bluuebunny/crossref_metadata_2025 huggingface dataset:
Chemotaxis-Related Factors Are Dysregulated In Bone Marrow Mesenchymal Stem Cells (MSCs) In Multiple Myeloma Patients
Authors: Lu Hua , Hu Huijin , Fei Xiaoming , Li Jianyong
Published: October 2019

Abstract 5010

Abstract Objective:

This study was aimed to investigate the mRNA expression levels of hepatocyte growth factor(HGF), stromal-derived factor-1(SDF-1), Chemokine (C-C motif) ligand 2 (CCL2) and interleukin-8(IL-8) in bone marrow mesenchymal stem cells (MSC) from multiple myeloma (MM) patients.

Methods:

The real time quantitative polymerase chain reaction(RQ-PCR)technique was used to detect the mRAN expression levels of HGF, SDF-1, CCL2 and IL-8 in bone marrow MSC from 20 newly diagnosed MM patients compared with 9 controls.

Results:

The results indicated that the mean mRNA expression level of HGF was up-regulated in MM patients, as compared with control (P < 0.01). However, the mean mRNA expression level of SDF-1 mRNA was down-regulated in MM patients, as compared with control (P < 0.05). There was no significant difference in the mRNA expression levels of CCL2 and IL-8 between MM and control cohorts (P > 0.05).

Conclusion:

The research suggests that multiple chemotaxis-related factors expression of bone marrow microenvironment cellular component are dysrelugulated in MM patients, which might result from the interplay between MM cell and MSC.

Disclosures:

No relevant conflicts of interest to declare.
Acadesine Inhibits Proliferation and Induces Apoptosis In Multiple Myeloma Cells, and Synergizes with Standard of Care Antimyeloma Agents
Authors: Hernández-García Susana , de Frias Mercè , Campàs Clara , Paiva Bruno , Ocio Enrique M. , Miguel Jesús F. San , Pandiella Atanasio
Published: October 2019

Abstract 5023

Multiple myeloma (MM) is a malignancy characterized by the accumulation of plasma cells. The disease represents the second most common hematologic malignancy and remains incurable, despite recent advances in its treatment. Therefore, studies to develop new therapies are still necessary, particularly in patients with bad prognostic factors, such as 17p deleted/p53 mutated patients. In this study we describe the preclinical activity of 5-Aminoimidazole-4-carboxamide-1–4-ribofuranoside (AICAR or acadesine) in multiple myeloma. Acadesine is an analog of AMP that is widely used as an activator of AMP-kinase (AMPK), a protein that regulates the responses of the cell to energy changes.

Acadesine induces apoptosis in different cell types including CLL, mantle cell lymphoma (MCL) and splenic marginal zone B-cell lymphoma (SMZL) cells and tumor cell lines, without affecting primary T lymphocytes. Thus, acadesine is a promising drug for the treatment of B-cell neoplasms. A clinical phase I/II study of acadesine is currently being performed in CLL patients.

We studied the effects of acadesine on the MTT metabolization of several multiple myeloma cell lines (MM1S, MM1R, RPMI-8266, RPMI-LR5, U266, U266-LR7, U266 Dox4, MM144, MGG, SJR, OPM-2, NCIH-929). Acadesine inhibited MM cell growth and induced apoptosis, with IC50 values in the micromolar range, and independently of the p53 mutational status.

Cancer treatment, including myeloma, is generally based on combinations of drugs with different mechanisms of action. Thus, we studied the effect of acadesine in double combinations with drugs used in myeloma therapy, such as dexamethasone, melphalan, doxorubicin, bortezomib, and lenalidomide. Analyses of these data using the Chou and Talalay method indicated that acadesine was synergistic with dexamethasone (CI values of 0.60), and particularly with lenalidomide (CI values of 0.42). These promising results with double combinations promoted the investigation of triple combinations in the MM1S cell line. Triple combination of acadesine plus dexamethasone plus lenalidomide or bortezomib notably improved the efficacy of the respective double combinations, being the combination of acadesine plus lenalidomide plus dexamethasone especially efficient.

Further studies to determinate the mechanism of action, and in vivo studies in MM1S xenograph are ongoing.

Disclosures:

de Frias: Advancell: Employment. Campàs:Advancell: Employment.
Analysis of the JAK2 Gene cDNA Full-Length In One Chinese Familial Myeloproliferative Disorders
Authors: Feng Ru , Hao Lixia , Zhang Yongmin , Wei Yongqiang , Huang Fen , Yin Changxin
Published: October 2019

Abstract 5056

Introduction:

JAK2V617F point mutation have been confirmed to be one of the major molecular mechanism of BCR/ABL negative myeloproliferative disorders(MPD). Besides, some other gene mutations such as JAK2 exon12, MPL W515L/K, c-mpl and EPOR have extended the scope of the research in this field. Most of the MPD patients are sporadic and there are seldom reports in Chinese familial MPD. 2008 ASH metting we have reported in a Chinese family of MPD's findings, the two brothers in our hospital diagnosis for MPD (one is a PV, another is ET), then we investigated the 15 members of the family. We discovered that there were three male members carried the JAK2V617F mutation in this family, including the two MPD patients and their father, which affected in two generations. All the family members were confirmed as BCR/ABL, MPL W515L/K, c-mpl, and EPOR negative. Subsequently, in order to understand the existence of family members in addition to the gene JAK2 V617F mutation, the existence of JAK2 gene mutations in other parts of the? if other mutations in existence and the high incidence of family members of MPD? We focus on the cDNA full-length of JAK2 gene to provide some theory basis on the pathogenesis in MPD.

Methods:

A total of 15 family members were enrolled in our study, including 2 brothers of MPD patients (the older one was thrombocythemia (ET), and another is polycythemia vera (PV)) and the other members in the same family. The mRNA of mononuclear cells from peripheral blood sample was extracted according to the manufacturer's instruction (TAKARA). RT-PCR and DNA sequencing have been used to analyze the cDNA full-length of the JAK2 gene.

Results:

All of the samples can be analyzed for JAK2 cDNA full-length. 3 members carried the JAK2V617F mutation (1849G®T) in this family, including the two MPD patients and their father. And the older brother was homozygous mutation and the other two were heterozygous mutation. All of the 15 samples were JAK2 exon12 gene mutation negative. 2 persons who were the male ET patient's children had a heterozygous mutation (380G®A) in JAK2 exon 3, caused a glycine-to-asparticacid substitution at position 127. Besides, 13 persons had 489C®T mutation in exon 4 and 14 persons had 2490G→A mutation in exon 17 in this family, But they were both same-sense mutation.

Conclusion:

It is necessary to do routine analysis of blood and other related inspection for MPD patient's family members, so as to make diagnosis earlier. However, we are not sure that the sequencing results are unique to all the familial MPD and need to be confirmed by more cases. We still do not determine the current discovery point mutations have biological significance, still need to be further explored.

Disclosures:

No relevant conflicts of interest to declare.
Long-Term Follow-up of Rituximab and Infusional Cyclophosphamide, Doxorubicin, and Etoposide (CDE) In Combination with HAART In HIV-Related Non-Hodgkin's Lymphomas (NHL)
Authors: Spina Michele , Jaeger Ulrich , Sparano Joseph A , Talamini Renato , Rossi Giuseppe , Vaccher Emanuela , Tirelli Umberto
Published: October 2019

Abstract 5072

Background:

The combination of Rituximab plus chemotherapy (CT) is more effective than CT alone in the treatment of high grade NHL. Objective: To report the long-term follow-up of CDE plus Rituximab in HIV-NHL.

Methods:

In June 1998, we started a phase II study using infusional CDE (Cyclophosphamide 187.5 mg/m2/day, Doxorubicin 12.5 mg/m2/day and Etoposide 60 mg/m2/day) administered by continuous intravenous infusion for 4 days every 4 weeks and Rituximab 375 mg/m2 i.v. on day 1. HAART was given concomitantly with CT.

Results:

Seventy-four patients (pts) have been enrolled. The median CD4+ cell count was 161 (range 3–691) and the median Performance Status was 1 (range 0–3). Diffuse large B-cell NHL was diagnosed in 72% of pts and Burkitt in 28%. Seventy per cent of pts had advanced stage (III-IV) disease and 57% of pts had an age-adjusted international prognostic index >2. Fifty-two out of 74 pts (70%) achieved a complete remission (CR), 4/74 (5%) had a partial remission and 18 pts progressed. With a median follow-up of 61 months, only 17% of CRs have relapsed and 41/74 pts are alive. The overall survival, disease free survival and time to treatment failure (TTF) at 5 years were 56%, 81% and 52%, respectively. Four cases of secondary tumors have been observed. No case of late pulmonary or cardiac toxicity has been reported.

Conclusions:

The combination of Rituximab and CDE in HIV-NHL treated concomitantly with HAART is very active. CR rate (70%) and TTF at 5 years (52%) are comparable to those observed in high grade NHL of the general population. Our data confirm that in HAART era a high proportion of HIV-NHL can be cured.

Disclosures:

No relevant conflicts of interest to declare.
Flow Cytometric Immunophenotypic Analysis of Cerebrospinal Fluid Cells In Hematological Malignancies
Authors: Ciriello Maria Matilde , Calcagno Lara , Callegari Tiziana , Gatto Simona , Primon Valeria , Arfini Carlo , Levis Alessandro
Published: October 2019

Abstract 5094

Background.

Leptomeningeal involvement is an adverse complication of haematological malignancies, acute leukemias and lymphomas. The diagnostic gold standard to identify cerebrospinal fluid (CSF) involvement is conventional cytological analysis, technique characterized by low sensitivity and specificity. Recent papers make evidence that the efficiency of CSF involvement detection could improve by use of Flow Cytometry (FCM) immunophenotypic analysis of CSF samples. We describe the FCM method used in our hospital and we compare the results with cytological findings.

Methods.

CSF samples should be processed immediately, within 1 hour after collection to avoid cell's deterioration; in B Non Hodglin Lymphomas (B-NHL) we use two steps FCM approach, in four color staining (FITC/PE/PerCP/APC). The first step was two tubes: kappa/lambda/CD45/CD19 and CD5/CD20/CD45/CD3 or CD3/CD20/CD45/CD10 on the basis of a previous know immunophenotype or not. If a pathological population is identified, we complete immunophenotype characterization with a second step, using antibody panels suitable for the suspected malignancy. In case of suspected meningeal relapse of Acute Leukemia we use a monoclonal antibody combination select for Minimal Residual Disease.

Results.

Between 2006 and 2009 we studied by FCM analysis 53 CSF samples of 36 patients. 7 CSF of 29 B-NHL newly diagnosed resulted positive at FCM analysis (24,1%), 1 CSF of 7 Acute Leukemias shows disease relapse. The conventional cytology was positive only in 1 case of B-NHL.

Conclusion.

Our study confirms that immunophenotypic flow cytometric analysis of CSF cells markedly improves sensitivity to identify CSF involvement in haematological malignancies.

Disclosures:

No relevant conflicts of interest to declare.
Acute T Cell Lymphoma/Leukemia (ATL) Cases for Notch1 and FoxP3 Expression and the Clinical Implications
Authors: Yan Wang Chun
Published: October 2019

Abstract 5096

[Abstract] Objective To investigate acute T cell lymphoma/leukemia (ATL) cases for Notch1 and FoxP3 expression and the clinical implications.

Methods

The expressions of Notch1 and FoxP3 in lymphoma and bone marrow specimens from 22 cases of ATL were studied by immunohistochemical analysis. The expressions of Notch1 and FoxP3 were analyzed by Image-ProPlus 6.0 and shown in integral optical density (IOD) (X±S).

Results

The neoplastic T cells of 90.91% of cases expressed FoxP3 and 72.73% of cases expressed Notch1, and 63.64% of patients had Notch1 and FoxP3 co-expression. IOD of FoxP3 and Notch1 were 13.57±11.38 and 8.42±3.48, respectively. The comparison of their IOD had no statistical significance (P=0.055).

Conclusion

The expressions of Notch1 and FoxP3 were high in ATL, which may be associated with the clinical outcome of ATL.

Disclosures:

No relevant conflicts of interest to declare.
Patients with Systemic Lupus Erythematosus (SLE) Having Developed Malignant Lymphomas. Continuing Complete Remission of Lymphoma Following Chemotherapy, but Not of SLE
Authors: Rossi Edoardo , Catania Gioacchino , Carella A. M. Michele , Marmont Alberto
Published: October 2019

Abstract 5098

Intoduction:

The development of malignant lymphomas, generally of the non-Hodgkin type (NHL), and with a preference to diffuse large cell B lymphomas (DLCBL), in systemic lupus erythematosus (SLE), has been proven and analyzed in an exhaustive recent literature. The combination of germline and somatic mutations, persistent immune overstimulation and the impairment of immune surveillance facilitated by immunosuppressive drugs, is thought to be at the origin of the increased lymphoma genesis. However the treatment and course of such affected patients is less known, and prognosis is generally estimated as poor. Materials: Out of 258 patients with complete/incomplete lupus and secondary antiphospholipid syndrome (APS) seen and treated at the institutional Day Hospital between 1982 and 2009, 6 developed lymphomas (4 DLCBL, 1 Hodgkin's and 1 indolent lymphocytic lymphoma).

Results:

The first 5 patients were treated with chemotherapy and achieved continuous complete remissions (CCR) with a follow-up comprised between 18 and 172 months. One patient achieved complete remission (CR) of both diseases. In the other 4 lupus serology (ANA, APA) persisted, with occasional lupus flares and vascular complications.

Conclusion:

While eradication of the last cancer stem cell is tantamount to cure in neoplastic disease, persistent autoantigenic overstimulation may contribute to the refractoriness of autoimmunity. The implications of these results for the increasing utilization of hematopoietic stem cell transplantation for severe autoimmune diseases (SADS), with lupus as a paradigm, are extensively discussed elsewhere (1).

(1) Rossi E, Catania G, Grassia L, and Marmont AM. Patients with systemic lupus erythematosus (SLE) having developed malignant lymphomas. Continuing complete remission of lymphoma following high-dose chemotherapy, but not of SLE. (submitted for publication)

Disclosures:

No relevant conflicts of interest to declare.
CRLF2 and JAK2 Mutations: Occurrence and Function In Pre-B ALL Cell Lines
Authors: Quentmeier Hilmar , Eberth Sonja , MacLeod Roderick AF , Nagel Stefan , Romani Julia , Zaborski Margarete , Drexler Hans G.
Published: October 2019

Abstract 5109

Thymic stromal lymphopoietin (TSLP), a cytokine produced by epithelial cells promotes early B-cell development and activates dendritic cells. It has recently been reported that a subset of B-cell precursor acute lymphoblastic leukemia (pre-B ALL) overexpresses the TSLP receptor CRLF2. CRLF2 overexpression is linked to translocations between sex chromosomes – localizing CRLF2 – and the immunoglobulin heavy chain locus on chromosome 14, or to an interstitial deletion on the gonosomes. Both events, translocation and deletion juxtapose CRLF2 to a different promoter (IgH or P2RY8). Performing quantitative real-time PCR we tested pre-B ALL and acute myeloid leukemia (AML) cell lines for overexpression of CRLF2. AML cell lines were included in the screening because we knew from an earlier TSLP project that the AML cell line MUTZ-3 is TSLP-responsive, and thus positive for the cytokine receptor. Three of 63 (5%) pre-B ALL cell lines tested (INC, MHH-CALL4, MUTZ-5) overexpressed CRLF2 mRNA. CRLF2-high cell lines carry a t(14;Y). With respect to the 58 AML cell lines tested: some expressed CRLF2 mRNA, but none of them rivalled the aforementioned pre-B cell lines. Pre-B ALL cell lines show the association between chromosomal CRLF2 aberrations and JAK2 pseudokinase domain mutations that has been described for primary pre-B ALL cells: cell lines MHH-CALL4 (JAK2I682F) and MUTZ-5 (JAK2R683G) and – newly described - also the CRLF2-high pre-B ALL cell line INC express a mutated version of Janus kinase 2 (JAK2R683G). We established a PCR based assay system that allowed for the rapid detection of the JAK2R683G mutation: none of the CRLF2-low or –negative pre-B ALL cell lines exhibited this mutation. All three CRLF2-high/JAK2mu cell lines showed high phosphorylation levels of the JAK2 downstream target STAT5. Inhibition of the JAK kinase led to dephosphorylation of STAT5. However, repression of 3H-thymidine uptake and induction of apoptosis by inhibition of the JAK2/STAT5 pathway was weaker in the JAK2mu pre-B ALL cell lines than in the JAK2V617F positive essential thrombocythemia-derived cell line SET-2. Provided that these results reflect the situation in primary cells, mutated JAK2 seems to be of lesser importance for growth and survival of pre-B ALL cells than for cells from myeloproliferative neoplasms. The CRLF2-high/JAK2mu cell lines INC, MHH-CALL4 and MUTZ-5 are promising model systems for the study of the roles of high-level CRLF2 expression and of JAK2 mutations in pre-B ALL.

Disclosures:

No relevant conflicts of interest to declare.
Expression of the Hypoxia-Inducible Factors In the AML and MDS: Is Not Associated with the Von Hippel-Lindau Gene
Authors: Tong Hongyan , Mei Chen , Li Kongfei , Jin Jie
Published: October 2019

Abstract 5114

The hypoxia-inducible factor (HIF) is a transcriptional factor with important roles in tumor biology, which activates transcription of genes encoding glucose transporters, glycolytic enzymes, and vascular endothelial growth factor. The VHL (von Hippel-Lindau) gene normally regulates ubiquitin mediated proteolysis of HIF-1a. VHL inactivation blocks HIF-1 proteolysis, resulting in increased HIF-1 expression. In the previous study, the expression of HIF-1a was detected in children ALL and NHL. To clarify the possible involvement of the HIF-1a and VHL gene in the development and progression of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), we analyzed the mutation and methylation of the VHL gene in 98 patients (54 AML and 44 MDS). The mutations in VHL gene was examined by direct exons sequence and the methylation status of the promoter region was determined using methylation-specific polymerase chain reaction (MSP). In addition, we examined the expression of HIF-1a in 44 of these patients (25 AML and 18 MDS) through the immunoblotting. VHL mutations were identified only in one patient with AML. However, no mutation was detected in the coding region of the VHL gene in MDS. Meanwhile the methylation of VHL gene was not found in the AML and MDS. But the expression of HIF-1a was found in 10 (66.7%) of 25 patients with AML and 5 (38.5%) of 18 patients with MDS. There is no significant correlation between the expression of HIF-1a and the VHL gene. Our primary date support that the HIF-1a may be involved in the development and progression of AML and MDS, but is not associated with the VHL gene.

Disclosures:

No relevant conflicts of interest to declare.
Are Beta-Receptor and Angiotensin-Blocking Drugs Protective Against Venous Thromboembolism (VTE)? A Population Based Case-Control Study
Authors: Ashrani Aneel A. , Geng Wencheng , Crusan Daniel J. , Petterson Tanya M. , Bailey Kent R. , Leibson Cynthia L. , Heit John A.
Published: October 2019

Abstract 5118

Background:

Increased factor VIII:C (FVIII:C) and hypofibrinolysis are VTE risk factors, and beta-blockers and angiotensin converting enzyme (ACE) inhibitors reduce FVIII:C and enhance fibrinolysis, respectively.

Objective:

To test the hypotheses that beta-blockers and ACE inhibitors reduce VTE risk.

Methods:

Using longitudinal, population-based Rochester Epidemiology Project resources, we identified all Olmsted County, MN residents with objectively-diagnosed incident VTE over the 13-year period, 1988–2000 (n=1306), and one to two Olmsted County residents per case matched on age, event year and duration of prior medical history (n=1500). For cases and controls, we reviewed their complete medical history in the community for previously-identified VTE risk factors (e.g., hospitalization with or without surgery, nursing home confinement, trauma/fracture, leg paresis, active cancer, superficial vein thrombosis and varicose veins), as well as body mass index (BMI), coronary artery disease (CAD), congestive heart failure (CHF), and the use of statins, beta-blockers, ACE inhibitors and angiotensin II receptor antagonist drugs. Using conditional logistic regression, we tested beta-blockers and ACE inhibitors/angiotensin II receptor antagonists for an association with VTE, both individually and after adjusting for age, BMI, previously-identified VTE risk factors, CAD, CHF and the use of statins.

Results:

Among cases and controls respectively, 191 and 173 received beta-blockers, and 171 and 154 received ACE inhibitors/angiotensin II receptor antagonists. Univariately, both beta-blockers (unadjusted OR=1.31; p=0.02) and ACE inhibitors/angiotensin II receptor antagonists (unadjusted OR=1.32; p=0.02) were modestly associated with increased VTE risk. However, after controlling for age, BMI, previously-identified VTE risk factors, CAD, CHF and the use of statins, beta-blockers (OR=1.06; 95% CI: 0.74, 1.51; p=0.75) and ACE inhibitors/angiotensin II receptor antagonists (OR=0.94; 95% CI: 0.65, 1.37; p=0.75) were no longer associated with VTE.

Conclusions:

Beta-blockers and ACE inhibitors/angiotensin II receptor antagonists do not appear to be protective against VTE.

Disclosures:

No relevant conflicts of interest to declare.
The Risk of Vascular and Thromboembolic Events Is Increased In Young Low-Risk Germ Cell Cancer Patients by Cisplatin Containing Chemotherapy
Authors: Solari Leticia M , Dollinger Kathrin , Heinz Jürgen , Reinhardt Heike , Rohdenburg Susanne , Engelhardt Monika , Waller Cornelius F.
Published: October 2019

Abstract 5119

Introduction:

It has been previously reported that both short and long term vascular and thromboembolic events (TEE) occur following cisplatin containing chemotherapy for germ-cell cancer (GCC) (Bokemeyer et al. 1998, Weijl et al. 2000, Diekmann et al. 2010). It has also been shown that patients with GCC are at higher risk of TEE than patients with non-GCC, while on cisplatin-based chemotherapy (Piketty et al. 2005).

Method:

We reviewed all GCC patients treated in our department from 2008 until 2010 with cisplatin-based chemotherapy and assessed the incidence of TEE. Cases were analyzed with respect to risk factors for vascular events (obesity, diabetes, nicotine abuse, prior cardiovascular disease, prior TEE), tumor characteristics (low- vs. intermediate/high-risk GCC) and age. In addition, further analysis of hemostasis laboratory tests of these patients is being performed.

Result:

Eight patients (8/50=16%) treated with cisplatin-containing chemotherapy developed TEE. Of these TEE, two were myocardial infarctions, four pulmonary embolisms, one patient showed thrombosis of the jugular vein and one a peripheral (femoral) arterial thrombosis. TE-risk factors included: nicotine abuse in two patients, obesity and nicotine abuse in one patient and no identifiable factors in the others (hemostasis laboratory tests are currently being performed). The median age of these 8 patients was 42 years (range 16–61). Patients were treated with PEB (cisplatin, etoposide, bleomycin) or PIV (cisplatin, ifosfamide, etoposide) initially applying continuous heparin prophylaxis with 15000IE/day (d), d1-5 only during PEB or PIV, but no low-molecular weight heparin (LMWH-) and/or aspirin (ASS-) medication for venous and arterial TEE prophylaxis thereafter. With these clinically very relevant and severe TEEs, we have adapted our PEB- and PIV protocols, adding a LMWH prophylaxis (enoxaparin 40mg d6-21 after chemotherapy).

Conclusion:

We observed an increased risk of major thromboembolic and vascular events with an incidence rate of 16% in GCC patients after cisplatin-containing chemotherapy. Prior studies have identified TEE-risks in GCC and with cisplatin-chemotherapy, albeit LMWH- and/or ASS-prophylaxis - without clinical trials - has not been recommended as yet. Prospective studies investigating thrombosis prophylaxis measures are currently performed in order to achieve the best standard of care for these patients. Currently an extensive hemostasis diagnostic testing from all patients with TEE is being performed.

Disclosures:

No relevant conflicts of interest to declare.
Simultaneous Occurrence of 2009 H1N1 Influenza and Server Acute Hemolytic Anemia —Three Cases
Authors: Wei Ju-ying , Jin Jie , Lou Yinjun
Published: October 2019

Abstract 5139

Most symptoms of 2009 H1N1 virus patients are mild, and most patients could recover well. Lymphopenia and thrombocytopenia are common in 2009 H1N1 patients. We observed that three patients who suffered from acute severe hemolytic anemia (Hemoglobin<60g/L), two of which(Case 1 and 3) also accompany with thrombocytopenia while suffered from H1N1. Two patients(Case 2and 3)got high fever and dark urine at the same time, and another patient(case1)suffered dark urine two days after cough. All patients were confirmed of H1N1 positive by RT-PCR test, while other lab tests and symptoms were consistent with hemolytic anemia. Two of them (case 2 and 3) progressed into hemolytic uremic syndrome and were treated with hemodialysis and methylprednisolone. Another patient (case 3) had been diagnosed with Paroxysmal nocturnal hemoglobinuria (PNH) for one year, and her hemoglobin had maintained about 110g/L, but lower to 45 g/L when she suffered from 2009 H1N1. All three patients were administrated with oseltamivir (75 mg, twice per day) for 5 days by oral and supportive therapy. The hemoglobin level of two patients (Case 1 and 2) increased to 100 g/L after three weeks gradually, and another PNH patient's hemoglobin returned to between 80 and 90g/L after two months. Our three cases had high fever and dark urine simultaneously, which indicated that 2009 H1N1 influenza may induce acute severe hemolytic anemia. Physicians need to be aware of this complication.

Disclosures:

No relevant conflicts of interest to declare.
Burr Red Blood Cells (BC) In Patients with Disseminated Intravascular Coagulation (DIC)
Authors: Patel Vipul R. , Khokhar Jatinder , Murthy Hemant S , Braverman Albert S.
Published: October 2019

Abstract 5141

Burr red blood cells (BC) in patients with disseminated intravascular coagulation (DIC).

Background:

Angiopathic hemolysis with schistocytes occurs in a minority of DIC patients. Burr cells (BC), are morphologically homogeneous, with a serrated membrane resembling that of red cells (RBC) in a hypertonic medium. They are associated with acute renal insufficiency, and have been observed in DIC.

Methods:

Criteria for patient inclusion were sepsis, often with multi-organ failure and hypotension. Of the 39 patients studied, 20 had DIC, based on platelet counts <100,000, and an International Society of Hemostasis and Thrombosis score of ≥5, derived from D-dimer or fibrin split product levels, prothrombin time elevations, and fibrinogen levels. The percentages of BC and schistocytes were determined by 1000 RBC counts, without knowledge of data concerning DIC. Patients' [Hb]'s, serum creatinine and blood urea nitrogen (BUN), were recorded.

Results:

Five of the 20 DIC patients succumbed to their acute illness, while 15 recovered without developing renal insufficiency. The BC%'s of the 20 DIC patients ranged from 0–80%, with a median of 18% and a mean of 27%; in the 19 patients without DIC the range was 0–45%, the median 6.4% and the mean 11% (p=0.046). The schistocyte percentages in patients with and without DIC ranged from 0.20–17, and 0–78; the respective medians and means were 1.1% and 1.0%, and 3.0–5.8%. The median and mean [Hb]'s of the patients with and without DIC were 9.3 and 11, and 9.6 and 10.3, respectively. BC% did not correlate with the [Hb]. The median creatinine levels of patients with and without DIC were 0.92 and 0.93 mg/dl, though the DIC patients' BUN's were higher (medians 31 vs 15). The BC% correlated with neither the BUN nor the BUN/creatinine ratio. The median and mean serum sodium levels in patients with and without DIC were 137.

Conclusion:

BC's are significantly more frequent in septic patients with DIC than in those without it. BC% did not correlate with anemia, implying that BC do not cause significant hemolysis, though there was a trend to lower [Hb] in the DIC patients. BC% was much higher than that of schistocytes in both groups, and schistocyte percentages did not correlate with DIC or BC%. Because of the DIC patients' higher BUN's and BUN/creatinine ratio's, we cannot exclude a role for the factors, which caused pre-renal azotemia in BC formation. BC formation may prove to be a criterion for DIC.

Disclosures:

No relevant conflicts of interest to declare.
Too Much Ferritin, Too Little Overload
Authors: Crisostomo Ricardo H. , Docena Christian P.
Published: October 2019

Abstract 5154

A healthy 42y/o, White, male, without any systemic illnesses or complaints presents with a serum ferritin of 1800ng/mL; the discrepancy between his optimal physical status and the laboratory result, poses a challenge to the most astute clinician. The fundamental goal of this abstract is to provide recognition and prevention to torment that patients with Hereditary Hyperferritinemia Cataract Syndrome (HHCS) are exposed to. The history will offer an important clue, of early onset cataracts. Our patient was guided by an older sibling who herself for many years underwent unnecessary clinical management for hemochromatosis. The presence of hereditary hyperferritinemia cataract syndrome (HHCS) has been defined in families all across the world and likely is under-diagnosed. Such a rare syndrome may never make it into a hematology practice and be missed while the focus is on the “panic” ferritin result. Our patient is part of a family who has had the syndrome since at least the mid 1800's. The scope of this disorder is bringing to reality that confirmation of genetic testing doesn't change management and that the only intervention will be ophthalmologic. The abundant testing for ferritin across medical specialties may cause a rise in HHCS cases, recognizing this syndrome early on will lead to protect patients from insistent and costly interventions. These patients tolerate poorly phlebotomies and will develop iron deficiency anemia. Although appropriate data is available regarding HHCS there is no relevant clinical data to support a poor clinical outcome, and the precise recognition this triad (cataract, asymptomatic patient and hyperferritinemia) will avoid worthless and costly investigations. In this case, a family member who was victimized by excessive workup, was able to research, discover the family mutation and offer this practitioner the tools towards practicing better patient care. In summary, Hereditary Hyperferritinemia Cataract Syndrome is a benign condition that requires avoidance of increased healthcare costs for the proband.

Disclosures:

No relevant conflicts of interest to declare.
Smarca5 Regulates Ctcf Recruitment to Chromatin, Including to Regulatory Loci Involved In Control of Globin Gene Expression In Erythroleukemia
Authors: Kapalova Martina , Kokavec Juraj , Curik Nikola , Burda Pavel , Skoultchi Arthur I. , Stopka Tomas
Published: October 2019

Abstract 5159

Transcription factor Ctcf (CCCTC-binding factor) represents a major regulatory component of epigenetic regulation by recognizing its unmethyled DNA binding sites, resulting in changes in expression of neighboring genes. Ctcf plays an important role in transgenerational genetic imprinting. Very little is known about its role in hematologic malignancies. Ctcf has been described to promote differentiation of human erythroleukemia K562 cells (Torano 2005). We studied Ctcf in mouse erythroleukemia (MEL) cells and found it is expressed at both the mRNA and protein levels. Using chromatin immunoprecipitation (ChIP), we found that Ctcf is recruited to the H19/Igf2 imprinting control region (ICR) and also to the promoters of the alpha globin genes (Hba-a1, Hba-a2) as well as the beta globin locus control region (LCR) in MEL cells. To determine the mechanism by which Ctcf interacts with chromatin, we tested its interaction with chromatin remodeling proteins that associate with these DNA targets, including the well known Imitation Switch (ISWI class) ATPase Smarca5 (Snf2h). Using coimmunopreciptiation and ChIP experiments we found that Smarca5 and Ctcf interact on DNA. Next, we used MEL cells expressing an inducible Smarca5 shRNA. Doxycycline induction of Smarca5 shRNA led to a 5-fold decrease in Smarca5 mRNA and protein levels within 48hrs. ChIP experiments demonstrated that depletion of Smarca5 was accompanied by loss of Ctcf from the aforementioned loci indicating Ctcf requires Smarca5 for its association with chromatin. Furthermore, this was followed by significantly decreased levels H19 RNA. Our data provide evidence that Smarca5 regulates Ctcf recruitment to chromatin, including to regulatory loci involved in controlling globin gene expression. (Grants # IGA 10310-3, MSMT 2B06077, GAUK 251070 45410, SVV-2010-254260507, NIH R01CA154239).

Disclosures:

No relevant conflicts of interest to declare.
Beta Thalassemia In Different Tribal Population In West Bengal, India
Authors: Majumdar Deboshree , Basak Jayasri , Chakraborty Abhijit , Mukhopadhyay Soma , Pal Nabamita , Mukhopadhyay Ashis
Published: October 2019

Abstract 5170

Background:

The Thalassemias are a group of anemia that results from a genetic defect whch reduces the rate of synthesis of normal globin chains. Beta Thalassemia results in severe microcytic, hypochromic, haemolytic anaemia among affected patients. It has emerged as one of the most common health problems among the tribal populations in West Bengal. A project entitled, “Status of Prevalence and Awarness Among Thalassaemia Population: Special reference to various tribes belonging in different Districts of West Bengal” has been sanctioned by the Department of Biotechnology, Govt. of India(BT/PR10336/SPD/24/293/2007). This study is aimed to observe the spectrum of various beta mutations among the tribes. In the screening schedule thalassemia awarness programme was conducted in various rural areas of West Bengal. We mainly confronted with the Sardar tribes in extreme south of West Bengal. In North Bengal there were Toto, Rabha, Oraon, Munda, Toppo, Baraik, Nagbanshi, Khalko, Kheria, Kerketta and Soren.

Method:

In the year 2009–2010, 1289 tribes were screened. Mass awarness programme were followed by collection of peripheral blood samples in EDTA vials and transported to the laboratory in ice packs. NESTROFT was done on spot using 0.36% Saline Buffer solution (Sodium chloride, Sodium dihydrogen phosphate, Disodium hydrogen Phosphate). Complete Blood Count was performed within 24 hours of collection. HPLC (High Performance Liquid Chromatography) was done to identify the beta samples. DNA was extracted from the blood of beta carrier and beta major and subjected to ARMS (Amplification Refractory Mutation System) PCR (Polymerase Chain Reaction) to detect the point mutations. Result:

Conclusion:

Molecular Characterization of Beta globin gene mutations among these tribes have confirmed the presence of the following mutations: IVS-1 nt5 (G>C), IVS-1 nt1(G>T), codon 15 (G>A), codon 26 (G>A), the mutation which leads to HbE, codon 19 (A>G), codon 17 (A>T), -28 (A>G). The most common mutation observed among Totos and Rabhas were codon 26 (G>A) of North Bengal. The prevalence of Beta carrier among them is negligible. The mutation IVS-1 nt5 (G>C) is prevalent among the Oraon, Nagbanshi and Sardar tribes of Bengal. The other mutations which are present among them are codon 15 (G>A) and codon 30 (G>C). There are two codon 17 (A>T) and one -28 (A>G) mutations which are present among Mundas. The rest of them are mutation IVS-1 nt5 (G>C). In few cases presence of sickle cell anemia was observed. Our results have added to the existing data on the common beta globin gene defects which are prevalent among the general population of West Bengal, India.

Disclosures:

No relevant conflicts of interest to declare.
A Novel Dendritic Cell Population Generated by Fusing Granulocyte-Macrophage Colony-Stimulating Factor and Interleukin-21 Induces Tumor-Antigen Specific Immunity
Authors: Williams Patrick , Bouchentouf Manaf , Rafei Moutih , Romieu-Mourez Raphaelle , Hsieh Hsiang-Chuan (Jeremy) , Boivin Marie-Noelle , Yuan Shala , Forner Kathy-Ann , Birman Elena , Galipeau Jacques
Published: October 2019

Abstract 5177

Introduction:

Cancer recruits the immune system to promote its growth and to inhibit reactions against itself. We generated a fusion of GMCSF and IL-21 (GIFT-21) with the aim of stimulating distinct, but complementary, elements of the innate and adaptive immune system against cancer. In a previous study, GIFT-21's aberrant interactions with its cognate receptors on macrophages resulted in an unanticipated pro-inflammatory response and tumor rejection in mice.

Results:

We further explored this phenomenon by treating mice with dendritic cells (DC) derived by treating monocytes with GIFT-21. B16 melanoma and D2F2/neu breast cancer growth was inhibited only in mice treated with a single injection of antigen naïve GIFT-21 DCs. This effect was lost in CD8-/- and CCR2-/- mice and when mice were treated with β2 microglobulin deficient GIFT-21 DCs, and we confirmed that GIFT-21 DCs migrated to and sampled from the tumors to present tumor antigens to CCL2 recruited CD8+ T cells via MHCI.

Conclusion:

When stimulated with GIFT-21 DCs, the immune system can identify cancer specifically, independently of cancer type. We conclude that GIFT-21 and its associated cellular products may serve as novel therapeutic platforms for the treatment of cancer.

Disclosures:

No relevant conflicts of interest to declare.
Defective Production, Turnover, and Secretion of the Platelet α-Granule Protein P-Selectin In Mice with Disrupted FOG1-NuRD Interaction
Authors: Wang Yuhuan , Meng Ronghua , Blobel Gerd , Marks Michael S. , Poncz Mortimer
Published: October 2019

Abstract 547

We reported recently that point mutations in the GATA-1 co-factor FOG-1 that disrupt binding to the NuRD chromatin remodeling complex display defects in the erythroid and megakaryocyte (MK) lineages. Homozygous FOG-1 mutant (ki/ki) mice display a Gray Platelet Syndrome (GPS)-like macrothrombocytopenia. Specifically, ki/ki platelets exhibit a paucity of α-granules and increased numbers of lysosomal-like vacuoles, reminiscent of the platelet morphology observed in human patients with GPS due to GATA-1 mutations. To understand the molecular basis for the phenotype in ki/ki platelets, we compared mRNA and protein expression of α-granule proteins in wildtype (wt) and ki/ki MK. P-selectin mRNA in ki/ki MKs was 3–5 fold reduced when compared to controls, while platelet factor 4 (Pf4), platelet basic protein (PBP) and von Willebrand factor (Vwf), were only mildly reduced. Strikingly, P-selectin protein levels were more severely reduced in ki/ki MK and platelets being nearly undetectable by Western blot and immunohistochemistry. In contrast PF4, PBP, and vWF protein levels were normal. P-selectin loss was MK-specific as it was expressed at normal levels in endothelial cells. The dramatic decrease in P-selectin protein in ki/ki MK relative to the more moderate reduction in mRNA was due to a >5-fold decrease in protein half-life, as measured by metabolic pulse-chase and immunoprecipitation experiments. This suggests that P-selectin in ki/ki MKs is largely mistargeted to lysosomes. Nevertheless, immunofluorescence microscopy and volume de-convolution analyses of ki/ki platelets showed that the remaining P-selectin resided in vesicular compartments that were distinct from Lamp1 (a lysosomal marker)-containing granules, indicating that a fraction of P-selectin remains within α-like granules. Interestingly, the reduction in P-selectin levels in ki/ki platelets was accompanied by a defect in agonist-stimulated degranulation. While stimulation of wt platelets with AYP, thrombin or phrobol myristate acetate induced α-granule, dense granule and lysosome degranulation, ki/ki platelets failed to do so or undergo platelet aggregation. In summary, the GPS-like phenotype in FOG-1/NuRD interaction-defective MKs involves reduced P-selectin mRNA production, accelerated turnover of P-selectin, abnormal granule formation, and defective degranulation in response to diverse agonists. These findings may partially explain patients having platelet α-granule deficiency associated with decreased P-selectin and selective impairment of thrombin-induced activation with GPS. Future work will determine whether similar defects occur in human GPS.

Disclosures:

No relevant conflicts of interest to declare.
Identification of a Discrete Subpopulation of T-Cells Over-Expressing HDAC11 with a TH17 Phenotype
Authors: Woan Karrune , Cheng Fengdong , Wang Hongwei , Rock-Klotz Jennifer , Wang Zi , Yu Yu , Seto Edward , Luetteke Noreen , Yu Xue-Zhong , Villagra Alejandro , Sotomayor Eduardo M.
Published: October 2019

Abstract 588

We recently defined a novel role of histone deacetylase 11 (HDAC11), the newest member of the HDAC family, as a negative regulator of IL-10 gene transcription in antigen-presenting cells (APCs).1 To better understand the role of HDAC11 gene expression in immune cells in vivo, we have utilized a BAC (Bacterial artificial chromosome) transgenic mouse in which the EGFP reporter gene was inserted downstream of the HDAC11 promoter region but immediately upstream of the HDAC11 coding sequence (TgHDAC11-EGFP mice).2 In the steady-state, macrophages and B-cells isolated from spleen of TgHDAC11-EGFP mice express low levels of HDAC11 as evidenced by a slight shift in EGFP fluorescence from background. In sharp contrast, we identified a discrete population (11.9%) of T-cells over-expressing HDAC11 as demonstrated both by flow cytometry for EGFP and by qRT-PCR for HDAC11, a majority of which were CD4+ T-cells. Sorting of this EGFP+, CD4+ T-cell population confirmed that the increased EGFP expression correlated with an increased HDAC11mRNA expression. Reminiscent of our prior data in APCs, the increased expression of HDAC11 in T-cells was also inversely correlated with IL-10mRNA expression. Further analyses revealed that in the absence of any stimulation or T-cell polarizing conditions, this EGFP positive population expressed significantly elevated levels of ROR-γt and IL-17 mRNA, markers specific for the TH17 subpopulation. Polarization of wild type CD4+ T-cells into functional TH17 cells was associated with reduction of HDAC11 expression, suggesting a potential role for HDAC11 in regulating T-cell function and/or activation, in particular within the TH17 subset. Further support for this regulatory role of HDAC11 has been provided by our additional findings that T-cells devoid of HDAC11 are indeed hyper-reactive in vitro and in in vivo models.

1. Villagra A, et al. Nat Immunol. 2009 Jan;10(1):92-100.

2. Gong S, et al. Nature. 2003 Oct 30;425(6961):917-25.

Disclosures:

No relevant conflicts of interest to declare.
Long-Term Prophylaxis In Children, Adolescents and Young Adults with Von Willebrand Disease – First Results of a Cohort Study
Authors: Halimeh Susan , Kruempel Anne , Rott Hannelore , Bogdanova Nadja , Budde Ulrich , Manner Daniela , Faeser Britta , Mesters Rolf M. , Nowak-Gottl Ulrike
Published: October 2019

Abstract 714

In children and young adults with von Willebrand disease [vWD] replacement therapy with von Willebrand factor (vWF)/factor VIII concentrates is increasingly performed as “prophylactic” therapy. Between 2000 and 2008, 82 consecutively enrolled children, adolescents and young adults with clinically meaningful bleeding episodes [spontaneous, peri- or postoperative] were diagnosed with vWD [type 1: 42/82; type 2: 24/82; type 3: 13/82; acquired: 3/82]. Here we report results of prophylaxic VWF replacement therapy in 32 patients [female: n=15; >Tanner 2: 9/15] with VWD type 1 [n=4], 2 [n=15] and 3 [n=13] in which a prophylactic treatment regimen was performed. Decision on prophylaxis was based on i) a pre-diagnostic bleeding score, and ii) on recurrent bleeds associated with microcytic anemia after diagnosis in patients with on-demand VWD therapy. Patients were treated with Humate P® (n=8; once switched to Wilfact®) or Wilate® (n=24). Median [min-max] dose was 40 (20-47) IU/kg, 23 patients were substituted twice weekly, 7 were treated three times, and two children 4 times per week. Within a six-months-period hemoglobin concentrations returned to normal values. Median [min-max] duration of prophylaxis was 3 years [1.0-9.0]. Recurrent bleeding episodes stopped in 31/32 patients, inhibitor development occurred in one. Following a 12-months observation period the bleeding frequency/bleeding score was significantly reduced, compared to pre-prophylaxis/pre-diagnostic values. The use of prophylactic VWF replacement therapy is an effective and well tolerated treatment modality, highly beneficial for children and young adults with vWD with recurrent bleeding events on on-demand therapy.

Disclosures:

No relevant conflicts of interest to declare.
Protective and Predisposing HLA Alleles In Dutch Classical Hodgkin Lymphoma Patients
Authors: Diepstra Arjan , Huang Xin , Nolte Ilja , Hepkema Bouke , Kooistra Wierd , Visser Lydia , Poppema Sibrand , Van Den Berg Anke
Published: October 2019

Abstract 749

Classical Hodgkin lymphoma (cHL) is characterized by an overwhelming reactive infiltrate and the involvement of Epstein Barr virus (EBV) as a causative agent in a proportion of patients. We previously did a population-based genetic screening study of the Human Leukocyte Antigen (HLA) region in Dutch cHL patients and found a strong association between HLA class I (HLA-A1) and susceptibility to EBV+ cHL. This association might be explained by the known lack of HLA-A1 restricted T cell responses to EBV latent antigens. We now performed a case-control study to determine allele frequencies of all major HLA class I and class II genes in this cHL population. HLA genotyping of 294 cHL patients was performed by a polymerase chain reaction-based sequence-specific oligonucleotide probe hybridization (PCR-SSOP) approach in Luminex assays. The resulting single nucleotide polymorphism (SNP) data were converted into two-digit HLA typing for the HLA class I and class II genes. Phenotype frequencies of serologically defined HLA-A, HLA-B and HLA-DR blood donors were retrieved from the database of the blood bank of the University Medical Center Groningen. Allele frequency differences between these controls and cHL patients (total group and EBV+ and EBV- subgroups separately) were analyzed by Chi-square tests. In addition, we analyzed the HLA genotyping data for every PCR-SSOP probe to assess potential differences between the EBV+ and EBV- cHL group using PLINK software. Frequencies of HLA-DR4 and HLA-DR7 were significantly decreased and HLA-B5 and HLA-B37 were significantly increased in cHL as compared to the controls. In EBV+ cHL, HLA-A1, HLA-B37 and HLA-DR10 frequencies were significantly increased and HLA-A2 was decreased. An increase in HLA-B8, an allele that can present EBV peptides and that usually is in strong linkage disequilibrium with HLA-A1, was not present in these patients. In EBV- cHL, HLA-DR5 was significantly more common and HLA-DR4 was underrepresented. The SNP analysis revealed significant differences for 16 HLA-A probes between EBV+ and EBV- cHL. Eight probes that represented a high susceptibility for EBV+ cHL were specific for HLA-A1, whereas the other 8 probes correlated with a low risk for EBV+ cHL and were specific for HLA-A2. Most of the corresponding SNPs had a presumed antigenic peptide or T cell receptor binding function. In conclusion, the current study demonstrates that certain HLA alleles are protective or predisposing for cHL with specific associations for the EBV+ and EBV- cHL subgroups. The genotype analysis indicates that the complete HLA-A1 and HLA-A2 alleles are more important than individual T-cell receptor or antigen binding polymorphisms for the association with EBV+ cHL.

Disclosures:

No relevant conflicts of interest to declare.