bluuebunny/crossref_metadata_2025 huggingface dataset:
Distinct Functions of pH-Sensing Histidine Residues in the Multimer Assembly and Storage of Von Willebrand Factor in Weibel-Palade Bodies
Authors: Huang Jing , Westfield Lisa A , Sadler J. Evan
Published: October 2019

Abstract 3311

The hemostatic function of von Willebrand factor (VWF) depends on the formation of disulfide-linked multimers. We have previously shown that several evolutionarily conserved His residues within the VWF propeptide are critical for pH-dependent VWF multimerization. For example, the charge-neutralization mutations H395A and H460A in the VWF D2 domain prevented multimer assembly, while the charge-stabilization mutation H460K preserved it, suggesting that protonation at these sites is essential. In this study, we investigated the role of these His residues in VWF targeting to storage granules and agonist-induced secretion. Full-length VWF was expressed in transiently-transfected HEK293 cells. If Weibel-Palade body-like storage granules (WPBs) were formed, their morphology was analyzed by immunofluorescence microscopy. A WPB was defined as “long” if length/width was > 2, or “round” if length/width was ≤ 2. To analyze the assembly of VWF multimers, we stimulated VWF secretion by incubating cells with 100 ng/ml of phorbol myristate acetate for 1 hour and collected cell lysates and supernatants for multimer gel electrophoresis. Expression of wild type VWF and VWF H460K induced the formation of approximately 60% long WPBs. Treatment of cells with monensin increases the pH of the Golgi, and as expected monensin reduced the storage of wild type VWF in long WPBs. However, monensin did not impair the storage of VWF H460K or reduce the fraction of long WPBs, indicating that a positive charge at residue 460 can sustain tubular storage of VWF despite neutralization of the late secretory pathway. Both WT VWF and VWF H460K were secreted with a normal distribution of multimers. VWF H395A and VWF H460A formed predominantly round pseudo-WPBs, with only 10–20% long granules, and these mutations also disrupted VWF multimerization. In contrast, VWF H395R and VWF H395A/H460R did not assemble multimers but nevertheless formed long WPBs. Insertion of an extra Gly between the vicinal cysteines in the D1 domain (159CGLC162 to 159CGGLC162) was reported to prevent multimerization but allow storage in long WPBs in AtT-20 cells (Mayadas & Wagner, PNAS 1992; 89:3531). We found that this mutation also inhibited VWF multimerization and resulted in the formation of long WPBs in HEK293 cells. These results indicate that VWF multimerization and packing in elongated WPBs both depend on the acidic pH of the late secretory pathway. However, these processes are independent, with distinct structural requirements, and they can be dissociated by mutations of specific His residues.

VWF construct Multimers Granule Shape Granule Shape (with monensin) Wild type Yes Long Round H395A No Round H395R No Long H395A/H460R No Long H460A No Round H460K Yes Long Long 159CGGLC162 No Long

Disclosures:

No relevant conflicts of interest to declare.
Incidence of Heparin Induced Thrombocytopenia in the Transplant Population: A Large Retrospective Cohort, Single Transplant Centre Experience
Authors: Hassan Syed , Khoulani Dania , Badami Ami , Alirhayim Zaid , Younes Mohamad A. , Kuriakose Philip
Published: October 2019

Abstract 3320

Objective:

Heparin induced thrombocytopenia (HIT), a prothrombotic complication of heparin therapy, can lead to serious thromboembolic events and cause significant morbidity and mortality. Its occurrence has never been studies in transplant patients, where use of heparin products is very common. We aim to study its prevalence in the transplant population at our institute.

Methods:

This is a retrospective cohort, single center study which looked into the clinical and laboratory database of all the patients that has undergone any kind of transplant at our institution over a period of 25 years (January 1985 - December 2010). In patients with clinical suspicious of HIT, a pre-test probability was calculated using the 4T scoring system. Results of the laboratory test like the ELISA HIT antibody (HIT ab) test and the functional serotonin release assay test (SRA) along with clinical manifestation of skin necrosis or thromboembolic events were reviewed.

Results:

Medical records of 2800 patients that has undergone transplant from January 1985- December 2010 were reviewed. HIT antibody assay was performed in 262 patients in which HIT was suspected. Of these, only 48 (18%) patients (mean age 57 ± 11 years, 71% women) had HIT ab positive, 9 were pre transplant recipient and remaining 39 were post transplant recipients. Baseline characteristics of the transplant population are illustrated in Table.1. Confirmatory test, SRA was performed in 8 HIT antibody positive patients, of whom only 4 were positive. The mean 4T score in HIT suspected patients was 3.7 ±1.3, while the score in HIT ab positive patients was 4.2 ± 1.2. Thrombotic complications were seen in 11(0.4%) patients, with the highest incidence rate of 1% in heart transplant recipients. No transplant patient had skin manifestations. Direct Thrombin inhibitor (DTI) was used only in 5 patients who had thrombotic events. No other complication or mortality was reported in any of the HIT ab positive transplant patients.

Conclusion:

To our knowledge, this is the first study of its kind that has shown very low incidence of HIT in transplant population. In conclusion, transplant patients can safely undergo any type of organ transplant, without having any peri or post operative complications or immediate mortality related to HIT.

Disclosures:

No relevant conflicts of interest to declare.
Primary Immune Thrombocytopenia Guidelines for Bone Marrow Aspirate Reduce the Diagnostic Rate of Myelodysplastic Syndromes
Authors: Vincelli Iolanda , Cufari Patrizia , Roda' Filippo , D'Errigo Maria Grazia , Oliva Esther Natalie , Nobile Francesco
Published: October 2019

Abstract 3328

Introduction:

About one quarter of patients with low risk myelodysplastic syndromes may present with isolated thrombocytopenia, defined by International Prognostic Scoring System (IPSS) as a platelet (PLT) count < 100 Gi/L. Primary immune thrombocytopenia (ITP) is defined as an acquired immune-mediated disorder characterized by isolated thrombocytopenia, PLT count <100 Gi/L, and the absence of any obvious initiating and/or underlying cause of the thrombocytopenia (Rodeghiero et al, Blood, 2009). Recent guidelines consider bone marrow (BM) aspirate informative in patients > 60 years of age, those with systemic symptoms or abnormal signs or in some cases where splenectomy is considered (Provan et al, Blood, 2009). We performed a retrospective chart analysis between January 2008 and July 2012 of all patients referred to our clinic for thrombocytopenia but with a PLT count 100– 149 Gi/L. The aim of the study was to evaluate whether BM aspirates in these patients be informative.

Results:

Twenty-six cases (18 males/8 females) of mean age 59 ± SD 17 years were evaluated at our clinic for a PLT count below normal lab range values, > 100 Gi/L. At the time of evaluation none had bleeding symptoms. PLT counts ranged from 101 to 149 Gi/L, mean 122 Gi/L. Five patients had an enlarged spleen. BM aspirates identified 20 patients with low IPSS risk MDS. Cytogenetics were obtained in 15 cases: 7 normal, 2 del20q, 6 –Y. Twelve patients were aged < 60; 8 (66%) were diagnosed with MDS. Amongst the 14 patients with an indication for BM aspirate (age > 60), 12 (86%) had MDS.

Conclusion:

The thresholds of PLT count 100 Gi/L and age 60 years for BM aspirate in patients with isolated thrombocytopenia reduces the diagnostic rate of MDS. In our retrospective review, 66% of patients (younger age and PLT > 100 Gi/L) would not have had MDS diagnosed. BM aspirate should be considered irrespective of age, since more than half of the patients in our case review had MDS with PLT > 100 Gi/L as a single cytopenia and age under 60.

Disclosures:

No relevant conflicts of interest to declare.
Activated Protein C Targets PI3K-p85/XBP1 Pathway to Inhibit Hyperglycemia Induced Endoplasmic Reticulum Stress in Diabetic Nephropathy
Authors: Isermann Berend H. , Wang Hongjie , Nawroth Peter P. , Madhusudhan Thati
Published: October 2019

Abstract 3354

Diabetic nephropathy (DN) is a multifactorial disease associated with substantial changes in the haemostatic system. A hallmark of diabetes induced haemostatic dysfunction is impaired thrombomodulin (TM) dependent-protein C (PC) activation. Impaired PC activation triggers glomerular podocyte and endothelial cell dysfunction, thus promoting DN. The intracellular mechanism through which loss of TM and PC activation contributes to DN is not known. Here we show that the haemostatic mediator activated PC (aPC) regulates cellular homeostasis by inhibiting hyperglycemia induced endoplasmic reticulum (ER)-stress in DN.

Hyperglycaemia was induced in wild-type (wt) mice or mice with altered activity of the TM-PC system (loss of function secondary to impaired PC activation (TMPro/LacZ) or gain of function with high aPC plasma levels (APChigh)). Subsets of diabetic mice were treated with the chemical ER-chaperone TUDCA. After 26 weeks of persistent hyperglycaemia markers of DN were determined and tissue samples were isolated for ex vivo analysis. Supplementary in vitro assays were performed in podocytes and endothelial cells.

Persistent hyperglycaemia in wt mice caused severe ER-stress and DN. Ex vivo analysis of transcription factors regulating the ER-stress response showed an increase of the ER-stress markers CHOP and ATF6, while nuclear translocation of the highly conserved transcription factor X-box binding protein-1 (XBP1) was reduced in DN. These changes were aggravated in diabetic TMPro/LacZ mice. Conversely, in a mouse model with constitutively higher aPC levels (APChigh mice) nuclear levels of XBP1 were normalized and expression of ATF6 and CHOP was reduced despite persistent hyperglycaemia. In addition, aPC reverses the pathological ER-stress alterations in diabetic TMPro/LacZ mice. Pharmacological inhibition of ER-stress by TUDCA normalized nuclear levels of XBP1, inhibited CHOP/ATF6 expression, and protected against DN in diabetic wt and TMPro/LacZ mice. In vitro hyperglycaemia inhibited nuclear translocation of XBP1 in endothelial cells and podocytes, the two cellular components of glomerular filtration barrier. Activated PC directly promotes the nuclear translocation of XBP1 in these cells, which is required to inhibit hyperglycaemia induced ER-stress. Deletion of XBP1 in podocyte or endothelial cells abolished the cytoprotective effect of aPC. Furthermore, aPC regulates the interaction of PI3K regulatory subunit p85α with XBP1 which is known to mediate its nuclear translocation and diminished hyperglycaemia induced ER-stress. These studies demonstrate that hyperglycemia induced ER-stress is causally linked to DN and establish a novel link between haemostatic system and ER function in regulating cellular homeostasis in chronic kidney disease.

Disclosures:

No relevant conflicts of interest to declare.
Crystal Structure of a Proteolytic Fragment of ADAMTS13 Reveals Alternative Disulfide Pairings in the Cysteine-Rich Domain
Authors: Skipwith Christopher G. , Stayrook Steven , Rottensteiner Hanspeter , Scheiflinger Friedrich , Zheng X. Long
Published: October 2019

Abstract 3363

Proteolytic cleavage of von Willebrand factor (VWF) by ADAMTS13 depends on its non-catalytic domain (or exosite) interactions. These exosites, particularly the residues between Tyr659 and Tyr665 and their adjacent residues Arg568 and Phe592 in the ADAMTS13 spacer domain, are the major targets of anti-ADAMTS13 autoantibodies in patients with acquired TTP. In an effort to further determine the potential exosite interactions between ADAMTS13 and VWF, we carried out crystal screening of a recombinant full-length ADAMTS13 purified from stably transfected Chinese hamster ovary cells. After over 900 conditions were tested, we obtained multiple well-formed and diffractable crystals. Upon X-ray diffraction, we discovered that only a proteolytic fragment of full-length ADAMTS13 (Met432-Arg670) had been consistently crystallized. Here, we report the structure of this non-catalytic ADAMTS13 fragment at 2.2Å. The fragment encompasses 8 residues from the first TSP-1 repeat, an entire cysteine-rich domain (CA and CB), and a majority of the spacer domain. While recombinant ADAMTS13 in the present study is fully glycosylated, the overall structure agrees very well with the analogous portion of the ADAMTS13-DTCS structure previously reported (RMSD = 0.02Å, 1817 atoms, 232 residues). A region within the CA domain does vary slightly from that in the ADAMTS13-DTCS (RMSD = 0.08Å, 379 atoms, 51 residues). As with the ADAMTS13-DTCS structure, our structure shows 3 intermolecular disulfide bonds in CA, in addition to 2 free cysteine residues. In this region, our structure shows differences in the disulfide bonding pairing, and a slight shift in the α4 helix from Gly479-Cys483. Specifically, our structure reveals an intermolecular disulfide bond between the residues Cys483 and Cys527 and Cys508 and Cys522. These disulfide pair patterns are different from those reported in the ADAMTS13-DTCS structure, in which disulfide bonds are formed between the residues Cys483 and Cys522 (4.6Å apart in our structure) and the residues Cys508 and Cys527 (3.8Å apart in our structure). Our results indicate that, despite the difference in conditions under which crystals of ADAMTS13 fragments are formed, the overall structure of the Cys-rich and spacer domains are quite similar. The difference in the disulfide bond pattering may provide a novel insight into the mechanism regarding the ADAMTS13 exosite interaction with VWF. Our ongoing work is aimed to determine the complex formation between ADAMTS13 and its substrate or autoantibodies.

Disclosures:

Rottensteiner: Baxter Innovations GmbH: Employment.
Prognostic Factors for Post-Thrombotic Syndrome in Children with Limb DVT: A Bi-Institutional Cohort Study
Authors: Lyle Courtney A. , Gibson Elizabeth , Lovejoy Amy , Goldenberg Neil A
Published: October 2019

Abstract 3401

Background:

Early identification of children with deep venous thrombosis (DVT) of the limb, whoare at heightened risk for post-thrombotic syndrome (PTS), is important in order to evaluate therapeutic interventions aimed at decreasing the risk and severity of PTS.

Objective:

We sought to evaluate prognostic factors for PTS in children following DVT of the limbs.

Methods:

In this bi-institutional mixed cohort study with prospective ascertainment of PTS using a validated pediatric instrument, we collected data on patient/thrombus characteristics, thrombophilia testing results, and outcomes in children (<21 years at event) diagnosed with acute limb DVT at Rady Children's Hospital of San Diego and Children's Hospital Colorado.

Results:

Median age at presentation was 13 years (range, 0–18 years). Cumulative incidence (i.e., risk of PTS) was 23%, at a median follow-up duration of 33 months (range: 13.2–65 months). The presence of a lupus anticoagulant by dilute Russell Viper venom time (dRVVT) testing within two weeks of DVT diagnosis was associated with markedly increased odds of developing clinically-significant PTS (OR 16.8, 95%CI (1.60–176.2); P=0.02). The presence of an infectious or inflammatory condition at DVT presentation was neither associated with PTS risk nor dRVVT positivity.

Conclusions:

An acutely positive dRVVT following diagnosis of limb DVT appears to be a significant prognostic factor for development of clinically significant PTS in children. Larger collaborative cohort studies are required to substantiate these findings, evaluate other prognostic factors, and determine whether the present association is modulated by persistent dRVVT positivity or beta-2-glycoprotein-I dependence.

Disclosures:

Goldenberg: Eisai Inc: Research Funding; Pfizer Inc: Membership on an entity's Board of Directors or advisory committees; CPC Clinical Research: Consultancy.
PRT064445 but Not Recombinant Fviia Reverses Rivaroxaban Induced Anticoagulation As Measured by Reduction in Blood Loss in a Rabbit Liver Laceration Model
Authors: Hollenbach Stanley J , Lu Genmin , Tan Siusze , Lee Gail , Athiwat Hutchaleelaha , Inagaki Mayuko , Sinha Uma
Published: October 2019

Abstract 3414

We used a modified rabbit liver laceration model to demonstrate the effects of PRT064445, a recombinant fXa derivative, to reverse rivaroxaban induced anticoagulation as measured by reduction of blood loss, decrease of unbound fraction of rivaroxaban in plasma and relevant pharmacodynamic markers: anti-fXa activity, prothrombin (PT) and activated thromboplastin (aPTT) times. Recombinant fVIIa (rfVIIa) was tested in the same model for comparison.

Anesthetized rabbits were administered vehicle or 1 mg/kg rivaroxaban via IV bolus injection. After 30 minutes to allow the rivaroxaban to biodistribute, vehicle, PRT064445 or rfVIIa was administered as a bolus injection followed by laceration of two liver lobes (5× each lobe: 1-cm long and 3-mm deep incisions with scalpel blade) and blood loss collected on pre-weighed gauze over 15 minutes. Blood loss due to rivaroxaban anticoagulation represented approximately 10% of the animal's total blood volume. Total rivaroxaban concentration in plasma was measured by LC-MS/MS. Free fraction of rivaroxaban (non-protein, non-PRT064445 bound) was assessed using equilibrium dialysis method followed by LC-MS/MS quantitation. Anti-fXa activity was measured using a modified chromogenic LMW heparin kit. PT and aPTT was measured using commercial reagents (HemosIL). Anticoagulation by rivaroxaban (1.65 μM average plasma concentration at 30 min time point) resulted in 2.3-fold and 1.9-fold prolongation of PT and aPTT, respectively, and increased blood loss by 3.2-fold over vehicle. PRT064445 (75 mg/rabbit) administration reduced blood loss due to rivaroxaban anticoagulation by >85% and decreased peak anti-fXa activity by 98%, PT by 74%, aPTT by 66% and the free fraction of rivaroxaban in plasma from 26% to <0.5%. In contrast, rfVIIa (150 μg/kg) had no effect on blood loss but reversed PT by 85% and aPTT by 54%. PRT064445 and rfVIIa alone had no effect on blood loss, but rfVIIa decreased PT by 35%.

These data demonstrate that PRT064445 can reduce blood loss due to rivaroxaban induced anticoagulation using a single bolus administration. Reduction of blood loss with PRT06445 correlated to the decrease in the free fraction of rivaroxaban in plasma as well as PD markers anti-fXa activity and PT, while rfVIIa decreased PT but had no effect on blood loss. These and our previous data with reversal of enoxaparin-induced anticoagulation in a rat tail transection model indicate that PRT064445 can be used as a universal antidote for both direct and indirect fXa inhibitors.

Disclosures:

Hollenbach: Portola Pharmaceuticals: Employment. Lu:Portola Pharmaceuticals: Employment. Tan:Portola Pharmaceuticals: Employment. Lee:Portola Pharmaceuticals: Employment. Athiwat:Portola Pharmaceuticals: Employment. Inagaki:Portola Pharmaceuticals: Employment. Sinha:Portola Pharmaceuticals Inc.: Employment.
Identification of Novel Adult Mesenchymal Progenitors That Can Contribute to HSC Niche
Authors: Hu Xingbin , Weng Lihong , Jung Xiaoman , Murakami Jodi , Kumar Bijender , Chen Ching-Cheng
Published: October 2019

Abstract 3467

The microenvironment (niche) cues hematopoietic stem cell (HSC) receives play an important role in the regulation of their decisions between self-renewal and differentiation. However, the cellular constitution of niches remains poorly understood. We identified three adult progenitor populations near the endosteum based on differential expression of cell surface markers, including CD166, CD146 and Sca1. Upon co-transplantation with fetal skeletal progenitors, Sca1+ progenitors can give rise to CD146+ and CD166+ stroma and phenotypically CXCL12 abundant reticular (CAR) cells in marrow. CD146+ and CD166+ progenitors, on the other hand, form bone without marrow cavity. Multiplex single cell qRT-PCR reveals all three progenitors expressed high levels of genes involved in HSC maintenance. In vitro co-culture assay demonstrated that all three progenitors could preserve HSC long-term multi-lineages reconstitution capability. Furthermore, disruption of stem cell factor (SCF) production in Sca1+ progenitor severely limits its ability to support HSC both in culture condition and after transplantation. Our results suggested that Sca1+, CD146+, and CD166+ mesenchymal progenitors and their progeny collaborate to provide supportive environment for hematopoiesis.

Disclosures:

No relevant conflicts of interest to declare.
CD19 Targeted Allogeneic EBV-Specific T Cells for the Treatment of Relapsed ALL in Pediatric Patients Post HSCT
Authors: Curran Kevin J. , Kernan Nancy A. , Wang Xiuyan , Taylor Clare , Doubrovina Ekaterina , Bartido Shirley , Sadelain Michel , O'Reilly Richard J. , Brentjens Renier J , Rivière Isabelle
Published: October 2019

Abstract 353

T cells can be genetically modified to target tumor antigens through the expression of a chimeric antigen receptor (CAR). CAR modified T cells targeting the CD19 antigen is a novel therapeutic approach for patients with relapse B-ALL following allo-HSCT. We have previously demonstrated donor derived Epstein-Barr virus specific cytotoxic T cells (EBV-CTLs) can be safely infused in patients following allo-HSCT. Following retroviral gene transfer with our CD19-specific CAR (19–28z), EBV-CTLs demonstrate in vitro cytotoxicity against CD19+ targets, retain EBV-specificity without allogeneic reactivity, and in vivo, eradicate systemic Burkitt lymphoma in a SCID/Beige murine model. Based on our preclinical studies we have initiated a phase I dose escalation clinical trial (NCT01430390) in pediatric patients with relapsed CD19+ B-ALL following allo-HSCT utilizing donor 19–28z+ EBV-CTLs. Three patients have received conditioning chemotherapy (fludarabine 25mg/m2 × 5 days) followed by infusion of CAR modified EBV-CTLs without infusion related toxicity. Subsequently, patient 1 and 3 safely received additional T cell infusions without (patient 1) and with (patient 3; cyclophosphamide) conditioning chemotherapy. Total CAR+ T cell dose for each infusion ranged from 1.4 – 4.1×105 cells/kg (1×106 total EBV-CTLs/kg per infusion). Patient 1 developed fever and biopsy proven skin GVHD (grade II) three days following his first infusion which resolved 2 weeks after starting topical steroids. Modified T cells were detected by Q-PCR in the bone marrow and peripheral blood up to 3 and 18 weeks respectively in patient 1 following first infusion, up to 2 weeks in the peripheral blood in patient 2, and have been below the limit of detection in patient 3. Patient 1 died from progressive disease 29 weeks following first infusion and patient 2 died from progressive disease 19 weeks post infusion. Patient 3, initially treated in remission following salvage chemotherapy, has no evidence of disease (NED) 15 months after last extramedullary relapse and 5 months post first infusion of 19–28z+ EBV-CTLs. These early results demonstrate the feasibility of allogeneic 1928z+ EBV-CTL infusions without the development of significant GVHD post allo-HSCT. Subsequent cohorts of patients will receive dose escalation of modified T cells and will be evaluated for safety, persistence of 19–28z+ EBV-CTLs, and for anti-tumor efficacy.

Disclosures:

No relevant conflicts of interest to declare.
Phenothiazines Induce Apoptosis in T-Cell Acute Lymphoblastic Leukemia by Activating the Phosphatase Activity of the PP2A Tumor Suppressor
Authors: Gutierrez Alejandro , Kentsis Alex , Pan Li , Baleydier Frederic , Marineau Jason , Grebliunaite Ruta , Kozakewich Elena , Reed Casie , Pflumio Francoise , Poglio Sandrine , Uzan Benjamin , Clemons Paul A. , VerPlank Lynn , An W. Frank , Burbank Jason , Norton Stephanie , Tolliday Nicola J , Steen Hanno , Bradner James E , Look A. Thomas , Aster Jon C
Published: October 2019

Abstract 3558

Despite the use of intensive and toxic treatment regimens, T-cell acute lymphoblastic leukemia (T-ALL) remains fatal in 25% of children and 60% of adults, highlighting the need for novel therapeutic strategies. We performed two complementary small molecule screens to identify novel T-ALL therapeutic agents: i) an in vivo zebrafish screen for small molecules toxic to MYC-overexpressing thymocytes, and ii) an in vitro screen for drugs that synergize with NOTCH inhibitors in human T-ALL cell lines. Hits common to both screens included perphenazine, an FDA-approved phenothiazine antipsychotic. Perphenazine potently induced mitochondrial apoptosis and had antileukemic activity in zebrafish T-ALL, in human T-ALL cell lines, and in primary human T-ALL lymphoblasts. However, the molecular target mediating the antileukemic activity of perphenazine appeared to be unrelated to any of its known cellular targets. To identify the relevant biologic target of perphenazine, we developed a novel method for drug target discovery, termed activity correlation proteomics, which combines native protein extraction of isotope-labeled proteomes, fluorous affinity chromatography, and quantitative mass spectrometry proteomics to relate phenotypic activities of drugs with their binding targets. Using activity correlation proteomics, we identified protein phosphatase 2A (PP2A) as the protein whose binding affinity for different phenothiazines most closely correlated with their antileukemic activity. Perphenazine was shown to activate PP2A in T-ALL cells, as assessed by rapid drug-induced dephosphorylation of multiple PP2A substrates including MYC, AKT, p70S6K, ERK, and BAD, and its effects were phenocopied by FTY720, a PP2A activator. Moreover, shRNA knockdown of PP2A scaffolding or catalytic subunits attenuated the activity of perphenazine, indicating that PP2A is required for its antileukemic activity. Our findings thus establish a novel strategy for pharmacologic activation of the PP2A tumor suppressor, and highlight its therapeutic potential in T-ALL and other cancers driven by hyperphosphorylated PP2A substrates.

Disclosures:

Off Label Use: Discovery of antileukemic activity of perphenazine mediated via activation of PP2A.
Maintenance Hypomethylating Therapy Post Allogeneic Stem Cell Transplantation Provides Favorable Progression Free Survival in High Risk Acute Myelogenous Leukemia or Non Responsive Myelodysplastic Syndrome
Authors: Sizemore Connie A. , Zhang Xu , Sanacore Melissa , Bashey Asad , Solomon Scott R. , Morris Lawrence E. , Holland H. Kent
Published: October 2019

Abstract 4468

Introduction

Patients with acute myelogenous leukemia (AML) or non responsive myelodysplastic syndrome (MDS) who fail to achieve a complete remission, have unfavorable cytogenetics or are refractory to therapy have a poor prognosis. Allogeneic HSCT is frequently considered a salvage option for these patients although remissions are short-lived. New strategies are needed for maintaining remission in this extremely high risk patient group. We hypothesized hypomethylating agents post allogeneic HSCT would enable patients to remain in complete remission.

Methods

Eleven patients were treated. Patient characteristics: median age, 48 years (range, 18–70 years), PBSC (n=9) or bone marrow (n=2); Diagnoses AML= 10, Non- responsive MDS=1, CIBMTR disease risk category: High [n=8], Intermediate [n=1], Low [n=2]; 36% of the patients had primary induction failure, 54% had complex molecular abnormalities with 18% of the patients having deletion 11q 23 molecular abnormality. Donors were unrelated (36%) and related (64%). All but 1 donor-recipient pairs were fully matched. Preparative regimen- busulfan based (16mg/kg or equivalent) (82%), TBI ≥ 12 Gy (9%) based or reduced-intensity haplo-identical regimen (9%). The median number of prior chemotherapy regimens was 2.5 (range, 2–6). Azacitidine (n=10) or decitabine (n=1) was selected at physician discretion as a planned prophylactic regimen post allogeneic HSCT engraftment. Initial starting doses were based on hematological conditions at the time of therapy initiation.

Results

Ten patients received azacitidine subcutaneously daily for seven days for a median of 5 cycles (range, 1–10 cycles) and one patient received decitabine 20mg/m2 intravenously for 5 days for 3 cycles. Median initial Azacitidine dose of 50 mg/m2 (range, 25–75mg/m2) daily were given at a median of 40 days (range, 38–101 days) post HSCT engraftment. Azacitidine doses were increased based on counts to a maximum dose of 75mg/m2. With a median follow-up of 24 months (range, 1.7–49 months) 6 patients are alive in complete remission. Twenty-four month progression free survival is 51% with overall survival of 76%.

Conclusion

Given the dismal results of progression free survival and overall survival following allogeneic HSCT in high risk AML or non responsive MDS, hypomethylating agents given post transplantation provide a valuable approach to prolonging remission.

Disclosures:

No relevant conflicts of interest to declare.
Decreased Pulmonary Function in Asymptomatic Long Term Survivors After Busulfan-Based Myeloablative Allogeneic Hematopoietic Stem Cell Transplant
Authors: Oh Annie L. , Patel Pritesh R. , Saraf Santosh , Sweiss Karen , Peace David , Quigley John G. , Mahmud Nadim , Dudek Steven , Rondelli Damiano
Published: October 2019

Abstract 4474

Pulmonary function post allogeneic hematopoietic stem cell transplant (HSCT) can be impaired by previous exposure to chemotherapy, infection, or underlying chronic graft versus host disease (cGVHD). In this retrospective study, we selected 21 long term transplant survivors with a median follow up of 48 months who had received a related (60%) or unrelated (40%) HSCT conditioned with a myeloablative busulfan-based regimen. All patients had routine pulmonary function tests (PFTs) repeated within two years from transplant and none had respiratory symptoms or a history of chronic lung infection at the time of analysis.

Median age was 45 years (range 18–54). Patients had a diagnosis of acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) (52%), acute lymphoblastic leukemia (ALL) (14%), chronic myeloid leukemia (CML) (14%), non-Hodgkin lymphoma (NHL) (9%), or chronic lymphocytic leukemia (CLL) (2%). Survival rate at the time of analysis is 90%.

We initially compared single parameters of PFTs including corrected diffusion limiting capacity of oxygen (DLCO), forced expiratory volume in one second (FEV1), forced vital capacity (FVC), FEV1/FVC ratio, and total lung capacity (TLC) obtained before and after transplant. Post transplant, all PFT parameters showed some degree of reduction compared to pre-transplant values (Figure 1). FEV1 and FEV1/FVC were significantly decreased (108 vs 92,p=0.04 and 86 vs 80%, p=0.04, respectively). Median values of corrected DLCO prior to and after transplant were 83% (range 55–148) and 77%(range 44–146) (p=0.2), respectively.

In order to test whether patients with >10% reduction of PFTs had an increased rate of overall cGVHD, we analyzed intra-patient changes in DLCO and FEV1 values. We then divided patients into groups with extensive, limited, or no cGVHD. No correlation was found between the degree of cGVHD and FEV1 or DLCO values.

A decrease in pulmonary function was detected in patients who received a busulfan based myeloablative HSCT despite the absence of lung infection or pulmonary cGVHD. This finding, however, did not affect long term survival.

Disclosures:

No relevant conflicts of interest to declare.
Haploidentical Transplantation (HAPLO) with Post-Transplant High-Dose Cyclophosphamide for Graft Vs Host Disease (GVHD) Prevention in the Treatment of High Risk Hematological Neoplasms
Authors: Gayoso Jorge , Kwon Mi , Serrano David , Balsalobre Pascual , Anguita Javier , Pascual Cristina , Pérez-Corral Ana , Buno Ismael , Martinez-Laperche Carolina , Castilla-Llorente Cristina , Heras Inmaculada , Pascual Maria Jesus , Caballero Dolores , Diez-Martin Jose Luis
Published: October 2019

Abstract 4545

Introduction:

Allogeneic transplantation is the only curative option in the treatment of multiple high risk hematologic neoplasms. Only 25–30% of patients have an HLA identical sibling donor and searching for a compatible unrelated donor or cord blood renders satisfactory results in around 60–70%. Haploidentical transplantation (HAPLO) offers a therapeutic alternative to more than 95% of such patients with the advantages of quick availability, easy programming and a committed donor.

Patients and Methods:

We evaluate the results of HAPLO with a reduced intensity conditioning regimen (Fludarabine 30 mg/m2 ×5 days (-6 to -2), Cyclophosphamide 14,5 mg/kg ×2 days (-6 and -5), IV Busulfan 3,2 mg/kg × 1–3 days (BUX, days -4 to -2) employing high doses of Cyclophosphamide post graft infussion (50 mg/kg days +3 and +4) as GVHD prophylaxis together with standard doses of cyclosporine and mycophenolate from day +5.

Results:

From Dec-2007, we have done 26 HAPLO in 4 spanish centers. Median age was 38 years (16–57), 20 were male and all were in advanced phases of their diseases (12 Hodgkin′s, 6 AML, 3 ALL, 2 MM, 1 MDS, 1 MF y 1 NHL). Previous autologous HSCT has been employed in 13 and allogeneic HSCT in 6 (2 MURD and 4 UCB). Disease status at HAPLO was CR in 8, PR in 14 and refractory in 4. Bone marrow was used in 16 and unmodified peripheral blood in 10. The haploidentical donor was patient′s mother (8), father (3), siblings (11) or other relatives (4). BUX was used in 1 dose (15), 2 doses (8) or 3 doses (2) and TBI 200 cGy in 1 case. Mean neutrophils engraftment was achieved on day +18 (13–26) and platelets >50K on day +27 (17–150) in all but 2 cases of graft failure (7.7%) due to progression (MF) or relapse (M7-AML). Main toxicities were grade 1–2 mucositis in 50%, febrile neutropenia in 75% and CMV reactivations in 58% with a 100 days NRM of 3.8% (1/26, VOD and MOF) and 10% NRM at 6 months (2/20). Grade II-IV acute GVHD appeared in 10/23 patients at risk (43%) and grade III-IV in 4/23 (17%). Chronic GVHD affected to 4/15 (27%), being extensive in 1/15 (6.7%). With a median follow-up of 9 months (1–38), 13/22 (59%) are alive in CR, progression or relapse has ocurred in 6/24 (25%). Immune reconstitution seems fast and complete in those evaluated.

Conclusions:

HAPLO with high-dose cyclophosphamide as GVHD prophylaxis is a useful alternative in the treatment of high risk hematologic tumours, with low toxicity, acceptable GVHD incidence and severity, long lasting remissions, and fast immunological reconstitution.

Disclosures:

No relevant conflicts of interest to declare.
Tandem Autologous Stem Cell Transplantation in Chemorefractory Multiple Myeloma
Authors: Garnett Catherine , Giles Chrissy , Ahmed Osman , Lasa Maialen , Auner Holger W. , MacDonald Donald , Marks Sasha , Marin David , Milojkovic Dragana , Pavlu Jiri , Bua Marco , Abdalla Saad , Kanfer Edward J , Apperley Jane , Rahemtulla Amin
Published: October 2019

Abstract 4554

High-dose chemotherapy followed by autologous stem cell transplantation (ASCT) is currently standard treatment for younger patients with multiple myeloma, resulting in improved survival and response rate compared to conventional chemotherapy. Disease relapse, however, remains almost inevitable and thus the role of two successive (tandem) autologous stem cell transplants has been evaluated in chemorefractory patients as a means of prolonging duration of disease response.

We retrospectively analysed the results of nine patients with chemorefractory disease treated at a single UK institution who received tandem ASCT between January 1998 and February 2009. There were six men and three women. Median age at diagnosis was 56 years (range, 42–65 years). Paraprotein isotype was IgG in eight patients and IgA in one patient. Median serum paraprotein level was 41g/L (range 12–73g/L) at presentation.

At time of 1st transplant six patients were in stable disease (SD) and three had evidence of progressive disease. Conditioning melphalan dose was 140mg/m2 in all but two patients who received 110mg/m2 and 200mg/m2. Median time between transplants was 3.7 months (range 2.3–6.4 months) with PR and SD being observed in 2/9 and 7/9 patients at time of 2nd transplant. None of the patients reached complete response (CR). One patient received melphalan 140mg/m2 prior to 2nd transplant. The remaining patients received melphalan 200mg/m2.

Median follow up after tandem transplant was 54.3 months (range 15.6 –143.6 months). No treatment related mortality was reported. At the time of analysis, six patients were still alive and under follow up with an overall survival (OS) figure for the group of 52% at 10 years from diagnosis (Figure 1). Median progression free survival (PFS) was 20 months from 2nd transplant (range 6.7–62.6 months) (Figure 2).

Tandem autologous stem cell transplant in chemorefractory patients has resulted in overall survival similar to autologous stem cell transplant in chemosensitive patients and should be considered in patients with chemorefractory disease.

Figure 1: Overall survival from diagnosis in patients receiving tandem autologous stem cell transplant for multiple myeloma Figure 1:. Overall survival from diagnosis in patients receiving tandem autologous stem cell transplant for multiple myeloma

Figure 2: Progression free survival following tandem transplant Figure 2:. Progression free survival following tandem transplant

Disclosures:

No relevant conflicts of interest to declare.
Clinical Features, Treatment Outcomes and Prognosis of Lymphoblastic Lymphoma: A Report On 52 Patients From a Single Center in China
Authors: Li Honghua , Zhao Yu , Huang Wenrong , Bo Jian , Wang Shuhong , Jing Yu , Gao Chunji , Yu Li
Published: October 2019

Abstract 4556

Objective:

To investigatethe clinical features and prognostic factors, compare the treatment efficiency of chemotherapy and hematopoietic stem celltransplantation (HSCT) in Chinese patients with lymphoblastic lymphoma (LBL).

Methods:

We retrospectively analyzed clinical features, outcomes and prognosis of fifty-two patients with LBL who underwent chemotherapy (n=31) or HSCT (n=21) from January 1998 to December 2010 in our hospital.

Results:

Conclusion:

LBL is more common in young men and most patients had advanced diease at diagnosis. Clinically, it often presents with mediastinal, bone marrow and CNS involvement. More intensive ALL-like regimens appear superior to NHL-like regimens. Compared with chemotherapy, HSCT may improve overall survival. The use of HSCT in CR1 produces a trend for improved PFS. T-cell immunophenotype, elevated LDH, disease status more advanced than CR and patients without HSCT are independently associated with inferior outcomes.

Disclosures:

No relevant conflicts of interest to declare.
Outcome of Pediatric Allogeniec Hematopoietic Stem Cell Transplantation for Nonmalignant Disease: A Single Center Experience
Authors: Abosoudah Ibraheem , Lashin Asem , Yassin Fawwaz , Al trabolsi Hassan , Bayoumy Mohamed
Published: October 2019

Abstract 4558

Background:

The aim of this study was to determine the outcome of pediatric allogeneic hematopoietic stem cell transplantation (Allo-HSCT) for nonmalignant disease in our center.

Method:

Data were retrospectively collected for all patients (aged 0–18 years) who received allogeneic HSCT between May, 2005 and December, 2011. Outcomes according to the type of transplant, diagnosis, and transplant-related complications are reported.

Result:

Allo-HSCT was performed in 17 patients, (11 male; 6 female). Bone marrow Failure (7;41%) was the commonest nonmalignant disease followed by B-thalassemia major (4;24%) and others (6;35%). Only HLA-matched family donors were used. Most patients were conditioned with Busulfan/Cyclophosphamide/ATG. GVHD prophylaxis comprised mainly of cyclosporine and methotrexate. The median time to neutrophil engraftment was 20 (9–27) days. Two patients experienced graft failure. Four patients had Grade (I–III) aGVHD and no patient had grade (IV). Four patients (23%) had chronic GVHD. No patient had sinusoidal obstruction syndrome (SOS). Five patients had reactivation of CMV infection, which was treated and resolved in all of them. Only one patient died of sepsis. Overall survival (OS) and event free survival (EFS) were 94%, and 88% respectively.

Conclusion:

In this cohort of patients, bone marrow failure was the main reason for transplantation. The early results of HSCT were promising and consistent with published international data. Similarly, graft failure remains a concern.

Disclosures:

No relevant conflicts of interest to declare.
Absence of Spred, a Negative Regulator of Tyrosine Kinase Activity, in Acute Myeloid Leukemia Patients
Authors: Rosso Valentina , Panuzzo Cristina , Bracco Enrico , Carturan Sonia , Campia Valentina , Favole Alessandra , Signorino Elisabetta , Niparuk Pimjai , Calabrese Chiara , De Luca Luciana , Caivano Antonella , Morano Annalisa , Frassoni Francesco , Saglio Giuseppe , Cilloni Daniela
Published: October 2019

Abstract 4616

Background:

The Sprouty family proteins, including Spred, were originally identified in Drosophila melanogaster as an antagonist of Breathless, the ortholog of Fibroblast Growth Factor Receptor (FGFR) in mammals. These proteins are inducible inhibitors of signalling induced by receptor tyrosine kinases. Their main function is to deregulate the RAS/MAPK and RAS/RAF/ERK signal pathways by physically interacting. The role of Spread proteins in haematological malignancies is still not been clarified.

Aim:

The aim of this study was to investigate a possible involvement of Spred in maintaining the aberrant TK signalling in patients affected by acute myeloid leukaemia (AML).

Methods:

Bone marrow (BM) cells were collected from 82 AML patients at diagnosis and 10 BM samples from healthy donors as control. In addition 20 patients were analyzed during follow-up at the time haematological remission. All the patients included had been characterized at the cytogenetic level by conventional karyotyping, and screened by reverse transcriptase-PCR for the presence of the most frequent fusion transcripts or mutations. We analysed Spread mRNA expression by RQ-PCR and the protein by Western blot and immunofluorescence assay. In addition gain of function experiments were performed by transfecting with Spred coding sequence different leukemic cells lines lacking Spred activity.

Results:

We found that Spread mRNA levels were significantly decreased in AML compared to healthy subjects (2−DDct = 0,1 in AML compared to 0,6 in controls) (p<0,001). Western blot and immunofluorescence confirmed the Spred downregulation at the protein level. Spread expression did not correlate to FAB subtypes or cytogenetic risk. Interestingly, when patients obtained the complete hematological remission (HR) the levels of Spred mRNA and protein were significantly upregulated to a level similar to healthy subjects (p=0.1 between HR and normal controls). Transfection experiments of Kasumi cell line with Spred coding sequence resulted in a decrease of proliferation of 53%, a colony growth reduction of 50% and a significant increase of apoptosis (p<0.001). Finally, in order to establish the molecular basis for Spred1 deregulation we analyzed the expression levels of miRNA-21 which is a known regulator of Spred and we found a correlation between the expression of miRNA-21 and Spred.

Conclusions:

A decreased expression and activity of Spred, a negative regulator of TK activity through Ras pathway, is implicated in sustaining the oncogenetic signalling and abnormal proliferation induced by tyrosine kinase proteins in acute myeloid leukemia patients.

Disclosures:

No relevant conflicts of interest to declare.
Tissue Factor Pathway Inhibitor 2 (TFPI2) Is Commonly Methylated in Multiple Myeloma
Authors: Hatzimichael Eleftheria , Dasoula Aggeliki , Kounnis Valentinos , Katsenos Andreas , Crook Tim , Briasoulis Evangelos
Published: October 2019

Abstract 4617

Background:

Tissue Factor Pathway Inhibitor 2 (TFPI2) is a member of the Kunitz-type serine proteinase inhibitor family that regulates extracellular tissue homeostasis by inhibiting matrix metalloproteinases. Transcriptional silencing of TFPI2 due to DNA methylation of the CpG island of its promoter has been reported in several tumors and has been suggested to facilitate tumor cell invasion and angiogenesis. We investigated the DNA methylation status of TFPI2 in multiple myeloma (MM) patients as part of a program of epigenetic profiling of multiple myeloma.

Methods:

Genomic DNA extracted from two human MM cell lines (U-266 and RPMI-8226) and bone marrow aspirate samples from a well chracterized series of MM patients was modified by sodium bisulphite using the EZ DNA methylation kit, ZymoResearch. Bisulfite modified DNA was used as a template for Methylation Specific PCR with primers specific for unmethylated and methylated alleles. Control methylated (CpG Genome™ Universal Methylated, Chemicon International) and unmethylated genomic DNA was included in each experiment. Chi-square test, logistic regression analysis and unpaired t-test were used to investigate associations between gene methylation and ISS stage, presence of extramedullary disease, bone disease, anemia (hemoglobin ≤10 mg/dL), renal failure, serum albumin and beta (2)-microglobulin level. Kaplan-Meier curves were used to estimate the probabilities of survival and the Log-rank test to assess the statistical significance of differences in event rates.

Results:

Forty six patients with MM (26 male, 20 female; mean age 64) and two human MM cell lines were studied. According to the International Staging System (ISS) 21 patients were classified as stage I, 10 stage II and 15 as stage III disease. Methylation in the CpG island of the promoter of TFPI2 was detected in 30 patients (65%, 95%CI= 52,6–78,7%) and in both cell lines (100%). Patients with methylated TFPI2 had significantly elevated beta 2 microglobulin levels (5553 vs 3787 mg/L mean values, p=0.0002) but we did not detect a statistically significant difference in overall survival among patients with methylated versus non methylated TFPI2. No other relevant correlations were detected.

Conclusion:

TFPI2 is commonly methylated in MM patients. Transcriptional silencing of TFPI2 may play a role in the mechanism of myelomatogenesis and its methylation status warrants further evaluation as a diagnostic co-marker for this disease.

Disclosures:

No relevant conflicts of interest to declare.