bluuebunny/crossref_metadata_2025 huggingface dataset:
Deferasirox In The Treatment Of Iron Overload During Myeloproliferative Neoplasms (MPN)
Authors: Latagliata Roberto , Montagna Chiara , Porrini Raffaele , Leonetti Sabrina Crescenzi , Niscola Pasquale , Ciccone Fabrizio , Spadea Antonio , Breccia Massimo , Rago Angela , Spirito Francesca , Cedrone Michele , De Muro Marianna , Montanaro Marco , Andriani Alessandro , Bagnato Antonino , Montefusco Enrico , Cimino Giuseppe , Alimena Giuliana
Published: October 2019

Background

Deferasirox is an oral iron chelator widely employed in the treatment of iron overload during thalassemic syndromes and myelodysplastic syndromes.

Aim

At present, very few data are available on the efficacy and safety of a treatment with deferasirox in patients with Ph- Myeloproliferative Neoplasms (MPN) and transfusional requirement.

Methods

To address this issue, we report here on 23 patients (M/F 17/6) with MPN and iron overload secondary to transfusional requirement enrolled in the database of our regional cooperative group who received a treatment with deferasirox. Of them, 20 had a primary Myelofibrosis, 2 a post Essential Thrombocythemia myelofibrotic phase and 1 a post Polycythemia Vera myelofibrotic phase.

Results

The main features of the patients at diagnosis and at baseline of deferasirox treatment are reported in the Table:

Treatment with deferasirox was started after a median interval from diagnosis of 9.8 months (IR 6.7 – 21.7) and from transfusion requirement of 7.3 months (IR 5.7 – 15.3). Starting deferasirox dose was 1,500 mg/day in 11 patients, 1,250 mg/day in 4 patients, 1,000 mg/day in 7 patients and <1,000 mg/day in 1 patient. All patients were evaluable for toxicity: extra-hematological toxicity was reported in 12/23 patients and consisted of gastro-intestinal symptoms in 6 patients, renal impairment in 4 patients and skin reactions in 2 patients. A dose reduction/temporary discontinuation was needed in 11 cases, but only 1 patient went off treatment due to recurrent renal toxicity. Eighteen patients were considered evaluable for response (≥ 6 months of treatment) and 5 were too early. As to the iron overload decrement, after a median treatment period of 14.4 months (IR 7.1 – 21.3) 2 patients achieved ferritin levels < 500 ng/ml, 7 patients ferritin levels < 1,000 ng/ml and 9 patients did not have any ferritin reduction. As to hematological improvement, 5/18 patients showed an unexpected and persistent rise of Hb levels > 1.5 g/dl, with disappearance of transfusional requirement in 3 cases.

Conclusions

Treatment with deferasirox is feasible and effective in MPN with iron overload. Moreover, also in this setting an hematological improvement can occur in a sizeable rate of patients.

Disclosures:

No relevant conflicts of interest to declare.
T-Cell Engineering For “off-The-shelf” Adoptive Immunotherapy
Authors: Poirot Laurent , Schiffer-Mannioui Cécile , Philip Brian , Derniame Sophie , Gouble Agnes , Chion-Sotinel Isabelle , Le Clerre Diane , Lemaire Laetitia , Grosse Stéphanie , Cheung Gordon , Arnould Sylvain , Smith Julianne , Pule Martin , Scharenberg Andrew
Published: October 2019

Adoptive T-cell therapies, where exogenous expression of a chimeric antigen receptor (CAR) confers cancer recognition, have shown significant promise in initial clinical trials. However, present adoptive immunotherapy Methods are limited by the need for manipulation of autologous patient T-cells. To permit such an approach in an allogeneic context, Transcription Activator-Like Effector Nucleases (TALENTM) have been used to simultaneously inactivate the endogenous T cell receptor and CD52, a cellular target for a lymphodepleting treatment. This approach reduces the risk of GVHD while permitting proliferation and activity of the introduced T lymphocytes in the presence of the immunosuppressive drug alemtuzumab. Electroporation of primary T cells with mRNA coding for the appropriate TALENTM result in double knock-out (dKO) frequencies of up to 70%. Furthermore, functional characterization demonstrates that the dKO cells are resistant to complement dependent lysis or in vivo depletion by alemtuzumab, and show no apparent potential for TCR-mediated activation. Finally, endowing the dKO cells with a CD19 CAR supports their capacity to kill CD19+ tumor targets as efficiently as unedited T-cells both in vitro and in vivo.

Disclosures:

Poirot: CELLECTIS THERAPEUTICS: Employment. Schiffer-Mannioui:CELLECTIS THERAPEUTICS: Employment. Philip:UCL Cancer Institute, London, United Kingdom: Employment. Derniame:CELLECTIS THERAPEUTICS: Employment. Gouble:CELLECTIS THERAPEUTICS: Employment. Chion-Sotinel:CELLECTIS THERAPEUTICS: Employment. Le Clerre:CELLECTIS THERAPEUTICS: Employment. Lemaire:CELLECTIS THERAPEUTICS: Employment. Grosse:CELLECTIS THERAPEUTICS: Employment. Cheung:UCL Cancer Institute, London, United Kingdom: Employment. Arnould:CELLECTIS THERAPEUTICS: Employment. Smith:CELLECTIS THERAPEUTICS: Employment. Pule:UCL Cancer Institute, London, United Kingdom: Employment. Scharenberg:CELLECTIS THERAPEUTICS: Employment.
The Pan-PIM Kinase Inhibitor LGH447 Shows Activity In PIM2-Dependent Multiple Myeloma and In AML Models
Authors: Garcia Pablo D , Langowski John L , Holash Jocelyn , Burger Matthew , Zang Richard , Zavorotinskaya Tatiana , Fanton Christie , Saci Abdel , Growney Joseph , Vanasse K. Gary
Published: October 2019

Background

PIM1, PIM2, and PIM3 are a closely related family of constitutively active serine/threonine kinases. PIM kinases were first identified as common integration sites in MMLV-induced murine T-cell lymphomas, with these viral insertions resulting in transcriptional activation and expression of the kinase. Similar unbiased insertional mutagenesis screens were used to demonstrate that the three kinases could substitute for each other in this oncogenic process, indicating that inhibition of all three PIM kinases may be necessary to achieve a therapeutic benefit. In human cancers, increased expression and activity of PIM kinases has been detected in many hematopoietic malignancies and solid tumors (prostate, lung, liver) and it is thought that this contributes to cancer cell survival and proliferation in these malignancies.

Methods & Results

We have developed a potent and specific pan-PIM inhibitor, LGH447, and shown that it is active in PIM2-dependent Multiple Myeloma (MM) cell lines by inhibiting proliferation, mTOR-C1 signaling and phosphorylation of BAD. In addition, LGH447 inhibited proliferation of several AML cell lines. Furthermore, we demonstrated that LGH447 inhibits tumor growth in several mouse subcutaneous xenograft models of MM and AML, where modulation of the pharmacodynamic marker phospho-S6 Ribosomal Protein was used to predict efficacy. We have also determined that LGH447 significantly reduced the bone tumor burden in a disseminated orthotopic human xenograft model of MM. Finally, we have demonstrated increased activity of LGH447 in combination with the PI3K inhibitor BYL719 in a MM model and with Cytarabine in an AML model.

Our Results have supported the clinical development of LGH447, a Pan-PIM inhibitor, in relapsed, refractory MM, and further suggest that it may be effective in the treatment of AML and other hematological malignancies, either as a single agent or in combination with other select therapeutic agents.

Disclosures:

Garcia: Novartis Institutes for Biomedical Research: Employment, Equity Ownership. Langowski:Novartis Institutes for Biomedical Research: Employment. Holash:Novartis Institutes for Biomedical Research: Employment, Equity Ownership. Burger:Novartis Institutes for Biomedical Research: Employment, Equity Ownership. Zang:Novartis Institutes for Biomedical Research: Employment, Equity Ownership. Zavorotinskaya:Novartis Institutes for Biomedical Research: Employment, Equity Ownership. Fanton:Novartis Institutes for Biomedical Research: Employment, Equity Ownership. Saci:Novartis Institutes for Biomedical Research: Employment, Equity Ownership. Growney:Novartis Institutes for Biomedical Research: Employment, Equity Ownership. Vanasse:Novartis Pharmaceuticals Corporation: Employment.
Clostridium Difficile As a Risk Factor For Hospital-Acquired Venous Thrombosis In Medical Inpatients
Authors: Merrill Samuel A , Desarno Michael , Houghton Damon , Winters John P. , Huston Christopher , Callas Peter , Repp Allen B , Cushman Mary , Zakai Neil A
Published: October 2019

Introduction

The American College of Chest Physicians and government agencies recommend risk stratification and pharmacologic prophylaxis to prevent venous thromboembolism (VT) in hospitalized individuals. While risk factor assessment is often completed at admission, clinical changes during hospitalization may influence VT risk. Based on clinical observation of coincidence of C. difficile infection and VT, we sought to determine if C. diff or clinical suspicion of C. diff are risk factors for hospital acquired VT in medical inpatients.

Methods

Derivation and validation cohorts for the Medical Inpatient Venous Thrombosis Risk Assessment Score were used. Patients were admitted to the medical services at Fletcher Allen Health Care, a 500 bed teaching hospital for the University of Vermont. For derivation, all cases of hospital-acquired VT between 2002-2009 were identified using ICD-9 codes and verified by chart review; cases were matched by admission year and medical service to controls; VT risk factors and C. diff testing and Results were confirmed by chart review. For validation, hospital-acquired VT between 2009-2012 were captured by ICD-9 codes with confirmatory imaging; testing and Results for C. diff were ascertained from the microbiology lab database; VT risk factors were ascertained by ICD-9 codes, electronic problem lists, vital sign data, and lab values. Logistic regression, accounting for VT risk factors (Table), was used to determine whether 1) testing for C. diff or 2) a positive confirmation for C. diff was associated with hospital-acquired VT.

Results

In the derivation analysis 299 cases of hospital-acquired VT were identified from 64,334 medical admissions and matched to 601 controls. In the validation analysis 120 hospital-acquired VT were identified among 20,946 admissions. In the derivation cohort there were 4,793 tests for C. diff and 478 confirmed cases; and in the validation cohort there were 1,708 tests for C. diff and 260 confirmed cases. After accounting for other VT risk factors in the derivation cohort, testing for C. diff was associated with an OR of 2.14 (95% CI 1.28, 3.61) for VT, positive C. diff with an OR of 3.23 (95% CI 1.00, 10.45) for VT, and negative C. diff with an OR of 1.95 (95% CI 1.09-3.46) for VT. These associations were confirmed in the validation cohort (see Table).

Conclusions

Both testing for, and a diagnosis of C. diff were associated with hospital-acquired VT in medical inpatients. This relationship could be a surrogate of antimicrobial therapy indicating another active infection or a general marker for systemic illness, direct causation appears less likely. These data suggest that VT risk is dynamic during hospitalization and that further studies incorporating dynamic VT risk assessment are warranted in medical inpatients.

Disclosures:

No relevant conflicts of interest to declare.
Acute Myeloid Leukemia Survival In Elderly Patients In The United States
Authors: Shah Binay K. , Ghimire Krishna B
Published: October 2019

Background

Survival in Acute myeloid leukemia (AML) has improved in younger patients over the last decade. Though chemotherapy has not changed significantly, better patient care may have improved survival in AML patients. This study was conducted to evaluate the relative survival rates in older AML patients over two decades in the United States.

Methods

Newly diagnosed AML (first primary only) in older (age≥75 years) patients were selected from the Surveillance, Epidemiology, and End Results (SEER) database. We analyzed SEER 18 registry database to evaluate relative survival rate in older (≥75 years) AML population diagnosed during 1992-2000. We selected AML patients from 13 SEER registries to compare RS during 1992-2000 and 2001-2009. The relative survival rates at 1 and 5 year were analyzed for various cohorts categorized by race (White, African-Americans (AA) & other), gender & age (≥75-84 and ≥85 years). The relative survival rates accompany standard error (SE). We used SEER-Stat, statistical software provided by NCI for statistical analysis.

Results

The relative survival rates improved significantly during 2001-2009 compared to 1992-2000 for all age groups, sex. For young elderly patients (≥75-84 years) RS increased from 13.1±0.8% to 17.4±0.9% at 1 year Z value=3.98, P<0.0001 and from 2.0±0.4 to 2.6±0.5%, Z value= 3.61, P<0.0005 at 5 years with improvement in median relative survival from 2.41 to 2.75 months.

Similarly, for very elderly (≥85 years) patients RS increased from 5.3±1.0% to 8.0±1.0%, Z value=3.03, P<0.005 at 1 year, 0.7±0.4% to 1.3±0.5% at 3 years Z value= 2.81, P=0.005, but no improvement seen at 5 years. There was significant improvement in RS from 1992-2000 to 2001-2009 in both sexes, caucasian and other races. There was no improvement in RS at 1 year and 5 year for older AA population. (Table)

Conclusion

The relative survival in elderly AML has increased significantly during 2001-2009 compared to 1992-2000. The RS improved both at 1 year and 5 years for young elderly patients. Improvement in RS was observed at 1 year and 3 years for very elderly (>85 years) AML patients.

Disclosures:

No relevant conflicts of interest to declare.
Prognostic Significance Of Systemic Inflammatory Factors In Patients With Diffuse Large B Cell Lymphoma Treated By R-CHOP
Authors: Yoh Kyung Ah , Lee Ho Sup , Park Lee Chun , Lee Eun Mi , Shin Seong Hoon , Ye Byeong Jin , Moon Joon Ho , Jo Jae-Cheol , Lee Won Sik , Shin Ho-Jin , Oh Sun Young , Kim Yang Soo
Published: October 2019

Background

Systemic inflammatory factors such as C-reactive protein (CRP), ¥â2-microglobulin (B2MG), and ferritin were documented an prognostic factor in patients with hematologic malignancies including lymphoma. The purpose of this study was to find the significance of systemic inflammatory factors for predicting survival outcome in patients with diffuse large B cell lymphoma (DLBCL).

Methods

A total of 188 patients who newly diagnosed DLBCL and received an rituximab combined chemotherapy at the South Korea between September 2004 and April 2012 were enrolled retrospectively in the current study. Pretreatment serum CRP, B2MG, and ferritin were measured within 4 weeks before the beginning of first line chemotherapy.

Results

The median age of patients was 58 years (range, 14-84 years) and the mean level of serum CRP, B2MG, and ferritin at pre-treatment were 2.37 mg/dL (range: 0.01 – 29.80), 2.64 mg/L (range: 0.71 – 24.01), and 285.01 ng/ml (range: 5.34-4872.40). Systemic inflammatory factors score were given 1 point if serum levels were more than normal range (CRP; 0.8 mg/dL, B2MG; 2.5 mg/L and ferritin; 220 ng/ml). Systemic inflammatory risk were divided into three groups according to systemic inflammatory factors scores ; low risk was 0 score, intermediate risk was 1 or 2 score, and high risk was 3 score respectively. 5-year progression free survival rates (PFS) were 66.9%, 65.0% and 10.6% in risk groups respectively (p < 0.001). 5-year overall survival rates (OS) were 74.4%, 48.8% and 19.8% in risk groups respectively (p < 0.001).

Conclusions

Systemic inflammatory factor (CRP, B2MG and ferritin) score were associated with survival outcomes in patients with DLBCL treated by R-CHOP. However, further studies are needed to confirm prognostic value of systemic factors such as CRP, B2MG and ferritin.

IPI, international prognostic index; LDH, lactate dehydrogenase; ECOG, Eastern Cooperative Oncology Group (ECOG) performance status; CRP, C-reactive protein; BM, bone marrow; ALC, absolute lymphocyte counts; CHOP-like (cyclophosphamide, adriamycin, vincristine, prednisolone); RCHOP (rituximab plus CHOP)

Systemic inflammatory factor (normal range); ¥â2-microglobulin (2.5mg/L), CRP (0.8mg/dL), ferritin (220 ng/mL)

Systemic inflammatory factor score : given 1 point if more than normal range

Low risk ; 0, Intermediate risk ; 1 or 2, High risk ; 3

Disclosures:

No relevant conflicts of interest to declare.
Intraperitoneal Administration Of Activated Protein C Prevents Post-Surgical Adhesion Band Formation
Authors: Dinarvand Peyman , Rezaie Alireza R.
Published: October 2019

Postoperative intraperitoneal adhesion bands are the most important causes of intestinal obstruction, pelvic pain and female infertility. Adhesion bands are induced by inflammatory responses elicited by injury following surgery. The objective of this study was to evaluate the effectiveness of activated protein C (APC), a natural anticoagulant and antiinflammatory serine protease in plasma, in preventing post-surgical intraperitoneal adhesion band formation. The protective effects of APC and the FDA approved agent, sodium hyaluronate/carboxymethylcellulose (HA/CMC, also called Seprafilm), were compared by administering the two drugs intraperitoneally into 30 male C57bl/6 mice. After 7 days, the pathological adhesion grades were scored by two scaling systems and the peritoneal fluid concentration of tissue plasminogen activator (tPA) as well as the concentrations of the proinflammatory cytokines, IL-1, IL-6, TNF-α, IFN-γ and TGF-β were evaluated. The inflammation scores of mice in all groups were also measured based on histological data obtained from peritoneal tissues. Relative to the HA/CMC group, intraperitoneal administration of APC led to a significantly higher reduction of adhesion band formation. Moreover, a markedly lower inflammation score was obtained in the adhesive tissues of the APC-treated group which correlated with significantly reduced peritoneal concentrations of IL-6, TNF-α, IFN-γ and TGF-β and elevated concentration of tPA in this group. These results suggest that in comparison to the FDA approved agent, Seprafilm, APC exhibits a markedly higher efficacy in preventing postoperative adhesion band formation. Thus, APC holds the potential to be therapeutically used as a novel anti-adhesive agent.

Disclosures:

No relevant conflicts of interest to declare.
Reversal Of Dabigatran Anticoagulation By a 4-Factor Prothrombin Complex Concentrate: Correlation Between Effects On Parameters Of Thrombin Generation and Hemostatic Effect In Vivo
Authors: Hoffman Maureane , Volovyk Zoya , Monroe Dougald
Published: October 2019

Dabigatran etexilate (Pradaxa®) is a prodrug that is metabolized to a small molecule direct thrombin inhibitor. It has the drawback that it is not readily reversible. Several studies have suggested that prothrombin complex concentrates (PCCs) might reverse the effects of dabigatran on laboratory assays. However, the effects of PCCs on laboratory assays do not seem to correlate well with their effects on hemostatic function in several animal models. We have previously reported the effects of a 4-factor PCC (KCentra®, CSL-Behring) on parameters of thrombin generation in a cell-based assay of thrombin generation (cell-based Calibrated Automated Thrombogram Assay, CAT®). This model allowed us to use varying levels of dabigatran and varying levels of PCC in the presence of freshly isolated platelets, a cell-associated source of tissue factor (TF) and plasma levels of coagulation factors. We found that dabigatran lengthens the lag, and reduces the total and peak levels of active thrombin in this model. KCentra can normalize the peak, rate and total amount of thrombin generated in the presence of dabigatran, but does not normalize the lag time. These effects were seen at all levels of dabigatran tested. A level of 1 U/mL KCentra® (equivalent to an dose of 50 U/kg in vivo) was able to restore the rate and peak levels of thrombin generation, as well as the area-under-the-curve, to normal levels. A level of 0.5 U/mL KCentra did not completely normalize the rate, peak and AUC.

We compared results in the cell-based CAT assay to results in a novel mouse saphenous vein bleeding model (Pastoft, et al, A sensitive venous bleeding model in haemophilia A mice: effects of two recombinant FVIII products (N8 and Advate) 2012). We found that an i.v. dose of 15 ug/kg dabigatran doubled the time to hemostasis from 80 sec + 9 in controls to 173 sec + 24. A dose of 25 U/kg KCentra partially corrected hemostasis and a dose of 50 U/kg completely corrected hemostasis in this model. These results are completely consistent with the results of the cell-based CAT assay.

While many workers report the peak or the area under the thrombin generation curve (AUC or ETP) as the key parameter in thrombin generation assays, it is not known which parameters reflect hemostatic function in vivo. Because the parameters of thrombin generation are not independent, it is very difficult to determine which of them are most closely related the hemostasis. However, our current data suggest that a 4-factor PCC is able to completely correct the time to hemostasis is a reproducible mouse model, even though it does not the lag time in an assay of thrombin generation

Disclosures:

Hoffman: Novo Nordisk: Consultancy, Research Funding; CSL-Behring: Consultancy, Research Funding; Boehringer Ingelheim: Research Funding. Monroe:CSL-Behring: Research Funding.
Circulatory Glycan-Modifying Enzymes As Systemic Regulators In Blood Cell Development
Authors: Lau Joseph
Published: October 2019

Regulating blood cell production is crucial to meeting physiologic demands and to maintaining hematopoietic equilibrium. Elucidating the systemic factors and how they contribute to the interaction of hematopoietic progenitors with their bone marrow microenvironments is crucial to the mechanistic understanding of hematopoietic regulation and development of novel therapeutic strategies. Glycosyltransferases are enzymes that normally reside within the intracellular secretory apparatus to assemble glycans on nascent proteins and lipids in transit, but they are also present in abundance in the extracellular spaces, especially in systemic circulation. One such blood-borne enzyme is the ST6Gal-1 sialyltransferase that mediates the attachment of sialic acids in a6-linkages to glycoproteins. By virtue of their presence on cell surfaces, sialyl-glycans, which are conserved structures in mammals, occupy the critical interphase between cells and their external environment. We hypothesize that remodeling of these cell surface glycans can drastically modulate the communication of systemic cues among marrow hematopoietic cells. A mouse model genetically modified for low circulating ST6Gal-1 has increased numbers of hematopoietic progenitors, increased ability to produce inflammatory cells upon acute challenge, and faster recovery from cyclophosphamide-induced myelosuppression. In ex vivo cultures, addition of ST6Gal-1 strikingly delays expansion and differentiation of murine LSK (Linneg sca-1pos cKitpos) and human CD34pos cells. We developed a subcutaneously implantable B16-F10 model genetically modified to overexpress circulatory ST6Gal-1 (B16st6gal1). Mice bearing B16st6gal1 had >10-fold elevation in circulating ST6Gal-1 levels, accompanied by >50% reduction in marrow granulocyte and B cell populations. B16st6gal1 bearing mice also had 2-fold increase in the number of Linneg cells, suggesting a blockade in development of progenitors into end-effector cells. Murine LSK cell surfaces are decorated with a6-sialic acids but they do not endogenously express ST6Gal-1. Bone chimeras show that the a6-sialic acids on LSK surfaces require the distally synthesized ST6Gal-1 enzyme in circulation. Taken together, our data indicates the glycan-modifying enzyme, the ST6Gal-1 in circulation, is a potent systemic regulator of hematopoiesis by remodeling of cell surface glycan architecture of marrow hematopoietic stem and progenitors. (Funded by NIH Program of Excellence in Glycosciences award P01HL107146 and NIH R01AI56082)

Disclosures:

No relevant conflicts of interest to declare.
LFA-1-Mediated Adhesion Of Human Hematopoietic Progenitors To Bone Marrow Stromal Cells Enhances B-Lineage Differentiation Toward CD19+ ProB Cells, While The Generation Of CD7+CD56-CD45RA+ Multipotent Lymphoid and CD10+CD19- B-Biased Progenitors Is Supported By Soluble Factors Produced From Stromal Cells
Authors: Minami Hirohito , Ohishi Kohshi , Nakamori Yoshiki , Masuya Masahiro , Katayama Naoyuki
Published: October 2019

The regulatory mechanism of human lymphoid differentiation remains less defined. Here we examined how bone marrow stromal cells regulate human early lymphoid differentiation, using human telomerized bone marrow stromal cells that support the generation of CD7+CD56- early T and CD10+CD19+ proB cells from human hematopoietic progenitors. To examine the role of direct contact between hematopoietic progenitors and stromal cells in lymphopoiesis, cultures were performed by inhibiting the cell-cell contact with microporous insert or by incubating hematopoietic progenitors with conditioned medium collected from stromal cell cultures. The separation suppressed B-lineage differentiation to CD10+CD19+ cells, while the generation of CD7+ cells was not significantly influenced. The CD7+ cells generated with or without direct contact with stromal cells similarly had multipotent differentiation capacity for T, B, NK, granulocytic, and monocytic cells but not for erythroid cells in various culture conditions. On the other hand, even CD10+CD19- immature cells had more limited differentiation capacity for T, B, and monocytic cells in various culture conditions, and mostly differentiated toward CD10+CD19+ proB cells on the stromal cells. By time course analysis after coculture on the stromal cells, CD7+CD10- followed by CD10+CD19- and then CD10+CD19+ cells were developed. Some portion of CD7+CD10- and most of CD7-CD10+CD19- cells, upon recultured on stromal cells, differentiated toward CD10+CD19+ cells, but such B-lineage differentiation on the stromal cells was diminished by reculture with conditioned medium. ICAM-1 was expressed on the telomerized stromal cells. Coculture on stromal cells in the presence of LFA-1 neutralizing antibody that blocks the binding to ICAM-1 inhibited the differentiation to CD19+ proB cells. Our findings show that stromal cells support the generation of CD7+ multipotent lymphoid and CD10+ B-biased progenitors by producing soluble factors, but enhances B-lineage differentiation toward CD19+ proB cells in part via LFA-1-mediated direct cell-cell contact.

Disclosures:

No relevant conflicts of interest to declare.
Primitive Erythropoiesis and Osteogenesis Are Differentially Impaired In Rpl5 and Rps19 Mutant Murine Embryonic Stem Cell Models Of Diamond Blackfan Anemia
Authors: Dulmovits Brian M. , Singh Sharon A. , Goldberg Tracie , Didier Sebastien , Lipton Jeffrey M. , Blanc Lionel , Liu Johnson M.
Published: October 2019

Diamond Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome characterized by red blood cell hypoplasia, congenital anomalies and cancer predisposition. In addition, short stature and poor skeletal growth are found in a subset of DBA patients, suggesting similar developmental abnormalities in erythropoiesis and osteogenesis in that subset. Furthermore it has been shown recently that osteoblasts secrete erythropoietin, linking the marrow niche to the modulation of erythropoiesis. DBA has been shown in the majority of cases to result from haploinsufficiency of large or small ribosomal subunit proteins. The p53 pathway, known to be activated by abortive ribosome assembly, contributes to the erythroid failure of DBA. We studied two DBA genotypes in vitro using murine embryonic stem (ES) cell lines harboring gene trap mutations in ribosomal proteins RPS19 and RPL5, respectively. Both mutants had decreased embryoid body (EB) formation, decreased definitive erythroid colony formation and similar p53-dependent primitive erythroid differentiation defects (see Figure A). Cell cycle analyses were normal in the Rps19 mutant ES cells, but there was a significant G2/M arrest in the Rpl5 mutant ES cells, which was unaffected by p53 knockdown. In addition, the Rpl5 mutant cells had a more pronounced growth defect in culture compared to the Rps19 mutant cells (Figure B). ES cells were differentiated, in vitro, to osteoblasts using established culture conditions, and confirmed both by morphology and molecular characterization (e.g. RUNX2 and Osteopontin). Following 14 days of osteogenic differentiation, bone mineralization was confirmed via Alizarin Red staining. A marked reduction in Alizarin Red staining was seen in the Rpl5 mutant cells while there was only a slight diminution of staining in the Rps19 mutant ES cultures (see Figure C). Therefore the erythroid differentiation defect appears similar in both the Rps19 and Rpl5 mutant ES cells. However the Rpl5 mutant appears to have a more severe phenotype at the ES stage, as evidenced by a pronounced p53-independent G2/M arrest and slower growth rate and subsequently during osteogenic differentiation. These data suggest an explanation for the more severe non-erythroid phenotype seen in a subset of DBA patients.

Disclosures:

No relevant conflicts of interest to declare.
AML1-ETO Represses Multiple Transcription Factor Induced CTSG Activation by Reducing H3 Acetylation
Authors: Tan Yun , Jin Wen , Wu Kang , Wang Kankan
Published: October 2019

Acute myeloid leukemia (AML) is often accompanied with the aberrant expression of transcription factors. In t(8;21) AML, the AML1-ETO fusion protein executes its critical role in leukemogenesis through the interference with hematopoietic transcription factors (TFs) including AML1, C/EBPα, PU.1 and c-Myb. These transcription factors cooperate to modulate hematopoiesis by regulating their differentiation-related target genes. In our previous work, we have identified that AML1-ETO suppresses the AML1-dependent transactivation of the gene encoding the neutrophil granule protease, cathepsin G (CTSG). However, the detailed mechanisms of AML1-ETO mediated transrepression, especially coordinated regulation of hematopoietic transcription factors, have not been characterized yet. To investigate the regulatory pattern of CTSG by hematopoietic specific transcription factors, we constructed a luciferase reporter containing the CTSG promoter and co-transfect it with AML1, c-Myb, C/EBPα or PU.1 to 293T cells. The results of luciferase assays showed that these TFs individually activated the CTSG promoter, and synergistic transactivation occurred between AML1 and c-Myb, C/EBPα and PU.1, and PU.1 and c-Myb on the CTSG promoter. Furthermore, AML1/ETO effectively suppressed the transcription factor-dependent transactivation and synergistic transactivation of the CTSG promoter. Chromatin immunoprecipitation assays further demonstrated that AML1-ETO coexisted with these TFs on the CTSG promoter in AML1/ETO-positive Kasumi-1 cell line, indicating AML1-ETO was tethered to the chromatin bound by these TFs. The data suggested that AML1-ETO might act as a negative regulator by interfering the normal function of hematopoietic TFs instead of competing for their binding. In addition, to reveal the underlying mechanism of AML1/ETO-mediated transcription repression at the epigenetic level, we examined the epigenetic status of the CTSG promoter in AML1-ETO negative and positive cells, and found the level of histone H3 Lys9 acetylation on the CTSG promoter was obviously lower in AML1-ETO positive cells than that in AML1-ETO negative cells. The data suggested that AML1-ETO might repress the gene transcription by changing the H3 acetylation status of its target gene. Collectively, our findings demonstrate that AML1-ETO represses the transactivation of the CTSG promoter mediated by multiple hematopoietic transcription factors through a decrease of H3 acetylation.

Disclosures:

No relevant conflicts of interest to declare.
Hhex Is a Critical Gene In The Development Of Normal and Malignant Lymphoid Cells
Authors: Goodings Charnise , Smith Stephen B. , Mathias Elizabeth , Smith Elizabeth , Tripathi Rati , Elliott Natalina E , Cleveland Susan , Dave Utpal P. , Du Yang
Published: October 2019

Hematopoietically expressed homeobox (Hhex) is a T-cell oncogene. It is frequently deregulated in murine retroviral insertional mutagenesis screens and its enforced expression induces T-cell leukemia in bone marrow transduction and transplantation experiments. We discovered that HHEX is a direct transcriptional target of an LIM domain Only-2 (LMO2)-associated protein complex. HHEX clusters with LMO2-overexpressing T-ALLs and is especially overexpressed in Early T-cell Precursor (ETP) – ALL where it is a direct transcriptional target of LMO2. To further understand Hhex's function, we induced a conditional knockout in floxed Hhex mice with the Vav-iCre transgene. Mice were viable and showed normal blood cell counts with highly efficient deletion of Hhex in all hematopoietic tissues. Thymocytes from conditional knockouts showed a normal pattern of development. Most impressively, Hhex conditional knockout markedly prolonged the latency of T-ALL onset in CD2-Lmo2 transgenic mice (figure 1). Hhex conditional knockouts (Hhex cKOs) also had a significant decrease in mature B cells in the spleen and bone marrow. Interestingly, hematopoietic stem and progenitor cells plated on OP9-GFP or OP9-DL1 stromal cells showed proliferative defects and incomplete differentiation towards both B and T lineage. Also under stress conditions such as sublethal irradiation and competitive bone marrow transplants, Hhex conditional knockouts show a marked defect in both B and T lineages but an increase in early progenitor populations. Our experiments show that Hhex is a critical transcription factor in lymphoid development and in LMO2-induced T-ALL.Figure 1Hhex conditional knockout markedly prolonged the latency of T-ALL onset in CD2-Lmo2 transgenic miceFigure 1. Hhex conditional knockout markedly prolonged the latency of T-ALL onset in CD2-Lmo2 transgenic mice

Disclosures:

No relevant conflicts of interest to declare.
Cytidine Deaminase Status As a Predictive Marker Of Response and Toxicity In Patients With Haematological Malignancies Treated With Azacytidine Or Cytarabine
Authors: Mercier Cédric , Fanciullino Raphaelle , Serdjebi Cindy , Ciccolini Joseph , Pourroy Bertrand , Milano Gerard , Ouafik L’Houcine , Costello Régis T
Published: October 2019

Background

Cytarabine and azacytidine are mainstays for treating haematological malignancies. As most nucleosidic analogs, both azacytidine and cytarabine are metabolized in the liver by an exclusive enzymatic step driven by cytidine deaminase (CDA). CDA expression polymorphism has been associated with clinical outcome in patients treated with gemcitabine.

Methods

We determined CDA levels in 39 adult patients (16F, 23M, mean age 77 years), treated mainly for AML and myelodysplastic syndromes with either aza-cytidine or a cytarabine-containing regimen. Response and treatment-related toxicities were monitored following current standards. In addition, impact of CDA status on azacytidine tolerance was evaluated in mice with or without CDA deficiency, as a proof of concept for the actual implication of metabolic deregulations in the toxicities observed in patients.

Results

In patients, mean CDA activity was 3.7 +/-2.8 U/mg (min: 1, max: 14.8 U/mg). Ten out of 39 patients (i.e., 25%) had low CDA activities and were considered as poor metabolizers (PM). Conversely, 8 patients (i.e., 20%) displayed high CDA activities (i.e., > 6U/mg) and were considered as high metabolizers (HM). PM showed severe toxicities, including two toxic-deaths. Conversely, HM showed little efficacy when treated with either azacytidine or cytarabine. In mice with CDA-deficiency, standard dose azacytidine led to profound and long-lasting neutropenia, as compared with normal mice. Drug monitoring confirmed that individuals with low CDA activity and toxicities showed higher concentrations of azacytidine as compared with normal individuals.

Conclusions

this study suggests that CDA status could be a relevant marker for predicting clinical outcome in patients treated with either azacytidine or cytarabine. CDA status could be further used as a covariate to tailor drug dosage so as to ensure an optimal efficacy/toxicity balance in patients with haematological malignancies.

Disclosures:

No relevant conflicts of interest to declare.
Second Primary Malignancies In Adult Acute Lymphoblastic Leukemia
Authors: Shah Binay K. , Ghimire Krishna B
Published: October 2019

Background

Pediatric acute lymphoblastic leukemia (ALL) patients have higher rates of second primary malignancies. There is limited data on second primary malignancies (SPM) among adult patients with ALL. This study was conducted to evaluate SPM in adult ALL patients using US Surveillance, Epidemiology and End Results (SEER) cancer registry database.

Methods

We analyzed the SEER 13 Registries using multiple primary standardized incidence ratio (MP-SIR) session. We analyzed secondary cancer rates among adult ALL patients during the period 1992 - 2010. We used SEER*Stat software provided by national cancer institute for statistical analysis.

Results

There were 3,259 adult (age ≥20 years) ALL patients reported in SEER database during 1992-2010. Among them, 65 ALL patients developed 75 second primary malignancies. Fifty-nine ALL patients developed 1 SPM each, 3 ALL patients developed 2 SPM each, 2 ALL patients developed 3 SPM each and 1 ALL patient developed 4 SPM. All site cancers were significantly higher among adult ALL patients compared to general population with observed/expected ratio (O/E): 1.47, p value< 0.05, an absolute excess risk of 24.43 per 10,000 populations. Similarly, oral cavity cancer, respiratory system cancer and hematological SPM were significantly higher in ALL patients than expected in general population. (Table)

Conclusions

Adult patients with ALL have higher rates of second primary malignancies compared to general population.

Disclosures:

No relevant conflicts of interest to declare.
Differentiation Induction In Acute Myeloid Leukemia Using Site-Specific DNA-Targeting
Authors: Palchaudhuri Rahul , Ang Kwan-Keat , Saez Borja , Sykes David B. , Verdine Gregory L. , Scadden David T.
Published: October 2019

Hoxa9 and Meis1 are overexpressed in >70% of acute myeloid leukemia (AML) and associated with poor prognosis and survival. Hoxa9 and Meis1 interact with DNA and PBX to achieve transcription of differentiation-blocking genes. We tested transcriptional repression at Hoxa9-PBX-Meis1 genomic binding sites to induce differentiation in a model of human AML

We designed a DNA-recognition strategy based on the known structure of the Hoxa9-PBX-DNA complex by fusing the DNA binding helices of Hoxa9 and PBX to create concise homeodomain fusion proteins that target the Hoxa9-PBX DNA recognition sequence. To confer transcription-repressing properties to the proteins, we attached a transcriptional repressor (sin3 interacting) domain and ectopically expressed this protein in Hoxa9-Meis1 immortalized murine progenitors. Introduction of this transcription repressor protein significantly enabled cell differentiation versus control (51.2% Mac-1high Gr-1high cells versus 11.3% for control). Multiple gene transcripts indicative of differentiation, such as GCSFR, myeloperoxidase, neutrophil elastase, and the calcium binding protein, S100A8, were also elevated in repressor-expressing cells. Furthermore, direct transcriptional targets of Hoxa9 (e.g. SOX2, CD34, FOXP1, FLT3R, DNAJC10) were down regulated in repressor-expressing cells. Importantly, a mutant repressor lacking the DNA-interacting amino acids did not affect transcription of Hoxa9 targets, demonstrating on-target specificity. Repressor-expressing cells also exhibited lower surface expression of c-Kit and Flt3 receptors and when transplanted into mice resulted in a significant increase in disease latency with a 94 day median latency versus 62 day latency for the control group (p value = 0.002).

Our results demonstrate that site-specific DNA-targeting using homeodomain fusion proteins can enable AML cell differentiation and significantly increase disease latency.

Disclosures:

Scadden: Fate Therapeutics: Consultancy, Equity Ownership.
CD34+CXCR4(CD184)+ Cells Differentiate Into Myeloid Dendritic Cell Progenitors
Authors: Donahue Robert E. , Jin Ping , Uchida Naoya , Bonifacino Aylin C. , Tian Yu , Klinman Johanna I. , Mattapallil Joseph J , Metzger Mark E. , Williams Ann , Malide Daniela , Tisdale John F. , Stroncek David
Published: October 2019

Dendritic cells (DCs) play a central role in innate immunity and adaptive immunity. DCs are antigen presenting cells capable of inducing primary T cell responses or facilitating self-tolerance. Myeloid DCs (mDC) express CD11c. They are further divided into Type 1 myeloid DCs (mDC1) which are CD1c+CD141+ and Type 2 mDCs (mDC2) which are CD1c-CD141+, and plasmacytoid DC which are CD11c- and express CD303. Plasmacytoid DCs are the main source of type 1 interferon upon infection. CD34+ cells are a heterogeneous population of cells which contain both hematopoietic progenitors and stem cells. The microarray signature of CD34+CXCR4 (CD184)+ cells in both human and non-human primates suggest that this cell population plays a role in innate immunity. The signaling pathway for the Triggering Receptor Expressed on Myeloid Cells 1 (TREM1) is the most up-regulated pathway in both human and non-human primate sorted CD34+CD184+ cells. The TREM family is involved in the amplification and regulation of inflammatory responses. Upon sorting both human and rhesus CD34+ cells into CD34+CD184+ and CD34+CD184-, we observe that CD34+CD184+ cells are both adherent and non-adherent in nature (Figure 1) and while maintained in Flt3-L, IL-3, and SCF form progeny which appear dendritic in nature (Figure 2). In addition, we have found the CD34+CD184+ subpopulation to be resistant to lentiviral vector transduction. Upon culturing in defined, serum free media supplemented with cytokines, the progeny of the CD34+CD184+ population using both flow cytometry and confocal microscopy are CD11c+ and contain both CD1c+ and CD1c- subpopulations (Figure 3) with a predominately CD80(B7-1)+, CD123(IL-3R)+, CD197 (CCR7)+ , and HLA-Dr+ immunophenotype, having variability in CD86(B7-2) +/-, CD141(BDCA-3/Thrombomodulin)+/- , and CD144 (VE-cadherin)+/- expression, and being negative for CD303(BDCA-2)and CD309(VEGFR). Of special interest is that the CD34+CD184+ progeny contain both CD1c-CD16+ and CD1c+CD16- subpopulations of mDC. CD1c-CD16+ mDC have been shown to have strong pro-inflammatory activity, whereas CD1c+CD16- mDC are mainly inducers of chemotaxis. The progeny of the CD34+CD184+ cells can be stimulated by LPS and IFNγ to produce IL-12 based on an enzyme-linked immunosorbent assay. These results demonstrate the importance of the CD34+CXCR4+ progenitor in mDC development and allow one to speculate on how this mDC progenitor might prove of therapeutic benefit in vaccine development and cancer therapy.Figure 1Human CD34+CD184+ Sorted PopulationFigure 1. Human CD34+CD184+ Sorted PopulationFigure 2Cultured Progeny CD34+CD184+Figure 2. Cultured Progeny CD34+CD184+Figure 3Non-Human Primate CD34+CD184+ Confocal MicroscopyFigure 3. Non-Human Primate CD34+CD184+ Confocal Microscopy

Disclosures:

No relevant conflicts of interest to declare.
Characterization Of a Weakly Expressed KIR2DL1 Allele
Authors: Li Hongchuan , Huehn Andrew R , Fellow Postbaccalaureate , Wright Paul W , Biologist Research , Cooley Sarah , Miller Jeffrey S. , Anderson Stephen K
Published: October 2019

The variegated expression of the human KIR family of class I MHC receptors provides an interesting model system for the study of stochastic activation of gene transcription. Previous studies have linked distal KIR promoter transcription to the initiation of KIR expression from the proximal promoter. In order to identify novel genetic alterations associated with decreased KIR expression, a group of 182 donors was characterized for KIR gene content, KIR transcripts, and FACS analysis of KIR surface expression. An individual was discovered that possessed a single copy of the KIR2DL1 gene but had a low level of gene expression by either FACS or Q-PCR.  Complete sequencing of the KIR2DL1 gene confirmed the presence of an intact coding region. Analysis of promoter elements revealed a cluster of three single nucleotide polymorphisms (SNPs) in the distal promoter approximately 1 kb upstream from the start of KIR2DL1 translation. These SNPs are also found in the distal promoter region of the non-transcribed KIR2DL5*002 allele as well as the KIR3DP1 pseudogene. One of these SNPs creates a functional binding site for the ZEB1 transcription factor.  Individuals possessing the ZEB1 site in their KIR2DL1 promoter produce high levels of a non-translatable distal KIR2DL1transcript that inhibits transcription from the proximal promoter, resulting in weak expression of this allele.

This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. Funded by NCI Contract HHSN261200800001E.

Disclosures:

Miller: Coronado Biosciences: Scientific Advisory Board Other.
Oncomirnas and Tumor Suppressors In Microvesicles From Four Types Of Cancer
Authors: Li Wenying , Xiong Wei , Chen Xiaomei , Huang Shiang , Huiyu Li
Published: October 2019

Objectives

Microvesicles (MVs) are small vesicles that are shed from almost all cell types including cancer cells into their surroundings. These secreted MVs bear surface receptors/ligands and miRNA of the original cells and play a role in cancer. Our aim in this study was to identify MVs miRNAs that might play important roles both in solid and non-solid tumors.

Methods

The MVs miRNA expression from 4 kinds of cancer cell lines (SMMC-7721, K562, Nalm6 and Jurkat) and healthy peripheral blood cells were analyzed by miRNA microarray. Quantitative real-time polymerase chain reaction (qRT-PCR) was further performed to verify the expression of 4 dysregulated miRNAs.

Results

The miRNA microarray revealed aberrantly expressed miRNAs of MVs between the four types of cancer cells and normal cells. In the MVs from cancer groups, 73 miRNAs were up-regulated and 148 were down-regulated in all the four types of cancer cell lines, indicating that solid and non-solid tumors shared some dysregulated MVs miRNAs. qRT-PCR verified statistically consistent expression of four selected miRNAs with microarray analysis. The top six shared upregulated MVs miRNAs in the four cancer cells lines were miR-1290, miR-1268, miR-1246, miR-125a-3p, miR-1305, miR-1226*. The top seven shared downregulated MVs miRNAs in the four cancer cells lines were miR-335, miR-1, miR-363, miR-122, miR-223, miR-338-3p and miR-340.

The general roles of the upregulated MVs miRNAs were oncomiRNAs in multicancer. MiR-1290 and its potential targets are associated with characteristics of estrogen receptor α-positive breast cancer. Up-regulations of miR-1290 impaired cytokinesis and affected the reprogramming of colon cancer cells. miR-1246 might serve as a likely link of the p53 family with Down syndrome and proposed a new p53-miR-1246-DYRK1A-NFAT pathway in cancer.

The functional roles of the downregulated MVs miRNAs were tumor suppressors in many kinds of cancer. MiR-335 acted as a candidate tumor suppressor and inhibited tumor reinitiation and represented an invasion suppressor gene by targeting Bcl-w in many kinds of cancer. HBx regulated miR-338-3p in HCC and miR-338-3p inhibited proliferation by regulating CyclinD1, and miR-338-3p suppressed the invasion of liver cancer cell by targeting smoothened gene in liver cancer. miR-340 inhibited of breast cancer cell migrations and invasions through targeting of oncoprotein c-Met and may play an important role in breast cancer progression.

Conclusion

Our study identifies oncomiRNAs and tumor suppressors in MVs from four types of cancer. These MVs miRNAs may contribute to the developments of solid and non-solid tumors and should be further evaluated as a novel biomarker for cancers and may provide potential targets for novel therapeutic strategies.

Disclosures:

No relevant conflicts of interest to declare.