bluuebunny/crossref_metadata_2025_split huggingface dataset:
Low Frequency of Molecular Alterations of H3.3-Atrx-Daxx Chromatin Remodeling Component Genes in Myelodysplastic Syndromes (MDS)
Authors: Attieh Youmna , Wei Yue , Yang Hui , Jia Yu , Zheng Hong , Fang Zhihong , Kantarjian Hagop M. , Garcia-Manero Guillermo 
Published: October 2019

Abstract
Abstract 3844
Novel sequencing technologies have allowed identification of a group of highly recurrent genetic mutations in myelodysplastic syndromes (MDS). Of importance, it has been noticed that a majority of these mutated genes in MDS encode important components of epigenetic regulation, including both DNA methylation and histone modifications. This phenomenon highlights the importance of epigenetic mechanisms in the pathogenesis of MDS. Recently, highly recurrent somatic mutations in the Histone H3.3-ATRX-DAXX chromatin remodeling pathway have been documented in pediatric glioblastoma (Schwartzentruber et al. Nature and Wu et al. Nature Genetics 2012), further supporting the importance of epigenetic regulation for tumorgenesis. We therefore examined potential genetic and epigenetic alterations of the same pathway in MDS. First, in a cohort containing 80 samples of MDS whole bone marrow mononuclear cell DNA (representative of both lower and higher risk disease), we performed Sanger sequencing covering genomic areas of reported mutations of H3F3A, H3F3B, ATRX, and DAXX in glioblastoma. Sequenced genomic areas included reported mutations in pediatric tumors: Lys27 and Gly34 of H3F3A and H3F3B; sequences upstream of and within the helices domain of ATRXX; and the whole coding sequence of DAXX. Overall, we only detected one mutation of H3F3A (K27N) in one MDS case (76 year old male with RA; INT-1; diploid). No other reported mutation of H3F3B, ATRX and DAXX genes was detected in any other patients of this MDS cohort. Because of the potential of epigenetic deregulation, we then examined status of DNA methylation for the promoters of ATRX and DAXX in MDS patients by bisulfite pyrosquencing. While no DNA hypermethylation of DAXX promoter was detected, 8 out of 40 (20%) patients had hypermethylation of the CpG island in the promoter region of ATRX. However, six of these eight patients were females. Based on reports of ATRX methylation in healthy females, it is likely that the 6 cases in female patients represent physiological × chromosome inactivation. Finally, we performed RT-PCR analysis using cDNA samples isolated from CD34+ hematopoietic stem cells of 40 MDS patients. Results indicated that expression of ATRX and DAXX were increased by 2 fold (p-value 0.07) and 5.2 fold (p-value 0.0003) respectively compared to control CD34+ cells. The implications of this phenomenon need to be studied further. Taken together, these results suggest that genetic mutations of the H3.3-ATRX-DAXX chromatin remodeling do not play a role in the pathogenesis of MDS.

Disclosures:
No relevant conflicts of interest to declare.
Regional and Racial Differences in Venous Thrombosis in the United States: The Reasons for Geographic and Racial Differences in Stroke (REGARDS) Study
Authors: Zakai Neil A , McClure Leslie A , Judd Suzanne E , Folsom Aaron R , Lutsey Pamela , Callas Peter , Safford Monika , Cushman Mary 
Published: October 2019

Abstract
Abstract 396

Introduction:
Venous thrombosis (VTE) is thought to be more common in African-origin (black) than Caucasian-origin (white) Americans, however most prior studies are based on administrative databases, are limited in geographic scope, or have few blacks. Studies of VTE with diverse racial and geographic participants and validated VTE events are lacking.

Methods:
Between 2003–07, 30,239 participants were enrolled in REGARDS, 55% were female, 41% were black, and 56% lived in the southeast (by design). Possible VTE events were identified by telephone interviews, review of reported hospitalizations, and review of deaths. VTE events were validated by physician review of hospital records, outpatient notes, imaging studies, and death records. VTE incidence rates by race and region controlling for age and sex were estimated using Poisson regression and racial and regional differences in VTE were evaluated using Cox proportional hazard models controlling for age, sex, region, obesity status, education, and income.

Results:
Among 27,807 individuals free of VTE at baseline, 268 incident VTE events occurred over a mean of 4.7 ± 1.6 years (Table). Blacks and whites had the same age and sex-adjusted VTE incidence (1.8 per 1000 person-years). When stratified by region, blacks living in the southeast had a higher incidence of VTE than whites (2.2 vs. 1.7 per 1000 person-years), while this was not the case in the rest of the US (1.5 vs. 2.0 per 1000 person-years) (Table). This difference in associations by race and region was statistically significant, such that the HR for blacks compared to whites was higher in the southeast (1.3) than in the rest of the country (0.8) even after adjusting for income, education, and obesity (all p-interaction ≤ 0.03) (Table). Further, blacks in the southeast had a higher VTE risk than blacks in the rest of the US (HR 1.5; 95% CI 1.0, 2.3) while there was no geographic disparity for whites (HR 0.8; 95% CI 0.6, 1.1) for the southeast vs. rest of the country.

Discussion:
For the first time, we demonstrate that region of residence significantly influences racial differences in VTE. Prior studies of racial differences in VTE have used administrative databases or studied blacks primarily from the southeast. In REGARDS, blacks in the southeast have higher rates of VTE compared to blacks in the rest of the US, and this is not explained by differences in income, education, and obesity. Whether these findings are due to differences in quality of care, genetic admixture, or novel VTE risk factors needs further study.

Disclosures:
Cushman: Beckman: Honoraria.
Enhancers of Polycomb EPC1 and EPC2 Prevent Accumulation of MYC to Sustain the Oncogenic Potential of MLL Leukemia Stem Cells
Authors: Huang Xu , Somervaille Tim 
Published: October 2019

Abstract
Abstract 400
Dysregulation of the structure and function of chromatin is common in myeloid malignancy. We used a lentiviral screening strategy to target for knockdown ∼270 genes coding for chromatin regulatory proteins in human THP-1 AML cells. THP-1 cells harbor a t(9;11) translocation which is the cytogenetic hallmark of MLL-AF9, found in ∼5% of patients with AML. The strategy identified the EP400 chromatin complex components EPC1 and EPC2 as critical oncogenic co-factors in MLL leukaemia. Knockdown of both EPC1 and EPC2 in human MLL-mutated cell lines and of Epc1 and Epc2 in experimentally initiated murine MLL-AF9 AML cells led to loss of AML cell clonogenic potential, due to induction of apoptosis. The phenotype was rescued by forced expression of human EPC1 or EPC2 in Epc knockdown cells. Epc1 and Epc2 knockdown MLL-AF9 AML cells failed to initiate AML upon syngeneic transplantation into secondary murine recipients, while control cells initiated short latency leukemia. Likewise, xenogeneic transplantation of EPC1 or EPC2 knockdown primary human MLL-AF9 AML failed to engraft recipients, while control cells which engrafted every recipient to variable extent. In complete contrast, knockdown of EPC1 or EPC2 did not adversely impact on either the in vitro clonogenic or in vivo repopulating potential of primary murine KIT+ or human CD34+ normal haematopoietic stem and progenitor cells. These data demonstrate a remarkable selective dependency of MLL-mutated leukemia stem cells, but not normal hematopoietic stem and progenitor cells, on Epc1 and Epc2.
EPC1 and EPC2 are part of an EP400 centred complex with a role in the DNA damage response. The complex is also known to associate with MYC. Consistent with a role for EPC containing complexes in the DNA damage response, Epc1 and Epc2 KD in both normal and leukemic cells induced γH2A.X. However, only in leukemia cells was a transcriptional signature of MYC activation noted together with accumulation of MYC protein, prior to induction of Bim and subsequent apoptosis. This observation reveals a novel mechanism operational in leukaemia cells but not normal myeloid cells by which protein levels of the critical cellular oncogene MYC are limited by EPC/EP400. This counterintuitive observation refines the paradigm for MYC in transformed cells: when the function of EPC and its associated EP400 complex components is inhibited, thus impairing the DNA damage response, MYC accumulates and sensitises cells to apoptotic death. Thus EPC1 and EPC2 are components of a complex which serves to prevent MYC accumulation in myeloid leukemia cells and their sensitisation to apoptosis, thus sustaining oncogenic potential. Therapeutic targeting of this complex may facilitate restoration of the natural tumour suppressor mechanisms that prevent cellular transformation by MYC and enhance chemosensitivity of AML cells.

Disclosures:
No relevant conflicts of interest to declare.
Endogenous Nras G12D/+ and G12D/G12D Distinctly Regulate Self-Renewal and Differentiation of Haematopoietic Stem Cells
Authors: Kong Guangyao , Wang Jinyong , Liu Yangang , Du Juan , Damnernsawad Alisa , Ranheim Erik A. , Ryu Myung-Jeom , Zhang Jing 
Published: October 2019

Abstract
Abstract 4086
How oncogenes regulate adult stem cells to promote tumorigenesis is poorly understood. We and others previously reported that recipient mice transplanted with Nras G12D/+ or Nras G12D/G12D bone marrow cells develop distinct haemtopoietic malignancies. Mice with G12D/+ cells predominantly develop a myeloproliferative neoplasm (MPN) closely resembling chronic myelomonocytic leukemia (CMML), while animals with G12D/G12D cells develop acute T- or B-cell lymphoblastic leukemia (TALL or BALL) and/or MPN, with varying penetrance, which appear to be regulated by the activity of G12D/G12D haematopoietic stem cells (HSCs). Consistent with this notion, we found that G12D/+ HSCs are required to initiate and maintain CMML-like phenotypes in recipient mice and serve as MPN initiating cells. Therefore, we further investigated how endogenous oncogenic Nras signaling regulates the self-renewal and differentiation of HSCs to promote leukemia development in different lineages of cells.
Here we show that G12D/+ signaling results in moderate hyperproliferation and increased self-renewal of HSCs, promoting expansion of myeloid progenitors and consequently myeloid malignancies. In contrast, G12D/G12D leads to excessive hyperproliferation, decreased self-renewal, and depletion of HSCs, which promote expansion of myeloid and lymphoid progenitors and subsequently malignancies in both compartments. Because leukemia development in Nras G12D/+ and G12D/G12D models is tightly associated with ERK1/2 hyperactivation in haematopoietic stem/progenitor cells (HSPCs), we studied the MEK/ERK signaling in HSCs and their downstream multipotent progenitors (MPPs) using a “HSC phosphor-flow” method we developed. Our data demonstrate that ERK1/2 is hyperactiavated in G12D/+ and G12D/G12D HSCs in a dose-dependent manner, while AKT is not affected in G12D/+ and G12D/G12D HSCs. In contrast, both ERK1/2 and AKT are not changed in G12D/+ and G12D/G12D MPPs. As expected, inhibition of MEK/ERK signaling by AZD6244 (a MEK1 inhibitor) rescues the HSC phenotypes and attenuates myeloproliferative neoplasm phenotypes in G12D/+ and G12D/G12D mice. Mechanistic analysis identifies that a cohort of MAPK pathaway genes regulating cell cycle and signaling are significantly differentially expressed in G12D/+ HSCs compared to control or G12D/G12D HSCs. Unlike the prevailing theory based on Ras overexpression studies, depletion of G12D/G12D HSCs is not associated with overexpression of cell senescence genes. Rather, the Wnt and Notch pathways are significantly downregulated in G12D/G12D but not G12D/+ HSCs. Therefore, we propose that endogenous Nras G12D signaling differentially regulates HSCs self-renewal and differentiation through a dose-dependent hyperactivation of ERK1/2.

Disclosures:
No relevant conflicts of interest to declare.
Inducible Costimulator (ICOS) Is up-Regulated in Dogs with Graft Versus Host Disease and Graft Rejection Following Non-Identical Hematopoietic Cell Transplantation
Authors: Graves Scott Stoll , Sato Masahiko , Loretz Carol , Diane Stone , Storb Rainer 
Published: October 2019

Abstract
Abstract 4109
Inducible co-stimulator (ICOS), a member of the CD28 family of costimulatory molecules, is induced on CD4+ and CD8+ T-cells following their activation. Evidence suggests ICOS functions as an essential immune regulator and ICOS blockade is a potential target for allogeneic transplantation. We have used the canine model to develop several strategies for hematopoietic stem cell transplantation that have been successfully translated into the clinic. Here we describe the production and testing of monoclonal antibodies (mAb) directed against canine ICOS and determined the expression profile of ICOS in dogs. Canine ICOS was expressed in an inducible pattern on up to 89% of T-cells activated by Con A, anti-CD3 plus anti-CD28 mAb and alloantigen stimulation. Immunosuppressive effects of ICOS blockade were observed in mixed lymphocyte reactions (MLR) using peripheral blood mononuclear cells obtained from dog leukocyte antigen nonidentical dogs. Significant augmentation of the immunosuppressive effects of ICOS blockade was observed in MLR when anti-ICOS was combined with suboptimal concentrations of cytotoxic T-lymphocyte antigen 4-Ig (CTLA4-Ig) or cyclosporine. ICOS expression was significantly up-regulated on T-cells collected from the peripheral blood, lymph nodes and spleen from dogs undergoing graft rejection or chronic graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. Dogs that remained mixed chimeric expressed ICOS at levels comparable to normal dogs. Analysis of the peripheral blood CD3+ T-cells isolated from dogs showed a significant up-regulation of ICOS expression 3 days but not 6–7 days before the diagnosis of chronic GVHD. Pharmacokinetic studies of 123I-labled anti-canine ICOS showed normal blood clearance profiles. These studies demonstrated that an antagonistic anti-canine ICOS mAb may have application in the prevention or treatment of GVHD in an outbred animal model shown to reliably translate novel hematopoietic cell transplantation therapies to the clinic.

Disclosures:
No relevant conflicts of interest to declare.
Use of FoxP3 Diptheria Toxin Receptor (Foxp3DTR) Donor T Cells to Define the Mechanism Involved in the Inhibition of GvHD by Azacytidine in Vivo
Authors: Cooper Matthew L , Choi Jaebok , Ritchey Julie , DiPersio John F. 
Published: October 2019

Abstract
Abstract 4113
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the primary treatment for high risk and relapsed hematological malignancies. However, the desired graft versus leukemia (GvL) effect is frequently accompanied by Graft versus Host Disease (GvHD) in which donor lymphocytes target host tissues, often resulting in significant morbidity and mortality. We and others have shown that the hypomethylating agent, 5-azacytidine (azaC), can mitigate GvHD in both murine preclinical transplant models and in human clinical trials while maintaining a robust GvL effect. Two mechanisms have been proposed to explain how azaC reduces GvHD: 1) the generation of suppressive regulatory T cells (Tregs, CD4+CD25+Foxp3+) through the in vivo conversion of alloreactive donor T effectors (FoxP3-) into suppressive regulatory T cells (Tregs, CD4+CD25+Foxp3+) via the pharmacologic hypomethylation of the Foxp3 promoter resulting in enhanced gene expression or,1 2) the direct anti-proliferative and pro-apoptotic effects of azaC on allogeneic T cells.2
To assess the importance of azaC-generated Tregs in ameliorating GvHD we obtained Foxp3DTR mice in which Tregs can be selectively ablated (>97%) following the administration of diphtheria toxin (DT).3 To induce GvHD, 1×107 panT cells obtained from Foxp3 DTR mice were injected (CD45.2+) via the lateral tail vein on day 11 post allo-HSCT (CD45.1+) into allogeneic recipients (Balb/c). Transplanted mice were treated with PBS (carrier) or azaC (2 mg/kg) (i.p.) on days +15, +17, +19, and +21, followed by injection (i.p.) of DT or PBS on days +16 +18 +20 (DT 10 μg/kg) and +22 (DT 50 μg/kg) to deplete azaC-induced Tregs. Complete depletion of Tregs by DT was confirmed by flow cytometery.
Mice injected with PBS (n=5) or DT (n=5) (without azaC) developed severe GvHD and died by day 26 post HSCT. In sharp contrast, 100% of mice (n=5) treated with azaC alone survived >100 days, displayed no visible GvHD related symptoms, and gained weight at a similar rate as T cell depleted bone marrow controls. The treatment of mice with azaC +DT (n=5) resulted in significantly prolonged survival compared to the PBS treated mice (p=0.003) but in contrast to the azaC group, failed to survive beyond day 60 (p=0.003). Furthermore, the azaC alone group had higher percentages of Tregs (CD4+/CD25+/FoxP3+, p=0.001), CD3+ T cells (p=0.002), and B220+ B cells (p=0.02) than the azaC +DT group (consistent with less clinical GvHD) and less gastrointestinal GvHD when graded by an independent pathologist. These results suggest that BOTH pharmacologic conversion of alloreactive FoxP3- donor T cells to FoxP3-expressing Tregs and direct toxicity to allogeneic T cells by azaC represent important mechanisms involved in the amelioration of GvHD by azaC in vivo.

Disclosures:
No relevant conflicts of interest to declare.
Chronic Myeloid Leukemia Survival in Older Population in Pre- and Post- Imatinib Era in the United States
Authors: Ghimire Krishna B , Shah Binay K. 
Published: October 2019

Abstract
Abstract 4237

Background:
Median age at diagnosis for CML is 64 years of age. CML survival in elderly population is not well studied. This study was conducted to evaluate the relative survival rates among CML patients older than 50 years in pre- (1991–2000) and post- (2001– 2009) imatinib era.

Methods:
We analyzed the Surveillance, Epidemiology, and End Results (SEER*Stat) 18 registry database to compare 3-year and 5-year relative survival rates among CML patients by gender and age groups (50–69, ≥70) from the pre- (1991–2000) to post- imatinib eras (2001–2009). We used Z-test in the SEER*Stat program to calculate the differences in relative survival rates among different cohorts.

Results:
The 3-year and 5-year relative survival rates for CML patients age ≥50 years in pre- (n=3,848) vs post- (n=6,501) imatinib era were: 44.1±0.9% vs 55.9±0.8%, p=<0.0001, Z-value=10.179 at 3-years and 31.4±0.9% vs 46.9±0.9%, p=<0.0001, Z-value=12.361 at 5-years. The 3-year and 5-year relative survival rates for old (50–69) patients in pre- (n=1,723) vs post- (n=3011) imatinib era were: 57.7 ± 1.2% vs 72.3 ±1.0%, p=<0.0001, Z-value=9.454 at 3 years and 44.8±1.3% vs 64.3±1.2%, P=<0.0001, Z-value= 11.365 at 5 years. The survival rates for elderly (≥70) patients in pre (n= 2,125) and post (n=3,490) imatinib era were: 32.4±1.2% and 41.3±1.1%, p=<0.0001, Z-value=5.806 at 3 years and 19.3±1.1% and 31.2±1.2%, P=<0.0001, Z-value=7.135 at 5 years respectively. Table 1 shows CML survival rates by age and sex in patients older than 50 years of age.

Conclusions:
This study showed significant increase in 3 year and 5 year relative survival rates in post- imatinib era among CML patients older than 50 in all cohorts examined. However, the improvement in survival rates is modest compared to published data from randomized clinical trials.

Disclosures:
No relevant conflicts of interest to declare.
Second Primary Malignancies in Chronic Myeloid Leukemia
Authors: Shah Binay K. , Ghimire Krishna B 
Published: October 2019

Abstract
Abstract 4247

Background:
Development of second primary cancers among CML patients is not well studied. With improvement in CML survival, long term risks of CML, including second cancers need to be evaluated. This study was conducted to evaluate second primary malignancies in CML patients using data from the US Surveillance, Epidemiology and End Results (SEER) cancer registries.

Methods:
We analyzed the Surveillance, Epidemiology, and End Results (SEER*Stat) database: Incidence - SEER 13 Regs Research Data, Nov 2011 Sub, Vintage 2009 Pops (1992–2009) using MP-SIR Session. We compared secondary cancer rate among adult CML patients (older than 20 years of age) during the period 1992 – 2009. We also evaluated the risk of secondary malignancies in pre- (1992–2000) to post- imatinib (2001–2009) eras. We used SEER MP-SIR session and Graph pad scientific software to calculate p value.

Results:
The total number of CML patients older than 20 years of age, reported during 1992–2009 period was 8,511. There were 4286 and 4225 CML patients in the pre- (1992–2000) and post- (2001–2009) Imatinib era respectively. All site secondary cancer (n=475) incidence was significantly higher among CML patients compared to general population with observed/expected ratio (O/E):1.28, p=< 0.05, an absolute excess risk of 31.39 per 10,000.
Similarly, all sites secondary cancer in post-(2001–2009) Imatinib era was significantly higher compared to pre- imatinib era with O/E ratio of 1.50 vs 1.13, p=0.03.

Conclusions:
This study showed that overall risk of second malignancies among CML patients is higher compared to general population. The risk of second primary malignancies is higher in post- imatinib era.

Disclosures:
No relevant conflicts of interest to declare.