bluuebunny/crossref_metadata_2025_split huggingface dataset:
Heparanase Regulates Response to Proteasomal Inhibition in Myeloma
Authors: Ramani Vishnu Prakash C. , Sanderson Ralph D. 
Published: October 2019

Abstract
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
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
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
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
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
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
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
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
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.