bluuebunny/crossref_metadata_2025 huggingface dataset:
Ferroportin Q248H Mutation Prevents Its Ubiquitination
Authors: Ammosova Tatiana , Ivanov Andrey , Nekhai Sergei A
Published: October 2019

Background

Ferroportin Q248H mutation is prevalent in African populations and leads to increased serum ferritin. Our recent study shows that ferroportin Q248H protein is resistant to physiologic hepcidin concentrations1. Also sickle cell disease patients with ferroportin Q248H heterozygote had lower serum ferritin concentration suggesting that the enhanced iron release by macrophages. Ferroportin glutamine 248 is located within the intracellular loop (residues 228-307), which is likely to be located in the cytoplasm. Recently ferroportin internalization was shown to be driven by ubiquitination of lysines lying within residues 229-269 including K229, K240, and K2472. The proximity of the K240 and especially to K247 to the Q248 residue suggests that a positively charged histidine in position 248 might change the overall negative charge of the 240eeetelkqlnlhk253sequence toward a more positive charge, which might affect ubiquitination and subsequent degradation of ferroportin. Here we analyzed and compared ubiquitination of WT and Q248H mutant ferroportin.

Results

WT ferroportin and Q248H mutant were expressed as EGFP-fusions in 293T cells and also combined with the expression of ubiquitin. Ferroportin was immunoprecipitated with anti-EGFP antibodies and analyzed by high resolution mass spectrometry using LTQ-Orbitrap. Phosphorylation and ubiquitination was determined using Proteome Discover and quantified using SIEVE 2.1 software.

Conclusions

WT ferroportin but not the Q248H mutant ferroportin was found to be ubquitinated on lysines 247 and 253 and also phosphorylated on Thr 144. Also WT ferroportin was found to associate with ubiquitine-conjugating enzyme E2 and ubiquitine protein ligase NEDD4. Thus hepcidin resistance of ferroportin Q248H could be due to its inability to undergo ubiquitination.

Acknowledgments

This project was supported by NIH Research Grants 8G12MD007597 and P30HL107253.

References

1. Nekhai S, Xu M, Foster A, et al. Reduced sensitivity of the ferroportin Q248H mutant to physiological concentrations of hepcidin. Haematologica. 2013;98(3):455-463.

2. Qiao B, Sugianto P, Fung E, et al. Hepcidin-induced endocytosis of ferroportin is dependent on ferroportin ubiquitination. Cell Metab. 2012;15(6):918-924.

Disclosures:

No relevant conflicts of interest to declare.
Myocardial and Hepatic Iron Overload and Cardiac Function In Sickle/Thalassemia Patients Of Italian Origin
Authors: Meloni Antonella , Ruffo Giovan Battista , De Marchi Daniele , Cardinale Antonio , Pietrapertosa Anna , Facchini Elena , Toia Patrizia , De Franceschi Lucia , Positano Vincenzo , Lombardi Massimo , Pepe Alessia
Published: October 2019

Introduction

Sickle-thalassemia results from the combined heterozygosity for sickle-cell and β-thalassemia genes. This study evaluates myocardial and hepatic iron overload and cardiac function in Italian patients and explores their correlation with transfusions, age and sex.

Methods

Fifty-nine sickle-thalassemia patients (29 males, mean age 35.6±14.1 years), enrolled in the MIOT network underwent magnetic resonance imaging (MRI). T2* value for all 16 myocardial segments and global heart T2* value were calculated. Hepatic T2* value was converted into liver iron concentration (LIC). Cine images were acquired to quantify biventricular volumes and ejection fraction (EF).

Results

55 (93%) patients had all segmental T2* values normal (>20 ms). Of the 4 patients with abnormal segmental T2* values, all showed an heterogeneous myocardial iron overload (some segments with T2*>20 ms and other with T2*<20 ms) and only one had a global T2*<20 ms. The mean global heart T2* value was 34.4±6.2 ms. The mean LIC was 5.9±6.5 mg/g/dw and 30 patients (50.8%) had a pathological value (≥ 3 mg/g dw). There was a statistically significant positive correlation between global heart T2* and age but with poor linearity (R=0.368; P=0.004) and there was not a significant correlation between age and LIC.

Males and females had comparable global heart T2* values and LIC values.

Twenty patients were regularly transfused, 32 received sporadic transfusions while 7 were not transfused. The comparison among the three groups is shown in Table 1. We did not find significant differences in the global heart T2* value while patients regularly transfused had significantly higher LIC than sporadically transfused patients. Biventricular volumes indexed by body surface area and ejection fractions were comparable among the groups.

Conclusions

In respect of MIO, the sickle/thalassemia patients are similar to patients with homozygous SCD for which iron overloading is relatively rare. Hepatic iron overload may develop also in no regularly-transfused patients, maybe due to increased absorption of iron from the digestive tract, characteristic of both SCD and thalassemia intermedia patients. This finding underlines the importance to monitor by MRI also no regularly transfused sickle/thalassemia patients.

Disclosures:

No relevant conflicts of interest to declare.
Left and Right Ventricular Wall Motion Abnormalities In Thalassemia Major Patients
Authors: Meloni Antonella , Giuffrida Gaetano , Paci Cristina , Sardella Leonardo , Quota Alessandra , Bitti Pier Paolo , De Franceschi Lucia , Salvatori Cristina , Vinci Valentina , Positano Vincenzo , Lombardi Massimo , Pepe Alessia
Published: October 2019

Introduction

Left ventricular (LV) and right ventricular (RV) wall motion abnormalities can be detected through a qualitative analysis of cine magnetic resonance (MR) images. Moreover, MR is the gold standard technique for the evaluation of myocardial iron overload (MIO), biventricular global systolic function and myocardial fibrosis. We investigated the relationships between LV movement abnormalities and MIO, LV function and myocardial fibrosis as well as between RV motion and function in thalassemia major (TM) patients.

Methods

CMR was performed in 1092 TM patients (537 male; 30.6±8.5 years) enrolled in the Myocardial Iron Overload in Thalassemia Network. Cine images were acquired to evaluate wall motion and to quantify biventricular volumes and ejection fraction (EF). For MIO assessment, a T2* multislice approach was used. To detect myocardial fibrosis, late gadolinium enhanced (LGE) images were acquired. For the LV the 16-segment model of the AHA/ACC was taken into account during image analysis: wall motion, T2* value and presence/absence of enhancing area were evaluated for each segment.

Results

Abnormal motion of LV was found in 66 (6%) patients (60 hypokinetic and 6 dyskinetic). Table 1 shows the comparison between TM patients with normal and abnormal LV motion. Patients with abnormal LV motion were older and had significantly lower global T2* value and significantly higher number of segments with T2*<20 ms. Left volumes and mass indexed by body surface area were significantly higher in patients with abnormal LV motion while the EF was significantly lower. LGE areas were detected in 196 patients (18%) and were predominantly located in the mid-ventricular septum. There was a significant correlation between LGE and abnormal LV motion.

Abnormal motion of the RV was found in 35 (3.2%) patients (29 hypokinetic, 5 dyskinetic and 1 akynetic). Table 2 shows the comparison between TM patients with normal and abnormal RV motion. Patients with abnormal RV motion were older and they were more frequently males. Right volumes were significantly higher in patients with abnormal RV motion while the EF was significantly lower.

Abnormal LV motion was not correlated with abnormal RV motion. Seventeen patients showed movement abnormalities in both ventricles.

Conclusions

Movement abnormalities in the left ventricle were not really frequent in TM patients but were associated with age, MIO, LV dilation and dysfunction, and myocardial fibrosis. Movement abnormalities in the right ventricle were less frequent compared to the left ventricle, but were associated with age, sex , RV dilation and dysfunction.

Disclosures:

No relevant conflicts of interest to declare.
Genetic Mosaicism and Hemoglobinopathies; Uniparental Isodisomy
Authors: Harteveld Cornelis L , Phylipsen Marion , Giordano Piero C
Published: October 2019

Background

Recently we discovered three independent cases of “severe late onset beta-thalassemia”, all presenting with the mild phenotype of beta thalassemia minor up to adult age and developing a severe transfusion dependent phenotype in the third and fourth decade of life when a presumed homozygosity for the beta-thalassemia mutation is observed. We demonstrate that uniparental isodisomy (UPD) of part of chromosome 11p, a mechanism also seen in 20% of Beckwith-Wiedemann syndrome patients, accounts for the observed mosaicism in all three independent cases. Clonal selection for hematopoietic stem cells containing the UPD for the mutant beta-globin gene during life may account for the progressive development of the disease. Recently we discovered another case showing a similar mosaic UPD of 11p, presenting as a regular beta-thalassemia carrier.

Method

Direct sequencing of the beta-globin genes. Multiplex Ligation dependent Probe Amplification (MLPA) analysis of the HBB (beta-globin) gene cluster. Affymetrix GeneChip Human Mapping 262K NspI array (Santa Clara, CA, USA). Illumina OmniExpress 730K SNP Beads array (San Diego, CA, USA).

Results

In all cases molecular analysis shows sequences in which a near to complete homozygosity for the beta-gene mutation in DNA extracted from leucocytes is seen. Loss of heterozygosity due to allele drop-out or a deletion of one allele was excluded by direct sequencing and MLPA analysis. Affymetrix and/or Illumina SNP-array analysis revealed incomplete homozygosity for SNP’s along almost the entire short arm of chromosome 11 containing the beta-globin gene, indicating mosaicism for a partial uniparental isodisomy of chromosome 11p. Three patients were born asymptomatic as beta-thal carriers and developed a severe blood-transfusion dependent beta-thalassemia major at different ages and with different percentages of mosaicism. The fourth patient however did not develop the clinical severity despite of an almost 50% mosaicism determined from the DNA isolated from leucocytes.

Conclusion

We demonstrate that uniparental isodisomy of part of chromosome 11 accounts for the observed mosaicism in all four independent cases. The most probable mechanism seems clonal selection for hematopoietic stem cells containing the uniparental isodisomy for the mutant beta-globin gene during life, this may account for the progressive development of the disease. However, there seems to be no correlation between the percentage of mosaicism measured in the DNA isolated from the white cells and the severity of the clinical phenotype related to the expression in red cells, which strongly suggests hematopoietic tissue heterogeneity for the observed UPD containing cell lineages. This may have serious consequences for disease prediction and counseling, as this is largely dependent upon DNA isolated from leucocytes.

Disclosures:

No relevant conflicts of interest to declare.
A Mechanism For Switch Of Integrin Signaling Direction and a New Anti-Thrombotic Strategy Through Selective Outside-In Signaling Inhibition
Authors: Shen Bo , Zhao Xiaojuan , O'Brien Kelly A , Stojanovic-Terpo Aleksandra , Delaney Michael Keegan , Kim Kyungho , Cho Jaehyung , Lam Stephen C.-T , Du Xiaoping
Published: October 2019

Antagonists of platelet integrin alphaIIbbeta3 are potent anti-thrombotics due to critical roles of integrins in thrombosis. However, integrins are also important in hemostasis, and thus integrin antagonists have potentially life-threatening bleeding side effect. It would be ideal if we can develop integrin antagonists without bleeding side effect. Integrins transmit signals bidirectionally. Intracellular signals activate integrin alphaIIbbeta3, leading to talin-dependent integrin ligation, which is required for platelet adhesion and initial hemostatic thrombus formation. Integrin ligation in turn mediates Galpha13/Src-dependent outside-in signaling that stabilizes and amplify thrombi, which is crucial for occlusive arterial thrombosis. Here we show that talin and Galpha13 bind to mutually exclusive but distinct sites in integrin beta3, and their bindings are dynamically regulated during integrin signaling. The first talin binding wave mediates inside-out signaling and also “ligand-induced integrin activation”, but is not required for early phase outside-in signaling. Integrin ligation induces talin dissociation and Galpha13 binding, which selectively mediates early phase outside-in signaling. The second talin binding wave is associated with late phase outside-in signaling and clot retraction. Based on these findings, we have designed a selective inhibitor of Galpha13-integrin interaction, which specifically abolished outside-in signaling without affecting inside-out signaling and integrin ligation. Strikingly, this inhibitor potently inhibits occlusive thrombosis in vivo, but has no effect on tail bleeding time, in contrast to the current integrin antagonists. Thus, we have discovered a mechanism for switching the direction of integrin signaling and a new anti-thrombotic that does not cause bleeding.

Disclosures:

No elevant conflicts of interest to declare.
Aberrant Type-2-Lactosaminoglycan Synthesis Severely Impairs Thrombopoiesis
Authors: Giannini Silvia , Begonja Antonija Jurak , Adelmann Max , Hoffmeister Karin M.
Published: October 2019

Platelet recovery following myelosuppressive/myeloablative chemotherapy is crucial to avoid bleeding complications of cancer treatment. Platelets are produced by bone marrow megakaryocytes (MKs), which develop and mature from hematopoietic stem cells (HSC). Mature MKs interact with sinusoidal bone marrow endothelial cells to form transendothelial pseudopods called proplatelets from which platelets are released into the bloodstream. Platelet survival is dependent on correct glycan expression. We here investigate the role of Type-2-Lactosaminoglycans (Type-2-LacNAc) in platelet production. beta1,4Galactosyltransferase 1 (beta4GalT1) is a major enzyme involved in Type-2-LacNAc synthesis which adds Galactose (Gal) to terminal N-Acetylglucosamine (GlcNAc) to form beta1,4Gal-GlcNAc (Type-2-LacNAc).beta4GalT1 deficient mice die in uthero between E15.5 and E16.5. A small percentage of beta4GalT1-/- mice survive until adulthood and they have severe macrothrombocytopenia but normal platelet clearance. Lethally irradiated wild type mice transplanted with beta4GalT1 deficient fetal liver cells failed to produce circulating beta4GalT1 deficient platelets, in marked contrast to beta4GalT1 deficient white blood cells, despite beta4GalT1 deficient MKs have been detected in the bone marrow of transplanted mice. beta4GalT1 deficient fetal liver MKs poorly produce proplatelets in vitro, following their normal maturation and differentiation, as judged by number, morphology, ploidy and expression of main surface glycoproteins. Our data strongly support the notion that glycosylation mediated by beta4GalT1 is crucial for platelet production in vitro and in vivo and demonstrate for the first time a role for post-translational glycan modification in platelet production.

Disclosures:

No relevant conflicts of interest to declare.
IL-1β Plays An Important Role On Thrombocytosis In An Immune Vasculitis Model Via Promoting Megakaryopoiesis
Authors: Yang Mo , Zhou Min , Li Su yi , Chong Beng , Li Xiao jing
Published: October 2019

Thrombocytosis and inflammation cytokines may be involved in the pathogenesis of vasculitis. Our previous study have showed that major inflammation cytokine IL-1β play an important role on in-vitro megakaryopoiesis (Yang M et al, Br J Haematol 2000). In this study, we investigated the changes of IL-1β and megakaryopoiesis and the effect of aspirin in an immune vasculitis model. Rabbit immune vasculitis model was established by intravenous injection of bovine serum albumin. In this model, platelet number and function of periphery blood, megakaryocyte number and the CFU-MK formation of the bone marrow, and serum levels of inflammatory cytokines were investigated. After treatment with BSA for 7 days, the platelet count, platelet aggregation and the expression of AnnexinⅤ were significantly increased in this vasculitis model group compared with normal control group (n=6). The serum levels of inflammatory cytokine IL-1β was also significantly higher in vasculitis model. There were positive correlations between platelet count and IL-1β levels (R=0.55), platelet aggregation and IL-1β levels (R=0.603). Treatment with aspirin (100 mg/kg/d) significantly decreased all these parameters, showing aspirin had anti-platelets and anti-inflammation effects. Our results also demonstrated that megakaryocyte number and the formation of CFU-MK were significantly increased in vasculitis group as compared to those in normal group. Treatment with aspirin significantly reduced the number of megakaryocytes and the formations of CFU-MK in bone marrow in this immune vasculitis model. Our study further demonstrated that IL-1β alone or in combination with TPO induced in-vitro CFU-MK formation. Using RT-PCR techniques, the mRNA of of IL-1 type I and type II receptors (IL-1 RI and RII) were detected in cultured CD61+ CD41+ cells and four megakaryocytic cell lines. The expression of IL-1 RI and RII was also confirmed by flow cytometry and immunofluorescence staining in bone marrow megakaryocytes. Moreover, the IL-1R bloker can reduced IL-1β induced megakaryopoiesis. This sudy showed that IL-1β may play an important role in the pathogenesis of immune vasculitis. Aspirin has anti-inflammation effects in this model which may be mediated via inhibiting megakaryopoiesis and platelet formation.

Disclosures:

No relevant conflicts of interest to declare.
Identification and Characterization By ChIP-Seq Of Genomic Sites Bound By E2A-PBX1 In Acute Lymphoblastic Leukemia Demonstrates Associations With p300 Recruitment, Transcriptionally Active Chromatin and Abundant Transcription
Authors: Nanan Kyster , LeBrun David P
Published: October 2019

E2A-PBX1 (EP1) is a chimeric oncogenic transcription factor expressed consequent to the 1;19 chromosomal translocation in cases of acute lymphoblastic leukemia (ALL). EP1 can induce transcription of reporter genes and EP1-driven oncogenesis requires direct binding of EP1 with the transcriptional co-activator and histone acetyltransferase p300. Therefore, we hypothesized that EP1 recruits p300 and other co-activators to cis-acting regulatory elements throughout the genome thereby inducing or maintaining transcription of target genes some of which contribute to the neoplastic phenotype. Here we have used chromatin immunoprecipitation followed by next generation DNA sequencing (ChIP-seq) to identify and characterize EP1-bound sites across the genome of the t(1;19)-associated, ALL-derived cell line RCH-ACV.

ChIP was performed with an anti-FLAG antibody using sheared chromatin prepared from RCH-ACV cells that stably expressed FLAG-tagged EP1; ChIP from parent RCH-ACV cells not expressing FLAG-EP1 served as a negative control for peak calling. Parallel immunoprecipitations were performed with antibodies for p300 and the chromatin marks H3K4me3, H3K4me1 and H3K27me3. Sequencing of DNA purified from the immunoprecipitated material and of total RNA (RNA-seq) was carried out commercially by BGI whereas bioinformatic analyses were performed in-house.

Bioinformatic analysis of data from replicate samples identified 3166 EP1 binding peaks across the RCH-ACV genome (irreproducible discovery rate threshold <0.01). Most EP1 binding sites were located in intronic (1408 sites) or intergenic (1346 sites) regions. Binding site consensus analysis showed overrepresentation of binding motifs for REST, CTCF, MYC, PAX5 and other transcription factors suggesting indirect recruitment of EP1 to DNA mediated by protein-protein interactions. EP1-bound regions were enriched for p300 binding (Figure 1), consistent with the documented importance of p300 recruitment in EP1 oncogenesis. A particular association with H3K4me3 relative to H3K4me1 or H3K27me3 (Figure 2) suggested association with active promoters. Three hundred and forty-two genes had EP1 binding sites within 1000 bp of their transcriptional start sites and these genes were associated with differentially abundant transcription (Figure 3, P<0.001). Querying the online Mammalian Phenotype Ontology tool with genes associated with EP1 binding generated terms that were obviously rich in phenotypes pertaining to B-lymphopoiesis.

In summary, our results suggest that EP1 recruits p300 and other co-activators to transcriptionally active chromatin in ALL cells. Results from studies currently underway to confirm the dependency of target gene expression and p300 recruitment upon binding of EP1 at specific binding sites will be presented.

Disclosures:

No relevant conflicts of interest to declare.
OSU-A9, An Indole-3-Carbinol Derivative, Induces Cytotoxicity In Acute Myeloid Leukemia Through Reactive Oxygen Species-Mediated Apoptosis
Authors: Bai Li-Yuan , Weng Jing-Ru , Wu Chia-Yung , Chiu Chang-Fang , Yeh Su-Peng , Liao Yu-Min
Published: October 2019

Introduction

Indole-3-carbinol (I3C) is a broadly targeted phytochemical shown to prevent carcinogenesis in animal studies and to suppress the proliferation of cancer cells of human breast, colon, prostate, and endometrium. Here we aim to test the anticancer effect of OSU-A9, an I3C derivative with improved potency, in acute myeloid leukemia (AML).

Materials and Methods

The in vitro activity of OSU-A9 was evaluated in AML cell lines (HL-60 and THP-1) and primary leukemia cells from 18 AML patients. THP-1 xenograft tumors in athymic nude mice was used for in vivo study.

Results

OSU-A9 mediates cytotoxicity in AML cell lines and primary leukemia cells from AML patients in a dose-responsive manner. The IC50 at 24 h for 18 patients was 1.63 μM. Normal human bone marrow cells were much less sensitive to OSU-A9 with an IC50 at 24 h greater than 8 μM. OSU-A9 causes cytotoxicity dependent on caspase activation, as evidenced by caspase-3 and PARP cleavage, and induces autophagy but not autophagic cell death. Interestingly, pretreatment of AML cell lines and primary AML cells with N-acetylcysteine or glutathione rescues them from apoptosis (and concomitant PARP cleavage) and Akt hypophosphorylation, implicating a key role of reactive oxygen species (ROS) in OSU-A9-related cytotoxicity. To investigate the anti-leukemia effect of OSU-A9 in vivo, fifteen male athymic nude mice were xenografted with THP-1 cells. Briefly, the anticancer utility of OSU-A9 is extended in vivo as it, administered intraperitoneally, suppresses the growth of THP-1 xenograft tumors in athymic nude mice without obvious toxicity. For biomarker analysis in the THP-1 xenografts, protein extracts were obtained from the tumors and immunoblotted for Akt levels. The tumors from OSU-A9 treated mice exhibited down regulation of Akt phosphorylation compared with those from placebo-controlled mice.

Conclusions

This study shows that ROS-mediated apoptosis contributes to the anticancer activity of OSU-A9 in AML cell lines and primary AML cells, and thus should be considered in the future assessment of its translational value in AML therapy.

Disclosures:

No relevant conflicts of interest to declare.
Functional and Phenotypic Characterization Of Acute Myeloid Leukemia By Analysis Of Diagnostic/Relapse Paired Samples
Authors: Ho Jenny M. , Shlush Liran I. , Mitchell Amanda , Marke Rene , McLeod Jessica , Minden Mark D. , Dick John E. , Wang Jean C.Y.
Published: October 2019

Treatment of relapsed acute myeloid leukemia (AML) results in lower complete remission rates compared to treatment of AML at diagnosis. Immunophenotypic changes in leukemic blasts are common from diagnosis to relapse (Baer et al. Blood. 2001), suggesting that the underlying biology of AML changes with disease progression. A better understanding of the biologic properties of AML cells at different stages of disease will facilitate the development of biomarker tools and more effective therapies. We therefore studied paired diagnostic/relapse samples obtained from AML patients. Cells from 11 paired samples were transplanted into immune deficient mice (NOD.SCID IL2Rg null) over a range of cell doses. In 10 of 11 patients, a leukemic graft could be generated after transplantation of lower cell doses from the relapse sample compared to the paired diagnostic sample. By limiting dilution analysis, leukemia stem cell (LSC) frequency was higher in relapse samples (1 in 5.8×102 to 1 in 2.4×106, median 1 in 2.0×103) compared to diagnostic samples (1 in 5.0×103 to 1 in 6.1×106, median 1 in 5.5×104); the fold increase in LSC frequency ranged from 2.2 to 745 (median 8.6). Multiparameter flow cytometric analysis carried out on 13 paired diagnostic/relapse samples demonstrated an increase in 2 known stem cell markers, CD34 and CD117, from diagnosis to relapse: CD34 was gained or increased at relapse in 7/13 (54%) of paired samples, while CD117 was gained or increased at relapse in 9/13 (69%) of paired samples. We plan to take a comprehensive approach to examine the surface marker expression of paired diagnostic/relapse samples by a high throughput flow cytometric screen (HTS) of both bulk and LSC-containing AML populations to identify markers that are altered at relapse. As a first step, we have performed HTS of 373 surface markers on 10 AML patient samples, including a relapse sample from a diagnostic/relapse pair. A significant proportion of markers (155/373, 42%) were expressed on less that 5% of cells in all 10 AML samples analyzed. We will therefore focus on the remaining markers in our HTS analysis of paired samples. Surface markers that are differentially expressed from diagnosis to relapse will be further characterized in order to gain insight into disease progression and identify potential therapeutic targets.

Disclosures:

No relevant conflicts of interest to declare.
Anticoagulation Prophylaxis With Enoxaparin For Patients With Acute Lymphoblastic Leukemia Receiving Asparaginase-Based Intensification Therapy
Authors: Sibai Hassan , Seki Jack T , Atenafu Eshetu G , Yee Karen W.L. , Schuh Andre C , Gupta Vikas , Minden Mark D. , Schimmer Aaron D. , Brandwein Joseph M.
Published: October 2019

Background

Venous thromboembolism (VTE) is a well-known complication in patients treated with asparaginase-containing regimens; the optimal preventative strategy is unclear. We assessed the safety and efficacy of low-dose low molecular weight heparin (LMWH) prophylaxis in adult patients with newly diagnosed acute lymphoblastic leukemia (ALL) treated with an asparaginase- based regimen.

Methods

Patients were treated with the DFCI 91-01 protocol from 2009-2012; patients age 60 and over received a modified version. All patients who were in complete remission and received at least 7 cycles (21 weeks) of the asparaginase-based intensification phase were evaluable for analysis. Patients already on anticoagulation for prior VTE were excluded. Patients received enoxaparin subcutaneously once daily, at a dose of 40 mg for patients weighing less than 80 kg and 60 mg for those 80 kg and over, beginning on day 1, cycle 1 of intensification until the completion of the entire 21-30 week intensification phase. Results were compared to a similar cohort of consecutive patients treated with the same protocol prior to 2009, without anticoagulation prophylaxis.

Results

There were 41 evaluable patients who received enoxaparin prophylaxis. The historical cohort (n=99) did not significantly differ from this group with respect to median age, weight and number of treatment cycles per patient. The mean enoxaparin dose administered was 0.62 mg/kg (range 0.39-1.05 mg/kg). As shown in Table 1, the overall rate of VTE was not significantly different than the historical non-prophylaxed cohort. Among patients receiving prophylaxis, there was a higher rate of VTE in patients who weight at least 80 kg, despite the higher enoxaparin dose used (p=0.036). There was no significant difference in the rate of VTE according to age or gender. Sites of VTE in the prophylaxed group included lower extremity (8 cases), sagittal sinus (2), subclavian line related (2), and pulmonary embolism (6). There were no major bleeding complications observed in the prophylaxed group (minor bleeding 3/41).

Conclusions

Prophylaxis with low-dose enoxarain during the intensification phase was safe, but did not show a significant benefit in reducing the rate of VTE. Prospective randomized studies are needed, using more intensive or novel prophylaxis strategies, in adult ALL patients treated with asparaginase-containing regimens.

Disclosures:

Off Label Use: enoxaparin for VTE prophylaxis in ALL.
Duration Of Neutropenia Is Associated With Reduced Relapse Risk In Children With De Novo Acute Myeloid Leukemia: A Report From The Children’s Oncology Group
Authors: Sung Lillian , Aplenc Richard , Alonzo Todd A. , Gerbing Robert B. , Meshinchi Soheil , Gamis Alan S.
Published: October 2019

Background

Children receiving treatment for de novo acute myeloid leukemia (AML) commonly experience infections and prolonged duration of neutropenia. There is great variability in these outcomes and pharmacogenomics may provide one mechanism to explain this variability. We hypothesized that relapse outcomes may be inversely related to the degree of toxicity.

Objectives

To describe the relationship between the number of sterile site infections and duration of neutropenia during the first four cycles of chemotherapy and the risk of recurrence and overall survival.

Methods

AAML0531 was a Children's Oncology Group (COG) randomized phase 3 clinical trial that included 1022 children with de novo AML enrolled between August 2006 and June 2010. Inclusion criteria were age ≥ 1 month to ≤ 30 years. For this specific analysis, we focused on non-Down syndrome patients who completed at least 4 cycles of chemotherapy on protocol therapy without recurrence on therapy and who did not receive HSCT in first remission. Because high-risk patients were allocated to best allogeneic donor HSCT, we focused on favorable and intermediate risk cytogenetic groups. The treatment protocol consisted of 5 cycles of intensive chemotherapy; patients were randomized to receive or not receive gemtuzumab ozogamicin (GMTZ) during Induction I and Intensification II.

The cumulative time to neutrophil recovery between start of Induction II and completion of cycle 4 (Intensification II) was examined; Induction I was not included since many centers did not wait for neutrophil recovery before starting Induction II. Relapsed risk and survival analyses were conducted from the end of Intensification II. Potential confounders explored for this analysis were gender, age, race, weight group, cytogenetic risk group and allocation to GMTZ.

Results

There were 569 patients included; 279 (49.0%) were male and 210 (36.9%) had favorable risk cytogenetics. There were 1116 sterile site infections; 735 (65.9%) were Gram-positive bacteria. The median cumulative time with neutropenia was 96 (range 46 – 204) days. The Table illustrates that infections did not significantly influence the risk of relapse or overall survival. However, increased duration of neutropenia was associated with a lower risk of relapse both in univariate and multiple regression models after adjustment for African-American race, favorable risk cytogenetics and GMTZ allocation.

Conclusions

Longer duration of neutropenia is associated with a reduced risk of relapse for children with favorable and intermediate cytogenetic risk AML. Toxicity may be mediated through pharmacogenomics which suggests that individualized chemotherapy dosing may be an effective strategy.

Disclosure:

No relevant conflicts of interest to declare.
Nanoparticle Encapsulated L-Asparaginase
Authors: Ortac Inanc , Ruff Laura , Yeh Yasan , Esener Sadik , Messmer Bradley T
Published: October 2019

Amino acid depletion with catabolizing enzymes is a potential therapeutic modality for many cancer types. L-asparaginase has been used to treat Acute Lymphoblastic Leukemia (ALL) for many decades and is part of treatment regimen that has a roughly 80% cure rate. The current frontline formulation is L-asparaginase from E.coli that has been conjugated to polyethylene glycol (PEG) for extended circulation half life and protection from immune responses. However, neutralizing antibodies still occur in a subset of patients, necessitating a switch to a different L-asparaginase, such as the less active enzyme from Erwinia chrysanthemi. This complicates therapy, risks severe allergic reactions, and if antibodies develop to the second enzyme the options become limited. The threat of immune reactions, as well as other side effects, has largely restricted L-asparaginase therapy to ALL, despite in vitro evidence that many other cancers might be sensitive to asparagine depletion.

Our group has developed a novel nanoparticle enzyme encapsulation technology that overcomes the limitation of immune responses to enzymes or PEG. These particles, termed Synthetic Hollow Enzyme Loaded Porous nanoShells (SHELS), are produced in a two-step process where the first creates silica shells with large pores through which the enzyme can be loaded, and a second sealing step that encapsulates the mesoporous shell and the loaded enzyme cargo with a nanoporous silica layer. The work below was done with 200nm particles that have a total shell wall thickness of approximately 10nm.

SHELS were loaded with L-asparaginase (Elspar). No loss of activity was seen when enzyme activity was measured in vitro. Groups of mice were injected IM with 5 IU of either free or SHELS encapsulated L-asparaginase. In each case, half of the mice were passively immunized with rabbit polyclonal anti-asparaginase antibodies to a blood concentration of ∼0.5 mg/ml. Serum asparaginase levels pre and post injection were measured by HPLC at 2, 5 and 8 days. Both free and encapsulated enzyme produced complete depletion of asparagine at day 2, but by day 5 asparagine levels had returned to baseline in the animals given free enzyme, whereas those given encapsulated enzyme maintained depletion at day 5 with restoration to baseline at day 8. There was no asparagine depletion in immunized animals with the free L-asparaginase, but SHELS encapsulated asparaginase produced an identical depletion both in the presence or absence of the neutralizing antibodies, indicating that the SHELS effectively shield the enzyme from the antibodies. Experiments in tumor models are ongoing as well as dose response and repeated administration studies.

Disclosures:

No relevant conflicts of interest to declare.
Zebrafish Xenotransplantation and Focused Chemical Genomics: A Preclinical Therapeutic Model For T-Cell Acute Lymphoblastic Leukemia
Authors: Bentley Victoria L , Veinotte Chansey J , Corkery Dale , Leblanc Marissa A , Bedard Karen , Weng Andrew P , Dellaire Graham , Berman Jason N
Published: October 2019

T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk subset of ALL, for which there is a need for new therapeutic strategies and efficient preclinical screening methods. We have pioneered an innovative zebrafish human cancer xenotransplantation (XT) model to examine drug-tumor interactions in vivo. T-ALL cell lines and primary patient T-ALL samples were microinjected into 48-hour zebrafish embryos, a stage at which the adaptive immune system has not yet developed. Fluorescent labelling of tumor cells prior to injection and use of casper pigment mutant fish facilitates evaluation of drug response both by direct observation in transparent fish and enumeration of human cells following embryo dissociation. Proliferation rates are rapidly determined by directly counting fluorescent cells using in silico-based programs and/or utilizing immunohistochemical approaches to distinguish human cancer cells from host cell populations. T-ALL cell lines harboring defined mutations in the NOTCH1, phosphoinositide 3-kinase (PI3K)/AKT and mTOR pathways differentially responded to targeted inhibition using the γ-secretase inhibitor Compound E, triciribine, and rapamycin, when xenografted into embryos, consistent with responses in vitro. Primary patient-derived T-ALL bone marrow samples similarly engrafted and proliferated in zebrafish embryos. Using this in vivo chemical genomic approach, a targetable mutation sensitive to γ-secretase inhibition was identified from the diagnostic bone marrow sample of a child with T-ALL, which was confirmed by exome Sanger sequencing, and validated as a gain-of-function mutation in the NOTCH1 gene by luciferase assay and Western blot. Focused chemical genomics using the zebrafish T-ALL XT model provides a means of tailoring therapy using a real time in vivo assay that more accurately recapitulates the tumor microenvironment than in vitro methods and more rapidly than mouse xenografts. Moreover, the efficiency and cost-effectiveness of this innovative platform provides a novel intermediary for the prioritization of much-needed drug candidates in the preclinical pipeline.

Disclosures:

No relevant conflicts of interest to declare.
High-Dose Vincristine Sulfate Liposome Injection (Marqibo®) Is Not Associated With Clinically Meaningful Hematologic Toxicity
Authors: Deitcher Olivia R. , Glaspy John , Gonzalez Rene , Sato Takami , Bedikian Agop , Segarini Karen , Silverman Jeffrey A. , Deitcher Steven R.
Published: October 2019

Background

The treatment of hematologic malignancy patients with heavily pre-treated, advanced, relapsed, or refractory disease and those with advanced age, active infection, cardiovascular disease, recent surgery, or poor performance status may require therapies with limited hematologic toxicity and associated peripheral blood cytopenias. Cancer and concomitant chemotherapy related anemia, neutropenia, and thrombocytopenia in patients with hematologic malignancy complicate evaluation of hematologic toxicity related to new drugs. Vincristine sulfate liposome injection (VSLI; Marqibo®) is a new drug developed to optimize vincristine (VCR) pharmacokinetics and facilitate VCR dose-intensification and densification. VSLI is active in untreated and relapsed lymphomas, and was recently approved in the United States for relapsed and refractory adult Philadelphia chromosome-negative acute lymphoblastic leukemia.

Methods

We assessed the hematologic toxicity of VSLI 2.25 mg/m2 administered every 14 days (Cohort 1) or 7 days (Cohort 2) in 54 patients with metastatic uveal melanoma, a cancer not known to involve the bone marrow. Cohort 1 patients could have received 1 prior systemic therapy. Cohort 2 patients must not have received any prior systemic chemotherapy, immunotherapy, cancer vaccine, or hepatic arterial chemotherapy for metastatic disease.

Results

Patients in Cohort 2 received a higher median number of VSLI doses (6 vs. 4) over a shorter median time period (5.7 weeks vs. 8.7 weeks) resulting in a larger median cumulative exposure (22.6 mg vs. 17.7 mg) and near doubling of median dose density (4.0 mg/week vs. 2.2 mg/week) compared to patients in Cohort 1. Despite greater VSLI exposure and dose density, Cohort 2 had a smaller decrease from baseline in median neutrophil count and greater increase from baseline in median platelet count compared to Cohort 1. Both Cohorts experienced similar gradual decreases in hemoglobin concentration over time that were attributed to cancer-associated anemia and frequent blood sampling. Hematologic adverse events (AEs) were uncommon and mostly Grade 1 or 2. There were no Grade 4 hematologic AEs.

Conclusions

VSLI at its approved dose resulted in a low incidence of clinically meaningful hematologic toxicity. A near doubling of median dose density did not have an impact on the reported incidence and severity of hematologic AEs. VSLI may be well suited for use alone or in combination with myelosuppresive drugs and in patients unable to tolerate peripheral blood cytopenias.

Disclosures:

Silverman: Talon Therapeutics: Employment. Deitcher:Talon Therapeutics: Employment, Equity Ownership.
The Polymorphisms In LNK Gene Correlated To The Clinical Type Of Myeloproliferative Disorders
Authors: Chen Yan , Hu Yang , Wu Xueqiang , Fang Fang , Liu Qian , Zhang Ying , Zhu Ping
Published: October 2019

Objective

LNK (lymphocyte adaptor protein) is an adapter protein negatively regulating JAK/STAT cell signaling pathway. In this study, we observed the effects of variations in LNK gene on the clinical type of myeloproliferative disorders (MPD).

Methods

A total of 285 MPD cases were recruited, including essential thrombocythemia (ET) 154 cases, polycythemia vera (PV) 76 cases, idiopathic myelofibrosis (IMF) 19 cases, and chronic myeloid leukemia (CML) 36 cases. Ninety-three healthy individuals were used as normal controls. V617F mutation in JAK2 was searched by allele-specific primers method, RT-PCR was used for the detection of BCR/ABL1 fusion gene, and mutations and variations in exons and their flanking sequences of LNK gene were examined by PCR-sequencing. Count data were analyzed by χ2 or Fisher exact test.

Results

The genotypes of 4 SNPs (rs3184504, rs111340708, rs78894077and rs7973120) could be identified. Missense mutations of A300V, V402M, and R415H in LNK were found in 8 patients, of whom 3 ET patients had homozygous A300V mutation combined with JAK2V617F mutation. The genotypes and allele types of the 3 SNPs (rs3184504, rs111340708 and rs78894077) correlated to the clinical type of MPD. For rs3184504, the allele frequencies of C/T were 0.5/0.5 in normal controls, 0.117/0.883 in ET, 0.132/0.868 in PV, 0.211/0.789 in IMF, and 0.944/0.056 in CML; T allele (p.262Trp) was significantly higher in ET, IMF and PV (P<0.01), and C allele (p.262Arg) was significantly higher in CML (P<0.01), as compared with the controls. For rs78894077, the allele frequencies of C/T were 0.097/0.903 in normal controls, 0.045/0.955 in ET, 0.086/0.914 in PV, 0.053/0.947 in IMF, and 0.167/0.833 in CML; T allele (p.242Ser) was higher in ET (P<0.05) than in normal controls. For rs111340708 which has 2 alleles of repeated sequences (TGGGG x5/TGGGG x4), the allele frequencies of TGGGG x5/TGGGG x4 were 0.462/0.538 in normal controls, 0.719/0.281 in ET, 0.533/0.467 in PV, 0.789/0.211 in IMF, and 0.639/0.361 in CML; TGGGG x4 allele was significantly lower in ET, IMF and CML than in normal controls (P<0.01). However, the allele frequencies of rs3184504 and rs111340708 were unrelated to the presence of JAK2V617F mutation and BCR/ABL1 fusion gene in MPD patients.

Conclusion

The polymorphisms of SNPs in LNK gene correlated to the clinical type of MPD.

Disclosures:

No relevant conflicts of interest to declare.
Safety and Efficacy Of Abbreviated Induction With Only 4 Cycles Of Fludarabine (F), Cyclophosphamide (C) and Rituximab (R) In Physically Fit Patients With Chronic Lymphocytic Leukemia (CLL) Who Achieve Early Complete Remission (CR) With Undetectable Minimal Residual Disease (MDR)
Authors: Pavlovsky Carolina , Pavlovsky Astrid , Fernandez Isolda , Galeano Adriana , Lastiri Francisco , Pavlovsky Santiago
Published: October 2019

Background

Chemoimmunotherapy with 6 cycles of FCR is considered standard therapy for physically fit patients (pts) with Chronic Lymphocytic Leukemia (CLL). Many pts are unable to complete planned treatment, due to treatment related complications. Levels of minimal residual disease (MRD) have been shown to correlate with PFS in previously untreated patients with CLL (CLL8, Boettcher S et al. Leukemia, 2009). Achieving a negative MRD is therefore a mayor endpoint in treatment.

Patients and methods

From 4/2003, 39 physically fit pts with CLL who had IWCLL-NCI criteria for initiating treatment started therapy with FCR in our institution. Eleven pts had previously received chlorambucil/prednisone and 28 were not previously treated. Median age at start of therapy was 63 years (34-80), Binet´s clinical stage were A/B: 22 pts (56%) and C: 17 (44%). The CD38 expression was positive (>7% of cells) in 23 (59%) and negative in16 (41%) of the pts.

After 4 courses of FCR response was assessed in peripheral blood (PB) or bone marrow (BM) using three colour flow Cytometry. Negative MRD was defined as < 0,1% of light chain restricted CD5+CD19+ B cells in PB and BM as assesed collecting 100000 CD19 cells in a three colour cytometer (FacsScalibur- blue laser ). All these patients stopped therapy after evaluation due to early CR with eradication of MRD.

Results

All patients had negative MRD in peripheral blood, 35 were also evaluated in bone marrow, 29 showed CR and 6 nodular partial remission (NPR). Neutropenia and infectious events grade 3-4 were observed in 24% and 7% of all the courses respectively. No pts died of toxicity.

After a median follow-up of 81 months (4.6-120), progression free survival (PFS) and overall survival (OS) at 72 months was 51% and 75% respectively. Five pts died of progressive disease and 3 of a secondary neoplasm.

Conclusion

Stopping therapy in patients who achieve negative MRD after 4 cycles of FCR is safe and induces durable remission with a PFS and OS of 51% and 75% at 72 months exposing them to less chemotherapy. Large randomized trials are necessary to confirm this data.

Disclosures:

Pavlovsky: Novartis: Speakers Bureau; BMS: Speakers Bureau.