks, but a majority of positive SNPs (88.9%) are located in Block 5 and 6, which significantly correlate with VWF antigen level. The O blood type contributes to ∼ 15% of variation in VWF antigen levels. The association for SNPs and Haplotypes grows stronger after ABO effects are removed. Among non-genomic factors, age and BMI are the most significant factors and contribute to 4.8% and 1.4% of variation in VWF antigen level. Conclusions: We have found a strongly positive association between VWF level and SNPs and haplotypes from a strikingly concentrated region of the VWF gene. This region encodes the D2, D' and D3 domains of VWF, including the propeptide and multimerization and Factor VIII binding sites. The physiological significance of these clustered SNPs on VWF synthesis or clearance remains to be further investigated. Our data suggest that this region plays an important role in regulating VWF antigen level and support the idea that combinations of SNPs in LD can provide additive affects.

Disclosures:

No relevant conflicts of interest to declare.
A Study of the Proliferative Effect on Acute Lymphocytic Leukemia Cell Lines by G-CSF and Synergetically Anti-Leukemic Effect on These Cell Lines by G-CSF Plus Cytotoxic Agent
Authors: Xue Shengli , Dai Lan , Chen Yan , Qiu Qiaocheng , Chen Suning , Tang XiaoWen , Sun Aining , Depei Wu
Published: October 2019

Abstract 4330

Objectives

To observe the proliferative effect on acute lymphocytic leukemia (ALL) cell lines caused by G-CSF, and to evaluate the synergetically anti-leukemic effect on these cell lines produced by Ara-C combined with G-CSF.

Methods

With fluorescence quantitation kit and flow cytometry, G-CSFR expression density of 5 ALL cell lines were detected, and expression density changes due to G-CSF stimulation were evaluated. After treatment by G-CSF of various concentrations, changes of cell cycle and cell proliferation in ALL cell lines were examined by DNA-PREP-TM kit and Cell Counting Kit (CCK-8), respectively. After treatment by different concentration's Ara-C combined with different concentration's G-CSF, ALL cell lines’ existing cell viability were detected by CCK-8.

Results

All 5 ALL cell lines expressed G-CSFR, 2 ALL cell lines including CCRF-CEM and 697 exhibited G-CSFR expression density up-regulation tendency after G-CSF's stimulation, while 3 T-ALL cell lines including MOLT-4, Jurkat,Clone E6-1, A3 showed G-CSFR expression density down-regulation tendency after G-CSF's stimulation, which was similar in neutrophil. Cell cycle analysis showed that S phase cell proportion in MOLT-4, Jurkat,Clone E6-1, A3 cell lines had a growing tendency after G-CSF’ treatment, while no similar tendency was observed in CCRF-CEM, 697 cell lines. A relatively proliferative advantage was found in MOLT-4, Jurkat,Clone E6-1, A3 cell lines after G-CSF's treatment, but only in MOLT-4 there was a statistically significance after 10ng/ml G-CSF's stimulation. A synergetically anti-leukemic effect was observed in MOLT-4, Jurkat,Clone E6-1, A3 cell lines after Ara-C's treatment combined with G-CSF.

Conclusions

The biological characteristics of ALL cell lines expressing G-CSFR was different from each other. Synergetically anti-leukemic effect produced by cytotoxic agent and G-CSF could occur in ALL cell lines which showed a growing S phase cell proportation after G-CSF's treatment. ALL cell's response to G-CSF, which means down-regulation of G-CSFR expression density, seems a promising indicator that a synergetically effect could be produced by Ara-C plus G-CSF. Before this indicator could be applied for guiding ALL patients’ management, clinical researches are needed to confirm it.

Disclosures:

No relevant conflicts of interest to declare.
Treatment of Primary Granulocytic Sarcoma of the Uterine Cervix: Reports of Three Cases
Authors: Jie Zhang , Jian Meng Xiao , Jin Ye Xiu , He Jing-Song , Huang He
Published: October 2019

Abstract 4346

Background:

Granulocytic sarcoma is a rare event with a poor prognosis which may herald, accompany or signal acute myeloid leukaemia. While many advances in diagnosis have been made, dilemmas remain concerning the diagnosis and treatment of this disease particularly when it arises in an unusual site such as the uterine cervix.

Case:

We describe here three female cases of primary granulocytic sarcoma in the cervix uteri who were 39, 44 and 41 years old, respectively. All the patients presented with irregular vaginal discharge or bleeding and postcoital pain. Biopsy of the vaginal wall or cervix was performed and multiple immunohostochemical analyses were obtained. Myeloid markers were strongly positive whereas lymphoid markers, epithelial markers as well as neuroendocrine markers were all negative, which confirmed the diagnosis of granulocytic sarcoma. The induction chemotherapy including homoharringtonine in combination with cytarabine (homoharringtonine, days 1–3; cytarabine days 1–7) or idarubicin with cytarabine (idarubicin, days 1–3; cytarabine days 1–7) was given followed by consolidation therapy. All the patients achieved complete remission after induction and two of whom remain in complete remission with normal complete blood counts, physical examination, abdominal ultrasonography and magnetic resonance imaging. One patient relapsed locally after six cycles of chemotherapy and progressed to acute myeloid leukemia after one year follow-up.

Conclusion:

Granulocytic sarcoma of the cervix is a rare entity and even in its localized form, early intensive therapy appears to be appropriate. Although this experience is confirmed to three patients, it may contribute to the design of prospective therapeutic studies in patients with this rare disease.

Disclosures:

No relevant conflicts of interest to declare.
Survival Outcomes of Adults with Acute Myeloid Leukemia – A Study of Native American, Hispanic, and Non-Hispanic Whites Treated In New Mexico (1986 to 2006)
Authors: Rivera Nancy , Armijo Beeling , Quintana Dulcinea , Abdul-Jaleel Maryam , Lee Sang-Joon , Wiggins Charles , Libby Edward
Published: October 2019

Abstract 4365

Purpose of study:

The three largest ethnic populations in New Mexico are Caucasians, Native Americans, and Hispanics. As the largest tertiary care center in the state, our institution offers treatment to significant numbers of all three groups. In addition, many residents of the state live in rural areas. The goal of this study is to identify potential differences in outcome between ethnic groups from urban or rural areas in patients with newly diagnosed acute myelogenous leukemia (AML) treated at this center from 1986–2006.

Methods:

A retrospective chart review was performed on patients identified through a New Mexico tumor registry search of all adult patients (≥ 18 years of age) with a new diagnosis of AML treated at this center from 1986 through 2006. The search identified 150 cases. Of those patients, 144 charts were available for review. Six charts could not be located. Electronic, microfilm and paper-based medical records were reviewed. Data was collected for race/ethnicity, sex, zip code, age at diagnosis, pathologic diagnosis, date of diagnosis, cytogenetics, treatments received, total number of chemotherapy cycles, achievement of complete remission, and overall survival from time of diagnosis and from achievement of remission after induction chemotherapy. Date of death was determined from the chart or from New Mexico tumor registry data. Cancer cases who resided in New Mexico counties with large population centers were designated as urban residents and the remaining cases were designated as rural residents. Statistical analysis was performed to examine the effect of the variables mentioned above on overall survival.

Summary of results:

The analysis included 144 patients (mean age 49.98, range 18–85). For those patients who received chemotherapy the standard treatment was 7+3 followed by high dose cytarabine (1-4 cycles) or similar regimen. There were no statistical differences between survival rates when comparing the three ethnic groups, Caucasians 78 (52%), Hispanics 43 (28%), and Native Americans 23 (15%) (P=0.2496). In agreement with previous reports, statistically significant differences were seen for treatment versus palliative care, cytogenetics and age. The study included 61 urban patients with a mean survival of 426 days and 83 rural patients with a mean survival of 278 days. Analysis revealed a statistically significance in survival for urban versus rural patients who achieved complete remission after induction chemotherapy (P=0.0443).

Conclusions:

Survival was improved for urban patients who achieved a complete remission after induction chemotherapy versus rural patients (P= 0.0443). Further study should be undertaken to identify the causes of this disparity.

Disclosures:

No relevant conflicts of interest to declare.