bluuebunny/crossref_metadata_2025_split huggingface dataset:
Use of Ubiquitin-Proteasome System Profiling for Differentiating Between Various Leukemic Processes.
Authors: Zhang Ke , Kantarjian Hagop M. , Ma Wanlong , Zhang XI , Wang Xiuqiang , Estrov Zeev , Yeh Chen-Hsiung , Sferruzza Anthony , O'Brien Susan , Keating Michael , Albitar Maher 
Published: October 2019

Abstract
Abstract 4712

The ubiquitin-proteasome system (UPS) plays a major role in cell homeostasis in normal and neoplastic states. Expression and function of the UPS system vary with the specific characteristics of individual cell types, suggesting that determination of UPS “signatures” could be useful in identifying various cell populations. Since direct analysis of cancer cells is often problematic, even in hematologic diseases, we explored the potential of using UPS signatures in plasma to differentiate between various leukemias. We first analyzed plasma UPS profiles of patients with acute myeloid leukemia (AML; n=111), acute lymphoblastic leukemia (ALL; n=29), advanced myelodysplastic syndrome (MDS; n=20), chronic lymphocytic leukemia (CLL; n=118), or chronic myeloid leukemia (CML; n=128; 46 in accelerated/blast crisis [ACC/BL], 82 in chronic phase), and 85 healthy control subjects. Plasma levels of proteasome, ubiquitin (poly-ubiquitin), and the 3 proteasome enzymatic activities (chymotrypsin-like [Ch-L], caspase-like [Cas-L], trypsin-like [Tr-L]) were measured. Specific activities were calculated by normalizing each of the 3 enzyme activities to the levels of proteasome protein in plasma (Ch-L/p, Cas-L/p, and Tr-L/p). These 8 variables were used in multivariate logistic regression models to differentiate between leukemic processes. UPS signatures provided clear differentiation between patients with a leukemic process and normal controls (AUC=0.991), using 6 different variables (Tr-L/P, Ch-L, Ch-L/p, Cas-L, Cas-L/P, ubiquitin). Distinguishing between acute (AML, ALL, MDS) and chronic (CML, CLL) processes was less efficient (AUC=0.853 using Tr-L, Tr-L/P, Cas-L/P, Ch-L/P, proteasome, Ch-L), likely due to the high proportion (36%) of CML patients in ACC/BL phase. However, UPS signatures generally yielded powerful differentiation between individual leukemias (Table). MDS was not well differentiated from AML (AUC=0.791), reflecting the significant biological overlap of these diseases. These data support the potential usefulness of the UPS profile to aid in the differential diagnosis of various leukemias.

Disclosures:
No relevant conflicts of interest to declare.
A New Accurate and Reproducible Method to Assess Telomere Length by Flow-FISH.
Authors: Brocardo Graciela Aparecida , de Godoy Carla Rosa Teixeira , Levy Débora , de Almeida Lis Vilela , Martinez Gracia , Chamone Dalton , Pereira Juliana 
Published: October 2019

Abstract
Abstract 4719

Background
Telomeres have been related to many diseases including cancer, medullar insufficiency and to understand aging. To measure the average length of telomere repeats have been described at least three methods as Southern blot and quantitative fluorescence in situ hybridization using either digital fluorescence microscopy (Q-FISH) or flow cytometry (Flow-FISH). After two independents groups (Hultdin et al., 1998 and Rufer et al., 1999) have described different protocols using Flow-FISH to measure telomere lengths, the Southern blot and Q-FISH methods have been less used. However, when we were performed Hultdin et al., 1998 or Rufer et al., 1999 protocols were found some facts that could cause mistakes in the final results. Afterward, many steps of these protocols were explored to become the Flow-FISH results more accurate and reproducible. In our laboratory we developed a unlike protocol to assess the average length of telomere repeats by Flow-FISH taking advantage of the both Hultdin et al., 1998 and Rufer et al., 1999 techniques that was named Modified Hultdin and Rufer protocol.

Methods
We compare the telomere length repeats measure by three different methods of Flow-FISH with or lacking standard beads for calibrate fluorescence intensity, control cell line 1301 and compensation by DNA index of G0/G1 cells.

Results
We demonstrated a linear relationship between the average length of telomere repeats by Hultdin et al., 1998 protocol and Modified Hultdin and Rufer protocol but showing that standard beads to quantify the fluorescence intensity was important to determine telomere length by Flow-FISH. However, the coefficient correlation (r) between the Rufer et al., 1999 protocol with Hultdin et al., 1998 and Modified Hultdin and Rufer protocols in our laboratory was poor, justifying of the internal control cell and compensation of the telomere length by DNA index of G0/G1 of unknown cells use.

Conclusions
We conclude that use of appropriate internal control cells with knowledge telomere length and DNA index is essential to improve accuracy and reproducibility of the telomere length measurement by Flow-FISH. Indeed the standard beads for fluorescence intensity quantification can allow intercenters comparison of the results.

Disclosures:
No relevant conflicts of interest to declare.
The Critical Requirement of the NKG2D Receptor On CD8+ T Cells in Killing Myeloma Cells.
Authors: Talebian Laleh X. , Fischer Dawn A , Szczepiorkowski Zbigniew M , Sentman Charles L , Meehan Kenneth R 
Published: October 2019

Abstract
Abstract 4733
Autologous stem cell transplant for myeloma improves survival, but the immunologic mechanisms accounting for this improvement are unknown. Our data indicate that NKG2D+CD8+T cells may be one reason for this benefit. NKG2D, one of four NK cell activating receptors, has been identified on some CD8+T cells that mediate TCR-independent and non-MHC restricted tumor cell killing.
We identified methods for ex vivo expansion of cyclophosphamide – mobilized blood progenitor cells (BPCs) from myeloma patients, as part of a clinical trial (Cytotherapy 2008). Mobilized BPC are cultured in serum-free media with IL-2 (50 IU/ml) and OKT3 (50ng/ml). After 7 days, the CD8+ T cells are isolated, evaluated and tested. Myeloma cells from patients' marrow aspirates are used as targets in cytotoxicity assays. Phenotypic analysis of the expanded cells and the patients' primary myeloma cells was performed using flow cytometry. Ex vivo expansion of cyclophosphamide-mobilized BPCs induces CD8+ T cells that acquire the NKG2D receptor during ex vivo expansion (P < 0.03). Three of the 6 known NKG2D ligands are strongly expressed on patients' myeloma cells, including MICA, ULBP1 and ULBP3. Using cytotoxicity assays, autologous effector cells recognize and kill the patient's autologous tumor cells (E:T 50:1, P< 0.001). When using K562 leukemia cells as targets (lack MHC class I), the NKG2D+CD8+ T cells kill K562, supporting an MHC class I-independent mechanism of tumor cell killing. Blocking the NKG2D receptor on CD8+ T cells prevents killing of autologous myeloma cells (P < 0.009). NKG2D-mediated cytotoxicity correlates with the amount of ligand expression on the target.
The NKG2D receptor on CD8+T cells recognizes ligands expressed on autologous myeloma cells. The NKG2D+CD8+T cells aggressively kill myeloma cells in a non–MHC restricted and TCR-independent manner. Blocking NKG2D on CD8+ T cells prevents killing of autologous myeloma cells. Since tumor cells often down regulate MHC Class I expression, the ability of NKG2D+CD8+ T cells to recognize and kill autologous myeloma cells in a non-MHC restricted manner may contribute to the beneficial effects in the treatment of myeloma. Ongoing experiments are testing these methods in mouse models and defining the molecular mechanisms involved.

Disclosures:
Szczepiorkowski: Cersus Corporation: Research Funding; CaridianBCT: Research Funding; BASF: Research Funding; Terumo Corporation: Research Funding; Fenwel, Inc - Scientific Advisory Board: Research Funding. Meehan:Berlex Pharmaceutical: Research Funding.
Effect of Reactive Nitrogen Metabolites and the Scavengers to NK Cell-Mediated Killing of K562 Cells.
Authors: Pan Jingxin , Pan Hongda , Zeng Yili , Guo Jianxin 
Published: October 2019

Abstract
Abstract 4740

Introduction
To explore the influence of the exogenous and endogenous Reactive Nitrogen Metabolites (RNM)as a NK cell supressor andthe effect of Tiopronin (TIP) A Glutamylcysteinylglycine (GSH) Adihydrochloride (DHT) as immunoadjuvant to reverse RNM's suppressing influence in NK cell-mediated killing of K562 cell line.

Methods
1.Exogenous ONOO- was administered in the NK cell and K562 cell culture system, Then TIPAGSH and DHT wereadministered in the cultivated systems espectively. The concentrations of RNM were measured after six hours and the corresponding changes of TNF-βand IFN-γ and K562 cell inhibition rate(KIR) were observed after 24 hours.2. IL-2 and PHA were administered as reactivators in the system of Monocytes(MO) plus NK cells(E) and K562(T) cells (E/T=10/1,E/MO=10/2). Different concentrations of TIP, GSH and DHT were administered in the cultivated systems then the Parameters of NK cell activity include KIR Aproductions of TNF-β and IFN-γ were measured on schedule.

Results
1.After exogenous ONOO- was administered in the NK cell and K562 cell culture system, The population and the activty of NK cells degraded significantly. When the TIPAGSH and DHT were administered in the systems, RNM productions dropped from 260.30 ± 9.51 umom/L to 178.65 ± 10.00 umom/L (P<0.05) A185.02± 8.17umom/L (P<0.05), 255.32 ± 11.93 (P>0.05) respectively. The percentage of live NK cells increase form 79.87 ± 1.03% to 91.13 ± 0.67% (P<0.05) A88.03 ± 1.46% (P<0.05) A82.6 ± 0.76% (P>0.05) respectively, KIR increase from 43.84 ± 0.19% to 59.35 ± 1.08% (P<0.05) A56.64 ± 0.83% (P<0.05) A51.41±1.47% (P<0.05) respectivelyGTNF-βAIFN-γ productions increased correspondingly. When TIP,GSH and DHT were administered in the cultivated systems mixing with NK cells,K562 cells,Monocytes,IL-2 and PHA, the trends of parameter changes we measured were like the result of part 1. With higher concentration of TIP and GSH,the RNM productions decreased accordingly, TNF-β and IFN-γ productions as well as KIR increased at the same time,while DHT can not clear RNM.

Conclusions
Exogenous ONOO-is deleterious to NK cells and K562 cells. Monocytes are the major resource of endogenous RNM. The RNM can disable NK cells in killing K562 cells. Both of TIP and GSH can protect NK cells via scavenging RNM and enhance the anti-neoplasm activity of NK cells. Meanwile,there was a dose-effect relationship between the doses of TIP and GSH and their capability of scavenging RNM,while the DHT cannot scavenge RNM. In scavenging RNM and up-regulating KIR,TIP is as good as GSH, but both of them are better than DHT. Therefore, TIP and GSH may substitude DHT as the immunoadjuvant during the adoptive immunotherapy in leukemia.

Disclosures:
No relevant conflicts of interest to declare.
Do Leucopenia Rates Change After Widespread Folic Acid Fortification in Randomized Controlled Trials of Breast Cancer Chemotherapy?
Authors: Zimmerman Collin T , Read William 
Published: October 2019

Abstract
Abstract 4741

Introduction
Folate is an essential nutrient functioning in a variety of one-carbon transfer reactions, is necessary for red blood cell formation and growth, and deficiency is known to cause megaloblastic anemia. The role of folate in prevention of neural tube defects has been the driving force in public health measures leading to large-scale food fortification in the United States starting in 1996. It is not known if folate fortification has an impact on hematologic toxicities of patients undergoing chemotherapy. The goal of this study is to compare rates of leucopenia reported in pre and post folic acid fortification randomized clinical trials of Adriamycin and Cyclophosphamide (AC) chemotherapy in breast cancer.

Methods
Randomized trials of breast cancer chemotherapy with AC conducted in the United States were searched for in the Medline database, bibliographies, and using Google Scholar. Studies were included if AC was used alone in any arm of study, at standard dosing. Only studies that included disease free survival (DFS), overall survival (OS), and rates of grade III/IV leucopenia for the AC group were included. Studies were eliminated if they included patients with known metastatic disease, “dose dense” or nonstandard AC regimens, or included growth factor support. Rates of phase III/IV leucopenia were grouped in pre and post 1996 folate fortification groups, and meta-analyzed in a random effects model using MIX, a comprehensive free software for meta-analysis of causal research data.

Results
Seven studies involving 14,386 patients were identified using the defined search methodology. There was no significant difference in phase III/IV leucopenia rates between prefolate (4.36% 95%CI 2.31-6.42%) and postfolate studies (7.07% 95%CI 5.41-8.73%).

Conclusions
This review disclosed no statistically significant difference in the rates of leucopenia in patients given AC for breast cancer before or after 1996. Postfolate studies did have 46% more leucopenia than those prior to folate fortification. The confidence intervals of the leucopenia rates overlapped but approached significance. This is contradictory to our theory that increased dietary folate might mitigate hematologic toxicities of chemotherapy. Further studies using individual patient data from institutions administering AC pre and post fortification would further elucidate the role of dietary folate in hematologic toxicities.

Disclosures:
No relevant conflicts of interest to declare.
A Research about Hyperthermia Effected On RPMI-8266 Multiple Myeloma Cells in Order to Enhance the Cytotoxicity of NK Cell.
Authors: Wang Xiuju , Yin Songmei , Ma Liping , Nie Danian , Xie Shangfeng , Li Yingqing 
Published: October 2019

Abstract
Abstract 4749

To compare the cytotoxicity of NK cells on RPMI-8266 multiple myeloma cells before and after thermotherapy, and to explore its mechanism.

MethodF
RPMI-8226 cell was heated for 2 hours at 42°C and after continous culturing for 48 hours, remove dead cells at lower-speed centrifugation. Detected cytotoxicity of NK cells against RPMI-8226 cells, combinig with or without thermotherapy, by LDH releasing assay at different effect-to-target cell ratios (E:T). Moreover compare the expression of HLA class I molecules and NKG2D ligands-MHC class I chain-related molecule A or B and human cytomegalovirus glycoprotein UL16 binding proteins, ULBPsFULBP1AULBP2AULBP3 on the surface of RPMI 8226 cells were analyzed by flow cytometry.

Result
As for E:T is 5:1, the cytotoxicity of NK cells combining with or without hyperthermia on the 8266 NK cells are (2.15 ± 0.32)% and (9.32 ± 1.27)% respectively; at E:T(10:1), cytotoxicities are (5.26 ± 0.84 )% and (20.37 ± 1.78)%; at E:T (20:1), they are(7.63 ± 1.05)% and (28.69 ± 2.25)%;at ET(40:1), they are (10. 18 ± 1.53)% and (35.64 ± 4.87)%. At the different target ratio, the cytotoxcity of NK cells combining with or without thermotherapy were significant statistical difference (P <0.05). Before hyperthermia, the expression of MICA, MICB, ULBP1, ULBP2 and ULBP3 cell surface molecules on RPMI-8266 is (2.82 ± 0.37)%, (6.74 ± 1.16)%, (3.26 ± 2.41)%, (5.42 ± 1.58)%, (5.72 ± 1.66)%; After hyperthermia, (3.18 ± 0.72)%, (7.83 ± 0.96)%, (19.77 ± 4.28)%, (5.23 ± 1.48)%, (21.42 ± 4.68)% respectively; before and after hyperthermia, all of RPMI-8266 cell surface NKG2D ligands were significant statistical significance (P <0.05). Before and after hyperthermia, the expression of 8266 cell surface HLA -± cell surface molecule were (92.25 ± 6.46)%, (89.38 ± 7.87)%; The difference between the two groups was no significant statistical significance (P> 0.05).

Conclusion
Hyperthermia can enhance cytotoxicity of NK cells on multiple myeloma RPMI-8266 cells through upregulating the expression of NKG2D ligand ULBP1 and ULBP3 molecule on RPMI-8266, while no effect on HLA ± molecules.

Disclosures:
No relevant conflicts of interest to declare.
C-MOPP: Results of a Forgotten Regimen for Follicular Lymphoma in the Era of Rituximab and PET.
Authors: Fesler Mark J. , Osman Medhat , Glauber James , Petruska Paul J. 
Published: October 2019

Abstract
Abstract 4755
Review Category: 623
Keywords: Follicular Lymphoma/ Chemotherapy/ Rituximab/ C-MOPP/Therapy

Introduction
A variety of options are available in first and second-line treatment of advanced follicular lymphoma. We report the efficacy and toxicity of an immunochemotherapy regimen, C-MOPP-R, in both upfront and salvage settings.

Patients and Methods
We retrospectively reviewed all thirty-five cases of follicular lymphoma treated with C-MOPP-R at our institution from 2000 through 2008. Excisional lymph node biopsies demonstrating follicle center cell phenotype with any follicular component were required. Patients received six planned 28-day cycles of rituximab 375mg/m2 IV day 1 & 8, cyclophosphamide 650mg/m2 IV day 1 & 8, uncapped vincristine 1.4mg/m2 IV day 1 & 8, procarbazine 100mg/m2 PO day 1-14, and prednisone 60mg/m2 PO day 1-14. All patients received pegylated filgrastim day 9 and prophylactic antimicrobials. Remission was assessed with PET, bone marrow biopsy/aspirate, and clinical assessment using the Revised Response Criteria for Lymphoma in all but one patient, who had computed tomography instead of PET. Most patients received consolidation therapy on a phase II pilot study, and progression-free and overall survival will be reported in the future.

Results
See table for baseline characteristics, response, and grade 3 & 4 toxicities. The overall response rate for de novo and relapsed patients was 100% and 76%, respectively. Seven patients did not complete six cycles of therapy, two patients due to progressive disease and five due to various toxicities at the judgment of treating physicians. Eight patients required cessation of vincristine during therapy for grade II neuropathy.

Conclusions
C-MOPP-R is an efficacious, tolerable immunochemotherapy regimen in de novo and relapsed follicular lymphoma. Response rates in an upfront setting are particularly high, and it represents a feasible regimen that should be considered in this disease.

Disclosures:
No relevant conflicts of interest to declare.
SRC Is a Critical Signaling Mediator in FLT3-ITD Positive AML.
Authors: Leischner Hannes , Grundler Rebekka , Albers Corinna , Illert Anna Lena , Spiekermann Karsten , Peschel Christian , Duyster Justus 
Published: October 2019

Abstract
Abstract 477
Activating mutations of FLT3 are frequent in patients with AML. Two types of mutations are most common: Internal tandem duplications (ITD) of the juxtamembrane domain in approximately 30% of patients and point mutations within the second tyrosine kinase domain (TKD) in about 7% of AML patients. Patients carrying the FLT3-ITD mutation have a significantly worse prognosis whereas FLT3-TKD mutations do not appear to influence the clinical outcome. Studies have shown that mice receiving a transplant of bone marrow expressing FLT3 ITD develop a myeloproliferative disease. In contrast, mice which were transplanted with FLT3 TKD infected bone marrow, suffer from a lymphoid disease. Thus, both FLT3 mutations seem to exert different biological functions. Interestingly, FLT3-ITD but not FLT3-TKD or FLT3-WT leads to a strong activation of the STAT5 signaling pathway. Therefore, STAT5 activation may be responsible for the observed differences in biology. Here we investigated the signalling pathways leading to STAT5 activation downstream of FLT3-ITD. FLT3-ITD does not bind STAT5 directly nor does it activate the classical JAK2 pathway. Instead FLT3-ITD utilizes c-Src to activate STAT5. Co-immunoprecipitations and GST pull downs revealed a strong and exclusive interaction between Src and FLT3 ITD, which is mediated by the Src-SH2 domain. This interaction is absent in FLT3-TKD or FLT3-WT after ligand stimulation. The sequence duplication in FLT3-ITD leads to additional potential Src-SH2 binding sites. We identified tyrosines 589 and 591 of FLT3-ITD to be essential for Src binding and subsequent STAT5 activation. Specific Src inhibitors or Src-siRNA blocked STAT5 activation and growth induced by FLT3-ITD but not FLT3-TKD. FLT3-ITD positive cells with a stable Src knockdown injected into syngenic mice led to a leukemic disease with a significant delayed onset and prolonged survival in comparison to the control group. Finally, a combination of FLT3 and Src inhibitors was tested. This combination was highly efficient in FLT3-ITD positive cells but not in FLT-TKD positive cells. Together these findings show that Src plays an important role in the signalling of FLT3-ITD but not FLT3-TKD. Thus, Src might be an interesting therapeutic target for FLT3-ITD positive AML.

Disclosures:
No relevant conflicts of interest to declare.
Rituximab Based Regimen May Decrease the Incidence of CNS Relapse in Patients with Diffuse Large B-Cell Lymphoma.
Authors: Mahrous Mohamed Mervat , Buckstein Rena , Piliotis Eugenia , Cheung Matthew C , Berinstein Neil 
Published: October 2019

Abstract
Abstract 4770

BACKGROUND
Relapse in the central nervous system (CNS) following initial treatment of diffuse large B-cell lymphoma (DLBCL) is an uncommon but fatal complication. However, the addition of rituximab improves the clinical outcome dramatically in DLBCL patients; its influence on CNS relapse is unproven.

Aim
This single centre retrospective study was conducted to investigate the incidence of CNS relapse, and to evaluate the impact of adding rituximab to standard CHOP (RCHOP) regimen without CNS prophylaxis in patients at risk of CNS relapse.

PATIENTS AND METHODS
All patients with DLBCL diagnosed from April 2002 to December 2007 at sunnybrook cancer center were retrospectively identified in the Cancer Database. Patients were included if they were >16 years old, had advanced stage (stage III /IV, or stage I /II with B symptoms, elevated LDH or bulky disease, were treated with RCHOP regimen with curative intent and were free of CNS involvement at diagnosis. CNS relapse was diagnosed by CSF cytology, radiology or clinically.

Results
A total of 155 patients were newly diagnosed with DLBCL and treated with RCHOP only. 22 pts were excluded, 20 had CNS prophylaxis and 2 pts had CNS involvement. 133 pts were eligible (69 male and 64 female) Median age was 64 (Age'60 was 59.4%). Stage III/IV was 69.9%. LDH was elevated in 59.4%. Bone marrow (BM) involvement and Extra nodal “>2 were 18.05% and 25.6% respectively. EN sites were: (liver 4.5%, Bone 6.8%, Pulmonary 4.5%, kidney 3.01%, cardiac 1.5%, intestine 2.3%, testicular 1.5%). The International Prognostic Index was high-intermediate/high in 55.6%. Pathologically transformed was 12.03% and were transformed from indolent histologies. BCL2 was positive in 65.4%, BCL6 was 48.9%, CD10 was positive in 49.6%, Ki-67 was >80% in 25%. All patients received RCHOP (Median 6 cycles, (range 2-8). Overall response (ORR) was 88.6%, CR/CRU 72.7% with a median follow up 24.6 months (range 2.6-75.5). 28 patients (21.05%) relapsed systemically. Two patients (1.5%) had a CNS relapse 1 brain parenchyma and 1 leptomeningeal one month after systemic relapse. The median time to CNS relapse was 10.4 mos (6.24-14.5 mos). In univariate risk factor analysis (LDH (p=0.8), IPI>3 (p=0.9), No of EN (p=0.9). Actuarial 5 y Overall Survival (OS) was 67.3% (95% CI (57-77%) and progression free Survival (PFS) was 65.7% (95% CI (52.3-78.6%).

Conclusion
Our data suggest that the addition of rituximab may reduce the risk of CNS relapse for poor risk patients likely through systemic control. Future prospective studies of rituximab-containing chemotherapies with CNS prophylaxis are warranted

Disclosures:
No relevant conflicts of interest to declare.