bluuebunny/crossref_metadata_2025 huggingface dataset:
ISWI Chromatin Remodeling ATPase Smarca5 (Snf2h) Is Required for Murine Erythroid Development and Globin Gene Regulation
Authors: Kokavec Juraj , Majumdar Romit , Kapalova Martina , Curik Nikola , Savvulidi Filipp , Necas Emanuel , Skoultchi Arthur I. , Stopka Tomas
Published: October 2019

Abstract 2062

Chromatin remodeling plays an important role during normal and malignant hematopoiesis. The chromatin remodeling associated Imitation Switch (ISWI class) ATPase Smarca5 is abundantly expressed in mammalian hematopoietic cells. Smarca5 levels are downregulated during erythroid terminal differentiation (Stopka 2000) and they are significantly increased in human acute myelogenous leukemia blasts (Stopka 2000). In addition, Smarca5 has been reported to interact with transcription factor GATA-1 (Rodriguez, 2005). Our previous studies showed that homozygous inactivation of Smarca5 leads to early embryonic lethality (Stopka 2003). To study the role of Smarca5 in hematopoiesis, we generated a conditional allele (cKO) of Smarca5 by introducing loxP sites around exon 5 in mouse ES cells and transmitted this allele to the mouse germline. Deletion of exon 5 mediated by Cre-recombinase leads to complete loss of production of Smarca5 protein. Deletion of Smarca5 in definitive hematopoietic progenitors using the Vav1-iCre transgene resulted in fetal anemia and death by E18. The block in erythropoiesis is characterized by accumulation of hematopoietic progenitors (Sca-1+) and progressive decrease of terminally differentiated Ter-119+ and Ter-119+ Cd71+ cells in mutant fetal livers. Colony forming assays indicate growth and differentiation defects in mutant cells compared to wild type littermates. Gene expression analyses of mutant fetal liver-derived hematopoietic cells indicate loss of expression of Gata1 and several of its gene targets including adult globins. Gata2 expression was unchanged. In turn, embryonic globins Ey and Zeta were significantly upregulated (~15 fold) in mutant fetal liver-derived hematopoietic cells, indicating a defect in hemoglobin switching. To summarize, our results show that Smarca5 plays important roles in erythroid differentiation and globin gene regulation. (Grant # IGA 10310-3, MSMT 2B06077, 0021620806, LC06044, SVV-2010-254260507, NIH R01CA154239).

Disclosures:

No relevant conflicts of interest to declare.
Neutrophil Proteinase 3 (PR3) Enhances Vascular Endothelial Cell Barrier Function
Authors: Kuckleburg Christopher , Newman Peter J.
Published: October 2019

Abstract 2108

Proteinase 3 (PR3) is a 29 kDa serine protease that is released from neutrophils (PMN) upon cellular activation. Following its release, PR3 can rebind to the neutrophil expressed cell surface protein NB1 (CD177). NB1 has recently been demonstrated to be a heterophilic binding partner for the endothelial cell junctional protein, PECAM-1. Therefore NB1/PECAM-1 interactions have the potential to localize PR3 to cell junctions, where it might play a role in facilitating neutrophil transendothelial migration, possibly by regulating vascular permeability.

To test the hypothesis that PR3 regulates endothelial cell barrier function, human umbilical vein endothelial cells (HUVEC) were cultured on transwell membranes, stimulated with IL-1β, and then incubated with NB1+ or NB1- PMN. Flow cytometric analysis revealed that transmigration alone resulted in a significant increase in PR3 expression on NB1+ but not NB1- PMN. Interestingly, NB1+/PR3+ PMNs promoted increased HUVEC barrier function compared to the NB1-/PR3- PMNs, as determined by electric cell substrate impedance sensing. Incubation with PR3 alone also resulted in an increase in HUVEC barrier function similar to a PAR2 peptide agonist (SLIGKV), but this was not observed in HUVEC incubated with the closely related neutrophil proteases HNE or cathepsin G. Both PR3 and SLIGKV dramatically inhibited RhoA phosphorylation, induced calcium signaling, and were able to significantly inhibit the ability of a PAR1 peptide agonist (SFLLR) to induce endothelial cell monolayer permeability.

These results demonstrate that the neutrophil expressed serine protease PR3 can play a role in enhancing endothelial cell vascular integrity and can antagonize vascular permeability induced by PAR1, possibly through regulation of RhoA activity.

Disclosures:

No relevant conflicts of interest to declare.
Regulation of Von Willebrand Factor A1 Domain by Mechanical Force and Neighboring Domains
Authors: Thomas Wendy E , Penkala Rebecca A , Hillenmeyer Elaine , Whitfield Matthew , Tu An-yue , Munday Adam D , Lenting Peter J , Kulman John , López José A.
Published: October 2019

Abstract 2117

Regulation of the bond between platelet glycoprotein (GP) Ibα of the GPIb-IX-V complex, and the von Willebrand Factor (VWF) A1 domain is critical to the balance between hemostasis and thrombosis, particularly in high shear conditions. The GPIbα-A1 interaction is known to be activated by shear stress and inhibited by neighboring domains in VWF, but the role of neighboring domains in the shear-dependence remained unknown. Here it is shown that platelet aggregation required shear stress in the presence of VWF proteins that contain the neighboring D′D3 domain (Plus D′D3 or plasma VWF) but that platelets aggregate spontaneously with a protein that lacks this region (Delta D′D3). Moreover, platelets and microspheres coated with the N-terminal 300 amino acids of GPIbα (GC300) bind to immobilized VWF in a shear-enhanced manner for Plus D′D3 but not for Delta D′D3. In single-molecule force spectroscopy experiments, the D′D3 domain decreased the number of GPIbα-A1 bonds that formed, but did not alter bond rupture force, consistent with the hypothesis that D′D3 shields the A1 domain. By expressing recombinant VWF fragments that contain the A1 domain and various lengths of the N-terminal region, we determined that most of the inhibition by the D′D3 domain was conferred by 23 amino acids in the linker between the A1 domain and the D′D3 domain. By anchoring the fragments to the surface in an oriented manner, we demonstrated that binding was much stronger when force was applied between GPIbα and the A1 C-terminus, than when force was applied between GPIbα and the A1 N-terminus, similar to what has been observed for integrins. Based on these results, we propose the following model for regulation of VWF by mechanical force. When multimeric VWF is stretched in flow, the D′D3 domains are pulled away from the A1 domains, exposing the latter to bind platelets. When force is applied between GPIbα and the C-terminus of A1, it induces an activating conformational change that could be analogous to that seen in integrins.

Disclosures:

No relevant conflicts of interest to declare.
Dynamics of Minimal Residual Leukemia After Combinations of the HyperCVAD Regimen with Imatinib or Dasatinib In Patients with Philadelphia-Chromosome Positive Acute Lymphoblastic Leukemia
Authors: Ravandi Farhad , Thomas Deborah A , O'Brien Susan , Faderl Stefan , Jorgensen Jeffrey , Luthra Rajyalakshmi , Jones Dan , Garris Rebecca , Cortes Jorge E. , Kantarjian Hagop
Published: October 2019

Abstract 2127

Background:

The addition of tyrosine kinase inhibitors imatinib and dasatinib to combination chemotherapy regimens has improved the outcome of patients (pts) with Philadelphia-chromosme-positive (Ph+) acute lymphoblastic leukemia (ALL). We examined whether the dynamics of minimal residual disease (MRD) measured by multiparameter flow cytometry (MFC) or reverse transcription quantitative polymerase chain reaction (RQ-PCR) was different among pts treated with the combination of hyperCVAD with imatinib or dasatinib.

Materials and Methods:

From April 2001 to September 2006, 54 pts with newly diagnosed Ph+ ALL were treated with the combination of hyperCVAD and imatinib; from October 2006 to July 2009, 42 pts were treated with the hyperCVAD and dasatinib regimen. The median ages for the two groups were 50 and 51 years [ranges, (27 - 84) and (21 - 78)]. Fifty one (94%) and 40 (95%) achieved complete remission (CR) on the two regimen and were followed by serial bone marrow assessments for MRD. MFC was performed using 4 or 6 color combinations of antibodies to lymphoblast and myeloid antigens (e.g., CD10, CD13, CD15, CD19, CD20, CD22, CD25, CD33, CD34, CD38, CD58, CD66c, and CD81), with a sensitivity of 0.01%. RQ-PCR for BCR-ABL was performed using TaqMan primer/probes for the e1a2, e13a2 (b2a2), and e14a2 (b3a2) BCR-ABL transcripts in a single tube with normalization to total ABL transcripts.

Results:

The dynamics of the MRD by RQ-PCR and MFC over the initial 18 month period after achieving CR are shown in the attached figures 1 and 2, respectively. The decline in the median value of BCR-ABL/ABL was more rapid with dasatinib, with more patients reaching the level of <0.01 at 3 and 6 months (p <0.05 and p=0.05, respectively). Similarly, a higher proportion of patients receiving dasatinib were negative by MFC at 3 and 6 months after therapy; 82% and 92% were negative at 3 months, and 94% and 100% negative at 6 months for imatinib and dasatinib, respectively.

Conclusions:

Dasatinib containing regimen produces a deeper and more rapid decline in the BCR-ABL and MRD by MFC in the first 6 months of treatment. Whether this would translate to an improved long-term disease-free and overall survival remains to be investigated.

Disclosures:

Ravandi: Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Speakers Bureau. Off Label Use: frontline use of dasatinib in acute lymphoblastic leukemia. Thomas: Novartis: Honoraria; Bristol-Meyer-Squibb: Honoraria; Pfizer:; Amgen: Research Funding. Cortes: Bristol Myers Squibb: Research Funding; Novartis: Research Funding. Kantarjian: Novartis Pharmaceuticals: Research Funding; Bristol Myers Squibb: Research Funding.
The Prophylactic Use of G-CSF During Induction and Consolidation of Adults with Acute Lymphoblastic Leukemia Is Associated with Reduced Risk of Relapse and Improved Leukemia-Free Survival. A Joint Analysis of Five Randomized Trials on Behalf of the European Working Group for Adult ALL (EWALL)
Authors: Giebel Sebastian , Thomas Xavier , Hallbook Helene , Geissler Klaus , Boiron Jean-Michel , Huguet Françoise , Smedmyr Bengt , Koller Elisabeth , Hellmann Andrzej , Holowiecki Jerzy
Published: October 2019

Abstract 2130

The use of granulocyte colony stimulating factor (G-CSF), either filgrastim or lenograstim, along with induction and consolidation of adults with acute lymphoblastic leukemia (ALL) was a subject of several trials documenting significant shortening of neutropenia, reduced rate of infections and better adherence to chemotherapy protocol. The above studies, however, were not powered to detect differences with regard to long-term outcome.

We performed a joint analysis of follow-up data from five multicenter prospective, randomized trials performed in France, Poland, Sweden, Austria and Australia. Among 347 adults with ALL (median age 33 years), 185 were assigned to receive prophylactically G-CSF while 162 patients were treated without G-CSF support. G-CSF was administered either in parallel to chemotherapy or in a sequential mode.

With the median follow-up of 3.2 years, the remission duration was significantly longer for patients receiving G-CSF compared to controls (33 vs. 17 months, p=0.007). The difference was particularly pronounced for T-ALL (median not reached vs. 13 m., p=0.01) and a trend was observed for B-ALL (22 vs. 17 m., p=0.11). The 5-years probability of leukemia-free survival (LFS) was 38% for patients assigned to the G-CSF arm and 23% for the remaining subjects (p=0.01). There was also a tendency to increased overall survival (OS) rate in favor of patients receiving G-CSF (45% vs. 39%, p=0.17), which reached statistical significance in a subgroup of T-ALL (54% vs. 33%, p=0.03). In a multivariate analysis adjusted to age, initial WBC and the presence of Ph chromosome the prophylactic use of G-CSF was associated with reduced risk of relapse (HR=0.65, p=0.01) and treatment failure (HR=0.7, p=0.02). The differences remained significant when the observations were censored at the time of alloHSCT.

We conclude that the prophylactic use of G-CSF during induction and consolidation of adults with ALL is associated with reduced risk of relapse and improved LFS. The possible explanation could be better adherence to chemotherapy schedules, as demonstrated in previous studies. Patients with T-ALL appear to particularly benefit from the use of G-CSF.

Disclosures:

No relevant conflicts of interest to declare.
VWF Binding to Types I, III or VI Collagen In the ZPMCB-VWD with Identification of Subjects with Selective Reduced Plasma VWF:CB
Authors: Flood Veronica H , Haberichter Sandra L , Friedman Kenneth D , Lentz Steven R , Gill Joan Cox , Montgomery Robert R
Published: October 2019

Abstract 235

Von Willebrand Disease (VWD) is a common bleeding disorder caused by quantitative (types 1 and 3) and qualitative (type 2) abnormalities in von Willebrand factor (VWF). Defective VWF binding to collagen (VWF:CB) has been identified in VWD patients, but the type(s) and amount of collagen vary between recommended assays. We measured VWF:CB using separate assays for types I, III, and VI collagen in plasmas from 233 healthy controls and 315 VWD index cases (261 type 1, 37 type 2 and 17 type 3) recruited into the Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCB-VWD). Additional studies included VWF antigen (Ag), VWF ristocetin-cofactor (RCo), VWF multimer, and VWF gene sequence analysis. VWF:CB was tested using ELISA assays with type I human placental collagen (5 ug/ml), type III human placental collagen (1 ug/ml), and by a commercial human placental type VI VWF:CB assay (Technoclone, Austria). Normal ranges were established for VWF:CB and VWF:CB/Ag ratios with the ZPMCB-VWD healthy control population. Three subjects (2 healthy controls and 1 type 1 VWD subject) were identified with reduced VWF:CB and reduced VWF:CB/Ag ratios to type VI collagen, despite normal results with type I and type III collagen. The two control subjects had VWF:CB/Ag ratios of 0.43 and 0.51 for type VI collagen. The patient with type 1 VWD had absent type VI VWF:CB and a VWF:Ag of 54 IU/dL with a history of clinical epistaxis and an EU Bleeding Score of 8 (normal less than or equal to 3). VWF sequencing demonstrated that these three individuals had an A1 loop polymorphism, R1399H, which has been previously reported as a VWF polymorphism. No other individuals in our study had this sequence change. Of particular note was that the VWF:CB/Ag ratios for types I and III collagen were each normal – suggesting a selective abnormality. As anticipated, collagen binding with all collagens was undetectable in the type 3 VWD subjects. Type 2A and 2B VWD subjects demonstrated reduced VWF:CB and VWF:CB/Ag ratios to all three types of collagen. Type 2M and 2N VWD subjects exhibited normal VWF:CB/Ag ratios to all three types of collagen, but one type 2M patient with an I1425F A1 loop mutation had a reduced VWF:CB/Ag ratio of 0.58. In the VWF A1 domain crystal structure, amino acid 1425 is in close proximity to amino acid 1399, suggesting the conformation of that region may be critical to type VI collagen binding to VWF. Since the frequency of the R1399H polymorphism is estimated to be 2% of the population (Sadler and Ginsburg, 1993), defective binding to type VI collagen may be an important contributor to the variability in the bleeding phenotype of VWD.

Disclosures:

Lentz: Novo Nordisk: Consultancy. Montgomery:GTI Diagnostics, Inc: Consultancy.
Autologous Stem Cell Transplantion for Adolescents and Young Adults with Relapsed and Refractory Hodgkin Lymphoma
Authors: Ali Muhammad , Schechter Tal , Panzarella Tony , Villa Diego , Kuruvilla John , Kukreti Vishal , Tsang Richard , Doyle John J , Crump Michael
Published: October 2019

Abstract 2402

Adolescents and young adults with Hodgkin lymphoma (HL) represent a unique patient population, facing cancer and the toxicities of therapy during an important, transformative period of their lives. The long term outcomes of AYA undergoing intensive chemotherapy and autologous stem cell transplantation (ASCT), both medical and psychosocial, have not been well studied. We assessed the outcome of AYAs undergoing ASCT to define the results of treatment, and assemble a cohort in which we can evaluate the effect of treatment for relapsed disease on personal developmental and psychological outcomes.

Between 1988 and 2009, 181 AYAs (median age 24, range 15–30y) with relapsed or refractory HL received salvage therapy followed by ASCT at Princess Margaret Hospital (PMH) and the Hospital For Sick Children (HSC). Patient characteristics: M:F 94:87; advanced stage (IIB-IV) at diagnosis 84%; first progression or relapse: 90%. All patients had complete or partial response to 2–3 cycles salvage chemotherapy (platinum-base, mini-BEAM or ifosfamide-vinorelbine), and underwent bone marrow harvest (n=83) or stem cell mobilization with chemotherapy and filgrastim (n=98). Intensive therapy: etoposide + melphalan (PMH) or cyclophosphamide, carmustine + etoposide (HSC). Patients with disease bulk >5cm at relapse received post-ASCT involved field radiation if tissue tolerance allowed.

Results:

After a median follow-up of surviving patients of 4 years (maximum 16 years), of 181 pts, there have been 105 events for progression-free survival (PFS) and 81 deaths. PFS at 5 years is 41% +/−4% (+/− standard error) and at 10 years is 36% +/−4% (median 1.6 years). Overall survival is 52% +/−5% at 5 years and 39% +/− 5% at 10 years(median 5.6 years). There have been no HL relapses beyond 39 months post-ASCT. Twenty patients (11%) died from treatment related-toxicity (10), second malignancies (5 AML, 2 NHL, 1 lung cancer) or unknown causes (2). Nine patients received an allogeneic stem cell transplant for relapse; 8 have died from progressive HL or treatment-related toxicity and one is alive in relapse.

Conclusions:

While highlighting the clear need for improved strategies for disease control for AYAs undergoing ASCT, this analysis has provided a cohort of patients in which to evaluate the long-term impact of intensive therapy on personal and social adjustment, and to identify opportunities for psychosocial intervention and health promotion.

Disclosures:

Kuruvilla: Hoffman LaRoche: Honoraria, Research Funding; Celgene: Research Funding; Amgen: Honoraria; Otsuka: Honoraria; Genzyme: Honoraria. Kukreti: Celgene: Honoraria.
MLL/AF4 Positive Acute Lymphoblastic Leukemia Has Resistance to Tumor Necrosis Factor-Alpha Caused by up-Regration of S100A6
Authors: Tamai Hayato , Miyake Koichi , Yamaguchi Hiroki , Okabe Masahiro , Dan Kazuo , Inokuchi Koiti , Shimada Takashi
Published: October 2019

Abstract 2477

Mixed-lineage leukemia (MLL)/AF4 positive acute lymphoblastic leukemia (ALL), which is common in infant leukemia, is associated with a poor prognosis even after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The resistance to graft versus leukemia (GVL) effect may be the possible reason of the poor effect of allo-HSCT on MLL/AF4 positive ALL. Cytotoxic effector mechanisms, which are mediated by tumor necrosis factor-alpha (TNF-alpha), Fas ligand (FasL), or perforin were reported to contribute to GVL effect. We analyzed the sensitivity to TNF-alpha of MLL/AF4 positive ALL cell lines (SEM and RS4;11) together with MLL/AF4 negative leukemia cell lines (MOLT4, Raji and K562) and found that the growth inhibition by TNF-alpha of MLL/AF4 positive ALL cell lines group was significantly lower than those of another MLL/AF4 negative leukemia cell lines group (P<0.01). To examine the possible mechanism of resistance to TNF-alpha of MLL/AF4 positive leukemia, we focused on S100A6 as a possible factor, since it was reported that expression of S100A6, which is a calcium-binding protein implicated in many cellular processes and often up-regulated in cancers limits apoptosis induced by TNF-alpha through p53 inactivation in cardiac myocytes. Western Blot analysis showed that significant up-regulation of S100A6 expression and inhibition of acetyl p53/p53 expression were observed only in MLL/AF4 positive ALL cell lines in the presence of TNF-alpha (5ng/ml)(P<0.01 for S100A6 and P<0.01 for acetyl-P53/p53). To confirm the effect of S100A6 on the resistance to TNF-alpha of MLL/AF4 positive ALL cell lines, we examined the apoptosis rate of MLL/AF4 positive ALL cell lines treated with small interfering RNA (siRNA) against S100A6. TUNEL assay showed that significantly increased rate of apoptosis under s100A6 siRNA in the presence of TNF-alpha (control siRNA VS s100A6 siRNA; 42.5%∓4.5% VS 90.0%∓5.0%, P<0.01 for SEM, 32.5%∓3.5% VS 87.5%∓5.0%, P<0.01 for RS4;11). Western Blotting also showed that these apoptosis were mediated by p53-Caspase8 pathway. Up-reguration of S100A6 and inhibition of p53-Caspase8 pathway were also confirmed in previously established MLL/AF4 transgenic mice. These results suggest that MLL/AF4 positive ALL escapes from TNF-alpha mediated apoptosis by up-regulation of S100A6 expression followed by interfering with p53 acetylation. Our present results suggest that S100A6 may be a promising therapeutic target for MLL/AF4 positive ALL by combinating with allo-HSCT.

Disclosures:

No relevant conflicts of interest to declare.
Impact of Protein Phosphatase PP2A on ATRA-Induced Activation of FoxO3a In Acute Promyelocytic Leukemia Cells
Authors: Sakoe Yasuhiko , Sakoe Kumi , Shimazaki Haruo , Kirito Keita , Komatsu Norio
Published: October 2019

Abstract 2492

Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia caused by reciprocal translocations of the long arms of chromosomes 15 and 17, which prevent cellular differentiation into mature neutrophils. The translocation of the promyelocytic leukemia (PML) gene on chromosome 15 and a retinoic acid receptor α (RARα) gene on chromosome 17 generates a PML-RARα fusion protein that inhibits PML-dependent apoptotic pathways in a dominant negative fashion. This fusion protein also blocks granulocytic differentiation by direct transcriptional inhibition of retinoic acid target genes. All-trans retinoic acid (ATRA) arrests cell growth, granulocytic differentiation, and apoptosis in APL cells via proteasome-dependent degradation of PML-RARα fusion protein and subsequent PML-nuclear body (NB) formation. Although PML is the essential component of PML-NBs and functions as a tumor suppressor, disruption of PML-NBs by the PML-RARα fusion protein inhibits endogenous PML tumor-suppressive functions in APL cells. Therefore, degradation of PML-RARα fusion protein and reorganization of PML-NBs during ATRA treatment are regarded as critical cellular responses, similar to the cell growth arrest and apoptosis of leukemia cells. Recently we demonstrated that FoxO3a (also named FKHRL1), a member of the Forkhead family of transcription factors, is a key molecule for the ATRA-induced cellular responses in APL cells (Blood 2010; 115: 3787–3795). In this study, we investigated the mechanism by which FoxO3a is activated by ATRA treatment in a human promyelocytic leukemia cell line NB4. Okadaic acid, a potent PP2A inhibitor, cancelled ATRA-induced dephosphorylation of AKT and its downstream molecule FoxO3a in NB4 cells. Knockdown of endogenous PP2A by siRNA significantly enhanced phosphorylation of both AKT and FoxO3a. These results suggested that PP2A is involved in ATRA-induced dephosphorylation of AKT and FoxO3a. Concomitantly, PP2AC, a catalytic subunit of PP2A, was dephoshorylated at tyrosine 307, and phosphatase activity of PP2A increased after ATRA treatment. Co-immunoprecipitation assay revealed that PP2A constitutively and directly binds to FoxO3a. Using artificial oligopeptides, we demonstrated that enhanced PP2A activity by ATRA directly dephosphorylates phosphothreonine 32 on FoxO3a. In addition, we found that 14-3-3 epsilon binded to phosphorylated FoxO3a in the cytoplasm in the absence of ATRA. After ATRA treatment, however, dephosphorylated FoxO3a dissociated from 14-3-3 epsilon and moved into the nucleus. Confocal microscopic analysis revealed that PP2A-FoxO3a complex partially co-localized with PML-NBs in the nucleus after ATRA treatment. Together, PML orchestrates nuclear networking with PP2A and FoxO3a for ATRA-induced granulocytic differentiation and apoptosis of APL cells.

Disclosures:

No relevant conflicts of interest to declare.
A Novel PHD2 Mutation Associated with Tibetan Genetic Adaptation to High Altitude Hypoxia.
Authors: Lorenzo V Felipe R , Simonson Tatum S , Yang Yingzhong , Ge Rili , Prchal Josef T.
Published: October 2019

Abstract 2602

The ability of the body to maintain oxygen hemostasis is essential for its survival. The body is required to adapt to the physiologic constraints imposed by a hypoxic environment in order to ensure optimal physiological functions. The study of indigenous high-altitude residents provides an opportunity to identify genes that may have play a role in hypoxia adaptation, and recent studies indicate that Tibetans have genetically adapted to the extremely hypoxic environment at high altitude. Interestingly, Tibetan adaption includes protection from polycythemia. We recently reported genome-wide scans of selection (Affymetrix Human SNP chips v6.0) for Tibetans (Simonson, Science July, 2010). At least 10 chromosomal regions were identified as likely targets for high-altitude adaptation, and two of these regions, EGLN1 (PHD2) and PPARA, were associated with protection against high-altitude polycythemia which is unique to this indigenous highland population. PHD2 is a negative regulator of the alpha subunits of HIF-1 and HIF-2 transcription factors regulating many essential functions including erythropoiesis, iron metabolism, development, in addition to other functions. A gain of function mutation of the alpha subunit of HIF-2 is associated with congenital polycythemia. We sequenced the exons of PPARA and PHD2 by direct sequencing and found two missense mutations in exon 1 of the PHD2 gene: the first is located within codon 4, and changes Aspartate to Glutamate (Asp4Glu) and the second is an unvalidated SNP (rs12097901) at codon 127 that changes Cysteine to Serine (Cy127Ser). The samples with high Hb show a heterozygous change, while the subject with normal Hb exhibited a homozygous change in both codons of the PHD2 gene.

The association of these two missense PHD2 mutations to hemoglobin level is now being evaluated by studies of a larger population of Tibetan natives and the functional effects on levels and activities of HIF-1 and HIF-2 genes evaluated by transfection of these mutated PHD2 cDNA constructs in reporter cells.

Table 1. List of samples and controls screened for mutation in PHD2. Sample Race Sex Hb PHD2 Mutation Asp4Glu Cys127Ser T03 Tibetan F 194 Hetero Hetero T43 Tibetan M 194 Hetero Hetero T48 Tibetan M 101 Hetero Homo T53 Tibetan M 159 Homo Homo Control Caucasian M ? WT WT Control Caucasian F ? WT WT

Disclosure:

No relevant conflicts of interest to declare.
Quantitation of EBV-DNA In Peripheral Blood In Hodgkin Lymphoma: Associations with Other Biomarkers and Patient Characteristics
Authors: Hohaus Stefan , Giachelia Manuela , Vannata Barabara , Santangelo Rosaria , Martini Maurizio , Cenci Tonia , Massini Giuseppina , Cuccaro Annarosa , Francesco D'Alo , Voso Maria Teresa , Fadda Giovanni , Larocca Luigi , Leone Giuseppe
Published: October 2019

Abstract 2678

The Epstein-Barr virus (EBV) is present in the malignant Hodgkin/Reed-Sternberg (HRS) cells of 20–40% cases of Hodgkin lymphoma (HL) in western countries. Detection and quantitation of cell-free plasma EBV-DNA has been proposed as a biomarker in EBV-associated malignancies, including HL. We analyzed the presence of EBV in HRS cells, peripheral blood compartments, and correlated these data to EBV serology, other circulating biomarkers and clinical characteristics. In a single institution cohort of 93 HL patients, EBV-DNA was quantitated using real-time PCR for the EBNA region in whole blood (WB, n=69), plasma (PL, n=75) and peripheral blood mononuclear cells (MNC, n=74). Cell-free DNA levels were determined using a real-time PCR for the globin gene (n=84), cytokine concentrations were measured in 85 patients. EBV status of HRS cells was studied using in-situ hybridization with EBER. EBV-DNA was positive in 32% WB, 21% PL and 20% MNC at HL diagnosis, with concordant WB and PL results in 86% of cases. EBER was present in HRS cells in 32% of cases. EBV-DNA copy number over 1500/ml was predictive for the presence of EBER in HRS cells in 86% and 88% WB and PL, respectively, while EBV copy numbers <1500 had only 55% and 57% PPV for WB and PL. Older patients (>50 years) were frequently EBER-positive (66%), with higher EBV copy numbers (median 12800/ml), while younger patients (<50 years) were less frequent EBER-positive (23%), and copy numbers were lower (median 1200/ml) (p=0.01). EBER-positive HL cases frequently had EBNA-1 antibody titers < 100 U/ml when compared to EBER-negative cases (44% vs. 13%, p=0.01), and there was a trend for a negative correlation between EBV copy number and EBNA1 titers (p=0.07). In patients with EBER+ HL the plasma EBV-DNA copy number correlated to the level of circulating cell-free DNA (rho=0.65), IL-10 levels (rho=0.87), IL-6 levels (rho=0.74) and inflammation parameters, including CRP (rho=0.79) and ESR (rho=0.57) (all p<0.02), and were higher in stage IV disease (p=0.02). In conclusion, a high EBV-copy number in whole blood and/or plasma at diagnosis is a good indicator for EBV-association of HL, and EBV copy numbers strongly correlate to other biological parameters of disease activity. Moreover, the inverse correlation between EBV copy number and EBNA1 antibody titers may indicate a reduction in immune surveillance favoring the expansion of EBV.

Disclosures:

No relevant conflicts of interest to declare.
Evaluation of the Prognostic Value of CD45RO+ and FOXP3+ Cells of the Micro-Environment In Classical Hodgkin Lymphomas Using Tissue Micro Array
Authors: Benet Claire , Ferrant Emmanuelle , Petrella Tony , Degrolard Emilie , Bastie Jean-Noel M. , Bonnotte Bernard , Casasnovas Rene-Olivier , Martin Laurent
Published: October 2019

Abstract 2687

Classical Hodgkin lymphomas (cHL) are B lympho-proliferative disorders that can be cured in 80% of cases. The goal of current research is to identify, at diagnosis, the 20% patients, who are likely to relapse or be refractory to treatment. As cHL have an extensive micro-environment implicated in tumour growth, the prognostic value (disease-free survival) of 2 important subsets of lymphoid cells (CD45RO+ effector memory T cells and FOXP3+ regulatory T cells) of the micro-environment has been evaluated using Tissue Micro Array (TMA).

Patients (n=96) treated and followed for cHL between 1998 and 2005 were included in this study. Six TMA with 3 spots per case, were built (3 formalin and 3 Bouin fixative). Immunostaining with anti-CD45RO (Dako, UCHL1, 1/400) and anti-FOXP3 (Abcam, 236A/E7 1/100) antibodies was performed on the Benchmark Ultra (Ventana). After scanning of the slides, the staining was quantified automatically using a function (pixel count) of Scanscope software (Aperio). ROC curves were used in order to calculate the best threshold for each marker, depending on the number and intensity of pixels. Survival curves were built according to these thresholds using the Kaplan Meier method and compared with the log-rank test. Then, a Cox model was used to estimate the risk of occurrence of an event in univariate and multivariate analyses.

The demographic characteristics of patients and sub-types of HL were not different in the << formalin >> and << Bouin >> groups. Median follow-up of patients was 69 months (1-135). In univariate analysis, low quantities of CD45RO+ cells was associated to a 40% 5-years progression free survival (PFS) compared to a 87% 5-years PFS for patients with high number of CD45RO+ cells (p=0.003), and low number of FOXP3+ cells was related to a 56% 5-years PFS compared to a 86% 5-years PFS for patients with high number of FOXP3+ cells (p=0.026). In multivariate analysis, taking into account the International Prognostic Score (IPS) and the expression of CD15 by neoplastic cells, the association of CD45RO and FOXP3 was the only independent prognostic factor identified (p<0.02 et RR=5.75).

This preliminary study underlines the importance of certain subsets of cells from the microenvironment of HL in the control of tumour growth. These results are encouraging. When used in association with validated criteria, they could make it possible to identify patients at risk of relapse and/or drug resistance. Nevertheless, due to the small number of patients in our study, these findings need to be validated in a larger and/or independent cohort.

Disclosures:

No relevant conflicts of interest to declare.
MMP-2 and MMP-9 Secreted by Leukemic Cells Increase the Permeability of Blood-Brain Barrier by Disrupting Tight Junction Proteins In Central Nervous System Leukemia
Authors: Feng Sa-ran , Chen Zi-Xing , Cen Jian-nong , Shen Hong-jie , Yao Li , Wang Yuan-yuan , Qi Xiao-fei , Chen Su-ning
Published: October 2019

Abstract 2694

Recurrence of acute leukemia (AL) in the central nervous system (CNS) confers a poor prognosis. However, little is known about the the underlying mechanisms of leukemic cell infiltration into the CNS. The blood brain barrier (BBB) makes CNS become a refugee to leukemic cells and serves as a resource of cells that seed extraneural sites. Tight junction in brain microvessel endothelial cells (HMECs) constituted by tight junction proteins is an important structure of BBB. Except the well known role of degrading extracellular matrix in metastasis of cancer cells, here we identify matrix metalloproteinase 2 (MMP-2) and MMP-9 secreted by leukemic cells are also associated with BBB opening by degrading tight junction proteins. We have successfully established an animal model of CNS leukemia by using a highly invasive human acute monocytic leukemia cell line SHI-1 in nude mice. Multiple organs including skull and brain were sectioned and determined histopathologically for leukemia cell infiltration. The fact that the down- regulation of ZO-1, Claun-5 and Occludin accompanied with up-regulation of MMP-2 and MMP-9 was correlated with BBB breakdown in mice with CNS leukemia was found when examined by laser scanning fluorescence confocal microscopy and gelatin in situ zymography. The treatment with MMP-inhibitor GM6001 could significantly reverse these changes in tight junction proteins. In an in vitro monolayer BBB model made by human encephalo-microvessel endothelial cells and matreil gel, MMP-2 and MMP-9 specifically down-regulated ZO-1, Claun-5 and Occludin. Knock-down or inhibition of MMP-2 and MMP-9 expression protected ZO-1, Claun-5 and occludin from degradation and alleviated the permeability of BBB. Our findings suggest that the degradation of tight junction proteins ZO-1, Claun-5 and Occludin by MMP-2 and MMP-9 secreted by leukemic cells constitutes an important mechanism in BBB breakdown in CNS leukemia. These studies provide a potent evidence for future pharmacological treatment to inhibit the CNS involvement in acute leukemia.

Disclosures:

No relevant conflicts of interest to declare.
Germline Genetic Variation and Treatment Response on CCG-1891
Authors: Sepe Dana , McWilliams Thomas , Chen Jinbo , Kershenbaum Aaron , Zhao Huaqing , La Mei , Davidas Meenakshi , Lange Beverly , Rebbeck Timothy R. , Aplenc Richard
Published: October 2019

Abstract 2711

Purpose:

Recent studies have suggested that polymorphisms in genes that encode enzymes involved in drug detoxification and metabolism may influence disease outcome in pediatric patients with acute lymphoblastic leukemia (ALL). We sought to extend current knowledge by using standard and novel statistical methodology to examine how frequency of polymorphism variants of certain drug metabolizing genes and gene pairs relate to relapse risk, toxicity, and drug dose delivery in standard risk ALL.

Methods:

We genotyped and abstracted chemotherapy drug dose data from treatment roadmaps on 557 patients on the Children's Cancer Group ALL study, CCG-1891. These data were analyzed with outcome data obtained from CCG. Fourteen common polymorphisms in genes involved in folate metabolism and/or phase I and II drug detoxification were evaluated individually by univariate analysis and clique-finding methodology was employed for detection of significant gene-gene interactions. Additionally, positive predictive value was calculated for those polymorphisms found to be statistically significant on univariate analysis.

Results:

After controlling for known risk factors, polymorphisms in four genes: GSTP1*B (HR=1.94, p=0.047), MTHFR (HR=1.61, p=0.034), MTRR (HR=1.95, p=0.01), and TS (3R/4R, HR=3.69, p=0.007), were found to significantly increase relapse risk. However, the positive predictive value of these genotypes was not statistically significant. One gene-gene pair, MTRR A/G and GSTM1 null genotype, significantly increased the risk of relapse after correction for multiple comparisons (p=0.012). Multiple polymorphisms were associated with various toxicities and there was no significant difference in dose of chemotherapy delivered by genotypes.

Conclusion:

These data suggest that various polymorphisms play a role in relapse risk and toxicity during childhood ALL therapy and that genotype does not play a role in adjustment of drug dose administered. Additionally, gene-gene interactions may increase the risk of relapse in childhood ALL and the clique method may have utility in further exploring these interactions.

Disclosures:

No relevant conflicts of interest to declare.
A Prognostic Score of Overall Survival In Adults with Acute Myeloid Leukemia
Authors: Al-Khabori Murtadha K. , Guyatt Gordon , Minden Mark D. , Yee Karen , Gupta Vikas , Schimmer Aaron , Schuh Andre , Brandwein Joseph
Published: October 2019

Abstract 2730

Background:

Acute myeloid leukemia (AML) is a malignant myeloid disorder with heterogeneous outcomes. A number of factors have been shown to be prognostic; age, white blood cell (WBC), prior malignancy, performance status (Eastern Cooperative Oncology Group; ECOG) and cytogenetics.

Methods:

We planned to develop and validate a prognostic score for the 5-year (y) overall survival (OS) of adults with AML receiving intensive induction chemotherapy. We used Cox model to estimate the regression coefficients and Kaplan-Meier to estimate the 5-y OS. We used Cox-Snell, Schoenfeld and deviance residuals for model diagnostics and bootstrap validation to estimate the performance measures; Harrell's concordance and deviance residuals.

Results:

We retrospectively analyzed 779 patients treated between 1998–2008, using a prospectively collected database. The median age was 58 y. Most patients had intermediate risk cytogenetics (61%) and good performance status (ECOG 0–1: 79%). The median follow up for the surviving patients was 26.7 months (95% CI 18.8–32.9 months). The 5-y OS was 26% (22- 30%). All variables were statistically significant in the multivariable Cox regression model; age (y) (Hazard Ratio, HR 1.02; 95% CI 1.018–1.034), WBC (1*10^9/L) (HR 1.004; 1.002–1.006), prior malignancy (HR 1.58; 1.26–2.00), ECOG (ECOG 2 HR 1.41; 1.06–1.88, ECOG 3–4 HR 9.99; 4.72–21.18) and cytogenetics (intermediate risk HR 2.49; 1.41–4.39, poor risk HR 4.74; 2.65–8.50). The score divided patients into four risk groups; good (n=47), intermediate (n=129), poor (n=198) and extremely poor (n=87). The estimated 5-y OS was 0.70 (95% CI: 0.53–0.81), 0.37 (0.28–0.46), 0.15 (0.10–0.21) and 0.03 (0.01–0.10) respectively. The model showed good discrimination with large differences between survival curves and good Harrell concordance of 0.69. It showed good calibration using Cox-Snell and deviance residuals. In the intermediate risk cytogenetic group, the model showed good discrimination with over 45% difference in 5-y OS between the good and extremely poor groups.

Conclusions:

Our study confirmed the prognostic impact of the 5 variables reported in the literature. Using these factors, we developed a score to predict long term OS that showed good discrimination and calibration. The score added further discrimination in the intermediate risk cytogenetic group. Prospective external validation of the score is needed.

Disclosures:

No relevant conflicts of interest to declare.
Overexpression of SIRT1, a Longevity Gene-Encoded Protein, and Induction of Apoptosis by Its Inhibition In Adult T-Cell Leukemia Cells
Authors: Kozako Tomohiro , Yoshimitsu Makoto , White Yohann , Aikawa Akiyoshi , Shoji Teruhisa , Honda Shinichiro , Shimeno Hiroshi , Soeda Shinji , Arima Naomichi
Published: October 2019

Abstract 2768

Adult T-cell leukemia-lymphoma (ATL) is an aggressive peripheral T-cell neoplasm developing after a long-term infection with human T-cell leukemia virus (HTLV-1), in which NF-kB is also implicated as an exacerbation factor. Despite recent progress in both chemotherapy and supportive care for hematological malignancies, the prognosis of ATL is still poor; overall survival at 3 years is only 24%. New strategies for the therapy and prophylaxis of ATL (e.g., vaccines and novel molecular target agents) are still required. SIRT1, an NAD+-dependent histone/protein deacetylase, plays a crucial role in various physiological processes, such as aging, metabolism, neurogenesis and apoptosis, due to its ability to deacetylate numerous substrates, such as histone and NF-kB. Here, we assessed how SIRT1 is regulated in primary ATL cells and leukemic cell lines. SIRT1 expression in ATL patients was significantly higher than that in healthy controls, especially in the acute type. Sirtinol, a SIRT1 inhibitor, induced significant growth inhibition or apoptosis in cells from ATL patients and leukemic cell lines, especially HTLV-1-related cell lines (S1T and MT-2). Sirtinol-induced apoptosis was mediated by activation of the caspase family, and inactivation of NF-kB, reducing IkBα phosphorylation. Interestingly, NAD+ augmented sirtinol-induced apoptosis following deacetylation of NF-kB via NAD+-dependent deacetylase. Thus, the SIRT1 inhibitor acted as a tumor suppressor, where NAD+ accelerated the SIRT1 inhibitor-induced apoptosis. These results suggest that SIRT1 is a crucial antiapoptotic molecule in ATL cells, and that SIRT1 inhibitors may be useful therapeutic agents for leukemia, especially in patients with ATL.

Figure 1. SIRT1 inhibitor reduces viability of leukemic cell lines. Cell lines were incubated at 1×105 cells/mL in the presence of various concentrations of sirtinol for 72 h. Proliferation of cell lines in the absence or presence of the indicated concentrations of sirtinol was assessed by WST-8 assay. Data are means ± S.D. from 3 independent experiments. Figure 1. SIRT1 inhibitor reduces viability of leukemic cell lines. Cell lines were incubated at 1×105 cells/mL in the presence of various concentrations of sirtinol for 72 h. Proliferation of cell lines in the absence or presence of the indicated concentrations of sirtinol was assessed by WST-8 assay. Data are means ± S.D. from 3 independent experiments.

Disclosures:

No relevant conflicts of interest to declare.
Short Course Adjuvant Chemotherapy for Localized Diffuse Large B-Cell Lymphoma After Complete Resection.
Authors: Jo Jungmin , Yoo Changhoon , Ahn Yongchel , Park Seong Joon , Lee Dae Ho , Kim Sang-We , Lee Jungsin , Suh Cheolwon
Published: October 2019

Abstract 2824

Introduction:

Diffuse large B-cell lymphoma (DLBCL) in general has been treated 6 or 8 cycles chemotherapy. Not a few patients have had adverse events during chemotherapy and stopped treatment because of treatment related toxicities. Favorable results were reported from NHL-BFM 90 trial on childhood and adolescents B-cell NHL with short intensive therapy courses depending on stage and tumor mass. Treatment was stratified into 3 risk groups and patients with initial complete resection received 2 therapy courses. We aimed to investigate effectiveness of short course chemotherapy for localized DLBCL after complete resection in adults.

Patients and Methods:

The records of Asan Medical Center registry for Non-Hodgkin's lymphoma were reviewed. Between March 2002 and March 2009, 18 patients were enrolled with DLBCL of stage I or II and complete resection before initial chemotherapy. All patients received 3 to 4 courses of rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone chemotherapy (R-CHOP) or CHOP without radiotherapy. Retrospective analysis of disease-free survival (DFS) and overall survival (OS) was performed.

Results:

The study cohort of 18 patients showed median age of 53.5 years (range 18–69 years) at diagnosis. Ten patients were males. The Ann Arbor stage was I in 14 patients, II in 4 patients. All patients of stage II had gastrointestinal lymphoma with regional lymph node involvement. Only 2 patients had B symptom. All patients were stratified into low-risk group according to international prognostic index (IPI). Most of patients (88.9%) had 3 cycles chemotherapy and 2 patients with 4 cycles. Fifteen patients were treated with R-CHOP and the others with CHOP. The median follow-up time was 27.5 months (range 6.1–100 months). The 2-year DFS was 100% and the 2-year OS was 100% in study cohort.

Conclusions:

It has been reported that 4-year OS of DLBCL was 86% in low risk group (0-1 IPI factor) treated with R-CHOP. Favorable result with short course chemotherapy showed in our study despite short follow-up time. On the result, we suggest reduced treatment for localized DLBCL after complete resection. However, prospective study is warranted to investigate effectiveness.

Disclosures:

No relevant conflicts of interest to declare.