bluuebunny/crossref_metadata_2025 huggingface dataset:
Placenta Derived Adherent Cells Modulate the Allogenic Mixed Lymphocyte Reaction Partially via an Indoleamine 2,3-Dioxygenase Mediated Mechanism.
Authors: Liang Bitao , Paludan Casper , Downey Matthew , Lewis Craig , Harbacheuski Ryhor , Rana Hemlata , Lugo Chris , Lu Xiaohua , Lakatos Tara , Wu Wei , Edinger James
Published: October 2019

Placenta Derived Adherent Cells (PDAC) are multipotent progenitor cells derived from human placental tissues. Previously we have reported that PDACs could suppress T-cell proliferation when added to in-vitro mixed lymphocyte reactions (PDAC-MLR) (Paludan C. et al, Blood. (ASH Annual Meeting Abstracts) 2006 108: Abstract 1737). Here we present aspects of the mechanism of this PDAC suppression property. We have found that PDACs modify cytokine production in the PDAC-MLR reaction in comparison to the MLR; TNF-α and IFN-γ are reduced 75% and 30% while TGF-β production is significantly increased. We have used a transwell assay system to investigate the cell-contact-dependency of the effects of PDACs on T-cell proliferation. The assay system comprised combinations of the MLR in the top chamber together with the PDAC-MLR, PDACs plus naive T cells or PDACs alone in the bottom chamber. Maximum inhibition of T cell proliferation of the MLR in the top insert could be achieved by placing the PDAC-MLR co-culture in the bottom chamber. Minimum suppression was obtained when placing PDACs plus naive T cells or PDACs alone in the bottom chamber. PDAC-MLR conditioned media could partially suppress the MLR reaction. Addition of L-tryptophan into the MLR with PDAC conditioned media completely abolished PDAC-induced suppression of T cell proliferation. Likewise, the addition of the 1-methyl tryptophan to the PDAC-MLR reaction could abolish the PDAC-induced suppression. These results suggested that the suppression of the MLR by PDACs was possibly due to the depletion of the essential amino acid L-tryptophan which could be due to up-regulation of indoleamine 2,3-dioxygenase (IDO). Quantitative RT-PCR analysis of IDO gene expression revealed that IDO was up-regulated by about 4000-fold when PDACs were co-cultured with activated T cells, but not when co-cultured with naive T cells. Experiments are ongoing to confirm the causative role of IDO, and other factors, in PDAC-suppression of T-cell proliferation. In summary, we believe that soluble factors including the production of pro-inflammatory cytokines may contribute to PDAC suppression of the MLR, that induction of soluble factors from PDACs is significantly augmented by T-cell activation and that IDO expression by PDACs during the PDAC-MLR reaction plays a significant and direct role in suppression of T cell proliferation by PDACs.
Der(20)idic(20)(p11)del(20)(q11q13) Is Seen Not Only in Myeloid Disorders but Also in Lymphoid Disorders: A Report of Further Seven Cases.
Authors: Li Tianyu , Xue Yongquan , Zhang Jun , Chen Suning , Pan Jinlan , Wu Yafang
Published: October 2019

As a newly identified cytogenetic abnormality, idic(20q−) has been reported in thirteen cases of myelodysplastic syndrome (MDS), one case of chronic myelomonocytic leukemia, and one case of acute myeloid leukemia (AML) since 2004. Here we report additional seven cases consisting of 3 MDS, 2 AML and 2 acute lymphocytic leukemia (ALL). A karyotype analysis using R-banding technique revealed an i(20q−) as a sole anomaly in 5 of them and as a secondary change in other 2 cases: one was a B-cell ALL whose leukemic cells at presentation had a karyotype of 45, XY, dic(9;20)(p11;q11). However, an idic(20q−) was found coexistent with the dic(9;20) as relapse; another was a Ph positive ALL whose leukemic cells simultaneously contained t(9;22)(q34;q11) and idic(20q−). A series of FISH using multiple probes such as the centromeric probes specific for chromosomes 9 and 20, the subtelomeric probe for 20q, the locus-specific probe for 20q12, the whole chromosome paint probes for chromosomes 9 and 22 and the translocation probe for t(9;22) demonstrated the idic(20q−) to be actually a der(20)idic(20)(p11)del(20)(q11q13), confirmed the dic(9;20)(p11;q11) in case 6 and revealed a BCR/ABL fusion gene in case 7. Among them, 2 MDS patients survived and anemic except one lost to follow-up, 2 AML and 2 ALL patients did not obtain complete remission and died with short survivals. Our study brings the total number of cases with idic(20q−) to 22 and suggests that the disease spectrum of idic(20q−) is not restricted to myeloid disorders, and it also includes ALL.
MicroRNA Profiling of EVI1 Deregulated Myeloid Leukemia.
Authors: De Weer An , Poppe Bruce , Mestdagh Pieter , Van Roy Nadine , De Paepe Anne , Verhasselt Bruno , Vandesompele Jo , Speleman Frank
Published: October 2019

Chromosomal rearrangements involving the EVI1 gene are a recurrent finding in malignant myeloid disorders. These translocations or inversions contribute to ectopic expression of, or to the formation of fusion genes involving the EVI1 gene. EVI1 transcriptional activation has been reported in up to 10% of acute myeloid leukemia (AML) patients, and is a diagnostic marker which predicts a poor outcome. Recently, microRNA deregulation was identified as a major contributor to cancer initiation and progression. Moreover, miRNA genes were shown to be directly regulated by activated proto-oncogenes. In this study, we investigated which miRNA genes are implicated in the transcriptional pathways governed by the EVI1 oncogene. A total of 384 miRNAs were profiled through automated qRT-PCR using high-throughput quantitative stem-loop RT-PCR (Applied Biosystems). In a first step, differential miRNA expression was determined in 5 EVI1 rearranged/EVI1 overexpressing samples, 5 non-rearranged/EVI1 overexpressing samples and 5 non-rearranged/non-EVI1 overexpressing bone marrow samples. Next, up and down regulated miRNAs were analyzed in the leukemic EVI1 overexpressing cell lines Kasumi-3 and UCSD-AML1 following transient EVI1 RNAi knockdown. We will present the detailed results of these profiling studies as well as the results of data mining for identification of putative EVI1 regulated miRNAs. Further functional studies will be performed in order to select validated miRNA target genes and to asses their contribution to the leukemic phenotype. We anticipate that the discovery of such miRNAs may provide new targets for therapeutic intervention.
ΔNp73 /TAp73 Expression Ratio Is Associated with Ara-C-Induced Apoptosis Resistance in Acute Myeloid Leukemia.
Authors: Araújo Antonio Roberto L. , Panepucci Rodrigo A. , Jacomo Rafael H. , Souza Guilherme A. , Araújo Amélia G. , Lima Ana Silvia G. , Garcia Aglair B. , Falcao Roberto P. , Rego Eduardo M.
Published: October 2019

The p73, a homologue to the tumor suppressor gene p53, is involved in oncogenesis, although its specific role remains unclear. This gene has two distinct promoters which encode for two protein isoforms with opposite transcriptional activities: full-length TAp73 and the ΔNp73, the latter differing by the lack of the N-terminal transactivating domain and, has a dominant-negative function towards p53 and TAp73 activity. In the present study, we have investigated, by Real Time PCR, the gene expression pattern of TAp73 and ΔNp73 in 147 samples from patients with de novo AML and 22 samples of CD34+ hematopoietic progenitor cells isolated from bone marrow of healthy donors. We detected a significantly higher expression of TAp73 and ΔNp73 genes in AML samples compared to normal hematopoietic progenitors. AML samples harboring the chromosomal translocations: PML-RARa, AML1-ETO and CBFβ-MYH11 presented significantly higher levels of the TAp73 than those negative for these rearrangements. In contrast, samples without these gene rearrangements expressed higher levels of the truncated form ΔNp73. No difference was observed among patients with PML-RARa, AML1-ETO and CBFβ-MYH11. In order to determine if the ratio between the relative expression of TAp73 and ΔNp73 genes was associated with differential susceptibility to pro-apoptotic stimuli, we performed an in vitro cytotoxicity assay using Ara-C. Blasts from 20 AML patients were incubated for 24 hours in a medium containing 100μg-ml of Ara-C and evaluated for apoptosis status using Anexin V and Propidium Iodite. The leukemic cells with a higher ΔNp73 / TAp73 ratio were significantly more apoptosis-resistant (Pearson’s R2: −0.607). We also assessed the protein expression by flow cytometry using saturating amounts of specific antibodies for each isoform and analyzing the mean fluorescence channel. Estimated protein expression corroborated the gene expression results. Our results suggest an association between the presence of PML-RARα, AML1-ETO or CBFβ-MYH11 chromosomal translocation and a higher TAp73 gene expression compared to ΔNp73 isoforms expression in AML samples. Moreover, the higher resistence to apoptosis detected in blasts with higher ΔNp73 / TAp73 values suggest that this pathway may contribute to prognosis.
Significant Activity of Dasatinib in CLL Demonstrated by Ex-Vivo Assay.
Authors: Sigal Darren S. , Nieva Jorge , Diver Jonathan , Romeo Elisa , Lee Francis , Saven Alan
Published: October 2019

Introduction: Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder characterized by the accumulation of mature monoclonal B-lymphocytes. Combination therapies produce high response rates, including complete responses, but all patients will eventually relapse. Novel therapies are needed. Ex-vivo chemosensitivity assays offer two distinct advantages for identifying new CLL therapies. First, agents can be screened without exposing patients to unnecessary risk. Second, no CLL clonogenic cell lines exist, requiring that patient samples be analyzed in an ex-vivo manner. Dasatinib is a potent inhibitor of Src, Abl, and several other tyrosine kinases. B-CLL has been characterized as having anomalous signaling through Lyn kinase, a member of the Src family. We sought to determine the single agent and combination ex-vivo activity of dasatinib in CLL samples.

Methods: We used an ATP-luminescent chemosensitivity assay (ViaLight, Lonza, Basel, Switzerland) to assess the activity of single agent dasatinib (0.01 nM to 1 μM), and dasatinib (1, 10, 100 nM) in combination with fludarabine (0.06 to 64 μg/ml), chlorambucil (0.2 to 102.4 μg/ml), or methylprednisolone (0.02 to 2,000 μg/ml) in CLL patient peripheral blood samples. Patients had a diagnosis of B-CLL, WBC count greater than 20 x 109/L, no chemotherapy within 14 days, and no antibody therapy within 28 days.

Results: Five patient samples were obtained; 4 from untreated patients and 1 from a patient being treated with fludarabine. Rai stages ranged from 0 to IV. Single agent dasatinib induced apoptosis in 3 of 5 CLL samples tested. The two samples without apoptosis were from untreated patients. IC50 values were >1 (no apoptosis), >1 (no apoptosis), 0.2, 0.2, and 0.04 (fludarabine treated patient) μM. The IC50 values of single agent fludarabine were 1.3, 0.18, 1, 0.22, and 3 (fludarabine treated patient) μg/ml, respectively. The IC50 values of fludarabine in combination with 100 nM dasatinib were 0.41, <0.06, 0.45, <0.06, and <0.06 (fludarabine treated patient) μg/ml, respectively.

Conclusions: In this ex-vivo CLL model, dasatinib has significant single agent activity and may be synergistic in combination regimens. All responses were achieved with clinically attainable concentrations of dasatinib. These ATP-luminescent chemosensitivity results provide the pre-clinical rationale for clinical studies of dasatinib in patients with CLL. Currently, two single agent dasatinib phase II studies are accruing patients and plans exist for a combination dasatinib study in CLL patients next year.
The Histone Deacetylase Inhibitor, Sodium Butyrate, Augments Chlorambucil Cytotoxicity in the Promyelocytic Leukaemia Cell Line, HL-60.
Authors: Kwa Faith , Kapuscinski Miroslav , Cole-Sinclair Merrole
Published: October 2019

Treatment of various haematological malignancies such as chronic lymphocytic leukaemia (CLL) with alkylating drugs (e.g. chlorambucil, CLB) fails to extinguish the leukaemic clone and often results in chemo-resistance. A possible mechanism of drug resistance in CLL B cells is p53 inactivation. The resistance of cells to standard chemotherapy may be ameliorated by agents that alter the chromatin remodelling processes, DNA methylation and histone acetylation (e.g. sodium butyrate, NaBu). We used the HL-60 cell line (which carries a non-functional p53 protein) as a cell model for resistant CLL B cells and determined the sensitivity of HL-60 cells to a range of concentrations of CLB (5μM,10μM and 20μM) and NaBu (0.5mM, 1mM and 2mM) by tetrazolium salt-based proliferation assays of cells incubated with the drugs over 4 days (days 0, 1, 2 and 3). Cells were resistant to 5μM CLB until day 3 where 56% of cells were killed. 10μM and 20μM CLB had very similar effects (p>0.05) with both treatments killing 83.2% and 87.3% respectively of cells by the end of the incubation. The cell death induced by NaBu was both time- and dose-dependent, with 2mM NaBu being the most effective treatment, while 0.5mM NaBu did not result in significant cell death (p>0.05). To investigate if the treatment of HL-60 cells with both drugs would enhance or have an additive killing effect on the cells, we tested combined treatment involving a series of lower drug concentrations of each of the two drugs (5μM/10μM CLB ± 0.2mM/0.5mM/1mM). All combined treatments using 5μM CLB were more effective in cell killing than when the cells were treated singly with CLB or NaBu (p<0.001). The most effective combined treatment was 5μMCLB+0.2mM NaBu (62% cell death) compared to only 10% and 40% cell killing caused by CLB and NaBu alone, respectively. In contrast, the effect of 10μM CLB was not augmented by NaBu in concentrations ranging between 0.2mM and 1mM (p>0.05). In conclusion, combined treatment involving NaBu increased the cytotoxic effect of low concentrations of CLB in p53-inactivated cells, implying the potential benefit of chromatin modifying agents in cytotoxic therapy. Further studies including the use of a wider range of concentrations of CLB with NaBu or other chromatin modifying agents are underway to investigate possible synergism in the killing of HL-60 cells by these agents.
Suppression of Angiogenesis and Resistance to Doxorubicin by the Thyroid Hormone Tetraiodothyroacetic Acid.
Authors: Mousa Shaker A. , O’Connor Laura , Aljada Ahmad , Davis Paul , Rebbaa Abdelhadi
Published: October 2019

Thyroid hormone has been recently shown to induce tumor growth and angiogenesis. These angiogenesis modulating activities are initiated at endothelial cell plasma membrane receptor via the integrin αVβ3, at or near the Arg-Gly-Asp (RGD) recognition site on the integrin. In the present study, we have investigated the effect of tetraiodothyroacetic acid (tetrac), a deaminated thyroid hormone analog that inhibits thyroid hormone-binding to the cell surface integrin, on angiogenesis and cancer cell resistance to doxorubicin both in vitro and in vivo. Two angiogenesis models were studied in which vascular endothelial growth factor, VEGF165 or basic fibroblast growth factor, FGF2 (1–2 μg/ml) or thyroid hormone, thyroxin (L-T4 or T3) were used either to induce tube formation in the human dermal micro-vascular endothelial cells (HDMEC), or to stimulate new blood vessel branch formation in the chick chorioallantoic membrane (CAM) models. In both models, Tetrac (0.1–10 μM) inhibited the pro-angiogenesis activity of VEGF, FGF2, L-T4 or T3 by more than 50% at 1.0 uM RT-PCR revealed that tetrac (1–3 μM) decreased abundance of angiopoietin-2 mRNA but did not affect the mRNA levels of angiopoietin-1, in VEGF-exposed endothelial cells, suggesting that specific angiogenic pathways are targeted by this compound. Additionally, microarray was used to examine changes in expression of Matrix Metalloproteinases (MMP) and Tissue Inhibitor of Metalloproteinases (TIMP) following VEGF treatment with and without tetrac. HDMEC cells treated with VEGF exhibited 3–5-fold increase in MMP-15 and MMP-19 expression and tetrac (3μM), inhibited expression of MMP-15 and MMP-19 by 3–9-fold, respectively. Expression of TIMP-3 was increased 5.4-fold following VEGF and tetrac treatment when compared to treatment with VEGF alone. This finding suggests that part of the mechanism by which tetrac inhibits VEGF-stimulated angiogenesis involves inhibition of certain MMPs and increase in TIMP expression. Investigation of the anti-proliferative function of tetrac was carried out using the αVβ3 expressing breast cancer cells MC7 and their drug resistant counterparts. Interestingly, proliferation of both cell lines was inhibited similarly by tetrac suggesting that this analog may circumvent drug resistance. In fact, tetrac was able to reverse resistance to doxorubicin in vitro and to suppress growth of doxorubicin resistant tumors in nude mice. Inhibition of the drug transporter p-glycoprotein was found to play a key role in mediating the action of tetrac. Taken together, findings presented in this study provide evidence that the anticancer function of tetrac can be attributed to its anti-angiogenic and drug resistance reversal activities.
Novel Deuterated Gemini-Vitamin D3 Analogs Have Potent Antitumor Activity.
Authors: Okamoto Ryoko , Saito Tsuyako , Haritunians Talin , O’Kelly James , Uskokovic Milan , Koeffler H. Phillip
Published: October 2019

The active form of vitamin D3, 1,25(OH)2D3, is an important regulator of calcium and bone metabolism. Also, it inhibits proliferation and induces differentiation of a variety of malignant cells. Here, we synthesized a new class of vitamin D3 analogs, which have a C-20 methyl group and a deuterium substituted basic side chain [Deuterated Gemini (DG)]. Six DG analogs were evaluated for their ability to inhibit growth of myeloid leukemia (HL-60), prostate (LNCaP and PC-3), lung (H520) and breast (MCF-7) cancer cell lines. All 6 DG analogs inhibited growth in a dose-dependent manner and had very similar potency. Most effective DG, BXL-01-0120, was over 70-fold more potent than 1,25(OH)2D3 in inhibiting 50% clonal growth (ED50) of HL-60, LNCaP, PC-3, H520 and MCF-7 cells with ED50s ranging from 10−11 M to 10−13 M. Focusing on this analog for further analyses, pulse-exposure, wash and culture experiments showed that 48 hours exposure of HL-60 cells to BXL-01-0120 (10−9 M) had potent anti-proliferative activity (82% growth inhibition compared to diluent control cells). Cell cycle assays found that BXL-01-0120 (10−10 M, 4 days) increased the percent of cells in the G0/G1-phase (61%, diluent control 47%) associated with a decrease S-phase (18%, diluent control 42%). BXL-01-0120 (10−10 M, 4 days) caused apoptosis in approximately 15% of cells. 1,25(OH)2D3 at the same concentration and duration changed neither the cell cycle nor number of cells undergoing apoptosis compared to control HL-60 cells. BXL-01-0120 (10−10 M, 4 days) strongly induced expression of the CD11b and CD14 (monocyte / macrophage differentiation markers) on HL-60 cells (87%); in contrast, 1,25(OH)2D3(10−10 M) stimulated a mean 43% of the cells to express these cell surface proteins. In summary, DGs strongly inhibited clonal proliferation, induced differentiation and cell cycle arrest in several types of cancer cells, especially HL-60 cells, suggesting that further preclinical and clinical studies to explore their anticancer potential are warranted.
Selenium Enhances the Anti-Angiogenic Effects of Bevacuzimab (Anti-VEGF Antibody) in Human Acute Myeloid Leukemia Xenograft Models.
Authors: Doro Nathaniel C. , Lal Deepika , Rustum Youcef , Wang Eunice S.
Published: October 2019

Selenium, a known antioxidant used in chemoprevention, has recently been shown to improve the efficacy of chemotherapy agents and mediate anti-angiogenic effects in preclinical solid tumor models. Prior data has also shown that sodium selenite can induce apoptosis of human acute myeloid leukemia (AML) cells in vitro. While the use of high non-toxic doses of selenium has been demonstrated to effectively enhance the therapeutic efficacy and selectivity of various anti-cancer drugs in multiple solid tumor xenograft models (Cao et al, Clin Canc Res10: 2561–2569, 2004), the in vivo effects of selenium compounds in hematological malignancies have not been extensively explored. Our laboratory has previously demonstrated the preclinical efficacy of VEGF inhibitors in human leukemia xenograft models. Here we investigated the in vitro and in vivo effects of seleno-L-methionine (SLM), alone and in combination with chemotherapy and anti-VEGF antibody, in human AML. The mechanism of action, dosage, and time course of SLM effects on AML cells (ML-2 and HL60) in vitro were determined using colorimetric (CCK-8) viability, tritiated thymidine proliferation, and Annexin-V flow cytometric assays. SLM therapy resulted in dose dependent inhibition of AML proliferation with marked induction of apoptosis and a 67–92% reduction in viable cells at 400–500 μM. Ten million AML cells (ML-2 and HL60-luciferase) were then subcutaneously engrafted into immunodeficient (SCID/SCID) mice. ML-2 tumor bearing mice were treated with PBS (vehicle), SLM (200 mcg p.o. daily), cytarabine (Ara-C 2 mg i.p. for 3 days), or combination SLM + Ara-C therapy for over two weeks. SLM showed no effective modulation of therapeutic efficacy and toxicity in AML tumor models, when combined with Ara-C. Neither SLM nor Ara-C alone or in combination resulted in significant reduction of AML growth. ML-2 and HL60-luciferase tumor bearing mice were then treated with PBS, SLM (200 mcg p.o. daily), anti-VEGF antibody bevacuzimab (BV 100mcg i.p. twice weekly), or SLM + BV for three weeks. Disease progression was monitored by tumor volume, end tumor weight, and bioluminescent imaging. Although SLM alone was ineffective in reducing HL60 leukemia growth, SLM + BV combination therapy effectively induced a 75% reduction in mean tumor volume after 21 days of treatment which was statistically greater than BV therapy alone (62% reduction). Synergistic effects of SLM+BV therapy was also confirmed by differences in mean tumor weights and bioluminescent imaging as compared with single agent BV and SLM treated mice. Similar results were seen in the ML-2 AML xenograft model.

Conclusions: SLM improved the anti-angiogenic and anti-tumor effects of anti-VEGF therapy with bevacizumab in human AML xenograft models. Additional studies investigating the mechanisms of action of these synergistic effects will be presented.
Quantitative Molecular Expression of the Immunoregulatory Enzyme Indoleamine 2,3-Dioxygenase in Acute Myeloid Leukemia Cells as a Possible Marker for Minimal Residual Disease Detection.
Authors: Aluigi Michela , Curti Antonio , Ottaviani Emanuela , Lucchetti Giovanna , Salvestrini Valentina , D’Addio Alessandra , Trabanelli Sara , Fiore Francesca , Massaia Massimo , Martinelli Giovanni , Baccarani Michele , Lemoli Roberto M.
Published: October 2019

The expression of the catalytic enzyme of tryptophan, indoleamine 2,3 dioxygenase (IDO) has been identified as a T-cell inhibitor effector pathway in different normal and neoplastic cells. We have recently shown that normal bone marrow (BM) cells, including hematopoietic CD34+ cells, express IDO mRNA only upon IFN-γ stimulation, whereas in a subset of human acute myeloid leukemia cells (AML) IDO is constitutively expressed both at molecular and protein level and induces immunological escape by promoting the generation of T-reg cells. To investigate whether the IDO transcript can be used as a marker for minimal residual disease (MRD) detection, we used a sensitive real-time reverse transcription-polymerase reaction assay (qRT-PCR) to quantify IDO mRNA levels in peripheral blood (PB) and BM samples of newly diagnosed AML patients. The level of IDO transcript was evaluated as IDO copy number/104 ABL copies. As control samples, we used normal PB and BM mononuclear cells (MNCs). Our data showed that normal BM and PB cells expressed minimal amount of IDO mRNA (range 100–1000). Among AML samples, we identified three subsets of patients according to IDO mRNA expression: 1) 33/74 (44.6%) IDO negative (i.e. range < 100), 28/74 (37.84%) IDO low (range 100–1000) and 13/74 (17.6%) IDO high (i.e. range > 1000). BM and PB AML blasts gave similar results. Assessment of protein expression and enzymatic activity was in accordance with molecular results. Some patients were evaluated for IDO mRNA expression before and after induction chemotherapy and the IDO levels were found to correlate with the reduction of BM blasts. Taken together, our qRT-PCR data demonstrate that

normal PB and BM cells are negative for IDO mRNA expression, which, in turn, is significantly up-regulated in a subset of AML patients (IDO high) and IDO mRNA expression correlates with tumor burden,

thus suggesting its possible role in the detection of MRD in IDO high patients.
Janus Kinase (JAK)-2 Does Not Play a Role in Bcr-Abl Signaling in Philadelphia Chromosome (Ph)-Positive (p190) Acute Lymphoblastic Leukemia (ALL) Cells.
Authors: Faderl Stefan H. , Van Quin , Koch Patricia E. , Harris David M. , Hallevi Inbal , Liu Zhiming , Wang Yongtao , Ferrajoli Alessandra , Estrov Zeev
Published: October 2019

Novel immunochemotherapy regimens combined with imatinib mesylate (IA) have significantly improved treatment outcome of Ph+ ALL. Nevertheless, most adult patients with Ph+ ALL relapse and succumb to their disease. Recent reports suggested that Jak-2 is engaged in the signaling of Bcr-Abl in chronic myelogenous leukemia (CML) cells. Because Jak-2 inhibitory agents are currently investigated in clinical trials, we sought to explore the role of Jak-2 in the signaling of Bcr-Abl in Ph+ ALL assuming that inhibition of Jak-2 might be beneficial in the treatment of Ph+ ALL. To do this, we used our Ph+ (p190) ALL cell lines Z-119 and Z-181 (Estrov et al. J Cell Physiol166: 618, 1996). We chose these cells because in both lines Jak-2 can be activated. Both Z-119 and Z-181 cells express granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors and GM-CSF activates Jak-2 and stimulates the proliferation of both cell lines. Using a clonogenic assay, we found that IA inhibited the proliferation of these cells at concentrations ranging from 50 to 500 nM. Because Bcr-Abl was found to activate the signal transducer and activator of transcription (STAT)-5 in CML cells, we used Western immunoblotting and found that IA inhibited the phosphorylation (p) of STAT5 in a dose-dependent manner in Ph+ ALL cells. To test whether JAk-2 plays a role in Bcr-Abl (p190) signaling we incubated Z-181 cells for 4 hours with or without 50, 100, 250, and 500 nM IA, extracted cellular protein and immunoprecipitated total STAT5 protein. Then, using Western immunoblotting we detected the Bcr-Abl p190 protein in all STAT5 immunoprecipitates and by using specific pSTAT5 antibodies, we demonstrated that IA induced a dose-dependent reduction in the levels of pSTAT5, but not of p190 protein, suggesting that the p190 Bcr-Abl kinase binds to and activates STAT5. Remarkably, neither Jak-2 nor pJak-2 was detected in either immunoprecipitate. To further delineate the role of Jak-2 in Bcr-Abl signaling we extracted protein from Z-181 cells and immunoprecipitated Jak-2. Neither Bcr-Abl nor STAT5 was detected in these immunoprecipitates, confirming that Jak-2 does not bind Bcr-Abl p190 protein and does not participate in the activation of STAT5. Taken together, our data suggest that Bcr-Abl (p190) binds and phosphorylates STAT5 whereas, Jak-2 is not engaged in Bcr-Abl (p190) signaling in Ph+ ALL cells.
Acute Myeloid Leukemia with Normal Karyotype: Analysis of FLT3, NPM1, MLL and CEBPA Gene Abnormalities.
Authors: Trigo Fernanda , Torres Fatima , Guimaraes Patricia , Silva-Coelho Pedro , Carvalhais Ines , Santo Ana Espirito , Melo Teresa , Castro Miriam , Fernandes Alexandra , Tavares Purificacao , Guimaraes Jose E.
Published: October 2019

Acute myeloid leukemia with normal karyotype (AML-NK) is markedly heterogeneous depending on the presence or absence of certain genetic alterations. We studied the prevalence of different abnormalities, namely of the FLT3, NPM1, CEBPA and MLL genes, in AML-NK patients and tried to correlate them with clinicopathological findings. Techniques used were as follows: FLT3-ITD (internal tandem duplications), PCR using fluorescent primers, analysis in automatic sequencer; FLT3-TKD (D385), PCR, fragment cut with EcoRV restriction enzyme run in agarose gel; NPM1, RT-PCR and automatic sequencing of a fragment containing exon 12; CEBPA, PCR and automatic sequencing of the entire coding region; MLL-PTD, RT-PCR with analysis in agarose gel. The following data from patients were recorded: sex, age, FAB classification, WBC and platelet count, hemoglobin and LDH level, response to induction, survival time and cause of death. The incidence of FLT3 alterations (all ITDs) was 10/23 (43.5%), varying from 1 to 10 ITDs; NPM1 was mutated in 12/24 cases (50.0%) and MLL-PTD in 2/24 patients (8.3%); for CEBPA, of the 10 cases in which all 3 amplicons (1A, 2B, 3C) were analysed, 6 had a mutation mostly in amplicon 2B (5 cases). More than one genetic abnormality was present in 9 cases: six showed coexistence of FLT3-ITD and NPM1 mutation, one of MLL and CEBPA mutations, one of FLT3-ITD and CEBPA mutation and one of FLT3-ITD, NPM1 and CEBPA. Regardind correlation with clinical characteristics, we observed that 72.7% of nucleophosmin mutated (NPM1+) cases were CD34 negative and 91.7% of CD34+ patients were NPM1 negative (p=0.003). There is a tendency, although not significant, for CEBPA+ cases to correlate with low WBC counts and for patients with high WBC count to be NPM1+ and FLT3-ITD+ (odds ratio of 3.0 and 3.33, respectively). All NPM1+ patients treated with appropriate induction therapy achieved complete remission; 22.2% of NPM1 negative patients were refractory to therapy. No significant correlation was observed between survival and presence or absence of any of the genetic alterations. This work was funded by an unrestricted grant from Roche Farmaceutica - Portugal.
Abnormal Expression of Receptor Tyrosine Kinase Mer in Adult Acute Leukemia.
Authors: Fan Lei , Xie Liqian , Shen Fei , Dai Lan , Xu Wei , Jianyong LI , Ruan Changgeng
Published: October 2019

BACKGROUND & OBJECTIVE Mer was found a proto-oncogene in some solid tumor. it belongs to Axl receptor tyrosine kinase family. This study was to detect Mer expression in 57 adult acute leukemia patients’ bone marrow (32 AML and 25 ALL patients) and normal control.

Methods Flow cytometry with anti-CD15, CD3, CD19 and CD14 antibody were used to detect Mer expression on granulocytes, T-lymphocytes, B-lymphocytes and monocytes in normal bone marrow. Mer expression on leukemia cells of patients’ bone marrow samples were also analysed by flow cytometry, and RT-PCR was used to verify parts of Mer mRNA level expreesion in mononuclearcell of bone marrow samples.

Results Flow cytometry showed that in normal bone marrow, there was no Mer expressed on granulocytes, T-lymphocytes and B-lymphocytes; and middle-expression on monocytes(37.5%). In all 32 AML patients, there were 23 Mer positive(71.8%), Mer expression level was high in 3 AML patients, middle in 8 AML patients, low in 12 AML patients and very low/negative in left 9 AML patients. Among 3 Mer high Expression AML patients, the FAB typping were all M1, and all of them had the high expression level of CD34 and CD117. In all 25 ALL patients, there were 16 Mer positive(64.0%), Mer expression level was high in 1 ALL patients, middle in 6 ALL patients, low in 9 ALL patients and very low/negative in left 9 ALL patients. The results of RT-PCR was similar to flow cytometry

Conclusion Mer was ectopic expression parts of adults acute leukemia patients, and may related to genesis and development of acute leukemia.
Molecular Remission in MLL/AF4-Positive Infant Leukemia Treated with the All Trans-Retinoic Acid Based MLL-Baby Protocol.
Authors: Tsaur Grigory , Semenikhina Elizaveta , Ivanova Anna , Nasedkina Tatiana , Popov Alexander , Shorikov Egor , Saveliev Leonid , Fechina Larisa
Published: October 2019

Statement. We recently developed a new treatment approach for infant leukemia with MLL rearrangements (MLL-Baby) that consists of intermittent pulses of all-trans retinoic acid (ATRA) and conventional chemotherapy. Here, we present data on the molecular remission status of patients with the MLL/AF4 rearrangement enrolled in this study.

Materials and methods. Six patients with MLL/AF4 rearrangement have been enrolled in the MLL-Baby study in Ekaterinburg and Moscow since September 2003. Median age was 6.5 months (range 1.5–10 months). All have had the BI immunophenotype. Leucocytes from bone marrow (BM) and peripheral blood (PB) were obtained. Quality and integrity of RNA was estimated using RNA 6000 Nano LabChip kit and 2100 Bioanalyzer (Agilent Technologies). Only RNA with RNA integrity number more than 4.5 was taken further. Qualitative PCR for MLL/AF4 fusion gene (FG) was performed either by nested PCR (4 patients) (A. Borkhardt et al, 1994) or by low-density biochips (2 patients). Conditions of qualitative real-time PCR (qRT-PCR) for MLL-AF4 FG detection were previously described (J. Gabert et al., 2003). β2-microglobulin was used as the control gene. Normalized copy number (NCN) of MLL/AF4 was calculated. Sensitivity of qRT-PCR for BM specimens was 1×10−4 – 6×10−5, for PB was 1×10−4 – 7×10−4. In accordance with the treatment design all patients were enrolled on the high risk (HR) schedule as approved by Local and Federal Ethics committees. Parents’ informed consent was obtained.

Results. We found 2 types of MLL/AF4 FG clearance. In 4 patients the elimination rate of FG transcripts was very high. In this group FG could not be detected after 1 course of ATRA that followed induction therapy or after HR 1 (I); in 1 patient with MLL/AF4, FG total clearance was achieved after HR block 3 (VI) respectively; 1 patient currently under treatment has few copies of MLL/AF4 after HR block 2 (V). There was no correlation between the rate of elimination of MLL/AF4 FG with age, initial WBC count or type of MLL/AF4 transcript. Once achieved, the molecular remission in 5 patients has remained PCR negative.

Conclusions. Achievement of molecular remission is one of the most important steps toward long-term survival and cure. Despite differences in the rate of elimination of MLL/AF4 FG, 5 of 6 patients treated on the MLL-Baby protocol have remained in hematological and molecular remission with a median follow-up 18 months (range 6–45 months). None have relapsed. Moreover, the youngest patient, a girl who was 2 months old at the time of diagnosis, has had the longest continuous molecular remission of up to 45 months. The 6th patient has single copies of MLL/AF4 after HR block 2 (V) and is currently receiving therapy.
Frequent Molecular Aberrations in Normal Karyotype (NC) Precursor B-Cell Acute Lymphoblastic Leukemia (pB-ALL) Detected by Comparative Genomic Hybridization.
Authors: Zeidan Amer , Gaile Dan , Conroy Jeffrey M. , McQuaid Devin E. , Wang Eunice S. , Deeb George , Wallace Paul K. , Sait Sheila N.J. , Nowak Norma J. , Wetzler Meir
Published: October 2019

The prognosis of adult NC pB-ALL has not improved over the last decade mainly because separation into distinct molecular subsets has been lacking. We conducted a proof-of-principle experiment to screen the genome of blasts from adult NC pB-ALL patients for novel genomic alterations using 19,000 bacterial artificial chromosomes (BAC) by array CGH and verified our results with gene expression profiling (GEP, Agilent oligonucleotide expression arrays) and fluorescent in situ hybridization (FISH) using the same BAC probes or commercially available probes if available. The median age at diagnosis of the 6 males and 4 females was 55 years; all were treated with a 5-drug induction regimen followed by intensive consolidation and maintenance regimens. Five achieved CR of whom 3 relapsed with complex karyotype; samples were available on 2 of the patients at relapse. Samples with <60% blasts (2 at diagnosis, 1 at relapse) were enriched for >95% purity by sorting according to their immunophenotype pattern. Only one case had a normal CGH pattern. A decreased copy number of p16 was found in 4 of the 10 diagnostic samples; 3 on whom mRNA was available demonstrated a decreased p16 mRNA expression by GEP. In addition, a loss at 9p21.2 and a loss and a gain at 8q21.2 were detected in one sample each; those were verified by FISH. Copy number losses were also observed across multiple samples at 1p34–1p36, 4p16, 5p15.33, 8q24, 9q34, 10q26, 11q12–11q13, 12q24, 14q32, 17p, 19p13.3, 22q11, and 22q13. Copy number gains across multiple samples were observed at 15q11.2 and 18q21. Those are currently being verified by FISH. Comparison of samples at diagnosis and at relapse revealed a disparate molecular pattern. In summary, enriching samples by sorting from NC pB-ALL to be studied by CGH is feasible; normal chromosome pattern does not correlate with normal molecular pattern; and diagnostic and relapsed samples have a different molecular pattern. Our work on 10 patient samples at diagnosis and 2 at relapse demonstrates the feasibility to detect specific molecular aberrations in NC pB-ALL. Since attaining good quality metaphase cells in ALL is often suboptimal, this work suggests that array CGH can complement conventional cytogenetic analysis to better characterize NC pB-ALL by identifying cryptic copy number changes. Work on a larger number of samples is warranted to determine the prognostic significance of the aberrations detected herein.
Correlation between High Expression of Natural Killer Related-Genes (NCAM/CD94) and Early Death during Induction in Children with Acute Myeloid Leukemia.
Authors: Nigro Luca Lo , Mirabile Elena , Munda Silvana , Bottino Daria , Manuela Tumino , Gino Schiliro
Published: October 2019

Death early (DE) during the induction phase of therapy is a rare but dramatic event in children with acute myeloid leukemia (AML). Recent reports emphasize that DE in patients with leukemia is related to aggressive disease, signaled by hyperleukocytosis, and to the early administration of intensive chemotherapy. Many efforts have been made to better characterize subgroups of patients with AML by defining biologic markers associated with aggressive disease. Even if little is known about the molecular mechanisms that regulate blast migration into tissues, adhesion receptors are likely to play an important role in this process. We have attempted to identify the molecular characteristics of an aggressive subset of pediatric patients with AML through a prospective evaluation of the CD56+ neural cell adhesion molecule (NCAM) and CD94 expression. For this purpose we developed an RT-PCR semi-quantitative methodology, using specific primers from NCAM, CD94 and β-actin as an internal quality and quantity control. We analyzed a total population of 44 children with AML, distributed as follow: 5 with M3v (group A), 9 with APL (group B) and 30 with non-APL AML (group C). We found a dramatic correlation in group A between high expression of NK-related genes and occurrence of early death. In groups B and C, primarily used as controls, we found a strong correlations between a high level of NCAM-CD94 expression and two cases with APL, three with FAB M5, two with FAB M2 AML, one case with FAB M4 and a child with dendritic cell leukemia (DCL). We primarily found that 9 out of 13 cases presenting with a high level of NCAM-CD94 expression died of disease-related events during the early days of induction. Our data strongly suggest that doses and timing of chemotherapy induction in children with AML need to be modulated because of the high risk of hemorrhagic events. These appear to be related to the presence of high concentration of adhesion molecules secondary to acute blast lysis and to the associated severe coagulopathy. We know of no pharmacological correctives for this phenomenon. It would be important to develop specifically designed molecules in order to disrupt the interactions with adhesion molecules, and thus to improve the clinical course of both children and adults with AML during the early days of induction.
The mTOR Inhibitor Rapamycin Demonstrates Activity Against AML in Combination with Imatinib Mesylate and with 5-Azacytidine.
Authors: Liesveld Jane L. , Rosell Karen , Lu Chaohui , Mulford Deborah , Walker Alison
Published: October 2019

The serine/threonine kinase mTOR (mammalian target of rapamycin) integrates pathways involved in cell cycle regulation and apoptosis and has been shown to be aberrantly activated in AML. mTOR (mammalian target of rapamycin) serine threonine kinase is involved in cell cycle regulation and apoptosis. mTOR inhibitors are a class of signal transduction inhibitors, originally developed as immunosuppressive agents, with antineoplastic activity in solid tumor models. Acute myelogenous leukemia blasts and stem cells have been reported previously to be inhibited by rapamycin, an mTOR inhibitor. As a single agent, rapamycin has minimal activity, however, suggesting that for meaningful clinical efficacy, it would require combination with other agents. We have therefore begun to examine its effects in combination with other signal transduction inhibitors and demethylating agents. Rapamycin by itself had minimal inhibitory effect on AML cell lines or primary AML samples. However, in combination with the c-kit inhibitor, imatinib mesylate, 2.5–25 uM, rapamycin demonstrated synergistic inhibition of AML cell lines such as MV411 as measured in a standard MTT assay (combination index 0.32 to 0.576 as determined with the Calcusyn program),, indicating strong synergism. In this cell line, rapamycin by itself demonstrated no significant inhibitory activity. Synergism with imatinib mesylate was also noted in the U937 and HL60 cell lines but not in the KG1a cell line. The combination of rapamycin and imatinib inhibited AKT phosphorylation to a greater extent than either agent alone in both the MV411 and U937 cell lines, but the combination did not effectively inhibit ERK phosphorylation. Inhibition of phosphorylation of p70S6 kinase by the combination of 20 uM imatinib mesylate and 80uM rapamycin did not occur to a greater extent than that seen with rapamycin alone, although phosphorylation of 4EBP1 was inhibited to a greater extent when both agents were added concurrently. When AML cell lines were exposed to 5-azacytidine alone, 1 to 16 uM, the IC50 was 4–8uM. 5-azacytidine increased apoptosis above control whereas rapamycin by itself had minimal effect on apoptosis as assessed in an Annexin V flow cytometric assay. In the U937 cell line, the combination index for the U937 cell line was 0.465 (10 uM 5-azacytidine and 100 nM rapamycin), indicating synergism. For MV411, the combination index was 0.736, for KG1a, 0.118, and for HL60, no synergism was noted. Exposure to the combination of 5-azacytidine and rapamycin resulted in greater suppression of AKT and 4EBP1 phosphorylation than did either agent singly. These data suggest that simultaneous targeting of more than one signaling pathway may improve inhibition of leukemic cell growth. Furthermore, combining signal transduction inhibitors with hypomethylating agents may also result in effective suppression of AML proliferation.
Discrimination and Quantification of Spectrally Similar Near-Infrared Probes by Time-Domain (TD) Optical Imaging in Acute Myeloid Leukemia Mouse Models.
Authors: McCormack Emmet , Belankov Alexandre , Mujic Maja , Couture Pierre , Gjertsen Bjorn T.
Published: October 2019

The use of whole-body optical imaging in the near-infrared (NIR) spectrum (650–1100 nm) employing fluorescently labelled reagents recognising cell-specific biomarkers of leukemia has become a standard modality in preclinical models of the human disease. A particular challenge is represented by leukemic infiltrates in liver and spleen, organs with high optical absorbance. While there are increasingly impressive arrays of fluorescently labelled biomolecules available for exploitation via optical imaging, the number of commmercially availible fluorophores for NIR imaging remain limited. In particular, simultaneous imaging of disease progression and functional imaging of more specific biological processes within the same sample is complicated by the requiste for multiple filtersets for fluorophores with similar spectral properties. Subsequent “bleeding” fluorescence through filtersets is unavoidable precluding ones ability to quantify specific fluorophores based on fluorescence. Similarly, descrimination of in vivo autofluorescence of similar spectral properties to commonly employed NIR dyes, consequent of ingested food comlicates contrast even further. More recently spectral imaging techniques have aided discrimination of fluorophores of similar spectral profiles however, these techniques attenuate much of the light reaching the detector. Time-domain (TD) optical imaging through the use of pulsed laser diodes and time resolved detector system, typically a photo-multiplier tube (PMT), has previously been demonstrated to distinguish between changes in physiological processes such as; tissue pH or calcuim concentration, based on changes in fluorescence lifetime of a fluorescently labelled probe. Here we demonstrate employing a single wave lenght TD optical imaging (eXplore Optix™, ART Inc) the potential to discriminate and quantify combinations of diverse NIR probes of spectrally similar properties but differing fluorescence lifetime on the basis of fluorescence lifetime in appropriate in vitro phantoms. Similarly, we illustrate the ability of this technique to discriminate between endogenous autofluorescence from administered fluorophores in vivo of leukemic cells in liver and spleen, and subsequent distinction of mixtures these fluorophores via their inherent fluorescent lifetimes in vivo.