bluuebunny/crossref_metadata_2025 huggingface dataset:
Assessment of Cross-Reactivity in Three Different Fecal Occult Blood Test Systems with Dabigatran and Dabigatran Etexilate: Identification of Useful Test Methods
Authors: Goss Ashley M , van Ryn Joanne , Schurer Johanna , Clemens Andreas
Published: October 2019

Abstract 4360

Anticoagulant therapy can elevate the risk of gastrointestinal (GI) bleeding and may affect its management. Fecal occult blood tests (FOBT) are non-invasive, widely-used tests that may be useful in detecting early and thus treatable stages of GI lesions or bleeding in patients on these therapies. The literature suggests that use of anticoagulants does not diminish the positive predictive value of FOBT, however a potential limitation and concern of these tests is the possibility for drug interactions with test components which may lead to false results.

Dabigatran is a new oral, reversible, direct thrombin inhibitor currently approved for stroke prevention in patients with atrial fibrillation. Dabigatran etexilate (DE) is the prodrug of the active ingredient dabigatran, and is converted by non-specific esterases in plasma and liver to dabigatran. Approximately 6.5% of DE in Pradaxa® is bioavailable and absorbed in the gut with the remaining eliminated in the feces. The purpose of this study was to investigate any potential cross-reactivity of dabigatran, DE or Pradaxa® with three commonly-used commercial FOBTs with different detection methods.

Common methods of detection in FOBTs used worldwide include guaiac oxidation in hemoccult tests, immunochromatographic methods with a membrane strip precoated with hemoglobin antibody, or the benzamidine method where heme in hemoglobin induces a blue/green color change. Examples of each method were tested. The Hemoccult II® SENSA® (Beckman Coulter Inc.), EZ DETECT (Biomerica Inc.), and BioNexia® Hb/Hp Complex Professionell (FROST Diagnostika GmbH) FOBT were used for this study and all tests were conducted according to manufacturer's protocol. DE, dabigatran or the contents of a single Pradaxa® capsule were suspended in a 0.5% cellulose solution. Vehicle solution was used as negative control and human blood used as a positive control.

Isolated human blood samples generated positive results as expected in all three FOBTs whereas the negative controls did not. Exposure to DE, dabigatran or Pradaxa® suspensions did not result in positive readouts with any detection method of the tested FOBTs.

In conclusion, these studies demonstrate that the prodrug DE and its active drug form dabigatran, as well as any further excipients in the Pradaxa® formulation do not cross-react with a set of commonly-used fecal occult blood tests. This supports the usefulness of these 3 tests in dabigatran etexilate-treated patients.

Disclosures:

Goss: Boehringer Ingelheim Pharmaceuticals Inc.: Employment. van Ryn:Boehringer Ingelheim: Employment. Schurer:Boehringer Ingelheim: Employment. Clemens:Boehringer Ingelheim (Anticoagulant Therapy): Employment.
Prevalence of Allo and Auto Antibodies in Adults with SCD
Authors: Kumar Mukesh , Shelia Palmer , Eckman James R.
Published: October 2019

Abstract 4379

Introduction:

Allo- and auto- antibody formation is one of the major and serious complications of RBC transfusion in patients with sickle cell disease. This problem appears to be more common than in other diseases requiring chronic transfusion that leads to acute and delayed hemolytic transfusion reactions, serious morbidity and occasional life threatening problems. Although there have been reports describing prevalence of immunization in SCD patients, data specific for adult population with SCD is lacking. We report the prevalence of allo- and auto-immunization in adult SCD patients.

Methods:

Patients aged 18 and above with diagnoses of SCD, confirmed by standard hemoglobin electrophoresis, who had at least one type and cross match done from 1995 through 2011 were considered eligible. To facilitate patient management all data on antibody identification, screening and transfusions were routinely recorded in the electronic database of the Georgia Comprehensive Sickle Cell Program at Grady Health System. We gathered red cell antigen phenotype, dates and results of antibody screens, antibody specificity, and demographics which include but not limited to age in years and gender of all transfused patients.

Results:

A total of 424 patients 230 females and 194 males were identified with SCD who have been tested for antibodies during this period. The average age of the patients is 34 (range 18–84). A total of 138/32.5% of patients had antibodies. The most common antibodies were E (14%), C (10%) and K (8%). This may be an under estimations because antibodies disappear over time without repeated exposure. A total of 82 (19.3%) of patients developed autoantibodies. Detailed results described in Table 1.

Conclusion:

Allo and auto antibody formation remains significant limiting factor in finding compatible blood resulting in delays in availability of blood for transfusion that can compromise patient care in SCD. With better supportive care and hydroxyurea, adult population is growing resulting in increased need for blood transfusion and risk of antibody formation. Our practice is to do limited phenotypic matching in all patients and extended phenotype matching once antibodies are produced. Despite this practice allo- and autoimmunization continues to be a significant problem. Studies need to be done to understand pathophysiology of antibody formation in SCD and prospective randomized trials are required to define best preventive clinical practices.

Disclosures:

No relevant conflicts of interest to declare.
Antithymocyte Globulin (ATG) Plus Cyclosporine (CsA) and Combined with Cord Blood Infusion Is Superior to ATG Plus Csa for the Treatment of Severe Aplastic Anemia
Authors: Fan Zhiping , Liu Qifa , Sun Jing , Jiang Qianli , Huang Fen , Zhang Xian , Xu Dan , Zhou Hongsheng , Xu Bing , Meng Fanyi , Liu Xiaoli , Feng Ru
Published: October 2019

Abstract 4397

Backgrouds and objectives:

Standard initial immunosuppressive therapy (IST) is ATG and CsA, which produces hematologic recovery in 60% to 70% of severe aplastic anemia (SAA), but the shortcoming is the long duration for hematologic response, the higher relapse rate and clonal evolution. In addition to stem cells, Cord blood contains plenty of mesenchymal cells, regulatory T cells, dendritic cells and hematopoietic factors. So we predicted that cord blood coinfusion could improve the effect of ATG plus CsA for treating SAA. The aim of the current study was to investigate the safety and effect of ATG plus CsA combined with cord blood infusion SAA.

Methods:

From August 2008 to May 2012, nine patients (6 males and 3 females), with a median age of 22 years (14–52) were diagnosed with SAA. They received an IST of rabbit ATG (3mg/kg/d, for 5 days) plus CsA (5mg/d, continuously), and then accepted a five or six loci matched cord blood infusion in 7–10 days after the end of ATG. The median nuclear cells were 4.02 (3.1–5.62) ×107/kg.

Results:

After a median duration follow-up of 38 (1–50) months, the response rate was 88.9% (8/9), the median duration of plantlet transfusion independence was 34(18–72) days and that of red blood cell was 30 (18–56) days, which were shorter than those of standard IST for SAA. Only 2 patients underwent serum sickness, 1 patients was diagnosed as having upper respiratory tract infection, and none underwent sepsis and invasive fungal infection. In addition, EB viremia was found in 3 patients, and all of them were cured by antiviral therapy. Of all 9 patients, only one showed evidence of partly engraftment (the percentage of cord blood was 24.6%) as determined by STR-PCR at 15 days after cord blood infusion, the others had no evidence of engraftment at 15days, 1 month and three months after infusion. No myelodysplastic syndrome or acute myeloid leukemia was found among the patients. No patient relapsed till now.

Conclusions:

ATG plus CsA and combined with cord blood infusion was safe, the rate of hematologic response was higher than traditional IST for SAA. The duration of transfusion independence was shorter than that of traditional IST.

Disclosures:

No relevant conflicts of interest to declare.
CD109 Deficiency Induces Preferential Proliferation of the Piga Mutant Leukemia Cell Line TF-1 in the Presence of TGF-β
Authors: Hosokawa Kohei , Katagiri Takamasa , Saito Chizuru , Espinoza Luis J. , Sasaki Yumi , Seiki Yu , Yamamoto Yasuhiko , Yamazaki Hirohito , Nakao Shinji
Published: October 2019

Abstract 4399

Background:

An increase in glycosylphosphatidylinositol-anchored protein-deficient (GPI-AP−) blood cells is a significant predictor of a good response to immunosuppressive therapy in patients with aplastic anemia (AA) and low-risk myelodysplastic syndrome (MDS). Immune mechanisms are therefore thought to be involved in the increase of GPI-AP− cells in such bone marrow (BM) failure, though the exact mechanisms are unknown. The lack of GPI-APs due to PIGA mutations may render hematopoietic stem cells (HSCs) resistant to myelosuppressive cytokines such as TGF-b. This may lead to preferential activation of PIGA mutant HSCs in immune-mediated BM failure, although little is known about the GPI-APs involved.

Objectives/Methods:

We assessed the roles of GPI-APs in TGF-b-mediated inhibition of cell proliferation, cell cycle, and signal transduction in the myeloid leukemia cell line TF-1, TF-1 with a PIGA mutation (PNH-TF-1), and TF-1 cells transfected with siRNA against CD109, a GPI-AP that serves as a TGF-b co-receptor in human keratinocytes. We used flow cytometry to study expression of CD109 in BM CD34 cells of healthy individuals and other hematopoietic cell lines.

Results:

TGF-b inhibited PNH-TF-1 proliferation to a lesser degree (percentage of inhibition, 19±13%) than wild-type TF-1 cells (67±3%) in an MTT-based proliferation assay. Transfection of PIGA into PNH-TF-1 cells restored GPI-AP expression as well as sensitivity to TGF-b (53±10% versus 19±13% in PNH-TF-1 cells). CD109 coimmunoprecipitated with TGF-b in TF-1 cells, and its expression was confirmed on BM CD34+ cells of healthy individuals, particularly on CD34+CD38+CD123−CD45RA− megakaryocyte-erythroid progenitor cells, and also on TF-1 and KG1a cells. CD109 knockdown rendered TF-1 cells less sensitive to TGF-b-induced inhibition of proliferation (26±10% versus 67±3%), TGF-b induced cell cycle arrest (a relative increase in the G0/G1 phase of the cell cycle, 6.9% versus 20.7%, Figure) and TGF-b-induced SMAD2 phosphorylation (a relative increase in pSMAD2 MFI, 1±0.3% versus 16%±3%) compared to non-transfected TF-1 cells.

Conclusions:

The lower sensitivity of PIGA mutant TF-1 cells to TGF-b compared to wild-type TF-1 cells is partially due to CD109 deficiency. This deficiency may also account for the preferential activation of PIGA mutant HSCs in immune-mediated BM failure, in which TGF-b suppresses activation of wild-type HSCs.

Disclosures:

No relevant conflicts of interest to declare.
Dasatinib Restores Full Donor Chimerism in Animatinib - Resistant Ph+ ALL Childwith E255K Gene Mutation Relapse Dafter Allo HSCT
Authors: Peng Ching-Tien , Tamamyan Gevorg , WU Kang-Hsi
Published: October 2019

Abstract 4827

Philadelphia chromosome positive acute lymphoblastic leukemia is uncommon among children and very tough to treat. With chemotherapy alone, only 20–30% of children are cured, but tyrosine kinase inhibitors (TKIs) plus chemotherapy and allogeneic hematopoietic stem cell transplantation in 1st complete remission has gradually high possibility of cure. Some recent papers also suggest that chemotherapy plus tyrosine kinase inhibitors (TKIs) may be the initial treatment of choice. Despite this, there is a few data about treatment after relapse.

We introduce a case of 15 years old boy with Philadelphia chromosome positive acute lymphoblastic leukemia early relapse after full-match related donor hematopoietic stem cell transplantation from his mother.

Both at the time of diagnosis and at relapse time the patient had various cytogenetic abnormalities and also at relapse he revealed with imatinib-resistant E255K gene mutation, which is a common mutation that occurs frequently after imatinib therapy in Ph-positive ALL patients is the glutamic acid to lysine mutation at codon 255.

After the relapse the boy received high-dose chemotherapy and dasatinib and obtained complete molecular remission and restored full donor chimerism. There were no observed side effects of dasatinib treatment. The patient is now still in complete molecular remission and is continuing maintenance chemotherapy with dasatinib.

Thus, this case suggests that chemotherapy plus dasatinibcould be a treatment of choice for the children with relapsed Philadelphia chromosome positive acute lymphoblastic leukemia after allogeneic hematopoietic stem cell transplantation, which alsohas E255K gene mutation and various cytogenetic abnormalities.

Disclosures:

No relevant conflicts of interest to declare.
Heparanase Regulates Response to Proteasomal Inhibition in Myeloma
Authors: Ramani Vishnu Prakash C. , Sanderson Ralph D.
Published: October 2019

Abstract 4902

Multiple myeloma is an incurable hematologic malignancy. In patients, even after several rounds of high-dose chemotherapy, myeloma tumor cells survive, proliferate aggressively and re-establish the active disease. Understanding the molecular mechanisms that aid myeloma cells in evading chemotherapy is therefore critical for curing this deadly disease. Studies from our lab have established heparanase, an endoglycosidase expressed highly by myeloma tumor cells, as a master regulator of aggressive myeloma disease where it drives tumor growth, angiogenesis, invasion, and osteolysis. However, the effect of heparanase on response to chemotherapy is unknown. Addressing this question, we have made two striking discoveries: i) treatment with proteasomal inhibitors dramatically elevates heparanase expression and secretion in myeloma, and ii) high heparanase expression significantly reduces therapy-induced apoptosis and cell death in myeloma. Proteasomal inhibition by either bortezomib or MG132 dramatically elevated the expression of heparanase and its secretion into the conditioned media. When this conditioned media was added to tumor cells that had not been exposed to proteosome inhibitors, the heparanase was readily taken up by the cells. This is consistent with previous studies demonstrating that exogenous heparanase can be taken up by myeloma tumor cells resulting in activation of aggressive tumor behavior.

Next, to test whether heparanase influences chemoresistance in myeloma, we evaluated the effect of bortezomib on inducing apoptosis of myeloma cells expressing either high (HPSE-high) or low (HPSE-low) levels of heparanase. Apoptotic cells were identified by staining for Annexin V and cell death was assessed by staining with propidium iodide. Results revealed that following treatment with bortezomib, HPSE-high cells had a significantly higher percentage of viable cells (Annexin V negative, PI negative) and a significantly smaller fraction of apoptotic cells (Annexin V positive) compared to HPSE-low cells. Similarly, even when myeloma cells were grown on fibronectin, cells expressing high levels of heparanase exhibited significantly decreased bortezomib-induced cell death. Consistent with the above findings, we found that several well established chemoresistant myeloma cell lines such as LR5, Dox 6 and Dox 40 all expressed high levels of heparanase. Together the above findings demonstrate that tumors respond to proteosome inhibitors by upregulating expression and secretion of heparanase and that tumor cells having high levels of heparanase resist proteosome inhibitor-induced apoptosis. These results indicate that heparanase may play an important role in chemoresistance of myeloma cells and that heparanase inhibitors used in tandem with other drugs may improve initial patient outcome and reduce incidence of relapse.

Disclosures:

No relevant conflicts of interest to declare.
Appearance of Monoclonal Plasma Cell Diseases in Whole Body MRI in 544 Patients and Correlation with Parameters of Disease Activity
Authors: Hillengass Jens , Kilk Kerstin , Listl Karin , Hielscher Thomas , Neben Kai , Hose Dirk , Ho Anthony D. , Weber Marc-André , Landgren Ola , Goldschmidt Hartmut
Published: October 2019

Abstract 4966

Focal lesions (FL) and diffuse tumor cell infiltration detected by whole body MRI (WB-MRI) have been demonstrated to be of prognostic significance for predicting progression free and overall survival in patients with monoclonal plasma cell diseases. In this trial we have assessed 544 unselected and untreated patients: 138 with monoclonal gammopathy of undetermined significance (MGUS), 157 with smoldering myeloma (SMM), and 249 with multiple myeloma (MM). WB-MRI was performed on two identical 1. 5 Tesla MRI-scanners with body array curls. Assessment of FL was done by two experienced radiologists blinded to the diagnosis of the patients in consensus. We found focal lesions in 23. 9%, 34. 4%, and 80. 3% of patients with MGUS, SMM and MM, respectively, and a diffuse infiltration was detected in 38. 4%, 45. 9%, 71. 5% of the corresponding patients. The differences between MGUS, SMM and MM patients were statistically significant (p<0. 0001). The presence of FL as well as the presence of a diffuse infiltration was correlated with an increased plasma cell percentage in bone marrow (p<0. 0001) and monoclonal protein concentration (p=0. 001). Further categorization of the diffuse infiltration patterns in WB-MRI into minimal, moderate, severe and “salt&pepper” patterns, was able to identify a significant correlation between both M-protein and plasma cell percentage in bone marrow as well as age. In MGUS and SMM patients, FL were more often detected in patients with a diffuse background, while in MM patients, FL were present at the same rate across the diffuse infiltration subgroups. In summary bone marrow infiltration in WB-MRI is significantly different between stages of plasma cell disease and correlates with established markers of disease activity.

Disclosures:

No relevant conflicts of interest to declare.
The Difference of Macrophage Involvement in Multiple Myeloma Patients with Distinct Prognosis
Authors: Yu Wu , Zhu Huangling , Long Li Xiang , Liu Ting
Published: October 2019

Abstract 4979

Recently the essential role of macrophages in the drug resistance or pathogenesis of plasma cell myeloma has gradually been identified and investigated. Here we analyzed patients diagnosed as multiple myeloma in West China Hospital, Si Chuan University, China in the past 3 years. The study involved 156 patients, aged 34–81 year old, mean age 60. 4 year old, male to female ratio is 1. 47:1, including 2 and 3 stages of myeloma according to International Staging System. The therapeutic response was evaluated on the basis of criteria of International Myeloma Working Group Uniform Response Criteria in patients who had completed 3 therapeutic dexamethasone- containing cycles. We observed the involvement of macrophages in bone marrow by immunohistochemical staining of anti-CD68 monoclonal antibody (DAKO). During the observation, two pathologists without any knowledge of clinical data find the “hot spot ” of myeloma cells and then enumerated the number of macrophage in “ hot spots ” under 400 magnification. Interestingly, patients with more macrophage involvement (>40/hp) in bone marrow showed poorer response (including PR, VGPR and CR after 3 cycles of chemotherapy) (31. 2%) to Dexamethasone-containing chemotherapy and higher incidence of disease progression-related deaths. On the contrary, the patients with lower macrophage involvement demonstrated much better response (91. 6%) to chemotherapy and persistent remission (figure 1 the response rate analyzed by macrophage involvement).

In summary, the difference of macrophage involvement in bone marrow of multiple myeloma patients with distinct prognosis suggested the macrophage inside bone marrow play a role in myeloma pathogenesis and therapeutic response.

Disclosures:

No relevant conflicts of interest to declare.
Clinical Significance of Immunoglobulin Isotype Switching in Patients with Multiple Myeloma
Authors: Jo Jae-Cheol , Yoon Dok Hyun , Kim Shin , Lee Kyoungmin , Kang Eun Hee , Jang Seongsoo , Park Chan-Jeoung , Chi Hyun Sook , Huh Jooryung , Park Chan-Sik , Suh Cheolwon
Published: October 2019

Abstract 4980

Background:

Multiple myeloma (MM) is characterized by clonal expansion of malignant bone marrow cells producing a unique monoclonal immunoglobulin. However, immunoglobulin isotype switching (IS) has been reported during follow-up of patients with MM. Its clinical significance has not been established well. We aimed to evaluate the clinical characteristics of the patients with IS and outcomes in those patients in a single center cohort.

Patients and Methods:

A total of 377 consecutive MM patients were treated at the Asan Medical Center between January 2002 and June 2012. We compared clinical characteristics and outcomes between those with and without IS.

Results:

Of the 377 patients, 34 (9%) demonstrated IS after a median 7. 9 months (range, 2. 2–95. 7 months) from diagnosis. These 34 patients with IS comprised 18. 2% (27/148) of patients treated with autologous stem cell transplantation (ASCT) and 3. 1% (7/229) of patients treated without ASCT (P<0. 001). Original immunological types were as follows: IgG (n=10), IgA (n=8), IgD (n=5), free kappa (n=4), and free lambda (n=7). Nine patients among them demonstrated second IS during follow-up. With a median follow-up times of 54. 1 months from the day of diagnosis in all study patients, the median OS has not been reached in patients with IS, and was 38. 3 months in patients with the absence of IS (P<0. 001). Multivariate analysis indicated that development of IS was a significantly favorable prognostic factor for predicting OS (Hazard ratio = 0. 19; 95% confidence interval = 0. 07–0. 53). ECOG PS 0–1 vs. 2–4, creatinine <2 mg/dL vs. ≥2 mg/dL, bone marrow plasma cells <50% vs. ≥50%, and ASCT done vs. not done were also independent prognostic factors for OS (P=0. 037, P<0. 001, P=0. 008 and P<0. 001, respectively). In the group of patients presenting with IS (n=34), differences in overall survival (OS) were not observed according to the timing of IS after diagnosis (<8 mo vs. ≥8 mo, P=0. 836), immunoglobulin isotypes (P=0. 176) and presence of second IS (P=0. 494), while ASCT was associated with longer survival (median OS; not reached vs. 65. 9 mo, P=0. 018).

Conclusions:

We confirmed higher frequency of IS in those who underwent ASCT and favorable prognosis in those experiencing IS. Its significance and underlying mechanism warrants further investigation.

Disclosures:

No relevant conflicts of interest to declare.
Clinical Comparison of the Freelite and N-Latex Serum Free Light Chain (FLC) Assays in the Diagnosis and Monitoring of AL Amyloidosis
Authors: Mapp Sally , Tate Jill , Pretorius Carel , Mollee Peter
Published: October 2019

Abstract 4991

Aims:

We compared a novel assay for FLC quantitation based on monoclonal antibodies (N Latex) to the established polyclonal antibody-based assay (Freelite) in AL amyloidosis.

Methods:

62 diagnostic samples from patients with AL amyloidosis were analysed, 32 of which also had a post-treatment sample. Serum FLC concentration was measured by Freelite (The Binding Site, UK) and N Latex (Siemens, Germany) immunoassays on a BNII nephelometer. Serum and urine immunofixation electrophoresis (IFE) was performed on Hydrasys gel systems (Sebia, France).

Results:

In the diagnostic samples: for AL of kappa type (n=18), the median involved FLC (iFLC) was significantly lower by the N Latex assay (289 vs 667mg/L, p=0. 0002) whereas in AL of lambda type (n=44) the values were similar (148 vs 161mg/L, p=0. 84). Measurable disease has recently been redefined as a difference between the involved and uninvolved FLC (dFLC) of > 50mg/L. By these criteria, 82% AL would be measurable by the N Latex assay compared to 89% by the Freelite assay. In terms of the diagnostic sensitivity, the FLC ratio was normal in 21% and 15% of patients by the N Latex assay and Freelite assays, respectively. The combination of serum and urine IFE with either FLC assay, however, allowed identification of the amyloidogenic clone in 98% producing comparable sensitivity for both methods.

For the 32 patients with monitoring samples the median overall survival was 50 months. The median reduction in dFLC was 40% for the N Latex assay and 61% for the Freelite assay (p=0. 03). This led to some differences in assigning response categories. In patients with measurable disease (dFLC>50mg/L), a partial remission (dFLC reduction >50%) predicted overall survival by the N Latex (n=25, 2yr OS 82% vs 27%, p=0. 0015) and Freelite assays (n=29, 2yr OS 75% vs 36%, p=0. 02).

Conclusions:

There are significant differences between iFLC as measured by the N Latex and Freelite assays, but overall the two assays have similar diagnostic sensitivity when used in combination with serum and urine IFE. In the monitoring context, preliminary data suggests that disease response as assessed by the N Latex FLC assay predicts overall survival. Because of the likely widespread introduction of this assay and the differences in absolute levels of iFLC measured by these assays, consensus criteria may need to be reconsidered.

Disclosures:

No relevant conflicts of interest to declare.
GPX3 Level in Serum of Diffuse Large B Cell Lymphoma Patients Is a Predictive Biomarker for Refractoriness Before Treatment
Authors: Villela Luis , Fajardo Oscar , Leon Darinka , Hernandez-Hernandez Jose A , Scott Sean , Encarnacion Sergio
Published: October 2019

Abstract 5102

Introduction.

Diffuse Large B-Cell Lymphoma (DLBCL) is the most frequent subtype of Non-Hodgkin Lymphoma in the world (35%). Despite of R-CHOP chemotherapy, patients with DLBCL continue to relapse/refractory after treatment. For this reason, the search for biomarkers that could predict refractoriness is necessary for personalized chemotherapies. There are some studies where antioxidant pathways are associated to refractoriness. Our group using a proteomic approach observed that GPX3 is a frequent protein observed in serum of DLBCL patients compared to healthy controls (72% vs. 12%; p=0. 009. Villela et al. Abstract EHA, 2012-only publication).

Aim.

To evaluate GPX3 levels as a biomarker for primary refractory in patients involved by DLBCL and before treatment.

Methods.

This study was registered and accepted for IRB's Institutional. From 40 DLBCL patients and 10 healthy controls were obtained serum previous being treated with R-chemo or Chemo alone or as part of donation. We used a commercial ELISA kit (USCNK, Life Science) for obtained GPX3 serum level. We evaluated classical clinical variables and compared to GPX3 level cut off that was obtained trough Area Under Curve (AUC) for primary refractory as dependent factor. Statistical analyses. Descriptive variable Chi2, quantitative variable T-Student or Independent-Sample Median-Test, Relative risk (RR) and confidence intervals 95% were calculated (CI95%).

Results.

Using proteomic approach, GPX3 was the most frequent protein observed in 72% of the cases, compared with the control group where was only observed in 12% (p=0. 009). These data were validating in 40 patients affected by Diffuse Large B Cell Lymphoma and 10 healthy people using ELISA Kit. We observed difference between GPX3 level in lymphoma patients vs. Healthy control [median (SD): 22(±5. 16) vs. 51(±22), p<0. 001). We obtained GPX3 cut off for AUC as a predictor for overall response vs. primary refractoriness (≥34. 75 mcg/dL vs < 34. 75 mcg/dL, respectively; AUC: 0. 67 (CI95%: 0. 5–0. 82). Variables associated to primary refractoriness vs. no refractoriness were GPX3 (44% vs. 17% respectively, RR=2. 55 [CI95%: 0. 87 to 7. 6]; p=0. 094) and no rituximab vs. rituximab combined in chemotherapy (45. 5% vs. 15. 4% respectively, RR=2. 95 [CI95%: 0. 97 to 8. 96]; p=0. 051. When we analyzed patients treated with vs. without rituximab depending GPX3 level (Cut off 34. 75 mcg/dL), we observed better response when patients received rituximab and had high level of GPX3 (67% vs. 33. 3%, RR= 0. 5 [CI95%: 0. 39 to 1. 21]; p=0. 085].

Conclusions.

The GPX3 and Rituximab use were a trend to predictive biomarker of refractoriness but more studies must confirm our data.

Disclosures:

No relevant conflicts of interest to declare.
Beta Thalassemia Trait: How the New Information Provided by the Routine Hematology Analysers May Help in Its Differential Diagnosis or Flagging
Authors: Barella Susanna , Simon-Lopez Ramon , Gaetano Nicola Di , Galanello Renzo
Published: October 2019

Abstract 5186

Introduction:

Beta Thalassemia (β-thalassemia) is one of the more common hemoglobinopathies worldwide, being the heterozygous variant, called Beta Thalassemia Trait, a benign variant, but important to diagnose, for genetic counseling, trying to avoid the homozygous variant, called major.

Diagnostic of Beta Thalassemia Trait:

Classic testing for β-thalassemia includes: hematologic testing of red blood cell indices, peripheral blood smear (prewsence of target cells and RBC with basophilic stippling, etc.), and qualitative and quantitative hemoglobin analysis. Have been proposed too Discriminant functions, like the one published many years ago, by England and Fraser.

Objective:

Recently have been developed new parameters and information in the new automated hematology analyzer called DxH8008™ from Beckman Coulter as @MSCV, @RSF, @MAF, @ LHD% and many morphological parameters for RBC and Reticulocytes calles Cell Population Data. All this parameters may be used to create flagging for laboratory use only (LUO) or Research use only (RUO). The purpose of this study is to investigate the possible use or utility of this new information for the screening/flagging of Beta Thalassemia Trait.

Patient and Methods:

We have collected 30 patients with Beta Thalassemia Trait. All of them were confirmed by red cell morphology, Hgb Electroforesis, cromatography in liquid phase in human whole blood for the determination of Hemoglobin A2, F, A1c, and identification of abnormal hemoglobins and DNA analysis (DNA Analysis by GAP-PCR). We have compared these patients with a control group (184 individuals) and with other anemias (see Table 1).

Results:

Using ROC analysis, the best parameters differentiating the Beta Thalassemia Trait from the normals were: MCV (AUC 1. 000), MRV (AUC 0. 999), @MAF(AUC 0. 999), @MCNRET (AUC 0. 997), RDW (AUC 0. 957), HGB (AUC 0. 915), RBC(AUC 0. 912).

Using ROC analysis, the best parameters differentiating the Beta Thalassemia Trait from other anemias (excluding normals) were: RDW-SD (AUC 0. 937), DF Eng-Fra (AUC 0. 779), RDW (AUC 0. 766), RBC (AUC 0. 734)

Disclosures:

Simon-Lopez: Beckman Coulter: @LHD, @MAF, @RSF, @LHD, @MAF, @RSF Patents & Royalties, Employment. Di Gaetano:Instrumentation Laboratory spa: Work for a distributor of Beckman Coulter Instruments in Italy Other. Galanello:Ferrokin: Research Funding; Apopharma: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau.
Targeting Deptor in Multiple Myeloma
Authors: Yang Yonghui , Gera Joseph , Lichtenstein Alan
Published: October 2019

Abstract 575

DEPTOR is an mTOR binding protein specifically over-expressed in some genetic subtypes of multiple myeloma (MM). DEPTOR binds mTOR in TORC1 and TORC2 complexes and inhibits their kinase activity. As a result, p70 and 4E-BP1 phosphorylation, substrates of TORC1, are decreased. However, TORC1/p70 down-regulation results in marked feedback activation of the PI3K/AKT pathway and AKT becomes activated even though DEPTOR restrains TORC2. Most importantly, DEPTOR knockdown is deleterious to over-expressing MM cell lines, resulting in apoptosis. We hypothesized that compounds preventing DEPTOR-mTOR association would have cytotoxic effects in these cell lines, comparable to DEPTOR knockdown. We, thus, developed a yeast two-hybrid assay where survival of yeast in histidine-depleted media depended upon an association between DEPTOR and mTOR in genetically engineered yeast. After demonstrating a robust yeast two-hybrid protein interaction, yeast were screened against the NCI small inhibitor library (>150,000 compounds) and six compounds were identified that inhibited the interaction. These compounds rapidly increased p70 phosphorylation in MM cell lines in a concentration-dependent fashion, consistent with an inhibition of DEPTOR's inhibitory effect on mTORC1. Co-immunoprecipitation confirmed the compounds inhibited binding of DEPTOR to mTOR without effects on an mTOR-RICTOR interaction. The compounds also significantly inhibited cell survival of cell lines and they were at least 10 × fold more effective against DEPTOR over-expressing MM lines (8226, OPM-2, MM1.S) versus non-expressing lines (U266, FR4, delta 47). These results support a drug development strategy for myeloma that focuses on preventing DEPTOR binding to mTOR within TORC complexes for subsequent anti-myeloma cytotoxic effects.

Disclosures:

No relevant conflicts of interest to declare.
A Screen for Small Molecules That Regulate Hepcidin Expression
Authors: Volovetz Josephine , Zhen Aileen W. , Gaun Vera , Patchen Bonnie , Fraenkel Paula G.
Published: October 2019

Abstract 611

Hepcidin, a peptide hormone produced in the liver, decreases intestinal iron absorption and macrophage iron release via effects on ferroportin. Hepcidin is a potential drug target for patients with iron overload syndromes because its levels are inappropriately low in these individuals. To generate a tool for identifying small molecules that modulate Hepcidin expression, we stably transfected human hepatocytes (HepG2) cells with a reporter construct containing 3 kilobases of the human Hepcidin promoter upstream of a firefly reporter gene. We then used high throughput methods to screen 10,360 chemicals in duplicate from the Harvard Institute of Chemistry and Cell Biology library for their effect on Hepcidin expression and cell viability. Regulators were identified as chemicals that caused a change >3 standard deviations above or >1.5 standard deviations below the mean of the other chemicals (z-score >3 or <-1.5), while not adversely affecting cell viability, quantified by a nonlytic fluorescence assay. Using these criteria, we identified 32 small molecules that upregulated and 3 that downregulated Hepcidin expression. Functional classification of the positive regulators indicated: 4 anti-inflammatory agents, 4 antimicrobials, 6 antineoplastic drugs, 6 kinase inhibitors, and 12 with other or unknown function. Of the positive modulators, two were flavones, consistent with our prior discovery that the isoflavone genistein upregulates Hepcidin expression. Of the negative regulators, one was a kinase inhibitor and two were of unknown function. Experiments are underway to characterize the mechanism of action of these regulators. The best candidates will subsequently be tested in mouse models of iron overload syndromes with the intention of developing new therapies for diseases in which Hepcidin is inappropriately regulated.

Disclosures:

No relevant conflicts of interest to declare.
HbF-Associated Genetic Variation Marks an Erythroid Regulatory Element Essential for BCL11A Transcription and Subsequent Stage-Specific Globin Expression
Authors: Bauer Daniel E. , Kamran Sophia C , Xu Jian , Lin Carrie , Shao Zhen , Fujiwara Yuko , Voit Richard A. , Porteus Matthew H. , Stamatoyannopoulos John A. , Orkin Stuart H.
Published: October 2019

Abstract 828

Genome-wide association studies (GWAS) have identified common genetic variation within a 14-kb segment of intron-2 of the BCL11A gene that is associated with the level of fetal hemoglobin (HbF) and the clinical severity of the β-globin disorders sickle cell disease and β-thalassemia. Functional studies have validated BCL11A as a major repressor of HbF. Here, we explore the consequences of genetic variation on BCL11A expression. The HbF-associated variants overlap BCL11A sequences that possess an evolutionarily conserved enhancer chromatin signature, including the presence of H3K4me1 and H3K27ac and the absence of H3K4me3 histone modifications. Moreover, these sequences show a unique pattern of DNase I hypersensitivity in erythroid precursors compared to other BCL11A-expressing lineages including fetal brain and B-lymphocytes. Occupancy of the major erythroid transcription factors GATA1 and TAL1 is observed at these DNase I hypersensitive sites (DHS). Chromosome conformation capture studies demonstrate that these DHSs form a long-range interaction with the BCL11A promoter.

To address the function of the putative regulatory element, we generated transgenic mice with the 12-kb intronic region attached to a minimal promoter and reporter gene. The element enhanced reporter gene expression in an erythroid-lineage restricted, definitive erythroid stage-specific manner. The minimal enhancer activity was mapped to the +58-kb DHS. Similar enhancer activity was observed in primary human erythroid precursors. To address the possible requirement of this element for BCL11A expression, we deleted the corresponding region in adult-stage murine erythroleukemia cells with TALEN-mediated gene targeting. Expression of BCL11A was dramatically reduced and embryonic globins Hbby and Hbb-bh1 were markedly derepressed with concomitant reduction of adult globin Hbb-b1. Analysis of heterozygous primary human erythroid precursors revealed that low-HbF haplotype alleles are preferentially occupied by GATA1 and TAL1 at sequences surrounding rs1427407. A composite half-E-box–GATA motif is disrupted at this SNP in the high-HbF allele. Furthermore, the low-HbF haplotype allele of BCL11A is expressed at a 1.7-fold higher level relative to the high-HbF allele. These data demonstrate that HbF-associated genetic variation in BCL11A modulates the function of a critical erythroid enhancer of BCL11A, and consequently expression of BCL11A itself. The quantitative level of BCL11A in turn regulates the relative expression of the stage-specific globin genes. The essential erythroid enhancer of the BCL11A gene might itself serve as a target for genetic manipulation as a novel approach to the human β-globin disorders.

Disclosures:

Porteus: Alex's Lemonade Stand Foundation: Research Funding.
Activation of A1 Domain Adhesiveness in von Willebrand Factor by Elongational Force
Authors: Springer Timothy A. , Kim Jongseong
Published: October 2019

Abstract SCI-16

Multiple mechanisms may contribute to activation of von Willebrand factor (vWF) adhesiveness by elongational flow at sites of hemostasis, including enhancement of A1 domain exposure within vWF concatamers and conformational change within the A1 domain or its complex with GPIbα. A receptor and ligand in a single molecule (ReaLiSM) containing the A1 domain, a flexible linker, and GPIbα fused in a single polypeptide and suspended between beads using DNA handles was interrogated with a laser trap. Two pathways for unbinding representing flexed and extended states were previously reported, with the flexed, more stable state predominantly at forces above 10 pN. vWD type 2B mutations in the A1 domain selectively stabilize the extended, high affinity state, whereas platelet-type vWD mutations in GPIbα stabilize both states. With ReaLiSM, we can also measure the kinetics and force-dependence of receptor-ligand binding. Remarkably, we also see two on-rates for receptor-ligand binding, with the faster on-rate predominating above 10 pN and the slower on-rate predominating below 10 pN. vWD type 2B mutations in the A1 domain selectively increased the fast on-rate, whereas platelet-type mutations in GPIbα increased both on-rates. Our results support force-dependent conformational change as one of the mechanisms that activates vWF in hemostasis.

Disclosures:

No relevant conflicts of interest to declare.
Biology of Myelodysplasia Clonality: Clinical Implications of Whole-Genome Sequencing
Authors: Walter Matthew J.
Published: October 2019

Abstract SCI-32

The discovery of acquired genetic mutations associated with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) has rapidly expanded with the use of next-generation sequencing technologies. The distinction between MDS and de novo or secondary AML (arising after MDS) and therapeutic recommendations are largely based on the presence of morphologic dysplasia and the percentage of bone marrow myeloblasts. Using whole genome sequencing to define the allele burden of hundreds of mutations, our group has shown that nearly all the bone marrow cells in seven patients with MDS and secondary AML were clonally derived (∼85%), regardless of the myeloblast count. We now have experimental evidence suggesting that the vast majority of mutations in these samples are randomly acquired during aging (passenger mutations) and carried forward in a cell when it is transformed. By using all the validated mutations in a sample (passengers and drivers) we found that five of the seven MDS samples harbored two or more malignant clones and that all seven contained a founding clone that gave rise to subsequent daughter clones. Thus, the MDS bone marrow samples are a mosaic of several tumors with unique sets of mutations each shaped by the acquisition of a series of mutations and clonal diversification. Each clone contained at least one coding gene mutation, and we identified 11 recurrently mutated genes in the seven samples, including recently described mutations in U2AF1, a spliceosome gene that is typically mutated in the MDS founding clone. In addition, the MDS founding clone always gave rise to the dominant secondary AML clone, suggesting that therapies targeting mutations in the founding clone may have benefit in treating both MDS and secondary AML. It is possible that therapeutic response and disease progression is driven not only by the presence of recurrent mutations, which have prognostic value, but also by the clone (i.e., founding and daughter) in which they arise. We are currently testing this hypothesis by monitoring the dynamic change in clone size, including driver mutation allele burdens, during treatment of patients with AML and MDS.

Disclosures:

No relevant conflicts of interest to declare.
Microbial Impact on Hematologic Homeostasis
Authors: Manz Markus G.
Published: October 2019

Abstract SCI-40

During systemic infection and inflammation, immune effector cells are in high demand and are rapidly consumed at sites of need. While adaptive immune cells have high proliferative potential, innate mature immune cells are mostly postmitotic and need to be replenished from bone marrow hematopoietic stem and progenitor cells. Indeed, severe clinical infection, particularly infections challenging the innate immune response, lead to an increase in hematopoietic differentiation and throughput in bone marrow, involving subsequent differentiation stages from hematopoietic stem cells, multipotent progenitors, as well as early-lineage and late-lineage restricted hematopoietic progenitors. A fundamental question is how the increased need is sensed and translated in enhanced production and how adequate levels of response are guided. Recent research has shed light on conserved intracellular and extracellular pathogen recognition receptors, such as Toll-like receptors, that are expressed on nonhematopoietic and hematopoietic effector cells and cause activation upon ligation. This activation results in production of hematopoietic growth, survival, activation, and migration factors operating at site on effector cells, but also at remote primary hematopoietic sites to increase production upon need. Recent research by several groups, including ours, surprisingly revealed that conserved pattern-recognition receptors are also expressed on hematopoietic stem and progenitor cells in bone marrow, implying a direct effect of systemically available ligands on these cellular populations. Indeed, it has been demonstrated that, for example, ligation of Toll-like receptor 4 by its cognate agonist lipopolysaccharide can lead to divisional activation, proliferation, lineage-directed differentiation, and migration of hematopoietic stem and lineage-restricted progenitor cells, all aimed at efficient contribution to immune responses and rapid reestablishment of hematopoietic homeostasis. The relative contribution of pathogen sensing by hematopoietic and diverse nonhematopoietic cells to appropriate hematopoietic responses, as well as the subcellular translation of the signals, is the focus of ongoing research. Also to be discussed will be how chronic infectious and inflammatory processes, which are frequently associated with aging, might impinge on hematopoiesis, potentially fostering hematopoietic stem cell diseases as exhaustion or transformation.

Disclosures:

No relevant conflicts of interest to declare.
Cyclophosphamide Related Cardiac Complication in Hematopoietic Transplantation: Analysis of 1091 Cases in Fukuoka Blood and Marrow Transplantation Group
Authors: Kohno Kentaro , Takase Ken , Kadowaki Masanori , Yamasaki Satoshi , Kamimura Tomohiko , Ohno Yuju , Eto Tetsuya , Miyamoto Toshihiro , Okamura Seiichi , Akashi Koichi
Published: October 2019

 Cyclophosphamide (CPM) is one of key drugs for conditioning therapy in hematopoietic transplantation in terms of anti-tumor effect as well as immune-suppressant activity. On the other hand, high dose CPM administration is related to rare but life-threatening cardiac complications, of which incidence and pathogenesis are not fully understood. We retrospectively reviewed the hematopoietic transplantation cases in Fukuoka Blood and Marrow Transplantation Group (FBMTG) during 2000/1/1 to 2013/7/31 and analyzed the cases with the cardiac complications.

 There were 1091 cases, which contained CPM administration in their conditioning regimens. Of which, 23 cases (2.1%) were considered as cardiac complications related to CPM. The mean age at transplantation was 51 (20-74). All of the cases underwent cardiac function surveillance with ultrasound echocardiogram and were administrated more than 45mg/kg/day of CPM. The clinical cardiac manifestation occurred one to seven (mean 3) days after the first administration of CPM. The electrocardiogram showed the statistically significant diminishment of SV1 + RV5 and increase of heart rate. Lowered ejection fraction was observed in ultrasound echocardiogram. Myocardial enzymes such as CPK, Troponins and H-FABP were not very reliable because some cases were negative especially in early phase. Fourteen cases (1.3%) developed acute circulatory failure soon after the first presentation of clinical manifestation and all of them end up with poor prognosis. On the other hand, of the 9 cases (0.8%) who survived first 20 days, 6 of them were alive at 100 days after the transplantation. The incidence of cardiac complication tends to be high in elderly patients especially when they were over 70.

 Cardiac complication related to CPM is rare but it remains to be fatal in present. The development of supportive care has provided higher limit of age that undergo hematopoietic transplantation. However, the administration of high dose CPM in conditioning regimen should be aware especially in elderly recipients.

Disclosures

No relevant conflicts of interest to declare.