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Background: Older patients encompass about 75 % of patients with acute myeloid leukemia (AML). Today therapeutic options to prevent relapse in older patients who managed to achieve complete remission (CR) after intensive chemotherapy are scarce. Recent randomized controlled trials (RCTs) have aimed to reduce the risk of relapse by means of post-CR maintenance therapy., Methods: We performed a systematic review and meta-analysis of RCTs comparing the efficacy and safety of maintenance with hypomethylating agents (HMA) vs. observation, conventional care or placebo in older patients with AML who are not candidates for allogeneic hematopoietic transplantation (allo-HCT). We searched Cochrane Library, PubMed and conference proceedings up to August 2021., Results: Six trials were included. Treatment with hypomethylating agents significantly improved overall survival (HR 0.80, 95 % CI 0.70 to 0.91, I 2 = 30 %), and disease control (HR 0.80, 95 % CI 0.70 to 0.91, I 2 = 0). A significant decrease was seen in both one year mortality (Risk Ratio [RR] 0.61, 95 % CI 0.48 to 0.77, I 2 = 0) and mortality at the end of follow-up (RR 0.77, 95 % CI 0.67 to 0.88, I 2 = 0). The rate of relapse at 6 months and at one-two years was lower in the HMA arm vs. control (RR, 0.59; 95 % CI, 0.47-0.72; RR, 0.74, I 2 = 0; 95 % CI 0.69 - 0.91, I 2 = 41 %, respectively). HMA were associated with a statistically non-significant increase in the risk of serious adverse events (RR 3.44, 95 % CI 0.93-12.74, I 2 = 80 %)., Conclusions: Our meta-analysis shows that in older patients who are not candidates for allo-HCT, maintenance therapy with HMA improves OS and disease control without a statistically significant increase in adverse events., (Copyright © 2021. Published by Elsevier Ltd.)

Technological advances have made it possible to offer home-based chemotherapy to patients without health care professionals being present. Prior studies on effects of home-based treatment lack inclusion of patients with hematologic malignancies. We present data from a multicenter single-arm feasibility and safety study of home-based intensive chemotherapy in patients with newly diagnosed acute myeloid leukemia and their quality of life and psychological wellbeing. This national study included patients from six sites in Denmark who received intensive chemotherapy on programmed CADD Solis infusion pumps through a central venous catheter and were also managed as outpatients during treatment-induced pancytopenia. Data are presented from 104 patients, receiving 272 treatments with 1.096 (mean 4.57, SD 3.0) home infusion days out of 1.644 treatment days (67 %). Sixty-two of 168 (36.9 %) reinduction and consolidation treatment cycles ensuing pancytopenia phases were solely handled in the outpatient clinic. Patients reported high satisfaction with home-based treatment, which had a positive influence on their ability to be involved in their treatment and be socially and physically active. No unexpected events occurred during the intervention. Overall, patients improved in all quality of life outcomes over time. Home-based intensive chemotherapy treatment was feasible and safe in this population. ClinicalTrials.gov identifier: NCT04904211., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

A decreased percentage of CD177 pos neutrophils is frequently present in MDS and AML and is a useful flow cytometry (FCM) marker for the identification of MDS. The underlying mechanism leading to the low percentage of CD177 pos neutrophils in MDS has not been explained. The aim of this study was to identify whether specific somatic mutations in myeloid neoplasms are associated with the low percentage of CD177 pos neutrophils. 507 myeloid neoplasms with one or more pathogenic molecular abnormality identified by NGS and in which CD177 expression was assessed were evaluated. Correlation with CD177 expression was determined for 39 variables (including genes mutated, diagnostic groups and gender) using a 40 % cutoff level for low CD177 expression. In multivariate analysis mutations involving NPM1 (OD 0.26), RUNX1 (OD 0.39), TET2 (OD 0.58), and U2AF1 S34F (OD 0.25) were associated with low percentage of CD177 pos neutrophils when all cases were evaluated. JAK2 (OD 2.5) alteration was associated with increased percentage of CD177 pos neutrophils. Differences were noted between diagnostic subgroups with no single mutation associated with decreased CD177 pos neutrophils in MDS and CCUS. The findings demonstrate an association between the percentage of CD177 pos neutrophils and somatically acquired mutations involving several genes., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Acute Myeloid Leukemia (AML) represents 1 % of all new cancer diagnosis made annually in the US and has a five-year survival of 30 %. Traditional treatment includes aggressive induction therapy followed by consolidation therapy that may include a hematopoietic stem cell transplant (HSCT). Thus far, HSCT remains the only potentially curative therapy for many patients with AML owing to the graft-versus-leukemia effect elicited by this treatment. The use of novel therapies, specifically immunotherapy, in the treatment of AML has been limited by the lack of appropriate target antigens, therapy associated toxicities and variable success with treatment. Antigenic variability on leukemia cells and the sharing of antigens by malignant and non-malignant cells makes the identification of appropriate antigens problematic. While studies with immunotherapeutic agents are underway, prior investigations have demonstrated a mixed response with some studies prematurely discontinued due to associated toxicities. This review presents a discussion of the envisioned role of immunotherapy in the treatment of AML in the setting of mixed therapeutic success and potentially lethal toxicities., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Both inhibitory and stimulatory effect of EGCG on cancer cells have been reported, which often is linked to receptor tyrosine kinase signaling. In this study, we present evidence that green tea extract and its chemical component, Epigallocatechin-3-gallate (EGCG), inhibit growth of human myeloid leukemia cells through the regulation of pRb synthesis and formation of pRb-E2F complexes. Addition of green tea extract to the culture of TF-1a and MV4-11 myeloid leukemia cells significantly inhibited their proliferation with a substantial portion of cell death being observed. The green tea extract and EGCG had no significant effect on the expression of G1 CDKs and the CDK inhibitors but downregulated the formation of pRb-CDKs. Surprisingly, the expression of pRb was markedly upregulated while the phosphorylation of pRb downregulated. The upregulation of pRb was blocked by pre-treatment with cycloheximide, a protein synthesis inhibitor, suggesting a requirement of protein synthesis. In agreement with these results, pRb-E2F complexes were upregulated and E2F DNA binding activity decreased. Since both TF-1a and MV4-11 are factor-independent cell lines, the upregulation of pRb-E2F complexes and inhibition of DNA binding activity by green tea extract is most likely through a receptor tyrosine kinase-independent pathway. We also found that the stem/progenitor cells derived from these two leukemia cell lines are more sensitive to the inhibitory effect of green tea extract. Our result suggests that concentrated green tea extract and EGCG may have potential for clinical investigation as an inducer of cancer cell death., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The association between cytomegalovirus (CMV) reactivation and relapse risk has not been evaluated in relation to T cell depletion strategies. We evaluated 93 patients who underwent allogeneic hematopoietic stem cell transplantation (HSCT) and analyzed the association between T cell depletion strategies with the cumulative incidence of relapse and CMV reactivation. A total of 33% of patients who received ATG vs. 34% who received alemtuzumab developed CMV reactivation. The cumulative incidence of relapse was 3% at 1year and 20% at 3 years in patients with CMV reactivation vs. 30% at 1year and 38% at 3 years in patients without CMV reactivation (p=0.02). When analyzed separately, this effect persisted in the myeloid, but not the lymphoid group. There was a numerical trend towards increased non-relapse mortality (NRM) in patients with CMV reactivation, especially in the myeloid group. The choice of T cell depleting agent and the rate of CMV reactivation were not associated with different overall survival (OS) rates. These results suggest that the choice of T cell depletion strategy may have similar effects on rates of CMV reactivation, disease relapse, and survival. Further studies examining these variables in patients not exposed to in-vivo T cell depleting agents may be of interest., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The presence of AML1-ETO (RUNX1-CBF2T1), a fusion oncoprotein resulting from a t(8;21) chromosomal translocation, has been implicated as a necessary but insufficient event in the development of a subset of acute myeloid leukemias (AML). While AML1-ETO prolongs survival and inhibits differentiation of hematopoietic stem cells (HSC), other contributory events are needed for cell proliferation and leukemogenesis. We have postulated that specific tumor suppressor genes keep the leukemic potential of AML1-ETO in check. In studying del(9q), one of the most common concomitant chromosomal abnormalities with t(8;21), we identified the loss of an apparent tumor suppressor, TLE4, that appears to cooperate with AML1-ETO to confer a leukemic phenotype. This study sought to identify the molecular basis of this cooperation. We show that the loss of TLE4 confers proliferative advantage to leukemic cells, simultaneous with an upregulation of a pro- inflammatory signature mediated through aberrant increases in Wnt signaling activity. We further demonstrate that inhibition of cyclooxygenase (COX) activity partly reverses the pro-leukemic phenotype due to TLE4 knockdown, pointing towards a novel therapeutic approach for myeloid leukemia., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

By grafting spontaneous leukemia tumor cells, the myeloid My2/De leukemia rat model was established. Death was caused by impaired functions of heavily infiltrated organs. In vitro culturing of tumor cells, blood and bone marrow counts and cytochemic reactions indicated the leukemic the origin resembling human myeoloblastic leukemia. Metastatic spread was followed after i.v. and i.p. injection, and by implantation of leukemia cells under the renal capsule of rats. Primary tumor and metastasis formation was visualized by (18)FDG or (11)C-methionine administration and MiniPET. The accumulation of radiotracers was measured in different organs and expressed as Differential Absorption Ratios (DARs). Subrenal implantation of My2/De cells resulted in their appearance in other abdominal organs and in parathymic lymph nodes. The release of tumor cells from the primary kidney to the peritoneum was mimicked by the i.p. administration of ink particles. Ink particles deposited in the abdominal organs and in the thoracal lymph nodes, preferentially in parathymic lymph nodes, confirming the notion of lymphatic spread of metastasis., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Regulation of ABCB1 (P-glycoprotein/Pgp) in AML was investigated. In a historical cohort with Pgp and transcriptional regulator expression profiling data available (n=141), FOXO1 correlated with Pgp protein expression. This was confirmed in an independent cohort (n=204). Down-regulation (siRNA) or hyperactivation (nicotinamide) of FOXO1 led to corresponding changes in Pgp. Low FOXO1 expression correlated with FLT3-ITDs (p<0.001) and siRNA inhibition of FLT3-ITD up-regulated FOXO1. As FOXO1 is a key growth regulator, it may underpin biological differences between Pgp-positive clones (low WBC and primary resistant disease) and clones with a FLT3-ITD (associated with a high WBC and early relapse)., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The revised IPSS (IPSS-R) was developed aiming at a better prognostication, taking into account patients treated with best supportive care. We herein validated this model on the basis of data from 1314 patients who received BSC only as well as patients who underwent induction chemotherapy (n=214) or allogeneic transplantation (n=167). We could demonstrate a clear distinction of the IPSS-R risk categories with regard to survival and risk of AML evolution in all patient cohorts. When comparing IPSS-R, IPSS, WHO prognostic scoring system (WPSS) and Duesseldorf score, the best results regarding the ability to predict survival were obtained by the IPSS-R., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Dose-intense etoposide-cyclophosphamide (D-I ECy) without stem cell transplantation has been used in salvage regimens for the treatment of resistant acute myeloid leukemia(AML). Previous D-I ECy studies classified AML according to FAB-criteria, before cytogenetic risk was found to be a major determinant of prognosis. Currently the influence of karyotype on response to D-I ECy is unknown. Thus, an observational study was conducted in thirty four patients treated with D-I ECy for resistant AML. The results show this regimen is moderately effective in achieving CR in relapsed AML patients, including those with age >60 and poor cytogenetic risk category., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Cytarabine combined with an anthracycline or an anthracenedione represents the usual intensive induction therapy for the treatment of AML. However, this protocol induces severe side effects and treatment-related mortality due to the lack of selectivity of these cytotoxic agents. In this paper, we present the study of the first galactosidase-responsive molecular "Trojan Horse" programmed for the delivery of doxorubicin exclusively inside AML blasts over-expressing the folate receptor (FR). This targeting system allows the selective killing of AML blasts without affecting normal endothelial, cardiac or hematologic cells from healthy donors suggesting that FDC could reduce adverse events usually recorded with anthracyclines., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Daunorubicin has historically been considered the anthracycline of choice at many cancer centers for the treatment of acute myeloid leukemia (AML). Drug shortages have required the substitution of daunorubicin with idarubicin. Randomized studies have shown idarubicin (10-12mg/m(2)) to be comparable or superior to standard dose daunorubicin (45-60mg/m(2)) for achieving complete remission (CR). Whether these results can be extrapolated to dose-intense daunorubicin (90mg/m(2)), recently shown to improve CR rates when compared to standard daunorubicin doses remains uncertain. This observational study was conducted at Northwestern Memorial Hospital (NMH) to compare CR rates. The results suggest idarubicin is equivalent to daunorubicin, and for some subsets of patients, idarubicin may have superior CR rates., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Spider venoms are a rich source of bioactive compounds with therapeutic potential. In traditional Chinese medicine, spiders and spider venoms have been used in the treatment of various ailments. In the present study, the venom of the spider Macrothele raveni potently suppressed cell growth in the myelogenous leukemia K562 cell line in a dose and time-dependent manner with an IC(50) of 5.1 μg/mL. The venom also had a low inhibitory effect on human lymphocytes with an IC(50) of approximately 36.4 μg/mL, indicating that the venom is relatively selective for leukemic cells. Venom treated K562 cells showed typical morphological indicators of apoptosis including condensation of nuclei and fragmentation of DNA. Annexin V-FITC and propidium iodide dual staining further demonstrated that the venom had potent apoptogenic activity. Venom treatment induced caspase 3 and caspase 8 activation in K562 cells and promoted PARP cleavage. The present results indicate that the venom of the spider M. raveni potently and selectively suppresses the growth of K562 cells by inducing apoptosis via caspase 3 and caspase 8 mediated signaling pathways., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Aberrant reactivation of Gli signaling has been described in a wide variety of human cancers and rapamycin can down-regulate Gli pathway in some solid tumors. In this study, we attempt to define the cytotoxic effect of Gli inhibitor on AML cells. And the regulation action of rapamycin on Gli in AML cells also has been assessed. Gli inhibitor GANT61 caused growth arrest and apoptosis in AML cells. Rapamycin decreased not only the Gli protein and mRNA expressions but also expression of the Gli-luciferase reporter in AML cells. Synergism effect between GANT61 and rapamycin was found in Kasumi-1, HL-60 and U937 cell lines. The results suggest that aberrant Gli activation is a feature of some myeloid leukemic cells and Gli activiation can be down-regulated by rapamycin., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In AML, prevention of GvHD leads to better tolerance of myeloablative therapy. 66 individuals with AML in CR underwent myeloablative conditioning and transplantation with allogeneic PBPC grafts. Median presentation age was 44.5 years. Karyotyping was intermediate in 48% and of unfavourable risk in 36%. For GvHD prophylaxis, PBPC harvests were incubated ex vivo with anti CD52 antibodies. TRM at day 100 and 1 year was 9% and 17%. At a median of 1018 days 65% are alive. Grade >1 GvHD was seen in 11%. GvHD and adverse karyotype were associated with treatment failure. In younger patients preservation of the dose intensity may improve cure rates., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Multipotent haematopoietic stem cells pass through stages of differentiation with the progressive loss of developmental options leading to the production of terminally differentiated mature blood cells. This process is regulated by soluble cytokines binding to a ligand specific cell surface receptor on a precursor cell. Key to signal transduction are tyrosine kinase proteins which can be divided into two sub families, the receptor protein tyrosine kinases which are transmembrane receptors and retain an intact catalytic kinase domain and the cytoplasmic tyrosine kinases which bind to cytokine receptors. Abnormalities of tyrosine kinase proteins are well recognised in myeloid malignancies, mutation in the cytoplasmic tyrosine kinase JAK2 (V617F) is key in the pathogenesis of myeloproliferative neoplasms, and translocations involving ABL key in the development of chronic myeloid leukaemia. However tyrosine kinase mutations are increasingly recognised to play a role in the pathogenesis of a wider range of haematological cancers. This review focuses on the role of deregulated tyrosine kinase genes either as part of novel fusion proteins involving FGFR1, PDGFRα, PDGFRβ, JAK2 and ABL, or as a consequence of point mutation in JAK1 or JAK2 in the development of precursor T and B lymphoid malignancies or mixed myeloid/lymphoid disorders. We also set out some of the postulated mechanisms which underlie the association of tyrosine kinase mutations with the development of lymphoid malignancy., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Cyclopamine, a plant-derived steroidal alkaloid, inhibits the hedgehog (Hh) signaling pathway by antagonizing Smoothened. This drug can induce the differentiation of myeloid leukemia cell lines and acute myeloid leukemia (AML) cells in primary culture. The treated cells were stained with Luxol-fast-blue, which is specific for eosinophilic granules. Ligation of CD44 with some specific monoclonal antibodies can reverse the differentiation of AML cells. Combined treatment with cyclopamine and a monoclonal antibody to ligate CD44 more than additively induced the differentiation of HL-60 cells. These results may provide useful information for the development of a CD44-targeted therapy in AML., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Patients with normal karyotype acute myeloid leukemia (NK-AML) without the FLT3 internal tandem duplication (FLT3-ITD) mutation account for approximately 30% of all AML cases, and exhibit a heterogeneous clinical outcome. Except for NPM1 mutations, prognostic factors in this subgroup of AML are still unclear. Here we explored the impact of immunophenotypic markers along with NPM1 mutations and clinical features on the outcome of 133 FLT3-ITD negative NK-AML patients. CD34 expression was associated with poorer complete remission (CR) rate, disease-free survival (DFS), event-free survival (EFS), and overall survival (OS), whereas CD56 expression adversely affected EFS and OS. In contrast, NPM1 mutations correlated with an improved CR rate, DFS, and EFS. Moreover, males experienced shorter DFS and EFS, while older patients (≥60 years) had shorter EFS. Multivariate analysis of age, gender, NPM1, CD34, and CD56 showed NPM1 mutation was an independent predictor of better CR rate, DFS, and EFS (P<0.001, P=0.003, and P=0.006, respectively). In addition, older age was associated with shorter DFS and EFS (P=0.045 and P=0.028, respectively), and CD56 positivity predicted shorter EFS (P=0.012). Our results confirm the favorable impact of NPM1 mutations and identify the adverse prognostic relevance of CD56 expression in this subgroup of AML., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

To determine the contribution of the common N-terminal truncation of NUP98 in NUP98-translocations resulting in acute myeloid leukemia, we have conducted a structure-function analysis of NUP98 in the context of NUP98-HOXA10HD, a novel, canonical NUP98-Hox fusion that significantly enhances the self-renewal capacity of hematopoietic stem cells and collaborates with Meis1 to induce AML in our mouse models. Our results identify that NUP98 functions by transcriptional activation likely by recruitment of CBP/p300 via its FG/GLFG repeats. In contrast, the functional interaction of NUP98 with Rae1 or the anaphase promoting complex appears non-essential for its role in NUP98-leukemogenic fusions., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

We have designed prodrugs that release nitric oxide (NO) on metabolism by glutathione S-transferases (GST). This design exploits the upregulation of GST in acute myeloid leukemia (AML) cells. O(2)-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K, a member of this class) has potent anti-leukemic activity. HL-60 myeloid leukemia cells were used for in vitro studies of the combination of JS-K with daunorubicin (DAUNO), cytarabine (ARA-C) or etoposide (ETOP) using the median effect method to determine synergistic, antagonistic, or additive effects. Combinations of JS-K added simultaneously, 2h before or 2h after the other compounds were used. JS-K and DAUNO were antagonistic in all three drug sequences. JS-K and ETOP were also antagonistic but to a lesser degree. JS-K and ARA-C showed strong synergy. The combination index 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. JS-K by itself induced DNA strand breaks at relatively high concentrations. However, at submicromolar concentrations, it significantly augmented ARA-C-induced DNA strand breaks. NMR spectroscopy revealed no evidence of chemical interaction between JS-K and the other chemotherapeutic agents. We conclude that ARA-C and JS-K have synergistic anti-leukemic activity and warrant further exploration in combination.

We analyzed the effect of (+)alpha-tocopheryl succinate (alpha-TOS) alone or associated with arsenic trioxide (ATO) or all-trans retinoid acid (ATRA) in acute promyelocytic leukemia (APL). alpha-TOS-induced apoptosis in APL clinical samples and in ATRA-sensitive (NB4) and ATRA-resistant (NB4-R2) APL cell lines. The effective dose 50% (ED-50) was calculated to be 71 and 58muM, for NB4 and NB4-R2, respectively. alpha-TOS neither induced nor modified ATRA-induced differentiation of APL cells, and did not affect the proliferation and differentiation of normal CD34(+) hematopoietic progenitors in methylcellulose assays. alpha-TOS exerted a moderate antagonistic effect to ATO-induced apoptosis when treatment was done simultaneously but when alpha-TOS was added 24h after ATO, an additive effect was observed. Our results support the concept of alpha-TOS as an anti-leukemic compound which spares normal hematopoiesis.

For about 40 years, the biology of human myeloid leukemia (ML) has been studied in different in vitro systems. Throughout this time, semisolid colony assays, Dexter-type long-term cultures and liquid suspension cultures have contributed to our understanding of the mechanisms involved in the origin and progression of this hematological disorder. By using such systems, it has been possible to identify the cells in which leukemia originates; to recognize a functional hierarchy within the hematopoietic system of leukemia patients; to identify factors, soluble and cell-associated, that regulate leukemic growth; and to study the effects of different antineoplastic drugs. Furthermore, in vitro systems for purging of leukemic cells have been developed. Still, many questions and problems remain unsolved regarding the biology of myeloid leukemia in vitro. This article presents a comprehensive review on the behavior of leukemic stem and progenitor cells, both from acute and chronic myeloid leukemia, in the different culture systems mentioned above.

Busulfan (Bu) resistance is a major obstacle to hematopoietic stem cell transplantation (HSCT) of patients with chronic or acute myelogenous leukemia (CML or AML). We used gene expression analysis to identify cellular factors underlying Bu resistance. Two Bu-resistant leukemia cell lines were established, characterized and analyzed for differentially expressed genes. The CML B5/Bu250(6) cells are 4.5-fold more resistant to Bu than their parental B5 cells. The AML KBM3/Bu250(6) cells are 4.0-fold more Bu-resistant than KBM3 parental cells. Both resistant sublines evade Bu-mediated G2-arrest and apoptosis with altered regulations of CHK2 and CDC2 proteins, constitutively up-regulated anti-apoptotic genes (BCL-X(L), BCL2, BCL2L10, BAG3 and IAP2/BIRC3) and down-regulated pro-apoptotic genes (BIK, BNIP3, and LTBR). Bu-induced apoptosis is partly mediated by activation of caspases; use of the inhibitor Z-VAD-FMK completely abrogated PARP1 cleavage and reduced apoptosis by approximately 50%. Furthermore, Bu resistance in these cells may be attributed in part to up-regulation of HSP90 protein and activation of STAT3. The inhibition of HSP90 with geldanamycin attenuated phosphorylated STAT3 and made B5/Bu250(6) and KBM3/Bu250(6) more Bu-sensitive. The analysis of cells derived from patients classified as either clinically resistant or sensitive to high-dose Bu-based chemotherapy indicated alterations in gene expression that were analogous to those observed in the in vitro model cell lines, confirming the potential clinical relevance of this model for Bu resistance.

Wilms' tumor gene 1 (WT1) is overexpressed in various hematological malignancies and has been proposed as a target for minimal residual disease (MRD) detection and for immunotherapy. Although WT1 is known as a key molecule for tumor cell proliferation, the expression pattern of WT1 in leukemic cells in dependency of proliferation has not yet been investigated. Furthermore, WT1 expression was mostly studied by reverse transcriptase PCR and the expression of WT1 protein has not been extensively studied. Here, we analyzed WT1 protein expression in the human myeloid leukemia cell lines K562 and HL-60 by indirect immunofluorescence and flow cytometry. Both cell lines exhibited varying nuclear WT1 immunoreactivity pointing to a cell cycle-dependent and/or proliferation-dependent WT1 expression. In rapidly proliferating cells high levels of WT1 protein were detected by flow cytometry. A reduced proliferation rate was associated with a low WT1 protein expression and an accumulation of cells in G(0)/G(1) phase. During G(0)/G(1) phase cells expressed WT1 at a lower level than in S or G(2)/M phase. Moreover, WT1 expression was diminished in all cell cycle phases in slowly proliferating cells. We conclude that WT1 protein expression is dependent on the cell cycle phase as well as on the proliferation rate. This finding might be relevant for MRD studies and immunotherapeutic strategies targeting WT1.

Neutrophils express functional toll-like receptor2 (TLR2) and 4 (TLR4) that are crucial for the production of inflammatory cytokines. Here, we show that dimethylsulfoxide-induced neutrophil-like differentiation of promyelocytic HL-60 cells (dHL-60) results in cells that respond to TLR2 and TLR4 ligands similarly to primary neutrophils. Consistent with the increased responsiveness of the cells to TLR2 ligand, the TLR2 gene was strongly up-regulated in dHL-60 cells. On the other hand, increased surface expression of LPS receptor complex, TLR4/MD2/CD14, was observed without affecting TLR4 gene expression. Thus, the data demonstrate a higher responsiveness of dHL-60 cells to TLR2 and TLR4 ligands because of increased TLR2 and MD2/CD14 gene expression.

Natural killer (NK) cells are potent effectors of innate antitumor defense and are currently exploited for immune-based therapy of human leukemia. However, malignant blood cells in acute myeloid leukemia (AML) display low levels of ligands for the activating immunoreceptor NKG2D and can thus evade NK immunosurveillance. We examined the possibility of up-regulating NKG2D-specific UL16-binding protein (ULBP) ligands using anti-neoplastic compounds with myeloid differentiation potential. Combinations of 5-aza-2'-deoxycytidine, trichostatin A, vitamin D3, bryostatin-1, and all-trans-retinoic acid, used together with myeloid growth factors and interferon-gamma, increased cell surface ULBP expression up to 10-fold in the AML cell line HL60 and in primary AML blasts. Up-regulation of ULBP ligands was associated with induction of myelomonocytic differentiation of AML cells. Higher ULBP expression increased NKG2D-dependent sensitivity of HL60 cells to NK-mediated killing. These findings identify NKG2D ligands as targets of leukemia differentiation therapy and suggest a clinical benefit in combining a pharmacological approach with NK cell-based immunotherapy in AML.

Differentiation-inducing therapy with the DNA-methylation inhibitor Decitabine (5'-aza-deoxycytidine) and histone deacetylase (HDAC) inhibitors are now considered in acute myelogenous leukemia (AML). We investigated the in vitro effects of Decitabine and two structurally unrelated HDAC inhibitors (Sodium 4-phenyl butyrate, Tricostatin A) on clonogenic AML cells. Based on morphological criteria we identified four major colony types: (i) non-erythroid colonies, (ii) erythroid colonies that were detected only for a subset of patients and could be further sub classified into mature and immature forms, and (iii) intermediate colonies. Erythroid differentiation was associated with low CD34 expression. The colonies showed differences in morphology, viability, cell cycle distribution and expression of differentiation markers. Both Decitabine and the two HDAC inhibitors altered AML cell expression of differentiation markers, whereas the drugs did not have any major influence on cell cycle distribution. However, the pharmacological effects differed between the four colony subsets, and differences were also detected between the two HDAC inhibitors. We conclude that clonogenic AML cells can be classified into well-defined subsets based on their differentiation, and these subsets differ in their biological characteristics as well as their response to pharmacological targeting of epigenetic regulation.

We evaluated the efficacy of FISH to detect chromosome anomalies in the evaluation of young (<60 years) patients with AML. Patients were enrolled in E1900, an ECOG clinical trial for AML. The protocol was designed to collect bone marrow or blood for both cytogenetic and FISH studies at study entry (diagnosis). FISH for each patient was performed and utilized eight probe sets to detect t(8;21), t(9;22), t(11;var), t(15;17), inv(16), +8, -5/5q, and -7/7q. We analyzed 237 specimens with complete cytogenetic and FISH results. Results for each specimen were classified by probe set into one of six categories. The concordance rate between cytogenetic and FISH results ranged from 98 to 100% for all probe sets and kappa analysis for concordance had a p-value of <0.0001. The high level of agreement between cytogenetic and FISH results demonstrate the accuracy of a panel of eight FISH probe sets for the detection of significant abnormalities in AML. Data from this investigation support the use of FISH as an adjunct method to increase the yield of useful cytogenetic results in large cooperative trials and demonstrate the potential of FISH as a follow-up study of minimal residual disease in ECOG trials.

The third-generation bisphosphonate zoledronic acid (ZOL) has recently been shown to be active against human tumour and leukemic cell lines. The purpose of this study was to evaluate the antileukemic potential of ZOL in acute myeloid leukemia (AML). We determined the lethal concentration 50% (LC 50) using the WST-1 assay of ZOL as being 287.9 microg/ml after 24 h and 108.3 microg/ml after 96 h in HL 60 cells and to be 382.4 and 43.2 microg/ml, respectively, in nine samples from patients with AML. The ZOL induced inhibition of proliferative activity of HL 60 cells could not be abrogated by the hematopetic growth factors G-CSF and GM-CSF. ZOL was found to by cytotoxic in HL 60 cells without activation of caspase 3. ZOL was not cross resistant with cytarabine as shown by the linear correlation of LC 50s. Both agents, however, exerted an additive cytotoxicity as revealed by isobologram-analysis and combination index. These data warrant further investigation of ZOL in the treatment of AML.

Platelet-activating factor (PAF) is a phospholipid mediator with potent immunoregulatory activities on mature leukocytes. PAF modulates leukocyte cytosolic Ca2+ concentration ([Ca2+]i) through a Gq mediated pathway. We highlight, for the first time, Gq transcripts, PAF receptor (PAF-R) transcripts and protein in blast cells of acute myeloid (AML) and lymphoid (ALL) leukemia patients. PAF stimulated [Ca2+]i in leukemic blast cells; PAF effects being prevented by a specific PAF-R antagonist. In conclusion, functional PAF-R are present in blast cells of patients with acute leukemia; a result that could be of physiologic importance regarding the important effect of PAF on leukocytes maturation and functions.

In studies of multiple myeloma cells, atiprimod was shown to block Stat3 activation and inhibited colony-forming cell proliferation. We hypothesized that atiprimod may also inhibit activation of intracellular signaling pathways in AML cells resulting in apoptosis and growth inhibition. We demonstrate that atiprimod inhibited clonogenic growth of AML cell lines and fresh AML marrow cells whereas it did not significantly affect growth of normal hematopoietic progenitors from marrow samples of healthy controls. Atiprimod decreased phosphorylation of Stat3 and Stat5, and protein levels of Jak2, whereas gene expression of Jak2 was not affected. Atiprimod further induced apoptosis by cleavage of caspase 3 and PARP. In summary, our data suggest that atiprimod has a significant antiproliferative and proapoptotic effect on AML cells. This effect may be facilitated by inhibition of the Jak-Stat signaling pathway. Further evaluation of atiprimod in clinical trials of AML should be considered.

UM384 cells, derived from the human myeloid leukemia U937 cell line, fail to differentiate in response to 12-O-tetradecanoylphorbol-13-acetate (TPA). Using cDNA microarray and real-time quantitative PCR (RT-QPCR) approaches, we observed a difference in the response to TPA treatment: all the genes from U937 cells were continuously modulated from 2 to 24h. In UM384 cells, 60% of the genes were transiently modulated at 2h, then returned to control levels at 24h. Moreover, HuR, an AU-rich element-binding protein (ARE-BP), was differentially located in the two cell lines. Therefore, a defect of mRNA stabilization could be responsible for the resistance of UM384 cells to TPA-induced differentiation, suggesting a possible role for the post-transcriptional regulation in the leukemogenesis.

The anti-epileptic drug valproic acid harbors anti-tumoral activity in solid and leukemic tumor cell models and is currently evaluated in clinical trials. However, the plasma trough concentrations obtained in patients by common anti-epileptic dose regimens are below concentrations required for exerting anti-tumor effects in vitro. Here, we describe the identification of three novel valproic acid derivatives with superior differentiation-inducing and anti-proliferative activities in K562 bcr/abl-positive chronic myeloid leukemia cells and HL60 promyelocytic leukemia cells at achievable therapeutic VPA concentrations. These compounds reveal potent inhibition of histone deacetylase activity, induction of p21Cip/Waf expression as well as low toxicity on CD34+ bone marrow cells.

The transformation site in acute myeloid leukemia (AML) has been proposed to be pluripotent hematopoietic stem cells (PHSCs), and the lymphoid development of leukemic PHSCs may be suppressed by leukemogenic events. Recent data from multiple laboratories have contradicted the current hierarchical model of hematopoiesis and indicated the presence of myeloid HSCs with minimal lymphopoietic potential (MyHSCs) in mice. Based on these findings and re-evaluating the published data on AML stem cells, we hypothesize that MyHSCs may be the transformation site in AML. If so, therapy targeting leukemic MyHSCs but sparing PHSCs is worth investigating.

Dimethyl sulfoxide (DMSO) is a widely used prototypical chemical inducer of cell differentiation. In the present study, the effects of DMSO on susceptibility of human myeloid leukemia U937 cells towards ligation of distinct death receptors (DRs) were investigated. DMSO sensitized cells towards induction of apoptosis by anti-Fas antibody, tumour necrosis factor-alpha or Apo2 ligand/TNF-related apoptosis-inducing ligand (TRAIL). Apart from increasing Fas levels, DMSO did not affect expression of proteins in death signal transduction, such as Bcl-2 family proteins, FADD, caspase-3 and -8, the inhibitor of apoptosis proteins (IAPs) or cFLIP(L). However, DMSO significantly potentiated mitochondrial membrane depolarization, suggesting that this mechanism might be involved in sensitisation of myeloid cells to DR-mediated apoptosis.

To better characterize acute myeloid leukemia (AML) development in non-obese diabetic (NOD)/severe combined immunodeficiency (SCID) mice, we transplanted samples from patients with AML or KG-1 and EOL-1 cell lines. We found 9/12 primary AML samples and both cell lines to engraft within 2-8 weeks, with 5-80% human cells in bone marrow. Compared with freshly isolated AML cells, percentages of human CD33+, CD38+, CD31+ CD13+ or CD15+ subpopulations increased after transplantation, whereas percentages of CD34+ cells decreased. Engrafted mice frequently showed expression of human endothelial cell markers. Thus, transplantation of human AML into NOD/SCID mice reveals expression of hematopoietic and endothelial differentiation markers.

Background and Objectives: A consecutive series of acute myeloid leukemias (AML) patients was analyzed in conditions which reduce the inter-assay variations (the same flow cytometer, the same observers and the same panel of monoclonal antibodies) in order to investigate the prognostic information provided by flow cytometry., Design and Methods: Two hundred and sixty-six bone marrow (BM) samples from 326 patients enrolled in the LMA-99 protocol from the CETLAM group were studied by multiparametric flow cytometry. Immunophenotyping studies were performed on erythrocyte-lysed BM samples. Antigen expression of leukemic cells was analyzed using triple stainings with fluorochrome-conjugated combinations of monoclonal antibodies., Results: CD2 was positive in 21 cases (8%); an associated inv(16) was detected in eight CD2+ cases (38%). Two-year overall survival (OS) rate for CD2+/inv(16)+ patients was 75%, whereas it was 0% for CD2+/inv(16)- patients and 47% for CD2- patients (p=0.0001). CD36 was expressed in 37% of patients (n=98). Two-year leukemia-free survival (LFS) rate was 34% for CD36+ patients and 55% for CD36- patients (p=0.001). In the multivariate analysis, CD2+ (RR=8.4; p=0.0001) and adverse karyotype (RR=10.2; p=0.0001) were associated with a lower CR rate, CD36+ (RR=1.5; p=0.03), CD2+ (RR=2; p=0.04) and adverse karyotype (RR=4; p=0.0001) were associated with a lower OS and CD36+ (RR=2; p=0.002) and adverse karyotype (RR=3.5; p=0.005) predicted a lower LFS., Conclusions: CD2+ patients had a very poor OS when CD2/inv(16)+ cases were excluded. CD36 and CD2 expression at diagnosis can provide prognostically important information in adult de novo AML.

12-O-Tetradecanoylphorbol-13-acetate (TPA) is being developed as a therapeutic agent by virtue of its being a potent modulator of signal transduction in pre-clinical models of AML [Strair RK, Schaar D, Goodell L, Aisner J, Chin KV, Eid J, et al. Administration of a phorbol ester to patients with hematological malignancies: preliminary results from a phase I clinical trial of 12-O-tetradecanoylphorbol-13-acetate. Clin Cancer Res 2002;8:2512-8]. In this report, we identify a subset of primary AML samples that undergoes apoptosis after exposure to TPA and demonstrate that TPA-induced cytotoxicity is associated with modulation of the ERK signaling pathway. Analysis of mitogen-activated protein kinase (MAPK) dual-specificity phosphatases (DUSP), as potential regulators of AML cell signaling, indicates that these genes are coordinately regulated and rapidly induced by TPA in primary AML cells. Therefore, TPA-induced primary AML cytotoxicity is associated with modulation of ERK signaling which may be partially mediated by regulation of phosphatase expression.

The correlation between drug sensitivity in vitro and the mutation status of the FLT3 receptor gene was evaluated in tumour cells from 17 previously untreated AML patients. Tumour cells with internal tandem duplication (ITD) in the FLT3 receptor gene were significantly more sensitive to the FLT3 inhibitor SU5614 than tumour cells with wild type FLT3. Combinations of SU5614 with etoposide and amsacrine showed better effect (p<0.05) compared with the respective single drugs. Our results suggest that the FLT3 inhibitor SU5614 may have a therapeutic potential, especially in combination with other cytotoxic agents, in patients with FLT3-ITD positive AML.

To identify prognostic factors alternative or additional to drug-resistance and apoptosis proteins, we studied the impact of the expression of heat-shock proteins (HSPs) in 98 newly diagnosed acute myeloid leukemia (AML). HSP27 was expressed by 39%, HSP60 by 26%, HSP70 by 58%, HSP90 by 41%, and HSP110 by 30% of cases. HSP expressions were correlated with that of differentiation antigens (CD34, CD14, CD15, CD33) and that of drug-resistance (MRP, MRK) and apoptosis (Bcl-2) proteins. HSP90 and HSP110 were correlated with FAB subtype and karyotypic grouping. Complete remission (CR) was obtained in 68 cases (69%). Median disease-free survival (DFS) of the 68 remitters was 18.1 months with a 3-year DFS rate of 41%. CR rates were higher in patients with lower expression of HSPs. Overall survival (OS) was significantly longer in patients with lower expression of HSPs. Cytogenetics, CD34 positive expression, MRK positive expression, and HSP110 positive expression remained as pejorative prognostic factors for OS in the multivariate analysis. When considering patients with intermediate risk cytogenetics, HSP110 and MRP positive expressions and CD33 negative expression were of poor outcome, while HSP27 and HSP60 positive expressions appeared of pejorative prognostic value in patients with unfavorable karyotypes.

The t(8;21)(q22;q22) rearrangement is observed in about 15% of acute myelocitic leukemia (AML) cases, while variant t(8;21) translocations are detected in 6-10% of AML patients positive for the 5'RUNX1/3'CBFA2T1 fusion gene. We report a detailed molecular cytogenetic analysis of a four-way variant t(8;11;16;21)(q22;q14;q12;q22) performed by fluorescence in situ hybridization with specific BAC and PAC clones. The study demonstrated the loss of several megabases belonging to chromosomes 11 and 16 whereas no deletion was detected on der(21). These findings suggest that a precise breakpoint characterization could identify submicroscopic genomic deletions whose meaning remains to be defined.

Exogenous expression of the transcription factor Scl (Tal1) in WEHI-3B D+ myelomonocytic leukemia cells interferes with their capacity to respond to all-trans retinoic acid (ATRA) induced differentiation; combination of ATRA with LiCl, however, circumvents the inhibition of differentiation produced by Scl. To gain information on the possible involvement of this transcription factor in the non-responsiveness of acute myelocytic leukemia (AML) patients to ATRA, we compared the endogenous expression levels of Scl and its transcription complex partners [i.e., Rbtn1 (LMO1), Rbtn2 (LMO2), Ldb1, and GATA family proteins] in leukemic blast cells from patients with AML and acute promyelocytic leukemia (APL), and determined the effects of lithium chloride alone or in combination with ATRA on the capacity of blast cells to differentiate during short-term ex vivo culture. Levels of Scl, Rbtn2, GATA1, and Ldb1 expression were comparable in AML and APL blasts, while the levels of expression of Rbtn1, GATA2, and GATA3 were absent or markedly lower in APL cells. Differentiation markers (cell surface myeloid antigens CD11b, CD15, CD14, and CD33) were also analyzed in blast cells. ATRA produced changes in at least one surface antigen differentiation marker in 89% of patient blasts, while LiCl caused such changes in 72% of the leukemic cells of patients. The combination of LiCl and ATRA induced the differentiation of leukemic blasts from 94% of patients. Although the expression of the transcription factors did not act as individual predictors of responsiveness or non-responsiveness to the inducers of differentiation, ATRA or ATRA plus LiCl, the addition of LiCl to ATRA increased the differentiation response over that of ATRA alone in a number of leukemic samples. These findings suggest that the combination of LiCl and ATRA may produce some clinical benefit in the treatment of the myeloid leukemias.

The prognosis for patients with relapsed/refractory AML, secondary leukemia and AML in older adults is extremely poor. An appealing alternative approach to intensive cytotoxic chemotherapy is to induce apoptosis with a novel agent. There is in vitro evidence that arsenic trioxide (ATO) has anti-proliferative and pro-apoptotic effects on myeloid leukemia cell lines. To evaluate efficacy and toxicities of ATO, we conducted a phase II trial including subjects with relapsed/refractory or secondary AML or age > or = 65 years with de novo disease. Eleven subjects were entered with a median age of 77 years (56-90) and a median total dose of ATO of 415.55 mg (91.5-793) with a daily dose of 0.25 mg/kg. Median survival following the first dose of ATO was 2.25 months (0.4-19). Myelosuppression was the major adverse effect, most likely due to disease progression rather than drug-related. All subjects had progressive disease. There was no direct treatment-related mortality. Based on this study, we do not recommend single agent ATO as a treatment option for AML.