bluuebunny/crossref_metadata_2025_split huggingface dataset:
Monitoring of Minimal Residual Disease in NPM1 Mutated Acute Myeloid Leukemia (AML) – a Single Center Experience in 27 Patients.
Authors: Dvorakova Dana , Racil Zdenek , Palasek Ivo , Protivankova Marketa , Jeziskova Ivana , Lengerova Martina , Mayer Jiri 
Published: October 2019

Abstract
Abstract 4672

Background
Mutations within NPM1 gene occurs in about 60% of adult cytogenetic normal AML (CN-AML) and represent the single most frequent molecular aberration in this subgroups of patients. These mutations usually occur at exon 12 and induce most frequently a net insertion of four base pairs.

Aims
To examine the applicability and sensitivity of DNA-based real-time quantitative polymerase chain reaction (RQ-PCR) with mutation-specific reverse primers and common minor groove binding (MGB) probe and to evaluate whether minimal residual disease levels are of prognostic relevance in CN-AML patients with NPM1 mutations.

Methods
Patients were treated within different AML trials and follow-up samples of peripheral blood or bone marrow were referred to perform an RQ-PCR. Samples were analysed at diagnosis, during, and after therapy. The NPM1 mutations were A (17 pts), B (1 pt), D (2 pts) and 7 patients with individual rare types. For all cases, levels of minimal residual disease were determined by DNA-based RQ-PCR with mutation-specific reverse primer, one common forward primer and one common MGB probe. The NPM1 mutation value was normalized on the number of albumin gene copies and expressed as the number of NPM1 mutations every 106 genomic equivalents.
This assay is highly specific as no wildtype NPM1 could be detected. Maximal reproducible sensitivity was 10 plasmide molecules per reaction.

Results
A total of 950 samples of bone marrow and/or peripheral blood from 27 patients have been analyzed. Twenty of 27 patients (74%) achieved molecular remission (MR), twenty-six of 27 patients (96%) achieved hematological remission (HR). 6 of 27 (22%) patients achieved HR without MR and one patient failed therapy. 8 of 20 patients (40%) with MR after treatment relapsed at molecular level and except one in all these patients hematological relaps occured (one patient is still in HR with bone marrow blast present, but < 5%). Considering relapsed patients, time from molecular to hematological relapse was 1 to 5 months (median: 3 months).
Considering all 14 patients with HR without MR (6 pts) or with molecular relapse (8 pts), in 11 of them hematological relaps occured (79%) and molecular positivity anticipating hematological relaps with median of 3,5 month (1-7 months). 3 of these 14 patients are still in HR.

Conclusions
Mutations within NPM1 gene are a sensitive marker for monitoring minimal residual disease in CN-AML patients. RQ-PCR using a MGB probe is an efficient approach to long-term follow-up of residual leukemia cells and frequent quantitative monitoring is useful for reliably predicting hematological relapse. Achievement of negativity appears to predict favorable clinical outcome.
This work was partially supported by research grant No. MSM0021622430

Disclosures:
No relevant conflicts of interest to declare.
Relationship Between CD56 Antigen Expression and Quantification of MDR1 Gene Expression in Patients with De Novo Acute Myeloid Leukemia(AML).
Authors: Xu Bing , Song Xiaoyan , Shi Pengcheng , Xiao Pengnan , Yu Zhengshan , Zhou Shuyun 
Published: October 2019

Abstract
Abstract 4677

Introdution
The expression of CD56 antigen and over-expression of the multidrug resistance gene 1 (MDR1) seems to confer the poor therapeutic outcome to AML. The aim of this study is to investigate the relationship between CD56 antigen expression and MDR1 gene expression in patients with de novo AML and explore the indicating effect of these two factors on clinical drug resistance.

Patients and methods
A real-time quantitative reverse transcriptase polymerase chain reaction method was established for detecting MDR1 expression levels and a three-color flow cytometry analysis with CD45/SSC gating was used to examine CD56 antigen expression in 79 patients with de novo AML.

Results
CD56 antigen was recorded in 19 out of 79 cases (24.1%) and particularly in those with M5 subtype and t(8;21) AML. Moreover, CD56 expression was significantly associated with unfavorable cytogenetic abnormalities (P< 0.05). Significantly higher percentage (57.1%, 4/7) of patients with t(8;21) demonstrated CD56 expression than those with favorable cytogenetic abnormalities (P< 0.05). CD56+ AML patients had higher incidence of splenohepatomegalia and level of lactate dehydrogenase than CD56- patients (P< 0.05). The median expression level of MDR1 was statistically higher in CD56+ AML patients than that in CD56- cases (P< 0.001) and 89.5% (17/19) CD56+ AML patients were found with high MDR1 expression. The CR rate in high MDR1 / CD56+ AML patients was significantly lower than that in low MDR1/ CD56- cases. (58.8% vs 89.2%, P< 0.01).

Conclusions
There is a linear correlation between MDR1 and CD56 expression in AML. This relationship may explain why CD56 expression is related to a poor prognosis in AML. Therefore both with high MDR1 expression level and CD56 antigen expression can identify AML patients with unfavorable outcome.

Disclosures:
No relevant conflicts of interest to declare.
Transforming and Tumorogenic Activity of JAK2 by Fusion to BCR: Molecular Mechanisms of Action of a Novel BCR-JAK2 Tyrosin-Kinase.
Authors: Cuesta-Domínguez Álvaro , Ortega Mara , Ormazabal Cristina , Santos-Roncero Matilde , Galán-Díez Marta , Steegmann Juan Luis , Arranz Eva , Figuera María Ángeles , Sayyah Jaqueline , Sayeski Peter P. , Vizmanos José Luis , Bueren Juan A. , Rio Paula , Fernandez-Ruiz Elena 
Published: October 2019

Abstract
Abstract 4683
Chromosomal translocations in human tumors frequently produce fusion genes whose chimeric protein products play an essential role in oncogenesis. Recent reports have found a BCR-JAK2 fusion gene in cases of chronic or acute myeloid leukemia, but the protein had not been characterized. We describe a BCR-JAK2 fusion gene by fluorescence in situ hybridization and RT-PCR amplification from bone marrow at diagnosis of a patient with acute lymphoblastic leukemia. After induction therapy, real time PCR showed persistent molecular response correlating with hematological remission maintained up to present. BCR-JAK2 is a 110 KDa chimeric protein containing the BCR oligomerization domain fused to the JAK2 tyrosine-kinase domain. In vitro analysis showed that BCR-JAK2 was constitutively phosphorylated and was located to the cytoplasm. BCR-JAK2 transformed the IL-3-dependent murine hematopoietic cell line Ba/F3 into IL-3 independent growth and induced STAT5b phosphorylation and translocation into the cell nuclei. The treatment with a JAK2 inhibitor abrogated BCR-JAK2 and STAT5b phosphorylation, leading to apoptosis of transformed Ba/F3 cells. To test whether BCR-JAK2 has tumorogenic ability in vivo, we performed experiments with nude mice, in which we injected subcutaneously cells transduced with the control vector and cells expressing BCR-JAK2. Notably, we only obtained tumors in the flank injected with BCR-JAK2 expressing cells, thus confirming the tumorogenic activity of the BCR-JAK2 fusion protein. We conclude that BCR-JAK2 is a new tyrosine-kinase that induces proliferation and cell survival, which can be abrogated by JAK2 inhibitors. In vitro studies demonstrate that BCR-JAK2 displays transforming activity. Moreover, the nude mice model reveals its ability to cause tumors.

Disclosures:
No relevant conflicts of interest to declare.
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
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
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
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
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
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
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.