bluuebunny/crossref_metadata_2025 huggingface dataset:
Diagnostic Cytogenetic Analysis in Pediatric Acute Myeloid Leukemia (AML): Evaluation of Methodological Issues with a Special Focus On Missing Data.
Authors: Athale Uma , Thabane Lehana , Ribeiro Raul C. , Barr Ronald
Published: October 2019

Abstract 4688

Background

Acquired clonal chromosomal alterations are common and considered to have prognostic implications in acute myeloid leukemia (AML). Although bone marrow cytogenetic analysis is required for the classification of AML subtypes and is often used for guiding therapy, the prognostic relevance of the cytogenetic abnormalities in children with AML remains controversial, limiting their value in risk-stratified therapy. Hence we reviewed the reports published by four major pediatric AML study groups to assess the strength of their evidence for the use of cytogenetics as a predictor variable in risk stratification of children with AML, focusing closely on the impact of missing data.

Methods

Reports of consecutive phase III clinical trials published by cooperative study groups that used cytogenetic features for risk stratification of children with de novo AML were reviewed for their methods of assessing the predictive strength of diagnostic cytogenetic analysis (predictor variable) for clinical outcomes, as well as the extent and impact of missing data. We focused on publications by the North American [Pediatric Oncology Group (POG) and Children's Cancer Group (CCG)] and the European [United Kingdom Medical Research Council (UK MRC) and Berlin-Frankfurt-Munster (BFM) group] consortia as they represent major pediatric AML study groups in their respective continents and each of these groups has conducted several multicenter pediatric AML studies with large sample sizes.

Results

During the period 1979-2003, four large pediatric AML study groups conducted 12 Phase III studies evaluating diagnostic cytogenetics. In all, 20 publications reporting the results of these trials were studied to evaluate the impact of diagnostic cytogenetics on clinical outcome. Across all study groups, large amount of cytogenetic data were missing (mean ± SD 31.5% ±17.5%; range 5.3% to 58.1%), but neither the causes of “missingness” nor the methods used to handle the missing data were reported or discussed. In addition, we found a lack of uniformity in the assessment of the predictor variable and outcome measures, lack of a priori estimation of sample size to address the impact of diagnostic cytogenetics and the absence of a validation process.

Conclusions

Missing data were a common but often unidentified problem in a series of large clinical trials testing various treatment strategies in children with AML. This problem, together with other methodological issues in the assessment of the predictor variable and clinical outcome measures, may have biased the estimates of the prognostic strength of diagnostic cytogenetic analysis.

Disclosures:

No relevant conflicts of interest to declare.
Expression and Role of APP Gene in Acute Myeloid Leukemia.
Authors: Meng Fanyi , Wang Wei , Huang Zoufang , Huang Ming , Liu Lixiang
Published: October 2019

Abstract 4691

Introduction

Amyloid precursor protein(APP) gene was increasingly expressed in solid tumors, promoted the proliferation of tumor cells and the overexpression of APP was a bad prognostic factor to oral squamous cell carcinoma. However, little has been known about the clinical significance and role of APP in acute myeloid leukemia(AML).

Methods

The expressions of APP mRNA in 85 AML patients and 20 nonmalignant hematological diseases that worked as control were measured by real-time PCR and the expressions of APP in AML cell lines were examined by real-time PCR and western blot. Small interfering RNAs(siRNAs) targeting APP gene were synthesized and transfected into HL60 cell by lipofectamine2000, after RNAi 24h, 48h and 72h, cell growth of HL60 was measured by trypan blue dye exclusion method and MTT, differentiation was observed by Wright-Giemsa staining, cell cycle was examined by PI/RNase staining, apoptosis induction was analyzed by Annexin V/PI and Hoechst33342 staining; apoptosis-related proteins NF-κB, bcl-2 and Caspase-3 were detected by Western blot after RNAi 48h; sensitivity of HL60 to adrimycin was measured by MTT.

Results

The expression of APP mRNA among AML subtypes was significantly different(P=0.019), M2 with t(8;21) was the highest expression subtype and M5b was the lowest. APP expression had no significant effect on AML clinical characteristic excepting AML subtypes. kasumi-1 was the highest expression cell in AML cell lines and U937 was the lowest(P<0.05), and the expression of APP in HL60/ADM was significantly lower than HL60(P<0.05). The APP expressions in AML cell lines was in agreement with its expressions in primary AML subtypes. After RNAi 24h, 48h, and 72h, no significant differences in proliferation, differentiation, apoptosis, cell cycle and sensitivity of HL60 to adriamycin were detected between interfering group and control groups.

Conclusions

The APP mRNA expression in M2 with t(8;21) was high and M5b was low. Down-regulation of APP expression had no significant effect on biological behaviour of HL60 and APP was not tightly related to pathogenesis of AML.

Disclosures:

No relevant conflicts of interest to declare.
Expression of Osteopontin in the Bone Marrow of Patients with Acute Myeloid Leukemia.
Authors: Liersch Ruediger , Bayer Michael , Biermann Christoph , Appelmann Iris , Schliemann Christoph , Schwoeppe Christian , Kessler Torsten , Mueller-Tidow Carsten , Berdel Wolfgang , Mesters Rolf
Published: October 2019

Abstract 4694

BACKGROUND AND OBJECTIVES

Osteopontin (OPN) is a secreted glycoprotein that is widely expressed in various kinds of cells and is involved in normal tissue remodelling processes as well as in certain diseases such as tumorigenesis and tumor metastasis. In the bone marrow (BM) OPN is predominantly secreted by osteoblasts and hematopoietic cells, which have been shown recently to express the OPN-binding integrins alpha4beta1 and alpha9beta1. In addition, OPN has been defined as an important factor for hematopoietic stem cells (HSCs). OPN suppressed the proliferation of HSCs in vitro and may regulate the hematopoietic stem cell pool. Increased serum OPN concentrations have been reported in chronic myeloid leukemia (CML), multiple myeloma (MM) and acute myeloid leukemia (AML).

DESIGN AND METHODS

We investigated the expression of OPN in newly diagnosed AML patients by immunohistochemistry (n=84), enzyme-linked immunoassays (ELISA) of blood /bone marrow sera (n=40) and on the RNA level by analyzing microarray data (n=261).

RESULTS

Expression of OPN was increased in AML patients bone marrow sera (ELISA) as well as in bone marrow blasts (IHC) Patients expressing high levels of OPN within the bone marrow (IHC: > 10 arbitrary units [AU]; ELISA: > 10 ng/ml) had significantly shorter overall survival (OS) than those with lower OPN levels. In contrast, blood OPN levels showed no predictive value. There was no correlation found between OPN expression and FAB-subtypes M0 to M7 or different karyotypes. Multivariate analysis identified the already known risk factors karyotype, blast clearance (day 16) and the level of OPN expression as independent prognostic factors for OS. Furthermore, analyses of microarray data from 261 patients of a different cohort confirmed OPN as a prognostic marker. In detail, high OPN expression demonstrated a negative predictive value for EFS and OS. Subgroup analysis revealed a significant difference in EFS and OS for OPN levels above the median in FLT3-ITD/TKD mutation negative leukemias, only. No difference was found in FLT3-mutated leukemias or in patients with favorable cytogenetics such as t(8/21) or inv (16).

INTERPRETATION AND CONCLUSIONS

These data provide evidence for OPN as prognostic marker in AML. OPN might be of pathogenetic relevance in AML. Although the mechanism is not yet understood modulation of the OPN axis might be a promising approach to improve the outcome of AML patients in the future.

Disclosures:

No relevant conflicts of interest to declare.
Constitutive Lymphoid Expression of the Nuclear Form of RNase H1 Is Associated with a Developmental Bottleneck at the Pro-B Cell Stage of B Cell Differentiation.
Authors: Gugliotti Lina A. , Sakhuja Kiran B. , Wang Hongsheng , Pinkhasov Julia , Love Paul E. , Cerritelli Susana M. , Morse Herbert , Crouch Robert J.
Published: October 2019

Abstract 4702

The development of B lymphocytes and the process of lineage determination are initiated by expression of a set of transcriptional regulators leading to V(D)J recombination events initiated by double-strand DNA breaks. Subsequently, these recombinations form DNAs that permit transcription of immunoglobulin genes and translation of the corresponding mRNAs, first by joining the V(D)J DNA sequences, then by recombination, that generates various isotypes of immunoglobulins by class-switch recombination (CSR). Formation of R-loops, regions containing RNA/DNA hybrid and a displaced single-stranded DNA, have been shown to lead to recombination in bacteria, yeast, HeLa and chick cells. Expression in each of these cases of excess ribonuclease H1 (RNase H1), a class of enzymes that degrade RNA in RNA/DNA hybrids, has ameliorated the deleterious effects and decreased recombinational events associated with R-loop formation. R-loops have been observed following transcription of the switch regions that occurs during CSR. The possibility that R-loops are important in V(D)J recombination has not been addressed, and whether ribonucleases H (RNases H) play a role in this process is still uncertain. Transgenic (TG) mice that overexpress RNase H1 in B and T cells (M27F7) were employed in this study. FACS analysis of hematopoietic cells from TG mice revealed a decrease in pre-B cells in the bone marrow. The data indicate a block at the pro-B to pre-B stage of B cell development, which may be the result of apoptosis due to the failure to generate a productive VH-D-JH rearrangement and expression of the pre-B cell receptor. A few B cells that successfully passed these checkpoints predominately differentiated into marginal zone and B1a cells in the peripheral lymphoid organs of the TG mice. These data suggest that R-loops are important in H chain gene rearrangement.

This research is supported by the Intramural Research Program of the National Institutes of Health, the Eunice Kennedy Shriver National Institute of Child Health and Human Development and the National Institute of Allergy and Infectious Diseases.

Disclosures:

No relevant conflicts of interest to declare.
The Change of Elf-1 Gene Expression Level in Hematological Malignancies.
Authors: Shen Qi , Zheng Haitao , Chen Shao hua , Yang Lijian , Li Yangqiu
Published: October 2019

Abstract 4704

Elf-1 (E-74-like factor 1), a special transcription factor of T cells, is a member of the Ets (E-26-specific) family. Elf-1 plays a critical role in the transcription of the TCR ζ-chain. Our previous studies showed that the low expression level of TCR ζ-chain in hematologic malignancies, in the present study, we farther analyzed the expression of Elf-1 in patients with hematologic malignancies. Real-time PCR with SYBR Green I technique was used for detecting Elf-1 expression level in peripheral blood mononuclear cells from 33 patients with acute myeloid leukemia (AML) [including 17 cases with AML-M2 subtype (M2), 6 cases with (M3), 10 cases with (M5)], 49 patients with lymphoid malignancies [including T-cell acute lymphoblastic leukemia (T-ALL) 7 cases, T-cell non-Hodgkin lymphoma (T-NHL) 6 cases, B-cell acute lymphoblastic leukemia (B-ALL) 10 cases, B-cell chronic lymphoblastic leukemia (B-CLL) 7 cases, B-cell non-Hodgkin lymphoma (B-NHL) 19 cases] and 20 healthy volunteers. β2-microglobulin gene (β2M) was used as an endogenous reference. Relative changes in Elf-1 expression level were used by the 2-ΔCt×100% method. We found that the expression level of Elf-1 in 33 cases with AML (13.518±19.197%) was significant higher than those in the healthy control (2.044±1.321%) (P<0.01). However, the expression level of Elf-1 had no significant difference among the three AML subtypes (P>0.05). The expression level of Elf-1 in both T-cell lymphoid malignancies group (12.050±10.334%) and B-cell lymphoid malignancies group (14.144±21.576%) was significant higher than those in the healthy control (P<0.01, P<0.01). Moreover, the expression level of Elf-1 was different in AML and the lymphoid malignancies groups (P<0.01). That was, the highest in the B-ALL group, the lowest in the B-NHL group. These results indicated that overexpression of Elf-1 is a common feature in peripheral blood from patients with both AML and lymphoid malignancies, as TCR ζ-chain was down regulation in AML and lymphoid malignancies, the increased expression of Elf-1 gene, which an upstream factor of the TCRζ-chain might indicate a feedback up regulation situation in patients with hematologic malignancies.

Disclosures:

Shen: The study was supported by grants from National Natural Science Foundation of China (No. 30871091): Research Funding. Zheng:The study was supported by grants from National Natural Science Foundation of China (No. 30871091): Research Funding. Chen:The study was supported by grants from National Natural Science Foundation of China (No. 30871091): Research Funding. Yang:The study was supported by grants from National Natural Science Foundation of China (No. 30871091): Research Funding. Li:The study was supported by grants from National Natural Science Foundation of China (No. 30871091): Research Funding.
Detection of ERG and MN1 Gene Expression in Adult Patients with De Novo Acute myeloid Leukemia(AML) by Real-Time Quantitative Polymerase Chain Reaction and Its Clinical Significance.
Authors: Xu Bing , Guo Xutao , Chen Guoshu , Song Xiaoyan , Shi Pengcheng , Xiao Pingnan , Zhou Shuyun
Published: October 2019

Abstract 4706

Introdution

Current study found that certain genetic abnormalities are related to the prognosis of AML, such as ERG and MN1 gene expression. However, little is known about the clinical significance of ERG and MN1 expression level in Chinese patients with AML and and their correlation.

Patients and methods

A real time quantitative reverse transcriptase polymerase chain reaction method was established for detecting ERG and MN1 gene expression levels in 87 de novo AML patients.

Results

The median expression levels of ERG and MN1 in AML patient were statistically higher than that in normal control group (P<0.001). ERG gene expression had no correlation with MN1 gene expression (P=0.194) in AML patients. ERG and MN1 gene expressions were equally distributed among the FAB subtypes (P=0.850 and 0.870). Spearman's rank correlation showed that leukocyte counts and lactate dehydrogenase were significantly related to high ERG expression (P=0.005 and 0.032), but not significant correlation was found between hemoglobin and platelet counts. The ERG expression level of cases with middle and low risk group was lower than that of cases with high risk group (P<0.036). There was no significant difference of CR rates in high and low ERG and MN1 expression groups after chemical theropy (P=0.968 and 0.695). During the follow-up of one year, the cumulative relapse rates of high ERG expression groups was significantly higher than that of low ERG expression groups(P=0.039), and high ERG expression cases have a significantly worse OS than low ERG expression cases(P=0.005).

Conclusions

There isn't a linear correlation between ERG gene expression and MN1 gene expression in AML. Overexpression of ERG gene is a independent prognositic factor for AML. Quantification of the two gene expression together is not more effective to the judgement of prognosis in AML.

Disclosures:

No relevant conflicts of interest to declare.
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 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 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 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 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 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 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 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 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 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.
Anti-CD20 Antibody GA101 in Combination with Chemotherapy or Flavopiridol in Mantle Cell Lymphoma.
Authors: Heinrich Daniel A. , Klein Christian , Decheva Kristina , Weinkauf Marc , Hutter Grit , Zimmermann Yvonne , Weiglein Tobias , Hiddemann Wolfgang , Dreyling Martin H.
Published: October 2019

Abstract 4781

Background

Mantle cell lymphoma (MCL) responds only transiently to conventional chemotherapy resulting in a dismal long-term prognosis. At a molecular level it is characterised by the chromosomal translocation t(11;14)(q13;q32), which leads to constitutive over-expression of the cell cycle regulatory protein cyclin D1. GA101 is a third generation, glycoengineered type II IgG1 anti-CD20 monoclonal antibody with superior direct cell death induction by targeting a type II epitope and enhanced antibody dependent cellular cytotoxicity (ADCC). High efficacy in lymphoma cell lines has led to combination experiments with various chemotherapeutic compounds or the CDK-inhibitor Flavopiridol.

Methods

Using a MCL cell line panel (Granta-519, HBL-2, Jeko-1, Rec-1 and Z-138) and a Diffuse Large B-Cell Lymphoma cell line (Karpas-422) we determined the effect of GA101 (1 μg/ml) monotherapy as well as in combination with Fludarabine (0,25 μg/ml), Bendamustine (5 μg/ml), Mitoxantrone (0,25 and 0,5 μg/ml) and Flavopiridol (100nM) on cell proliferation and viability. Trypan-blue exclusion tests were used to analyze cell viability at 0h, 24h, 48h and 72h. The panel of MCL cell lines was treated to determine potential synergism of agent combinations. Accordingly, fractional product was calculated: synergism > 0,1; additive effect -0,1<x<0,1; antagonism < -0,1.

Results

After mono-exposure with GA101 (1 μg/ml), Granta-519 and Rec-1 showed the highest sensitivity (Granta: 65-75% cell reduction, Rec-1: 30-45%). Intermediate results were achieved for HBL-2 (20-30%), Z-138 and Karpas-422 (10-15%), Jeko-1 (5%). Fludarabine alone resulted in a 20-40% cell reduction. Bendamustine showed a higher efficacy in Jeko-1, Rec-1 and Z-138 (40-90%) than in Granta-519, Karpas-422 and HBL-2 (10%). Mitoxantrone treatment demonstrated a high impact on all cell lines (80-95% cell reduction). Flavopiridol induced a 65-85% cell reduction in Jeko-1, Rec-1 and Karpas-422in comparison to 30-45% in Granta-519, HBL-2 and Z-138. Additional experiments showed additive effects of all GA101 combinations resulting in 40-80% cell reduction (Fludarabine), 30-90% (Bendamustine), 85-95% (Mitoxantrone) and 60-80% (Flavopiridol).

Conclusions

These in vitro results demonstrate that the anti-CD20 monoclonal antibody GA101 alone or in combination with various chemotherapeutical compounds or the CDK-inhibitor (Flavopiridol) show a promising efficacy in MCL cell lines (additive in combination), supporting the clinical evaluation of such an innovative immuno-chemotherapy in mantle cell lymphoma.

Disclosures:

Klein: Roche (Glycart): Employment, Equity Ownership, Patents & Royalties. Weinkauf:Lilly Deutschland GmbH: Research Funding. Hutter:Lilly Deutschland GmbH: Research Funding. Zimmermann:Lilly Deutschland GmbH: Research Funding. Dreyling:Roche: Honoraria, Research Funding.
Protein Tomography Analysis Shows That CD20 Molecules Bound by Different Type I and Type II Anti-CD20 Antibodies Are Organized in Different Protein Complexes.
Authors: Niederfellner Gerhard J , Braennstroem Annika , Lindstrom Frida , Jansson Magnus , Lundin Marika , Schaefer Wolfgang , Mundigl Olaf , Umana Pablo , Klein Christian
Published: October 2019

Abstract 4782

Ramos cells were labeled with Type I (Rituximab and Ocrelizumab) and Type II (B1/H299 and GA101) anti-CD20 antibodies and then fixed and stained with marker gold labeled 2ary antibodies. Protein complexes connected to marker gold were analyzed by Protein TomographyTM at SIDEC and the corresponding 3D-structures visualized (∼100 structures per antibody). Less than 5 % of the refined structures were dimeric. While most (60 – 90%) CD20 molecules were present in tetramers or even higher order defined complexes, a sizeable proportion was also engaged in large protein networks (11 -38%). For the multimeric complexes, we could clearly distinguish between extended (or open) and ring-like (or closed) conformations. Although cells had been labeled with an excess of antibody, as suggested by FACS binding curves, the antibodies bound CD20 monovalently in most refined structures. Bivalent binding was overall more prevalent with Rituximab and Ocrelizumab than with B1 and GA101 (1/3 vs 1/6 structures). The proportion of CD20 molecules present in ringlike complexes was higher for the Type II than for the Type I antibodies. In co-localization experiments, Rituximab-CD20 and GA101-CD20 complexes were also found to only partially colocalize, while a large fraction of the two antibodies were found in separate cell surface compartments. These findings suggest that the different antibodies favor different CD20 conformations that seem to be associated with different protein complexes and might form the basis for initiation of different downstream signaling processes.

Disclosures:

Niederfellner: Roche: Employment. Braennstroem:SIDEC: Employment. Lindstrom:SIDEC: Employment. Jansson:SIDEC: Employment. Lundin:SIDEC: Employment. Schaefer:Roche: Employment. Mundigl:Roche: Employment. Umana:Glycart: Employment, Equity Ownership, Patents & Royalties. Klein:Roche: Employment, Equity Ownership, Patents & Royalties.
Cholesterol Regulation and Statin Therapy in Patients with Waldenstrom's Macroglobulinemia.
Authors: Ioakimidis Thea , Patterson Christopher J , Hunter Zachary , Soumerai Jacob , Manning Robert , Warren Diane L. , Ghobrial Irene , Sheehy Patricia , Treon Steven
Published: October 2019

Abstract 4789

Patients with WM commonly display low cholesterol levels, particularly LDL and VLDL levels. Given these observations, we examined their levels in 110 patients with WM, and observed decreased total cholesterol (<160 mg/dL), LDL (<100 mg/dL) and VLDL (<40 mg/dL) levels in 41 (37.3%), 68 (61.8%) and 90 (81.8%) patients, respectively. Importantly, median serum IgM levels which serve as a surrogate marker for WM disease burden were higher among patients with hypocholesterolemia (3440 vs. 1587 mg/dL; p=0.0004). Longitudinal follow-up of progressing as well as responding patients demonstrated an inverse correlation between serum IgM and cholesterol levels, which was not an artifact of laboratory testing. Importantly, significantly lower serum IgM levels (885 vs. 1960 mg/dL; p=0.004) were observed among those patients who were on a statin lipid-lowering agent. We therefore investigated the in vitro potential of statins to mediate anti-WM activity and showed that simvastatin acted a potent inducer of WM cell apoptosis through inhibition of geranylgeranylated proteins. Given these observations, we investigated the potential of simvastatin to suppress disease progression in WM patients with slowly progressing disease who would otherwise have been on watch and wait.

Patients and Methods

Eighteen patients, 8 of whom were previously untreated, and who were asymptomatic with slowly progressing disease were enrolled in this study. The median age for these patients was 67 (range 42-88 years), median IgM was 2,610 (571-5,880 mg/dL), and median hematocrit was 35.4 (range 30.2-41.4%). Patients received Simvastatin at 20 mg daily for the first week, then dose escalated weekly by 20 mg a day to a maximum of 80 mg daily by week 4. Patients were maintained on therapy until progression.

Results

With a median follow-up of 6 months (range 3-18 months), 7 patients have demonstrated objective disease progression. No objective responses have been observed in any treated patients. Among non-progressing patients, serum IgM levels (2,610 vs. 2545 mg/dL) and hematocrit (35.4% vs. 34.5%) remain stable (p=NS). Grade 2 toxicities included elevation in CPK (n=1), and musculoskeletal pain (n=3). No grade 3 toxicities were observed.

Conclusions

Simvastatin does not produce objective responses in patients with WM, though may have potential for suppression of disease progression in some patients with WM.

Disclosures:

No relevant conflicts of interest to declare.