bluuebunny/crossref_metadata_2025 huggingface dataset:
Tumor Marker CA 15-3 in Hematological Malignancies.
Authors: Saouli Zoi , Papadopoulos Athanasios , Kaiafa Georgia , Girtovitis Fotios , Charisopoulos Georg , Kontoninas Zisis , Zantidis Anestis , Andreadis Charalambos
Published: April 2020

Introduction: CA 15-3 is a glycoprotein expressed in several adenocarcinomas, especially of the breast. It is used to detect recurrent or metastatic disease. Elevated levels can also be found in adenocarcinomas of the ovary, lung, pancreas, and colon, and are also related to benign breast or ovarian disease, endometriosis, hepatitis, pregnancy and lactation. To our knowledge with the exception of multiple myeloma, there are no references for the significance of the CA 15-3 in hematological malignancies.

Aim of our study was to evaluate the levels of CA 15-3 in patients with various hematological malignancies.

Material and Methods: 84 patients with hematological malignancies were tested: MDS 27 pts, non Hodgkin lymphoma 12 pts, Hodgkin’s lymphoma 3 pts, chronic lymphocytic leukemia 13 pts, acute leukemia: 7 pts, multiple myeloma 8 pts, chronic myeloid leukemia 3 pts and other chronic myeloproliferative disorders 11 pts. 55% of the patients were men with average age of 55 (17–87) and 45% were women with average age of 52 (35–64). A group of 45 healthy volunteers’ blood donors was also tested. Immunoradiometric assay (IRMA) has been used to determine the circulating levels of the CA 15-3 marker.

Results:

None of the healthy volunteers had elevated Ca 15-3 levels. Among the 84 patients, 31 (36,9%) had elevated levels of CA 15-3. In MDS 10/27 pts, in Non Hodgkin Lymphoma 5/12 pts, in Hodgkin Lymphoma 1/3 pts, in Chronic Lymphocytic Leukemia 5/13 pts, in Multiple Myeloma 4/8 pts, in acute leukemia 1/7 pts and in other myeloproliferative disorders 4/11 pts. The CA 15-3 levels in hematological patients were higher than the ones in the healthy group. The difference was statistically significant (p <0.0001), (Table 1). 31 patients who where either untreated or had recurrent disease (subgroup a), had elevated CA 15-3 levels (36,8 ± 8,9 U/ml) while the rest 53 patients who were under therapy or were in remission (subgroup b) had normal levels (14,3 ± 5,2 U/ml). A statistically significant difference between the two subgroups was observed, (p <0.0001), (Table 2). The low risk MDS patients (RA, RARS) had normal level of CA 15-3, while the high risk MDS patients (RAEB, RAEB-t, CMML) had high levels of CA 15-3.

Conclusions: CA 15-3 can be an indicative marker of the activity of a hematological malignancy. Also, it might be of value in monitoring hematological diseases and response to therapy.

Table 1 Ca 15-3 (U/ml) Patients (total, n=84) 23 ± 12,3 Normal Subjects (n=45) 14,1 ± 4,6 p < 0.0001 Table 2 Ca 15-3 (U/ml) Subgroup a 36,8 ± 8,9 Subgroup b 14,3 ± 5,2 p < 0.0001
ASPP2 Haploinsufficiency Promotes Tumor Formation in a Mouse Model.
Authors: Kampa Kerstin M. , Acoba Jared D. , Chen Dexi , Beemer Kelly , Gay Joel , Zhu Zhiyi , Padiernos Emerson , Fan Alice , Felsher Dean , Corless Christopher , Naumovski Louie , Lopez Charles D.
Published: April 2020

ASPP2 interacts with the tumor suppressor protein p53 and promotes damage-induced apoptosis in part through stimulation of p53-mediated apoptosis. We have previously demonstrated that low ASPP2 levels correlate with poor clinical outcome in patients with diffuse large B-cell lymphoma treated with anthracycline-based chemotherapy. Moreover, reduced ASPP2 expression has been demonstrated in other tumor types. These findings led us to hypothesize that ASPP2 may function as a tumor suppressor. To further explore this, we targeted the ASPP2 allele in a mouse by homologous recombination using a knockout vector that replaced exons 10–17 with a neoR gene. Two separate ES clones were used for blastocyst injections to generate several chimeras that were used to generate ASPP2 heterozygous mice. ASPP2+/− mice appear developmentally normal and reproduce. However ASPP2−/− mice could not be generated. Genotype analysis as early as Ed 6.5 did not detect ASPP2−/− embryos---which implies an early embryonic lethal defect in the homozygote. ASPP2+/− (n=135) and ASPP2+/+ (n=63) sibling mice were observed for spontaneous tumor formation. Overall median tumor-free survival was 117 weeks in the ASPP2+/− mice verses 125 weeks in the ASPP2+/+mice (p = 0.035 log-rank test). Overall tumor incidence (at 115 weeks) for ASPP2+/− and ASPP2+/+ mice was 43% and 22%, respectively. The incidence of tumor types, from all tumors detected, was similar between ASPP2+/− and ASPP2+/+ mice: 34% versus 33% (lymphoma), 18% versus 14% (sarcoma), and 47% versus 52% (carcinoma), respectively. Compound p53+/−;ASPP2+/− mice did not exhibit accelerated tumor formation relative to p53+/−;ASPP2+/+ mice. Additionally, a tet-Myc:ASPP2+/− lymphoma mouse model did not exhibit accelerated lymphomagenesis. However, preliminary data suggests that ASPP2+/− mice may have an increased incidence of irradiation-induced leukemia/lymphoma when compared to ASPP2+/+ mice, and confirmatory studies are ongoing. In response to ionizing radiation, doxorubicin, or serum-starvation, preliminary analysis reveals a G0/G1 checkpoint defect in ASPP2+/− MEFs compared to ASPP2+/+ MEFs. Our results provide in vivo evidence that ASPP2 can function as a tumor suppressor. Further studies are underway to determine the mechanism of this observation.
Synergistic Anticancer Effects between Curcumin and Doxorubicin in Drug Sensitive and Resistant Human Pediatric Leukemic Cell Lines.
Authors: Ramachandran Cheppail , Nair P.K. Raveendran , Escalon Enrique , Melnick Steven J. , Khatib Ziad
Published: April 2020

Curcumin (diferuloyl methane), the yellow colored chemopreventive agent from turmeric has anti-tumor, anti-inflammatory and anti-oxidant effects. The synergistic effect of curcumin and doxorubicin on cytotoxicity in leukemic cell lines was investigated preclinically in vitro for their eventual use in combination therapy in pediatric patients. Curcumin is cytotoxic to both sensitive (CEM) and resistant (CEM/VLB) cell lines with an IC50 value of 12 μM. Multi-drug resistant (MDR) cell line (CEM/VLB) showed 36.4-fold doxorubicin resistance based on DOX IC50 values of 0.14 μM and 5.1 μM for CEM and CEM/VLB cell lines. Analysis of cytotoxicity data by CalcuSyn program showed that curcumin and doxorubicin combination treatments are synergistic with combination index values less than 1 at IC50 (CEM=0.82; CEM/VLB =0.81) and IC75 (CEM=0.60; CEM/VLB=0.83) levels. Curcumin and doxorubicin induces G2/M arrest and apoptosis in tumor cells and the apoptosis data correlated with cytotoxicity values in both sensitive and resistant cell lines. The combination index values on the percentage of apoptosis induced by curcumin and doxorubicin combination treatments were less than 1 indicating the synergistic effect of both these agents on apoptosis. Doxorubicin treatment up regulated NF-κB activity in both CEM and CEM/VLB cell lines, although the level of up regulation is higher in CEM (2-fold) than CEM/VLB (1.5-fold) cell lines. Curcumin, on the other hand, is anti-inflammatory because of its significant inhibitory effect on NF-κB activity. Curcumin inhibited 80% and 50% of NF-κB activity in CEM and CEM/VLB cell lines in 24 h. These results showed that curcumin can be used either as a single or adjuvant agent along with doxorubicin because of the synergistic effects on apoptosis and cytotoxicity.
Study on RNA Interference Reversing Multidrug Resistance in K562/A02 Resistant Leukemia Cell.
Authors: Gong Yuping , Gu Ling , Liu Ting
Published: April 2020

Multidrug-resistance (MDR) is a major hindrance to successful chemotherapy. Among different mechanisms responsible for MDR, cellular over-expression of P-glycoprotein (Pgp) plays an important role. Pgp is an energy dependent drug efflux pump that effectively transports a broad of commonly used chemotherapeutic drug out of the cells and leads to resistance to the chemotherapeutic drugs. Use of modulators is main approach to overcome MDR. Although, in vitro and in vivo preclinical studies have yielded encouraging results, the clinical reversal of resistance by common modulators such as verapamil, quinine, and cyclosporine A has been difficult due to the toxic side effects. Antisense technology offers an alternative approach to overcome MDR. RNA interference (RNAi) is the best one of all antisense technologies. RNAi acts through a post-transcriptional targeting of mRNA for degradation, resulting in sequence-specific post-transcriptional gene silencing. Short hairpin RNA (shRNA) vectors can be transfected into the cells to stably express siRNA. In our preliminary study, eukaryotic shRNA expression vectors aimed at mdr1 mRNA target sequences were cloned and transfected into drug resistance cell line K562/A02 by liposome-induced gene transfection. The mdr1 mRNA was identified by real time RT-PCR, the function of P-gp was measured by a daunorubicin (DNR) efflux assay, and the sensitivity of cell lines to doxorubicin (ADM) was detected by an MTT assay. Our results showed that two mdr1-targeted shRNAs could down-regulate mdr1 mRNA and P-gp expression, with mdr1 mRNA reduced by 86% and 88%. The intracellular DNR increased after RNAi treatment, with the daunorubicin efflux ratio at 60min were 13% and 22%, compared with control (40%~45%) (P<0.05). The MTT assay demonstrated the relative reversing efficiency to doxorubicin to be 84% and 77%.

Conclusion: RNA interference can effectively reverse mdr1-mediated multidrug resistance in resistant leukemic cell line.
Assessing DNA Damage Ex Vivo Induced by Chemotherapy Targeting Topoisomerase Inhibitors in Acute Leukemias.
Authors: Halicka Dorota H. , Ozkaynak Fevzi M. , Seiter Karen , Kajstura Malgorzata A. , Liu Delong , Tanaka Toshiki , Traganos Frank , Darzynkiewicz Zbigniew
Published: April 2020

Our previous experiments on leukemic cell lines demonstrated that the intensity of immunofluorescence (IF) that is proportional to the level of the phosphorylated histone H2AX (γH2AX) reveals the frequency of DNA double-strand breaks (DSBs) in chromatin induced by DNA topoisomerase I and II (topo I and II) inhibitors. The aim of the present study was to explore whether ex vivo assessment of the extent of DNA damage in leukemic blasts induced during chemotherapy is feasible. The long term goal is to develop a rapid assay to assess the effectiveness of chemotherapy early in the course of treatment of leukemia. Mononuclear cells were isolated from blood samples of 1 AML and 10 ALL patients with peripheral blasts, 4 of whom were, at diagnosis, high-risk ALL while 4 were in relapse. The median age of the patients was 17 yrs (2–25 yrs) and all were entered into an anthracycline-containing drug regimen. Samples were collected:

prior to drug administration; and, one hour after completion of the drug infusion.

The extent of DNA damage was assessed by analysis of histone H2AX phosphorylation occurring at sites of DSBs, which could be detected immunocytochemically and measured by multiparameter flow cytometry in conjunction with DNA content. In the only patient not responding to treatment, one of 4 relapsed ALL patients, there was no induction of DSBs and no activation of ATM. In all patients phosphorylation of ATM appeared to be strongly linked to induction of DSBs after treatment targeting topo inhibitors. In a pilot study of 3 adult patients with acute leukemia (two AML and one ALL in relapse) treated with the topo II inhibitor Mitoxantrone (MTX), ATM activation was strongly linked to induction of DSBs 1 h post drug infusion. ATM activation and induction of DSBs was several times higher in two adult responders in comparison to one non responder to therapy. A new approach to detect DNA damage in leukemic cell by flow cytometry using gating strategy which combines CD45 expression on the cell surface with side scatter (SS) is being developed and was applied in 5 cases. This approach will be useful when the proportion of blasts in peripheral blood is low. Although the number of patients is very small in this study, the results suggest that a cytometric assay that reveals the extent of DNA damage based on detection of H2AX phosphorylation during induction therapy targeting topo I and II inhibitors may provide an early prognostic marker.
ARRY-429520, a KSP Inhibitor with Efficacy in Animal Models of Solid and Liquid Tumors.
Authors: Woessner Rich , Corrette Chris , Allen Shelley , Hans Jeremy , Zhao Qian , Aicher Tom , Lyssikatos Joe , Robinson John , Poch Greg , Hayter Laura , Cox April , Lee Patrice , Winkler James , Koch Kevin , Wallace Eli
Published: April 2020

Kinesins are eukaryotic microtubule-associated motor proteins. One of these, kinesin spindle protein (KSP) is a mitotic-specific kinesin that plays a key role in spindle pole separation and production of the bipolar spindle, as well as centrosome separation and maturation. As KSP is expressed predominately in proliferating cells and is absent from postmitotic neurons, inhibition of KSP has the potential to provide the antitumor activity of a mitotic inhibitor while avoiding peripheral neuropathy. ARRY-429520 is a member of a series of KSP inhibitors discovered and optimized by structure-based design. It is a potent inhibitor of human KSP (IC50 6 nM), with an EC50 of 1.5 nM for cellular phosphorylation of histone H3 (a pharmacodynamic marker for accumulation of cells in mitosis), and in vitro antiproliferative IC50s ranging from subnanomolar to low single digit nanomolar across a varity of human and murine tumor cell lines. We report here the in vivo characterization of this compound. ARRY-429520 inhibits histone (H3) phosphorylation in tumor xenografts , showing that the compound has pharmacodynamic activity in vivo. As a result, it is a highly efficacious inhibitor of the growth of tumor xenografts. At its maximally tolerated dose in mice of 25 – 30 mg/kg i.p. on a Q4Dx3 schedule, ARRY-429520 caused tumor regressions, with some complete responses, in subcutaneous mouse xenograft models of leukemia: HL-60 (acute promyelocytic leukemia) and K-562 (chronic myelogenous leukemia). The compound was also highly efficacious against other tumor types, including HT-29 (colon), HCT-116 (colon), and A2780 (ovarian). ARRY-429520 also showed activity in intravenous leukemia models. The molecule possess desirable drug-like properties, including high aqueous solubility (> 4 mg/ml at physiological pH), low CYP inhibition (> 25uM for 3A4, 2C9, 1A2, 2D6, and 2C19), and pharmacokinetics favorable for an IV-infused targeted chemotherapeutic.
Preanalytical mRNA Stabilization of Whole Bone Marrow Samples.
Authors: Langebrake Claudia , Guenther Kalle , Lauber Juergen , Reinhardt Dirk
Published: April 2020

Background: Gene expression profiling is a useful tool for the diagnosis and basic research of cancer. One of the major limitations of this approach is that already a short-term storage of native specimens of peripheral blood (PB) or bone marrow (BM) and/or the method of RNA isolation has significant influence on gene expression due to gene induction, repression and RNA degradation. The objective of the current study was to investigate the influence of a newly developed RNA stabilization and preparation system for BM specimens (PAXgeneTM Bone Marrow RNA System), focusing on RNA-yield, RNA-integrity and stability of gene expression profiles over time of BM sample storage.

Patients and methods: 180 RNA samples that have been processed from 45 heparinized BM specimens of unselected children with acute leukemia at initial diagnosis, during treatment or without pathological findings in hematopoiesis were analyzed. Immediately after collection, heparinized BM specimens were divided into two PAXgeneTM RNA tubes and two standard tubes, each. RNA isolation using either the PAXgeneTM protocol (P) or a reference protocol (R) was performed after 2 hours (P0 and R0) or after 48 hours (P2 and R2).

Results: The overall RNA yield (normalized to 1×107 leukocytes) was not different in all four isolation procedures, however the integrity of the RNA using the PAXgeneTM system was significantly higher at both time-points than that of the reference system: 8.6±0.2 (P0) vs. 6.8±0.4 (R0), p=0.0003 and 8.1±0.2 (P2) vs. 6.7±0.5 (R2), p=0.008. For the stabilized samples, we found very good pairwise correlation in gene expression for either gene at P2 compared to P0: GATA1 89±6%, RUNX1 83±10%, NCAM 69±12% and SPI1 89±6%. However, there were significant differences in two of the analyzed genes using the reference RNA isolation procedure: 40±6%, p<0.001 (GATA1) and 47±8%, p=0.005 (NCAM), respectively. Comparing the two RNA isolation procedures, there are significant differences in the expression levels of the analyzed genes.

Discussion: The PAXgeneTM system is appropriate for the stabilization of gene expression levels in BM samples after short-term storage. However, as in some genes the reference RNA isolation procedure resulted in similar gene expression levels, the use of a stabilization reagent has to be carefully evaluated within the preanalytical handling of the samples. Our analysis has shown that it is not suitable to compare RNA expression levels derived from different isolation procedures. In conclusion, the PAXgeneTM system is able to stabilize RNA from clinical BM samples while being suitable to isolate high quality and quantity RNA.
Study of FLT3 Internal Tandem Duplication (FLT3-ITD) in Patients with Acute Promyelocytic Leukemia (APL).
Authors: Hu Jiong , Zhu Yong-Mei , Liu Yuan-Fang , Zhang Su-Jiang , Shen Zhi-Xiang
Published: April 2020

Objectives To evaluate the prevalence and association with clinical features of FLT3 internal tandem duplication (FLT3-ITD) in patients with newly-diagnosed acute promyelocytic leukemia (APL).

Methods A total of 103 APL patients were screened by polymerase chain reaction (PCR) for FLT3-ITD.

Results FLT3-ITD mutations were identified in 19.4% (20/103) patients. The FLT3-ITD was associated with short/variant form of PML-RARα isoforms (p<0.0001). Among 20 patients with FLT3-ITD, 16 patients presented with short, 2 with variant and 2 with long form of PML-RARα isoforms. The expression level of FLT3 via quantitative real-time RT-PCR was significantly increased in patients with FLT3-ITD (p<0.05). Patients with FLT3-ITD also presented significantly increased initial peripheral white blood cell (WBC) count (p<0.01), especially in patients with short/variant PML-RARα isoforms (p=0.015). For patients with long form PML-RARα, there was no significant difference in initial WBC count. For clinical outcome, 90% (18/20) of FLT3-ITD positive patients obtained complete remission and 16 evaluable patients (2 lost follow-up) remained in first remission with relatively short follow-up (median 26 months, 11-47), which was not significantly different from patients without FLT3-ITD.

Conclusion FLT3-ITDs were frequently identified in patients with newly diagnosed APL. FLT3-ITD was associated with short/variant form of PML-RARα fusion gene and increased initial WBC. No significant impact of FLT3-ITD on treatment outcome was observed with limited follow-up.

Association of FLT3-ITD and PML-RARα isoforms in patients with APL No FLT3-ITD+ FLT3-ITD− Long 57 2(3.5%) 55(96.5%) Short 41 16(39.0%) 25(61.0%) Variant 5 2(40%) 3(60%) Total 103 20(19.4%) 83(80.6%)

FLT3 expression in different patient sample FLT3 expression in different patient sample FLT3 expression in different patient sample FLT3 expression in different patient sample
The Clinical Significance of Programmed Death1 Ligand-1 Expression on Leukemia Cells in Adult T Cell Leukemia Patients.
Authors: Yoshimitsu Makoto , Kozako Tomohiro , Fujiwara Hiroshi , Arima Naomichi , Tei Chuwa
Published: April 2020

[Purpose and background] Adult T-cell leukemia/lymphoma (ATLL) caused by HTLV-I virus infection is one of the most aggressive lymphoid malignancies with short prognosis, of which best-reported median survival by combined chemotherapy is 13 months. Even at the disease onset, opportunistic infections are frequently complicated. Thus, host immune system both against tumor and infection is considered to be severely impaired in ATLL patients. However precise mechanism underlying that immunodeficiency still remains undetermined. Recently the lines of evidence that co-inhibitory molecules;programmed death-1/programmed death-1 ligand (PD-1/PD-L1) pathway in T-cell activation seems to play a crucial role in tumor escape from host immune surveillance system have been accumulated. In this study, we investigated the expression of PD-L1 on ATLL cells (CD4+/CD25+), and as their counterparts, CD4+ lymphocytes from HTLV-I asymptomatic carriers (ACs) to define their clinical significance.

[Patients and Methods] Peripheral blood CD4+ lymphocytes from 11 ATLL patients (9; untreated, 2; progressive state), 12 ACs and 2 healthy donors (HDs) were investigated for PD-L1 expression. We simultaneously measured co-inhibitory receptor, PD-1, expression on CD8+ HTLV-I Tax-specific cytotoxic lymphocytes (anti-Tax CTLs), as a marker of immunity against HTLV-I.

[Results] PD-L1 expression was detected on CD4+ lymphocytes in 3 of the 11 ATLL patients and 2 of them were in progressive state. In contrast, none of the ACs or HDs expressed PD-L1 on CD4+ lymphocytes, while those cultured with IL-2 for 48hs transiently expressed PD-L1. Anti-Tax CTLs were detected in 6 of the 11 ATLL patients and all of those CTL expressed PD-1 simultaneously. Anti-Tax CTLs were not detected in the 2 cases in progressive state. Anti-Tax CTLs were detected in 9 of 12 ACs, and PD-1 also co-expressed on those CTLs.

[Conclusions] CD4+ ATLL cells in progressive state expressed cell surface PD-L1, but not in untreated patients or ACs. This is a striking finding that exclusively aggressive form of ATLL tumor cells expressed cell surface PD-L1, which renders tumor cells to evade from host immune surveillance systems. Nonetheless large scale studies are needed to confirm these findings. In addition, Interestingly, co-inhibitory molecule PD-1 expressed on anti-Tax CTLs in not only ATLL patients but also ACs. Because PD-1 expression of CTL means “exhausted”, our observation suggests that anti-HTLV-I virus immunity is already exhausted both ATLL patients and ACs. Further experiments are warranted to define the significance of the correlation between PD-L1 on ATLL cells and PD-1 on anti-HTLV-I CTL in ATLL patients and ACs.
Alterations of Cell Signaling Pathways in Pediatric B-ALL Patients.
Authors: Accordi Benedetta , Espina Virginia , te Kronnie Gertruuy , Liotta Lance A. , Petricoin Emanuel F. , Basso Giuseppe
Published: April 2020

The outcome for children with Acute Lymphoblastic Leukemia (ALL) has improved in the last few decades with current therapy resulting in an event free survival exceeding 75%. Despite of this, 25% of patients still relapse or resist to conventional therapies. Proteomics, in particular the Reverse Phase Protein Arrays (RPA), offers a new approach to study both the potential molecular mechanisms of drugs chemosensitivity versus resistance, and identify new drug targets in the patients that do not respond to therapy. RPA, which can quantitatively measure dozens to hundreds of phosphorylated kinase substrates from only a few thousand cells, can be used to profile the working state of cellular signaling pathways in a manner not possible with gene microarrays, since post-translational modifications cannot be accurately portrayed by global gene expression patterns alone. We employed RPA to study the phosphorylated cellular pathways in bone marrow aspirate samples collected prior to treatment. The phosphorylation status of 95 key signaling proteins, including pro-survival, pro-apoptotic and those involved in cell cycle control were analyzed from 120 pediatric B-ALL specimens collected at the Pediatric Oncohematology Laboratory (University of Padova) over the past 15 years. Molecular network analysis and phosphoprotein profiling were performed using commercially available software. Our results indicate that there may be a difference between the molecular networks of activated kinase substrates within leukemic patients depending on disease state, genotype, or response therapy. The discovery of the molecular mechanisms related to drug resistance could play an important role for individualizing therapy and may reveal new strategies to improve treatment outcome.
Double Minute Chromosome in Acute Myeloid Leukemia.
Authors: Amin Alpesh J. , Shaw Maria , Tadros James , Benn Howard , Maroules Michael
Published: April 2020

Introduction: Double minutes (dmin) are small chromatin particles that lack a centromere and represent a form of extrachromosomal gene amplification. They have been found in a variety of solid tumors but are rare in hematological malignancies. C-MYC and MLL are the most common genes amplified on the dmin in AML. Here we report a case of AML in which FISH did not demonstrate C-MYC or MLL gene amplification on the dmin.

Case Report: A seventy-seven-year-old man was diagnosed with acute leukemia. Histology and flow cytometry (blasts positive for CD13, CD33, CD34, HLA-DR, CD15, CD117 and cytoplasmic MPO) were consistent with M2 AML. Chromosome analysis revealed abnormal karyotype with hypodiploidy 42–44 and loss of chromosome 5, 7,13,17,18, and Y. Structural rearrangements involving chromosomes 2,7,11,19 and 20 in addition to 2 marker chromosomes and dmin. FISH did not reveal C-MYC gene amplification. FISH did reveal MLL gene amplification, but not on the dmin. The patient received induction chemotherapy, and expired on day 14 due to cardiac complications.

Discussion: Double minutes are a mechanism for upregulated oncogene expression. After the development of molecular technology, FISH, it became possible to identify the amplified gene. A previous review (Thomas et al. Double minute chromosomes in monoblastic (M5) and myeloblastic (M2) acute myeloid leukemia: two case reports and a review of literature. Am J Hematol 2004 Sep;77(1):55–61), found C-MYC to be amplified on the dmin in 25 cases, MLL was found to be amplified on the dmin in 5 cases, and only 3 cases in which neither gene was found to be amplified on the dmin. Double minutes are associated with advanced age at presentation, a more aggressive clinical course, poor outcome with therapy, and shortened survival. The survival in most cases is reported to be less than 12 months.
Can Fibroblasts Be Reprogrammed into Macrophages?.
Authors: Feng Ru , Graf Thomas
Published: April 2020

Previous work showed that B cell precursors can be reprogrammed into functional macrophages by the enforced expression of the bZip transcription factor C/EBPalpha. The efficient activation of myelomonocytic genes, such as Mac-1, required the co-operation with endogenous PU.1 (Xie et al. 2004), reflecting the fact that many myelomonocytic genes are regulated by a combination of the two transcription factors. We therefore asked: Is C/EBPa and PU.1 sufficient to convert non-hematopoietic cells into macrophages? To test this, NIH-3T3 cells were co-infected with PU.1-GFP and C/EBPa-hCD4 retrovirusesor control vectors encoding the indicators GFP and hCD4 only. Uninfected cells in the retrovirus treated cultures served as additional controls. Our results showed that ~25% of the PU.1 only infected cells express Mac-1 and that this percentage could be increased ~3 fold by co-expression with C/EBPa. In addition, most cells also expressed CD45 and some expressed F4/80 antigen. The PU.1 infected and the double infected cells, but not the C/EBPa only infected cells, also expressed a number of other myelomonocytic genes as detected by RT-PCR. These included CSF-1R (M-CSFR), GM-CSF Ralpha, Lysozyme, CD32, PYK2 as well as endogenous PU.1. The PU.1 induced reprogramming of fibroblasts required the DNA binding and transcription activation domains, but not the PEST domain of the transcription factor. To test whether the reprogrammed cells have functional macrophage properties, we generated two stable cell lines co-expressing C/EBPa and PU.1 delta PEST (wild type PU.1 is toxic in long-term cultures). These cells were morphologically altered, ingested carboxylated particles, and expressed functional Fc-gamma receptors but were unable to phagocytize antibody coated red blood cells. Remarkably, the two cells lines acquired CSF-1 dependence for growth. In accordance with this finding they exhibited a 10–15 fold reduction of CSF-1 production compared to NIH3T3 cells. The response observed was not restricted to fibroblast cell lines since both embryonic and adult fibroblasts could also be partially reprogrammed by co-infection with PU.1 and C/EBPa in that they expressed Mac-1, CD45, F4/80 and IA MHC antigens. In conclusion, enforced expression of PU.1 and C/EBPa converts fibroblasts into macrophage like cells, indicating that the combination of these two transcription factors is sufficient to regulate the majority of genes that define the myelomonocytic phenotype.
CD34 Expression and Clinical Outcome of Childhood Acute Leukemia.
Authors: Kim Nam Kyun , Won Sung Chul , Lyu Chuhl Joo , Oh Seung-Hwan , Kim Hwang Min
Published: April 2020

Purpose: CD34 is a surface glycophosphoprotein which is expressed on early hematopoietic stem cells. It has been thought that leukemic blast with CD34 expression was associated with poor prognosis. In this study, correlation between CD 34 expression and clinical outcome of childhood acute leukemia was explored.

Method: Seventy-six patients who were diagnosed as acute leukemia in Department of Pediatrics, Yonsei University Medical Center, Seoul, Korea were enrolled in this study. All patients were analyzed retrospectively. Myeloid markers expression in acute lymphocytic leukemia (ALL) as well as CD34 presentation in ALL and acute myeloid leukemia (AML) were analyzed. Three-year event-free-survival (EFS) was calculated according to whether those antigens were expressed or not.

Result: Fifty-eight and eighteen patients were ALL and AML, respectively. Median follow up period for all patients was 2.2 years (0.1–4.6 years). Boys and girls were 33 and 25 in ALL patients, respectively. In childhood AML, 11 and 7 patients were male and female, respectively. EFS for all patients with acute leukemia was 74.1± 5.4%. Three-year EFS of AML with or without CD34 expression was 83.3±15.2 % and 46.7±15.4 %, respectively. But, the difference was not different statistically (P=0.164, Fig. 1). In ALL, Three-year EFS was 80.7±8.9 % and 78.8±7.2 % for group of CD34 negative and positive, respectively (p=0.998, Fig. 2).

Conclusion: According to data, CD34 expression of AML in children might contribute to the survival rate, however EFS was not different statistically. We think that because of small number of cohort, the statistical results were not significantly different. In ALL, there were no statistically significant differences in EFS between CD34 expression or not.

Fig. 1. Event-free survival for acute myeloid leukemia according to CD34 presentation Fig. 1. Event-free survival for acute myeloid leukemia according to CD34 presentation Fig. 2. Event-free survival for acute lymphocytic leukemia according to CD34 presentation Fig. 2. Event-free survival for acute lymphocytic leukemia according to CD34 presentation
The Expression and Regulation of ADAMTS13 in Hematopoietic System.
Authors: Liu Fang , Bai Xia , Dai Master Lan , Dong Ningzheng , Ruan Changgeng
Published: April 2020

Background and Objective In 2001, VWF-cleaving protease was identified as ADAMTS13, a member of the ADAMTS family of metalloprotease. ADAMTS13 is now found as the mainly cleaving enzyme of VWF in plasma. It is synthesized predominantly in the liver, although variable expression has been observed in other issues including bone marrow. Although ADAMTS13 was detected only in platelet, not other blood cells, it is unsure what kind of situations would be when we come to immature hematopoietic cells. So we investigated ADAMTS13 in cell lines such as M07e, MEG01, HEL, k562, SHI-1, NB4, MR2 etc., to see if there is ADAMTS13 produced by blast cell of megakaryocyte and granulocyte and so on.

Methods We determined ADAMTS13 cDNA by RT-PCR and ADAMTS13 expression by flow cytometry and western blotting with some mono-clone antibodies against ADAMTS13. During all those test, we use human platelets as positive control.

Results ADAMTS13 was detectable in all those cell lines. It was highly expressed in M07e and MR2, and a bit lower in MEG01 and HEL, and much lower in k562, SHI-1 and NB4. While to our surprise, ADAMTS13 was up-regulated when NB4 was treated by 1μmol/L ATRA for 72 hours. When CD11b was rising from 21% up to 95% in NB4 after treatment, expression of ADAMTS13 followed by from 24% to 80%.

Conclusions Now we know what kinds of cells may express ADAMTS13 in bone marrow, the major part of it is localized in megakaryocyte. ADAMTS13 can highly expressed in NB4 after treatment with ARTA.
The Experience on Multiparametric Molecular Diagnostic Approach of Pediatric Acute Lymphoblastic Leukemia (ALL) in Greece.
Authors: Parcharidou Agapi , Sambani Constantina , Stavropoulou Christina , Paterakis George , Belesi Chrysoula , Papadhimitriou Stefanos , Tsitsikas Constantinos , Georgakakos Vassilios , Papadakis Vassilios , Polychronopoulou Sophia
Published: April 2020

The rate of success in the treatment of pediatric acute lymphoblastic leukemia (ALL) has been increased steadily during the last decades. The five years’ event free survival rate is nearly 80% for children with ALL. Attempts to boost cure rates further with the use of hematopoietic stem cell transplantation have improved for some but not all, subtypes of ALL. The best hope for continued progress lies in a better understanding of the pathogenesis, the basis of resistance to chemotherapy, and finally better organized clinical trials. The present study has been based on organizing and exploring new clinical correlations among clinical data obtained from molecular genetic profile, in vitro chemosensitivity and genetic polymorphisms of detoxifying enzymes. During the last 3 years 43 newly diagnosed ALL patients, 27 boys and 16 girls, aged 23 months to 14 years old were included in this study. Bone marrow and/or peripheral blood samples were studied for karyotyping aberrations. The presence of the specific translocations t(12;21), t(9;22), t(4;11) and t(1;19) was investigated using RT-PCR and FISH. Furthermore, FISH was also used for the detection 9p deletions and MLL rearrangements. Immunophenotype of blasts and DNA index were studied by flow cytometry. In vitro chemosensitivity studies were performed by the MTT assay (ELISA).The GSTT1 genetic polymorphism (null genotype) was detected by multiplex PCR and NQ1 genetic polymorphism was detected by PCR -RFLPs. A cytogenetic/molecular result was achieved in 39/43 patients. Structural or numerical aberrations were detected in 7/39 patients. 9/39 patients were positive for the TEL/AML1 (23%) and 3/39 for the BCR/ABL fusion genes. One patient showed only one MLL allele, no patient had MLL rearrangement and 5/19 patients presented 9p deletion. A null GSTT1 genotype was observed in 5/43children (11,62%) and 14/43 patients were heterozygotes for NQ1(32,5%). 8/36 patients presented in vitro chemoresistance and 8/43 patients had DNA index >1 (18,6%).In our series of patients the frequency of t(12;21) does not seem to differ significantly from the literature data. The patients who showed chemoresistance had also unfavorable prognostic markers according to cytogenetic/molecular diagnostic data or clinical characteristics. The number of the patients is low to correlate detoxifying enzymes to toxicity or response during treatment. The multiparametric diagnostic approaches in pediatric ALL seem to be of great importance in diagnosis and tailored therapy leading to high rates of cure. Our center’s effort is the optimal characterization of the pediatric ALL profile in Greece by the use of multiparametric diagnostic methods targeting a better outcome.
Bi-Allelic Involvement in AML: Examples of 16q Aberrations in Two Cases.
Authors: Howard Julie , Brazma Diana , Vaughan Beverly , Nacheva Elisabeth P.
Published: April 2020

Acute Myeloid Leukaemia is a heterogeneous clonal disorder of the bone marrow, which has characteristic acquired chromosomal abnormalities. Cytogenetic rearrangements of chromosome 16 have a well-established association with AML subtype M4Eo although they have also been described in other AML FAB types. Patients with rearrangements involving CBFb (16p13) and MYH11 (16q22) are associated with a more favourable prognosis. We describe two cases of AML where both homologues of chromosome 16 are involved in separate rearrangements; this to our knowledge has not been reported previously. Further more both patients have had previous treatment and thus the abnormality of the second homologue of chromosome 16 is thought to be treatment related, in-spite of which they have responded well and are both currently in remission. In case 1 (male, AML M5a) it is chromosome16 at band q22 that is involved, inv(16)(p13q22) and t(16;17)(q22;q11), while in case 2 (female, t-AML) chromosome 16 band q24 appears rearranged in der(16)t(8;16)(q1?3;q24) and t(16;21)(q24;q22). Extensive molecular cytogenetic investigations including mapping are presented and candidate gene aberrations discussed.
Differences in Proteasome Activity in Plasma of Patients with Hematologic Malignancy.
Authors: Ostrowska Halina , Kloczko Janusz , Klimiuk Miroslawa , Sokolowski Jaroslaw , Blusiewicz Eliza
Published: April 2020

The proteasome-dependent proteolytic pathway plays a crucial role in pathogenesis of hematologic malignancy, and as such it is a novel target for therapeutic drugs. High proteasome levels measured by conventional technique ELISA have been demonstrated in human malignant cells and in the plasma of patients with some hemopoietic malignancies and solid tumors. The objective of this study was to evaluate the activity of the 20S proteasome in the plasma from patients with hematologic malignancy.

The study included normal subjects (n=15) and untreated patients with acute lymphoblastic leukemia (ALL, n=13), acute myeloblastic leukemia (AML, n=21), multiple myeloma (MM, n= 26) and chronic lymphocytic leukemia (CLL, n=30). The 20S proteasome activity in plasma (50μg of protein) was assayed spectrofluorimetrically using synthetic peptide substrate, Suc-Leu-Leu-Val-Tyr-AMC (100μmol/L) in the presence of 0.03% SDS that selectively activates the chymotrypsin-like activity of 20S proteasome. Enzyme units (U) were expressed as pmol AMC/min.

The average activity of 20S proteasome in plasma from normal subjects was found to be 4.42 ± 0.56U/mg. The highest activity of the 20S proteasome was found in plasma from patients with ALL with a median of 55,5 U/mg (range 10.5–225.6 U/mg, p<0.01). High proteasome activity was also observed in plasma from patients with AML with a median of 28.6 U/mg (13.0–64 U/mg; p<0.01). In patients with MM, the average plasma proteasome activity was 11.1 ± 3.1 U/mg (range 8–18.8 U/mg; p<0.01). In contrast, in patient with CLL plasma proteasome activity was similar to that in normal subjects (4.2± 1.1; p=0.05). There was a significant positive correlation with plasma lactic dehydrogenase activity (LDH) (a global marker of cell lysis), but not with white blood cell (WBC) and blast cell counts in all the examined groups of patients.

The obtained results indicate the presence of high level of 20S proteasome in the plasma of patients with ALL, AML and MM, but not with CLL, which has potential chymotrypsin-like activity.
DEAH-Box Splicing Factor Gene, Prp16 Amplification in Acute Myeloid Leukemia.
Authors: Ma Yupo , Barnett Tammy , Chai Li , Yang Jianchang , Alipio Zaida , Pei Lin , Amin Hesham M. , Fink Louis , Di Chunhui , Yan Hai , Ward David
Published: April 2020

Aberrant mRNA splicing has been observed frequently in solid tumors and shown to play a functionally significant role in tumorgenesis. Here we demonstrate that the DEAH-Box splicing factor gene, Prp16, is amplified in fresh human acute myeloid leukemia (AML) and in established AML cell lines. Prp16, an RNA-dependent ATPase required for pre-mRNA splicing, maps to chromosome 16q22, a region frequently altered in AML. Amplification of the Prp16 gene was initially detected using digital karyotyping, a powerful technique for analyzing genome-wide alterations in DNA copy number and verified by quantitative real-time PCR. Analysis of mRNA expression profiles revealed that Prp16 transcripts were present at high levels in 22 of 39 cases of AML (56%) and in both AML cell lines examined. There was a strong correlation between gene amplification and mRNA expression levels. To our knowledge, this is the first demonstration linking aberrant expression of a splicing enzyme to leukemogenesis. The classification and clinical outcome of the AML cases is being correlated with the presence or absence of Prp16 amplification. The identification of leukemia-specific splicing event(s) may provide a novel target for therapeutic intervention.
Prognostic Factors and Outcomes of Patients with Acute Myeloid Leukemia (AML) Receiving Non Intensive Chemotherapy (NIC) or Best Supportive Care (BSC).
Authors: Baz Rachid , Rodriguez Cristina , Jawde Rony Abou , Rybicki Lisa , Kalaycio Matt , Advani Anjali , Sobecks Ronald , Sekeres Mikkael A.
Published: April 2020

Background: AML in older adults poses therapeutic challenges, as many patients are not candidates for intensive chemotherapy (IC), and it is not clear that IC provides a survival benefit compared to NIC/BSC. While risk factors and outcomes of patients treated with IC are well-defined, few data support similar prognostication in patients treated with NIC or BSC.

Methods: We conducted a retrospective review of all patients with AML receiving NIC or BSC at the Cleveland Clinic between January 1997 and December 2005. NIC consisted of treatment with hydroxyurea, low-dose cytarabine, or azacitidine. The choice and decision of therapy was dictated by patient and physician preferences. Data on known prognostic factors (age, white blood cell count (WBC) at diagnosis, cytogenetic risk groups (as defined by CALGB 8461) and AML etiology (de novo vs. secondary AML)) were collected and controlled for in multivariable analyses, and survival determined using the log-rank test, measured from the time of diagnosis. Race and residential zipcodes were obtained from information disclosed by patients in their electronic medical records. Average annual household income in a zipcode was obtained using a web-based database, www.melissadata.com.

Results: Fifty-three patients were identified. The median age was 74 years (range 33–92), and 26 patients (49%) were female. Eight patients (15%) were African American (AA), while 44 patients were Caucasian (C) and 1 patient was neither AA nor C. The median WBC at presentation was 6.0 ×103/μL. Cytogenetics were favorable in 1 patient (2%), intermediate in 10 (19%), unfavorable in 19 (36%), and unknown in 23 (43%). Most patients (60%) had secondary AML. The median average annual income was $39,332 (range 23,375–82,806). The median survival was 54 days (95% CI 33–80 days) for all patients, and the 1 year survival was 7%. Known prognostic factors for AML (WBC at presentation, de novo vs. secondary AML, cytogenetics, and age at diagnosis) were not predictors of overall survival in multivariable analyses in patients not receiving intensive chemotherapy, though higher WBC count was predictive in univariable analyses. Race was a predictor for outcomes, with AA patients experiencing a worse median survival than their C counterparts (22 days versus 64 days, log rank p=0.03, figure 1) despite similar baseline characteristics. SES, however, had no impact on outcome.

Conclusions: AML patients receiving NIC or BSC have a median survival of almost 8 weeks, with AA patients experiencing worse outcomes. A small but significant percentage of patients remain alive at one year. Prognostic variables useful in treatment decision-making in patients receiving IC are less valuable for NIC/BSC patients. This information can be used for patient counseling and to help guide treatment decisions.

Figure Figure
Mechanism Study of HL-60 Leukemic Cells Proliferative Inhibition Induced by Chinese Medicinal Herbs Mixture Jieduweikang Pill.
Authors: Zhang Ri , Wu Pin , Zhu Zi-ling , Cen Jiannong , Wu De Pei
Published: April 2020

Background. It’s not surprising that the traditional Chinese herbal remedy is yielding useful drug leads. The mixture composed of thirteen Chinese medicinal plants---jieduweikang(JDWK) pill including Qingdai, Qinghao, Huangchi, Baihuasheshecao, Banzhilian and so on has been shown the clinical antitumor effects in China. We here report the mechanism investigation of JDWK pill cytotoxicity to leukemia cell line HL-60.

Methods By using the method of serum pharmacology, the serum containing the JDWK pill was collected from rats. The MTT and tumor colony formation were used to determine the effects of JDWK pill on proliferation of HL-60 cells. NBT reduction, DNA electrophoresis, flow cytometry and RT-PCR were used to analyze the differentiation and apoptosis of HL-60 cells after treating with JDWK pill. Elisa was used to assay the secretion of VEGF from HL-60 cells.

Results The liquid growth and colony formation of HL-60 cells were evidently inhibited by the serum containing JDWK pill and the cells were shown characteristic DNA “ladder” on agarose gel electrophoresis and more than 20% apoptotic cells in DNA histogram as well as the downregulation of bcl-2 gene related to apoptosis in a concentration-dependent manner, compared with the control. NBT reduction assay indicated that JDWK pill with low concentration may induce myeloid terminal differentiation of HL-60 cells and the level of VEGF was obviously decresed.

Conclusion The JDWK pill can exerts the its anti-proliferative effect on the leukemic HL-60 cells through inducing apoptosis relating to the downregulation of Bcl-2 gene and anti-angiogenic effects with higher concentration and inducing myeloid differentiation with lower concentration.