bluuebunny/crossref_metadata_2025 huggingface dataset:
Epigenetic-Based Treatment Induces Apoptosis in Leukemic Cell Lines.
Authors: Carnicer Maria J. , Lasa Adriana , Serrano Elena , Pena Jorge , Remacha Angel F. , Aventin Anna M. , Nomdedeu Josep F.
Published: April 2020

Pharmacological treatment of cancer cells with demethylating agents and histone deacetylase inhibitors synergistically reactivates the transcription of previously silenced genes.

The aim of this study was to investigate the antileukemic properties of a DNA mehtyltransferase inhibitor 5-aza-2′-deoxycytidine (5-Aza-dC),an histone deacetylase inhibitor Trichostatin A (TSA) and trans-retinoc acid (ATRA), alone or in combination. The effects of these drugs on apoptosis, cell cycle progression, cell-survival pathways and restoration of proliferation-associated genes silenced by aberrant epigenetic mechanisms were assessed. Four human leukemic cell lines were used: lymphoid cell-lines REH (Tel-Aml1+) and DAUDI (c-myc) and myeloid cell-lines Kasumi-1(Aml1-Eto+) and NB4(Pml-Rara+). The cells were cultured for three days. Cell viability, percentage of apoptosis, cell cycle and apoptosis-controlling proteins were examined by multiparametric flow cytometry, western blotting and a RT-PCR low-density array (LDA)containing 48 probes. Combined 5-Aza-dC and TSA treatment induced a high degree of apoptosis and affected the cell-cycle in all the cell lines analyzed. Increased levels of cleaved PARP were detected after the double treatment and paralleled those of caspase-3 except for DAUDI cell-line where there was no cleavage of procaspase 3 suggesting that this treatment induced a death-pathway independent of caspase-3 activation. Furthermore, ATRA alone has a limited capacity to induce apoptosis in all the tested cell-lines.

The genes upregulated following the combined epigenetic-active treatment in DAUDI were; PRKCG, TNFSF10, TNFRSF10B, MAPK8, CASP9, APAF1, PER1, CDKN1A, CCND1. In the REH cell line were; PRKCG, TNFRSF10A, TNFRSF10B, MAPK8, CASP8, CASP9, APAF1, CASP3, AKT1, PIK3CG, BIK, PRKCG, MCL1, CCND1, CCND2, EZH2, PER1, GCLC, GSTP1. In the KASUMI-1 cell line were; PRKCABP, TNFSF10, TNFRSF10A, TNFRSF10B, FAS, CFLAR, CASP8, APAF1, CEBPA, CCND1, CDC25C, CDKN1A, EZH2.For the NB4 cell-line were: PRKCG, PRKCABP, AKT1, TNFSF10, CFLAR, CASP8 and CASP9. We can conclude that combined treatment with demethylating agents and histone deacetylase inhibitors may be active in a wide range of human leukemias. The potential use of this combination in the commonest form of pediatric ALL warrants further investigation.
Synergy Studies between the Nitric Oxide (NO) Donor JS-K and Other Anti-Leukemic Agents.
Authors: Shami Paul J. , Eddington Jordan K. , Udupi Vidya , Kosak Ken M. , Saavedra Joseph E. , Keefer Larry K.
Published: April 2020

NO induces apoptosis in Acute Myelogenous Leukemia (AML) cells. Glutathione S-Transferases (GST) play an important role in multidrug resistance and are upregulated in 90% of AML cells. We have designed a prodrug class that releases NO on metabolism by GST. O2-(2,4-Dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K, a member of this class) has potent antileukemic activity (Molecular Cancer Therapeutics 2:409–417,2003). We sought to identify synergistic interactions between JS-K and other antileukemic agents. The human HL-60 myeloid leukemia cell line was used for in vitro studies of the combination of JS-K with Daunorubicin (Dauno), Cytarabine (Ara-C) or Etoposide (Etop). Drugs were added to cells in logarithmic growth. Cell viability was determined 3 days later using the CellTiter 96 assay (Promega, Madison, WI). The median effect method of Chou and Talalay was used to determine synergistic, antagonistic, or additive effects with the CalcuSyn software (Biosoft, Ferguson, MO). Combinations of JS-K added simultaneously, 2 hours before or 2 hours after the other compounds were used. Drug combinations were added in increments at a fixed concentration ratio. The 50% growth inhibitory concentrations (IC50) for Dauno, Ara-C, Etop, and JS-K alone were 0.07, 0.18, 0.79, and 0.30 μM, respectively. JS-K and Dauno (Dauno:JS-K fixed concentration ratio = 1:5) were antagonistic in all 3 drug sequences. The Combination Index (CI) at the 50% fraction affected was 12.5 ± 1.6, 10.7 ± 0.41, and 7.2 ± 0.46 for simultaneous, JS-K first and Dauno first additions, respectively. JS-K and Etop (Etop:JS-K fixed concentration ratio = 2:1) were antagonistic but to a lesser degree. The CI at the 50% fraction affected was 3.03 ± 0.26, 2.2 ± 0.33, and 1.7 ± 0.082 for simultaneous, JS-K first and Etop first additions, respectively. JS-K and Ara-C (Ara-C:JS-K fixed concentration ratio = 1:1) showed strong synergy. The CI at the 50% fraction affected was 0.37 ± 0.23, 0.24 ± 0.27, and 0.15 ± 0.11 for simultaneous, JS-K first and Ara-C first additions, respectively. We conclude that Ara-C and JS-K have synergistic anti-leukemic activity and warrant further exploration in combination.
The Transcription Factor ZBP-89 Controls Generation of the Hematopoietic Lineage in Zebrafish and Mouse Embryonic Stem Cells.
Authors: Li Xiangen , Xiong Jing-wei , Shelley Carl Simon , Park HeiYoung , Arnaout M. Amin
Published: April 2020

Hematopoietic development is closely linked to that of blood vessels and the two processes are regulated in large part by transcription factors that control cell fate decisions and cellular differentiation. Both blood and blood vessels derive from a common progenitor, termed the hemangioblast, but the factors specifying development and differentiation of this stem cell population into the hematopoietic and vascular lineages remain ill defined. Here we report that knockdown of the Krüppel-like zinc finger ZBP-89 (ZNF148) in zebrafish embryos results in a bloodless phenotype, caused by disruption of both primitive and definitive hematopoiesis, leaving primary blood vessel formation intact. Expression of the primitive (Scl, Lmo2, Gata2, Gata1) and definitive (Runx1, c-Myb) hematopoietic stem cell lineage markers in ZBP-89 morphants were significantly decreased while expression of the lymphopoiesis lineage markers Ikaros and Gata3 was unaffected. These developmental HSC defects are rescued by injection of human ZBP-89 RNA, SCL RNA or ZBP-89 cDNA under control of the zebrafish Flk1 promoter. Injection of ZBP-89 RNA into cloche zebrafish embryos, which lack the hematopoietic and endothelial lineages, rescued hematopoiesis but not vasculogenesis. Overexpression of ZBP-89 in wild type zebrafish expanded hematopoietic progenitors (Scl+, Lmo2+, Gata1+) but reduced the endothelial cell markers FLK1 and TIE1. Forced expression of ZBP-89 in mouse embryonic stem cell cultures also induced the expansion of hematopoietic progenitors (c-KIT+, SCA1+) and definitive hematopoietic cell population (CD45+), but inhibited sprouting angiogenesis and the CD31+, VE-Cadherin+ endothelial cell population. These findings establish a unique regulatory role for ZBP-89, positioned at the interface between early blood and blood vessel development.
The Study of the Expression of TNF in Bone Marrow Specimens and Survival in Patients with Acute Myelogenous Leukemia.
Authors: Shum Merrill K. , Hageboutros Karine , Skaff Ziad , Kankipati Shoba , Hageboutros Alexandre
Published: April 2020

Background: The objective of the current study was to assess the correlation between the expression of TNF from bone marrow specimens and survival in patients with acute myelogenous leukemia (AML).

Methods: Immunohistochemical analysis of bone marrow biopsies from 10 patients recently diagnosed with AML was performed using the dakocytomation LSAB + system -HRP kit, to stain for TNF binding. TNF-α and TNF-β expression were analyzed for color intensity and assigned a score from (0–5+) with 5+ being bright and over-expressed.

Results: Bone marrow specimens from 4 women and 6 men (n=10), median age 34 (range 19–61 years), all diagnosed with AML, revealed high TNF-β expression (3, 4, 5+) in 8/10 patients and high TNF-α expression (3, 4, 5+) in 6/10 patients. Median survival was 5.5 (range 1–54 months). Fisher’s exact tests for paired samples showed no correlation between predictors (age, sex, survival) and outcomes (TNF-α, TNF-β). Pearson Chi-square tests showed no correlation between predictors (age, sex, survival) and outcomes (high TNF-α / high TNF-β expression, mixed TNF-α /TNF-β expression, low TNF-α / low TNF-β expression) [See chart below]. However there was an association seen with younger males expressing both high TNF-α and high TNF-β and older women expressing low TNF-α and low TNF-β. Interestingly, the eldest patient who expressed TNF-β 4+ and TNF-α 3+ also survived the longest. He achieved clinical remission after standard induction chemotherapy, but relapsed 8 times over a course of 54 months. During each clinical remission, the patient was treated with infliximab (a TNF-α blocker) for his Crohn’s disease.

Conclusion: Level of expression of TNF-α and TNF-β of bone marrow biopsies from our AML patients showed no correlation with age, sex or overall survival given the small sample size. A large, prospective trial will be needed to determine the significance of TNF-α or TNF-β expression with survival and whether TNF-α blockade alone or in combination with chemotherapy plays a role in the treatment of patients with AML over-expressing TNF-α.

SUMMARY OF ASSOCIATIONS OUTCOMES TNF α+TNFβ* TNF α** TNF β** *Pearson Chi-square with 2dF **Fisher’s Exact Test Sex 0.43 0.57 0.47 PREDICTORS Survival 1.00 1.00 1.00 Age 0.26 0.52 0.42
HLA-DRB1 Analysis in Hairy B-Cell Lymphoproliferative Disorder (HBLD): A Japanese Type of Benign Persistent Polyclonal B-Cell Lymphocytosis.
Authors: Nakahashi Hirotaka , Karasawa Masamitsu , Hashimoto Yoko , Irisawa Hiroyuki , Yokohama Akihiko , Saitoh Takayuki , Handa Hiroshi , Matsushima Takafumi , Murakami Hirokazu , Tsukamoto Norifumi , Nojima Yosihisa
Published: April 2020

Two types of benign persistent polyclonal B-cell Lymphocytosis have been reported: persistent polyclonal B-cell lymphocytosis (PPBL) in Western countries and hairy B-cell lymphoproliferative disorder (HBLD) in Japan. PPBL is characterized by B lymphocytosis with binucleated lymphocytes, polyclonal IgM, isolated splenomegaly, female sex, cigarette smoking and the presence of HLA-DR7 allele. Most patients with HBLD have atypical lymphocytes with microvilli and prominent membranous ruffles, serum polyclonal IgG, and splenomegaly. Only 9 patients with HBLD have been reported to date, and all of these patients are Japanese. Here, we report three new cases of HBLD. We performed HLA-DRB1 subtyping since the association between HBLD and HLA-DR4 has been suggested: 4 of 5 HBLD patients examined demonstrated the HLA allele. We also analyzed HLA-DRB1 alleles in a patient with hairy cell leukemia (HCL) and 3 with HCL Japanese variant (HCL-JV) to examine the possibility that polyclonal cell expansion in HBLD may be followed by transformation to these lymphoproliferative disorders. If the transformation were to occur, the patients should share the same HLA-DR allele.

Expanded B-lymphocytes in all HBLD cases displayed CD5− CD10− CD11c+ CD19+ CD20+ CD23− CD25− and CD103− cell surface phenotype and all patients exhibited a polyclonal increase in serum IgG. Phase-contrast microscopic and scanning electron microscopic examinations demonstrated lymphoid cells with many long surface microvilli. Analysis of immunoglobulin VH gene rearrangements showed a polyclonal pattern. Therefore, we diagnosed these patients as HBLD. HLA-DRB1 subtype analysis strengthened the close relation between HBLD and HLA-DR4: 2 patients having HLA-DRB1*0405 and one HLA-DRB1*0407. In the meanwhile one each patient with HCL and HCL-JV had HLA-DRB1*0405 allele.

Although the exact mechanism underlying the association between HBLD and HLAB1*04 needs to be elucidated, the presence of this HLA might be useful to establish correct diagnosis of the disease, and thus avoid patient exposure to unnecessary treatment. One each patient with HCL and HCL-JV has HLA-DRB1*0405, but a definitive conclusion cannot yet be drawn due to high HLA-DRB1*04 frequency in Japanese population (25.4%).
Significance of Marrow Angiogenesis Factors in Patients with Acute Myelogenous Leukemia.
Authors: Lin Liang-In , Lee Cheng-Yeh , Hu Chung-Yi , Tang Jih-Luh , Tien Hwei-Fang
Published: April 2020

Bone marrow (BM) neoangiogenesis plays an important role in the pathogenesis of acute myelogenous leukemia (AML). Paracrine exchange of growth factors among AML blasts, endothelial cells, and other marrow compartments is believed to contribute to the pathogenesis of AML. The vascular endothelial growth factor (VEGF) and the angiopoietin (ANG) family are the two major classes of growth factors associated with angiogenesis. In the present study, BM plasma from 52 AML patients and 20 normal donors were investigated. We measured the marrow concentrations of seven molecules associated with angiogenesis, VEGF, VEGF/PlGF, VEGF-C, VEGF-D, ANG-1, ANG-2, and Tie-2, by using enzyme-linked immunosorbent assay (ELISA). Compared to normal donors, the marrow levels of VEGF/PlGF, ANG-2, and Tie-2 were increased in patients with AML (p<0.005, <0.0001 and <0.0001, respectively). On the contrary, VEGF-C and ANG-1 levels were decreased in patients with AML (p<0.0001 and <0.0005, respectively). Although the values were not completely normalized, sequential study revealed that at complete remission (CR) period, VEGF/PlGF, ANG-2, and Tie-2 levels were decreased (p<0.005, <0.005 and =0.02, respectively), while VEGF-C increased (p=0.04). Kaplan-Meier survival curves showed that the subgroup of patients with lower Tie-2 level (29 ng/ml) had a longer median survival time than those with higher Tie-2 level (18.9 months versus 2.5 months, p<0.005). In addition, subgroups of patients with higher VEGF/PlGF level (1 pg/ml) and VEGF-D level (350 pg/ml) also had a longer median survival time than those with lower levels (20.8 months versus 7.2 months, p=0.03 and 18.9 months versus 3.4 months, p=0.03, respectively). Taken together, there were significant differences in VEGF/PlGF, VEGF-C, ANG-1, ANG-2, and Tie-2 expressions between AML patients and normal donors. Expressions of VEGF/PlGF, VEGF-D, and Tie-2 might be prognostic markers in AML patients.
Prognostic Significance of FLT3 Mutations in Paired Initial and Relapse Marrow Samples of Acute Myeloid Leukemia Excluding Acute Promyelocytic Leukemia.
Authors: Cho Young-Uk , Chi Hyun-Sook , Jang Seongsoo , Park Chan-Jeoung , Lee Je-Hwan , Lee Kyoo Hyung , Lee Jung-Hee , Lim Ho-Jun , Seo Jong-Jin
Published: April 2020

Fms-Like tyrosine kinase 3 (FLT3) mutations are related to poor prognosis in acute myeloid leukemia (AML), with the exception of acute promyelocytic leukemia (APL), in which the prognostic significance of this mutation is not firmly established even though it is more frequent than other FAB subtypes. We investigated FLT3 internal tandem duplications (FLT3/ITD) and/or D835 point mutations in both initial and relapse marrow samples from 120 AML patients, excluding APL. FLT3/ITDs were found in 18 initial and 16 relapse samples (15.0 and 13.3%, respectively). D835 mutations were found in one initial sample and 4 relapse samples. Two cases acquired FLT3/ITD at relapse, 4 cases lost mutations at relapse, and one had a different allele of FLT3/ITD at relapse. Patients who had FLT3/ITDs at initial diagnosis showed shorter median duration from diagnosis to relapse and shorter overall survival compared with those without mutations (median duration to relapse: 8.3 and 11.0 months, respectively, P = 0.074; overall survival: 12.2 and 20.8 months, respectively, P = 0.058). These findings were more pronounced when the FLT3/ITDs were acquired at relapse or discrepant patterns were noted between diagnosis and relapse (median duration to relapse: P = 0.047 and 0.044, respectively; overall survival: P = 0.002 and 0.038, respectively). In conclusion, the present study showed that patients with AML had heterogeneous patterns of FLT3/ITD or D835 mutations, either acquisition or loss of the mutations at relapse. The overall survival and relapse-free survival times were significantly shorter in the mutation acquisition group than in those with mutation loss at relapse.
Human Plasmacytoid Dendritic Cell like Leukemia Cell Line (PMDC05) Established from a Patient with CD4+CD56+ Acute Leukemia.
Authors: Narita Miwako , Tochiki Nozomi , Watanabe Norihiro , Saitoh Anri , Hashimoto Shigeo , Tsuchiyama Junjiro , Furukawa Tatsuo , Aoki Sadao , Fuse Ichiro , Aizawa Yoshifusa , Takahashi Masuhiro
Published: April 2020

Human dendritic cell precursors are commonly divided into two distinct subsets: myeloid DC and Plasmacytoid DC (pDC). The pDC, which show plasma cell like morphology, have been defined as the population that produce a large amount of type I interferon in response to viruses. The surface phenotypes of human pDCs are defined as CD4+, DC11c−, CD45RA+, IL3Rα (CD123)+, CD1c (BDCA-1)−, CD303 ((BDCA-2)+ and lineage negative. On the other hand, leukemia/lymphoma cells in CD4+CD56+ leukemia/lymphoma have been proposed to be of pDC lineage. CD4+CD56+ pDC leukemia/lymphoma are a rare hematological malignancy, totally only about 100 cases in the world by the literatures. We established a pDC like leukemia cell line (PMDC05) from leukemia cells of a patient with CD4+CD56+ acute leukemia. PMDC05 showed a complex hypoploid chromosomal abnormalities (44, XY) including add(5)(q22), add(15)(q26) and del(15)(q11q15), which is identical to original leukemia cells. Abnormalities including 5q and 15q are reported as the frequent aberrations in CD4+CD56+ pDC leukemia/lymphoma. PMDC05, which morphology was similar to plasma cells, was positive for CD4, CD56, CD123, CD33, CD86, HLA-ABC, HLA-DR, CD1a, CD40, and CD83 but negative for linage markers. Cytokine receptors for GM-CSF, IL3Rα and IL-6Rα were positive on PMDC05. The expression of Trail and Flt-3L was positive. By the culture with IL-3, CPG-A/B, GM-CSF, molecules associated with antigen presentation such as CD1a and CD40 were up-regulated. Besides, the addition of LPS increased the expression of CD40, CD80 and CD83 on PMDC05. PMDC05 by itself possessed a potent antigen presenting ability to naïve T cells and the treatment of PMDC05 with IL-3, CPG-A/B, or GM-CSF enhanced the antigen presenting ability to naïve T cells. TLR7, TLR 8 and TLR 9 as well as TLR1, TLR2, TLR4 were demonstrated to be expressed on PMDC05 by RT-PCR and RQ-PCR showed that the expression of TLR7 and TLR9 was most characteristic. λ-like 14.1 and preTα was also demonstrated to be expressed on PMDC05 by RT/RQ-PCR. PMDC05 possessed an ability to uptake the antigens like FITC-dextran and lucifer yellow. Although IFN-α was not identified to be secreted from PMDC05 by the stimulation of influenza virus, IFN-γ and TNF-α was demonstrated to be secreted to the similar level in pDC, which was examined simultaneously with PMDC05 by CBA assay. These data demonstrated that newly established leukemia cell line PMDC05 is involved in pDC lineage and PMDC05 provides invaluable tools not only for the elucidation of pathophysiology of CD4+CD56+ leukemia/lymphoma but also for the investigation of differntiation and regulation of pDC. In addition, PMDC05 could be applied for generating tumor-specific CTL clone, which may be used for anti-tumor cellular immunotherapy.
Expression of Activating Natural Killer Cell Receptor Ligands in Childhood Acute Lymphoblastic Leukemia.
Authors: Mura Ulrike , Pfeiffer Matthias , Handgretinger Rupert , Lang Peter J.
Published: April 2020

Activating and inhibitory cell surface receptors regulate natural killer (NK) cell effector functions. The extent of expression of their activating ligands on target cells probably plays a critical role in tumor immune surveillance, but the data of this expression on blasts of leukemia patients are still poor. We examined blasts of children with ALL for the activating human NKG2D ligands MICA, MICB, ULBP1, ULBP2 and ULBP3, and for the ligands of the activating human natural cytotoxic receptors (NCRs) NKp30, NKp44 and NKp46. Using ligand specific mouse monoclonal antibodies and flow cytometry, we screened 24 children (23 common-ALL, one pre-T-ALL) for the expression of NKG2D ligands and 15 children (all common-ALL) for NCR ligands. In 13 patients (all common-ALL), also the density of the NKG2D ligands was determined by quantitative flow cytometry. Considering cells positive for a particular ligand in case of a two fold increase of median fluorescence above negative control, 38 percent of the patients were positive for one or more NKG2D ligands, while only 13 percent expressed one or more NCR ligands at significant levels. ULBP1 was most frequently expressed (29 percent of patients positive), while no patients were positive for ULBP2 and NKp44 ligands. ULBP3 was positive in 17 percent of the patients, NKp30 and NKp46 ligands in 13 percent, MICA and MICB in 4 percent. The patient with pre-T-ALL was positive only for ULBP1. So ULBP1 was expressed more frequently, while the other NKG2D ligands were expressed less frequently in children than reported for adult leukemia patients before (Salih et al. Blood.2003;102:1389–1396.). The density of detected NKG2D ligand molecules was always rather low. For MICA the maximum were 1700 molecules per cell in a single patient, for MICB 900, for ULBP1 1100, and for ULBP3 1000 molecules per cell. In summary, blasts of pediatric ALL patients displayed low or negative surface levels of ligands for the human activating NK cell receptors NKG2D, NKp30, NKp44 and NKp46.

QUALITATIVE LIGAND EXPRESSION QUALITATIVE LIGAND EXPRESSION QUANTITATIVE LIGAND EXPRESSION QUANTITATIVE LIGAND EXPRESSION
Evolution of FLT3 Internal Tandem Duplication at the Diagnosis, Relapse, Remission or Induction Failure during the Treatment in Acute Myeloblastic Leukemia.
Authors: Bang Soo-Mee , Ahn Jeong Yeal , Park Jiyoon , Park Se Hoon , Nam Eun Mi , Cho Eun Kyung , Shin Dong Bok , Lee Jae-Hoon , Yoo Soo Jin , Jeon In Sang , Kim Yeo-Kyeoung , Kim Hyeoung Joon , Kim Hee-Nam , Lee Il-Kwon , Kang Hyoung Jin , Shin Hee Young , Ahn Hyo Seop
Published: April 2020

The FLT3 is class III fms-like tyrosine kinase. FLT3 mutations are one of the most frequent genetic changes at diagnosis and have been reported to be of prognostic significance in acute myeloid leukemia. FLT3 internal tandem duplication of the juxtamembrane domain (FLT3 ITD) was analyzed by PCR of genomic DNA at diagnosis or during the follow-up in patients with acute myeloid leukemia (AML). Total 223 patients were diagnosed as AML from Mar. 1996 till Aug. 2005. Their median age was 34 years-old (range: 0–86). The number of male or female patients was 116 or 107. Their FAB classifications were M0/1/2/3/4/5/6/7/RAEB/biphenotypic in 6/23/85/20/32/21/13/16/2/5 patients, respectively. Cytogenetic data at diagnosis were available in 182 patients; good/ intermediate/poor risk in 51/107/24 patients, respectively. Among the 190 patients receiving induction therapy, 147 patients achieved hematologic remission with a remission rate of 77%. Median event-free (EFS) and overall survival were 9 (95% confidence interval: 8–12) months and 14 (95% CI: 11–14) months, respectively. The incidence of FLT3 ITD at diagnosis was 13% (29/223). FLT3 ITD was more prevalent in M0~4 subtypes and older age. Among non-M3 population (N=203), patients with FLT3 ITD had a significantly short EFS when compared with those without ITD (p=0.0454). Among the 52 relapsed patients, 9 patients had a FLT3 ITD at diagnosis. Six patients showed reappearing of ITD and 3 patients remained negative at relapse. One patient acquired new ITD at relapse among 43 relapsed patients who had not baseline ITD. Tested 100 samples of 92 patients at remission were all negative for FLT3 ITD. Among the 28 patients at induction failure, one patient showed persistent ITD, one another re-appeared ITD after relapse, and the other one newly-developed ITD. Two others showed disappearance of ITD despite of persistent disease. Leukemic clones with FLT3 ITD may be susceptible to conventional chemotherapeutic agents and have no value for minimal residual disease. Therefore, effective induction and consolidation therapy could convert the poor prognosis of AML with FLT3 ITD.
Allosensitized Memory CD4 T Cells Induce Chronic Graft Versus Host Disease.
Authors: Dutt Suparna , Tseng Diane , George Tracy I. , Ermann Joerg , Liu Yinping , Fathman C. Garrison , Strober Samuel
Published: April 2020

It has been reported that naive CD4+CD62LhiCD44lo T cells induce severe GVHD in a complete MHC mismatched allogeneic model of mouse bone marrow transplantation (BMT), but that effector memory CD4+CD62LloCD44hi T cells obtained from normal donors do not induce GVHD. The lack of GVHD-inducing capacity of effector memory cells from unprimed donors may reflect their lack of previous exposure to host alloantigens. We tested this hypothesis in a complete MHC mismatched allogeneic model of BMT by comparing the ability of effector memory T cells obtained from untreated C57BL/6 donors and donors immunized against host BALB/C alloantigens to induce GVHD. C57BL/6 donors were immunized by injecting 50 x106 host BALB/C spleen cells i.p. and after one week with 10x 106 cells. Both the unprimed and alloantigen primed CD4+ T cells expressed similar levels of lymph node homing chemokine receptor CCR7, and activation markers like CD69 and CD25. We sorted naive (CD62LhiCD44lo) and effector memory (CD62LloCD44hi) CD4+ T cell subsets from C57BL/6 donor mice four weeks after immunization, and compared their alloreactivity to BALB/C in an in vitro MLR. Interestingly effector memory CD4+ T cells from primed mice produced significantly higher levels of IFNγ compared to the effector memory from unprimed donors. We found that CD62LloCD44hi cells from unimmunized donors failed to induce GVHD in 85% of the hosts over 100 days while CD62LloCD44hi cells from immunized donors caused progressive weight loss and death in 100% of hosts (p <0.001). Whereas naive CD4+ T cells from unimmunized donors accumulated rapidly in the lymph nodes and spleen of irradiated hosts, effector memory CD4+ T cells had markedly reduced accumulation in these tissues. Furthermore, at day 6 after transplantation effector memory CD4+ T cells from primed mice, showed hundred fold higher accumulation in the host liver compared to unprimed effector memory donor CD4+ T cells. Long term surviving hosts transplanted with primed effector memory cells showed histopathological features of chronic GVHD in liver characterized by portal tract inflammation and lymphocyte infiltration with bile duct injury. In conclusion, memory CD4+ T cells from donors immunized to host alloantigens are able to induce chronic GVHD , but memory cells from unimmunized donors do not.
Knockdown of the Nuclear Oncogene SKI Inhibits Flt3-ITD Induced Signaling in 32D - Flt3-ITD Cells.
Authors: Hagner Nicole M. , Burchert Andreas , Eilers Martin , Neubauer Andreas
Published: April 2020

Ski is a nuclear oncogene implicated in repressing TGF-β signaling. Recently, we found that Ski is overexpressed in AML, especially in AML with deletion of chromosome 7 or 7q−. We also showed that Ski inhibits myeloid differentiation by preventing retinoic acid signaling in U937 cells. It is unclear, however, how Ski exerts its oncogenic effect in myeloid leukemia cells. In order to address this question, we took advantage of the factor-independent murine myeloid progenitor cell line 32D, which is rendered factor independent via expression of Flt3 internal tandem duplication (Flt-3-ITD). We found that Ski protein expression is upregulated in Flt3-ITD transfected 32D cells (32D-Flt3) when compared to vector transfected 32D control cells (32D-V). Ski upregulation is reversed by treatment with specific Flt3-ITD inhibitor PKC412. As Flt3-ITD transformation is known to mediate factor-independent activation of STAT5 we investigated STAT5 phosphorylation status in these cells after treatment with PKC412 or knockdown of Ski with specific siRNA targeted to murine Ski (mSki). As expected, expression of phosphorylated STAT5 (pSTAT5) as measured by Western blot and flow cytometry was strongly reduced after PKC412 treatment. Interestingly, a similar reduction of pSTAT5 expression was also achieved by siRNA treatment against mSki but was not seen in non-silencing siRNA treated 32D-Flt3 cells. In parallel, transfection of siRNA against mSki, but not of non-silencing RNA led to significant inhibition of cell proliferation in 32D-Flt3-ITD cells and to a much lesser extent in 32D-V- cells. Together, these data suggest that in in addition to AML with −7 / del7q, Ski may also be implicated in AML with Flt3-ITD, as it regulates Flt3-ITD induced STAT5 phosphorylation, which itself is a key transcription factor in leukemogenesis.
Hypoplastic Acute Myeloid Leukemia.
Authors: Park Hye-Won , Lee Je-Hwan , Choi Seong-Jun , Lee Jung-Hee , Seol Miee , Lee Young-Shin , Ryu Seong-Gil , Kim Keun Hee , Jang Seongsoo , Seo Eul-Joo , Park Chan-Jeoung , Chi Hyun-Sook , Lee Jung-Shin , Kim M. Wookun , Lee Kyoo Hyung
Published: April 2020

Hypoplastic variant of acute leukemia is rare and almost always has a myeloid phenotype. We retrospectively investigated the incidence, clinico-laboratory characteristics, and clinical outcomes of hypoplastic acute myeloid leukemia (HAML), which was defined by bone marrow hypocellularity (less than 40% of cellularity in trephine biopsy) with increase of bone marrow blasts (20% or more) and peripheral blood leukopenia (less than 4,000/μl). Between 1989 and 2005, 740 patients were diagnosed as having AML at the Asan Medical Center and the definition of HAML was satisfied in 24 (3.3%). Patients with HAML showed a higher median age (67.5 vs. 44.0 years; P<0.001), a higher frequency of CD34 (100% vs 61.7%; P=0.007) and MDR (90% vs. 58%; P=0.043) expression and lower survival rate (6.7% vs 26.7% at 5-year; P=0.035) compared to other AML patients. The age range of 24 HAML patients, 16 males and 8 females, was 19 to 86 years, and 17 patients (70.8%) were 60 years or more. The results of cytogenetic analysis were available in 16 patients: 1 in good risk group, 14 in intermediate risk group, and 1 in poor risk group. Induction chemotherapy was given to 14 patients (58.3%): standard induction chemotherapy with AI or AD regimen (AI/AD) in 9 patients and low-dose cytarabine (LDAC) in 5. Complete remission (CR) was induced in 7 (50.0%) of 14 patients: 44.4% for AI/AD and 60.0% for LDAC. The CR rate was lower in patients with higher LDH over 400 U/L (25.0% vs. 100%; P=0.014). A median overall survival (OS) was 131 days. Seventeen patients died, two were lost to follow up, two were alive without disease, and one was alive with disease. Both patients, who were alive without disease, received allogeneic hematopoietic cell transplantation (HCT). Age (P=0.007) and LDH (P=0.016) were significant prognostic factors for OS. Our results suggest that HAML represents a poor prognostic group of patients. Prospective study is warranted for establishment of optimal treatment strategy including LDAC and HCT.
Obesity in Newly Diagnosed Childhood Acute Promyelocytic Leukemia.
Authors: Feusner James , Kim Haesook , Gregory John , Alonzo Todd , Woods William , Weinstein Howard , Tallman Martin
Published: April 2020

Obesity has been reported in several series of adults with acute promyelocytic leukemia (APL) (Estey et al., Leukemia11:1661–1664,1997; Douer et al.,Blood87:308–313,1996). Speculation regarding causation has included an association with female gender and/or an increased incidence of this disease in peoples of Latin extraction (Douer, Blood 1996). To date there have been no published reports of evaluation for obesity in children with APL. We report herein the results of an evaluation of body size of children enrolled on the first North American Intergroup trial of APL (INT 0129)(Tallman et al., N Engl J Med337:1021–1028,1997). This study was open for patient (pt) enrollment from April 1992 to February 1998. Fifty-three children (aged 1.4–18 years) with confirmed t (15;17) APL were eligible and evaluable. Body mass index (BMI) was calculated and compared to normals according to data from the 2000 CDC report (Ogden et al., Pediatrics 109:45–60,2002). Those pts who were obese (BMI > 95th centile for age and gender) were compared to the others on this trial. Three pts were excluded: 2 were too young for BMI assessment (< 2 years of age) and 1 insufficient body size data. Of the 50 evaluable pts, 26% (13/50) were obese. This compares to the expected overall incidence of 11% in the healthy pediatric population (Troiano et al., Pediatrics 101:497–504,1998). Comparisons of the obese pts to the rest on INT 0129 are shown:

Obese compared to non-obese pts Age (med) Sex Hispanic CR DFS-10 yr * Survival-10 yr * *Kaplan-Meier estimates Obese (13) 12.7 yrs 54% F 8% 85% 55% 77% Not Obese (37) 11.8 yrs 62% F 16% 84% 34% 57%

There were no statistically significant differences in CR (p>0.99), DFS (p=0.56), or survival (p=0.31) between the obese and the non-obese. Our data confirm a higher than expected incidence of obesity in children with APL, as previously reported in some adult series. Although the number of pts is relatively small, there is no suggestion of an increased representation of females or Hispanics in this subgroup of APL. Also of interest, the outcomes for obese children in this trial were no worse than for the non obese. The association of obesity at diagnosis with APL appears to be a real one, and deserves confirmation via meta-analysis of other pediatric trials, and investigation as to possible causal links with APL.
Increased Risk of PICC Line Thrombosis in Acute Promyelocytic Leukemia.
Authors: Gatt Moshe E. , Levin Moshe
Published: April 2020

Introduction: Central venous access catheters are being used at increasing rates in the practice of hematology. Although significant complication rates in patients with solid tumors exist, studies have shown a lower incidence of complications in hematology patients. Recently, we noted an increased incidence of percutaneously inserted central catheters (PICC line) thrombosis in patients with acute promyelocytic leukemia (APL).

Methods: We undertook an evaluation of the medical records of all adult patients with acute leukemia (AL) from the year 1995 to 2005 at our two hematology centers. All indwelling catheters (of which most are PICC lines) at our institutions are inserted by interventional radiologists under fluoroscopic visualization. The catheters are flushed with saline and heparin prophylaxis was not routinely used during this evaluation period. All thrombotic episodes were diagnosed based on clinical and ultrasound duplex studies. Data was collected in reference to catheters, thrombotic and infectious complications, and relationship to diagnosis and treatment.

Results: Of 368 evaluated patients with a central venous catheter and AL, there were 23 incidents of clinically apparent catheter thrombosis (6.2%). The subgroup incidence of catheter thrombosis was nine of 237 (3.8%) patients with acute myelogenous leukemia not including APL (AML), seven of 104 patients (6.7%) with acute lymphoblastic leukemia (ALL), half of the ALL episodes were associated with catheter sepsis, and seven of 25 (28%) with APL. Comparison of the APL group with the AML group is significant at p < 0.03.

Conclusion: There appears to be an increased incidence of PICC line thrombosis in APL patients. This may be secondary to the institution of all trans retinoic acid (ATRA) treatment and the thrombophillia of APL. In ALL patients the slightly raised incidence may be due to the thrombophillia of asparaginase or the increased infectious complications of high dose steroids associated with ALL treatment. Based on these findings we would recommend heparin prophylaxis for APL patients with central venous access lines. Following this evaluation we instituted heparin prophylaxis for newly diagnosed APL patients and found no evidence of clinically apparent thrombosis in 8 consecutive patients. There were no episodes of increased bleeding in these 8 APL patients. Further analysis of the data will be presented.
Clinical Significance of Free Circulating CD33 in Patients with Acute Myeloid Leukemia and Myelodysplastic Syndrome.
Authors: Hodge Menna , Giles Francis , Abdool Adam , O’Brien Susan , Keating Michael , Jilani Iman , Estrov Zeev , Estey Elihu , Kantarjian Hagop , Albitar Maher
Published: April 2020

CD33, a 67-kDa sialoglycoprotein expressed on the cell surface of monocytic/myeloid lineage and early hematopoietic progenitor cells, is frequently expressed in patients with acute myeloid leukemia (AML). Gemtuzumab ozogamicin (GO), an immunoconjugate consisting of a humanized anti-CD33 antibody and a cytotoxic compound (N-acetyl-γ-calicheamicin dimethylhydrazine), targets CD33 and has shown promising results in patients with AML. No evidence of a relationship between the levels of CD33-positive leukemic cells and clinical response has been found. We investigated the possibility that cell-free circulating CD33 (cCD33) might be useful as a marker of clinical behavior. We used a newly developed bead-based immunoassay to measure cCD33 in the plasma of patients with AML (n = 97) or myelodysplastic syndrome (MDS; n = 44). All patients were treated with standard therapy including idarubicin and ara-C. cCD33 levels were significantly higher in patients with MDS (median, 1600 U/μL; range, 102–791,350 U/μL) than in those with AML (median, 2709 U/μL; range: 62–263,349 U/μL) (P = 0.004). High-risk cytogenetic abnormalities were associated with higher cCD33 levels in patients with MDS (P = 0.04) but not in patients with AML (P = 0.72). cCD33 levels correlated with WBC count and % monocytes in patients with AML (R >0.35) but not in patients with MDS. cCD33 levels correlated with clinical behavior only among AML patients with intermediate-risk cytogenetic abnormalities (n = 56); those with cCD33 levels above the median had longer survival (P = 0.04). These data confirm the presence of cCD33 in AML and MDS and also suggest that cCD33 can be used as a tumor marker in patients with AML. Although further study is needed for confirmation, cCD33 appears to result from turnover of leukemic cells, may play a role in patients being treated with GO, and should be considered in the pharmacokinetic and pharmacodynamic studies of such therapy.

cCD33 in AML patient with intermediate cytogenetic abnormalities cCD33 in AML patient with intermediate cytogenetic abnormalities
Clinical Characteristics of JAK2 V617F Mutation Positive Acute Myeloid Leukemia.
Authors: Nahajevszky Sarolta , Andrikovics Hajnalka , Matrai Zoltan , Lovas Nora , Lueff Sandor , Masszi Tamas
Published: April 2020

Chronic myeloproliferative diseases (CMPD) are clonal disorders of pluripotent hematopoietic stem cells. Acquired JAK2 V617F point mutation has recently been identified as disease causing activating genetic abnormality in classic BCR-ABL negative CMPD (80% of polycythemia vera, 35% of essential thrombocythemia and 50% of chronic idiopathic myelofibrosis cases). JAK2 V617F is rare in other myeloid stem cell disorders like acute myeloid leukemia, chronic myelomonocyter leukemia or myelodysplasia with reported frequency of 3–8%. Between January 2001 and December 2005 155 consecutive adult patients [87 females and 68 males, median age of onset was 49±14 (range 18–83) years] were diagnosed with AML in our institute. Peripheral blood or bone marrow samples drawn at the time point of diagnosis were analyzed for the presence of JAK2 V617F by allele-specific PCR. JAK2 V617F mutation was present in 5 patients (3 males and 2 females; 3.2%). 3 of the 5 patients had prior history of CMPD, while 2 patients were diagnosed with de novo AML. The clinical characteristics and laboratory features of JAK2 V617F positive patients are shown in Table 1. FLT3 internal tandem duplication, FLT3 tyrosine kinase domain mutations, AML1-ETO, CBFB-MYH, PML-RARA rearrangements or nucleophosmin mutations were not present at the time point of AML diagnosis. In the case of patient 1, thrombocytosis was present prior the diagnosis of AML, and bone marrow biopsy revealed grade 3 fibrosis at diagnosis of AML, suggesting the presence of an atypical CMPD with the coexistence of del(5q) MDS. Patient 2 had no remarkable disease in his previous medical history. Induction therapy resulted in complete hematological and cytogenetic remission with persistent JAK2 V617F positivity. 10 month later clinical features of CMPD (elevated white blood cell count, left shifted peripheral blood smear, hepatosplenomegaly) appeared. In conclusion, these two cases suggest that acute myeloid leukemia with JAK-2 V617F mutation in fact corresponds to the blastic transformation of a clinically atipical chronic myeloproliferative disorders.

Clinical characteristics and laboratory features of JAK2 V617F positive patients Case Sex AML subtype Age of onset (years) FAB subtype Cytogenetic abnormality Therapy Overall Survival (months) 1 F de novo 52 M1 del(5q) DNR+ara-C, HDara-C 23 2 M de novo 65 M4 t(13;17) DNR+ara-C, HDara-C 12 3 F CMPD blastic transformation 65 M4 trisomy (1q) Supportive 18 4 M CMPD blastic transformation 70 M4 not available Supportive 3 5 M CMPD blastic transformation 71 M4 not available Supportive 6
t(8;21) Positive AML with Favorable Clinical Characteristics, in a Child with Ependymoma after Combined Treatment with Neuroaxis Irradiation and Topoisomerase II Inhibitors.
Authors: Papadakis Vassilios , Papadhimitriou Stefanos , Paisiou Anna , Parcharidou Agapi , Tourkantoni Natalia , Paterakis George , Belesi Chrysoula , Sambani Constantina , Polychronopoulou Sophia
Published: April 2020

The extensive use of etoposide has been implicated in the development of secondary acute myelogenous leukemia (AML) with unfavorable prognostic characteristics and most often involvement of the 11q23 locus. The case herein being presented exhibits favorable characteristics and poses the question whether it represents a secondary malignancy or a second primary event. A five year old boy was diagnosed with a mass of the posterior fossa representing anaplastic ependymoma of the brain stem (medulla oblongata) without CNS nor distant metastases. After partial resection of the tumor, the patient received 53 Gy to the tumor bed and 30 Gy to the neuroaxis followed by temozolamide p.o. for 4 cycles. Following this treatment, symptoms improved, he was ambulatory and back to school. Two years later he relapsed with unresectable tumor and received etoposide (VP-16) at 100 mg/m2/day for 21 days of 28 cycles, with a palliative intent. The treatment proved efficacious to him and he continued to receive VP-16 for 3 years in total. Six years after initial diagnosis and 3 years on VP-16, he developed ecchymoses, with thrombocytopenia (PLT: 15 k/uL) and leukocytosis (WBC: 25.8 k/uL). Bone marrow evaluation revealed infiltration with AML M2 blasts (>80%), MPO+ (93%), CD 34+ (58%), CD15+ (19%), CD71+ (33%), CD38+ (87%), CD33+ (47%) and CD123+ (52%), karyotype with detection of t(8;21). Bone marrow RT-PCR confirmed the presence of AML1-ETO hybrid gene, the absence of the MLL-AF4 product, and the absence of 11q23 involvement. Results were also confirmed by FISH technique. The patient was started on chemotherapy according to the AML-BFM 98 protocol. Disease remission was rapidly achieved (day +15 BM with no blasts) and treatment schedule was prompt, for the first 4 months of treatment. Severe, long-lasting marrow aplasia followed and pulmonary Aspergillosis was evident (compatible findings on CT and detection of Aspergillus specific proteins in the plasma by PCR), requiring assisted ventilation. However the patient succumbed 5 months following the diagnosis of AML. Childhood ependymomas have a dismal prognosis despite intensive treatment. This child exhibited a similarly good treatment response either to the ependymoma or to the later evolved AML. This AML1-ETO positive AML either representing a second primary malignancy or a rare type of secondary, therapy induced leukemia, is not a common and well described event and it certainly did not present, in our case, the usual dismal course of a secondary AML. Of importance our patient achieved and remained in remission with minimal treatment, for over 5 months, verifying the good prognosis of AML1-ETO positive AML either as a primary or a secondary event.
Compassionate Use of Intrathecal Depot Cytarabine (DepoCyte®) in Central Nervous System (CNS) Involvement in Acute Lymphoblastic Leukemia (ALL) or Lymphoblastic Lymphoma in Spain: Report of 10 Cases.
Authors: Sancho Juan-Manuel , Giraldo Pilar , Romero Maria-Jose , Martin-Reina Victoria , Gimenez Eugenio , Ruiz Elena , Josefa Plaza , Monteserin Carmen , Sanchez-Garcia Eva , Martino Rodrigo , Sarra Jose , Palomera Luis , Ribera Josep-Maria
Published: April 2020

The efficacy of intrathecal (IT) depot formulation of liposomal cytarabine (DepoCyte®) in the treatment of CNS involvement in patients with ALL has been only demonstrated in individual cases or series with a small number of patients. A retrospective review was performed of all cases in which IT depot cytarabine was employed as compassionate therapy of CNS involvement of ALL and lymphoblastic lymphoma in Spain. From February 2004 to March 2006, 10 cases (6 females) were recorded (ALL, 6, lymphoid blast crisis [BC] of Ph’ CML, 2, and lymphoblastic lymphoma, 2). The median (range) age was 31 (5–50) yr. Three patients (2 with ALL and another with BC-CML) had leukemic meningeosis at the time of diagnosis: 2 received IT depot cytarabine as adjuvant therapy after triple IT therapy (TIT) (BC-CML) and TIT plus cranial irradiation (ALL), whereas the remaining case (ALL) only received IT depot cytarabine. The remaining 7 patients (ALL, 4, lymphoblastic lymphoma, 2, and BC-LMC, 1) presented CNS relapse. CNS prophylaxis in these patients consisted of TIT plus cranial irradiation (2 patients with ALL), TIT (2 patients with ALL and 2 with lymphoblastic lymphoma), whereas the patient with BC-LMC did not receive any prophylaxis. IT depot cytarabine was administered as the only treatment for CNS relapse in one patient with ALL, as treatment of a second CNS relapse in 2 ALL patients and as adjuvant to other therapies in 4 patients (to TIT in other patient with ALL, to TIT plus radiotherapy in the patient with BC-CML, and to radiotherapy in the 2 patients with lymphoblastic lymphoma). In the 4 evaluable cases (one patient with ALL and CNS involvement at diagnosis and 3 patients with ALL and CNS relapse, 2 with second CNS relapse) treated with IT depot cytarabine as the only drug, clearance of blasts in cerebrospinal fluid (CSF) was observed with sustained response in two (at 7 and 22 months). The remaining 2 patients presented neurologic and systemic progression at 3 and 2 months, respectively. The median number of IT depot cytarabine administrations was 4 (1–9). Concurrent systemic dexamethasone therapy (4 mg twice/day/5 days) was administered with each dose of IT depot cytarabine. Side effects included headache (5 patients), dizziness (2) vomiting (2) and nausea (1). Depot formulation of cytarabine was effective in achieving CNS remission in ALL patients with CNS involvement or relapse. The administration of depot formulation of cytarabine was well tolerated. This justifies the development of a clinical trial to evaluate the efficacy and safety of IT depot cytarabine in meningeal involvement of ALL.