The lipid metabolism is important in the regulation of cell proliferation. We have examined effects of a fatty acid analogue, tetradecylthioacetic acid (TTA), on the functional phenotype of native, human AML cells. TTA inhibited AML blast proliferation in the presence of single cytokines (GM-CSF and SCF: P > 0.05, 35 patients with detectable proliferation) and a combination of cytokines (P < 0.005, n = 21). This antiproliferative effect was generally stronger than for the normal fatty acid palmitic acid (PA). Both TTA and PA increased the secretion of tumor necrosis factor α (TNFα) (P < 0.05, 27 patients with detectable cytokine release), but only PA increased interleukein 1β (IL-1β) release (P < 0.005, n = 34). AML blast populations varied significantly in their levels and activities of metabolites and enzymes characterizing oxidative status and fatty acid metabolism, and there was no significant correlation between the intrinsic oxidative status and the effects of PA and TTA on blast proliferation. Although TTA reduced the proliferation of mitogen-stimulated normal T cells derived from healthy individuals (P < 0.05, n = 8), no adverse effects were seen on peripheral blood cell counts (reticulocytes, platelets, total white blood cells, differential leukocyte counts) for healthy volunteers receiving TTA (oral administration of 1000 mg/day for 7 consecutive days). Our results suggest that TTA can inhibit AML blast proliferation through pathways that are unrelated to autocrine cytokine secretion and intrinsic oxidative status.
A variety of fatty acids and derivatives have antiproliferative effects in different cancer cell models. The mechanisms behind such growth inhibition seem to depend on both cell type and chemical structure of the compound. Long chain saturated fatty acids, such as palmitic acid (PA), have been reported to induce apoptosis via increased ceramide production in murine hematopoietic cell lines (LyD9 and WEHI-231).1 Polyunsaturated fatty acids (PUFAs) have been demonstrated to reduce proliferation and induce cell death in studies with leukemia cell lines.2 Antiproliferative effects of PUFAs may be due to influence on the cellular redox situation,3,4 blocked cycline production,5 or inhibition of oncogene expression.6 Furthermore, lipid-mediated cell death has been linked to fatty acid-induced accumulation of triacylglycerols.7,8
Some fatty acids and derivatives function as ligands for various nuclear receptors that may participate in the regulation of proliferation and differentiation. For instance retinoids, such as all-trans retinoic acid (ATRA) which is employed in AML therapy, are ligands for retinoic acid receptors (RARs). RARs dimerize and act in concert with retinoid X receptor (RXR)9 which is also able to dimerize with peroxisome proliferator activated receptors (PPARs),10 another family of nuclear receptors that are activated by fatty acid derivatives. PPARs are primarily involved in the regulation of lipid metabolism but they have also been demonstrated to participate in the control of differentiation and proliferation.11,12
Tetradecylthioacetic acid (TTA) is a fatty acid analogue that has been reported to be a potent ligand and activator of the PPARs.13,14,15,16 TTA has the structure of a long chain fatty acid with a sulphur atom inserted in 3-position from the carboxylic end. Following cellular uptake, TTA is converted to its CoA-thioester17 which can be incorporated into different classes of lipids, especially phospholipids.18 The sulphur insertion prevents TTA from being β-oxidized in mitochondria and peroxisomes, but TTA undergoes sulphur- and ω-oxidation leading to secretion via urine.19 In rats, TTA has hypolipidemic effects, presumably due to increased hepatic oxidation of normal β-oxidizable fatty acids.20 TTA induces proliferation of peroxisomes and mitochondria,21,22,23 which may lead to alterations in the redox situation in the cells. Administration of TTA to cell cultures has demonstrated that this 3-thia fatty acid inhibits proliferation of malignant cells from various origins.16,24,25,26,27 The mechanisms behind the antiproliferative effects of TTA are still elusive.
Reduced proliferation has been observed for cells treated with superoxide dismutase (SOD) inhibitors,28 and the cellular glutathione content appears to be associated with the action of apoptogenic drugs such as arsenic trioxide29,30 and cytarabine.31 Hence, the intrinsic antioxidant capacity of leukemia cells may be a determinant for their sensitivity to compounds that inhibit proliferation through mechanisms related to oxidative status.
In this study we investigated the effects of TTA on cytokine-dependent proliferation and cytokine secretion in cultures with native AML blasts. We also investigated the intrinsic redox situation and activities of central enzymes in fatty acid oxidation in order to decide whether these characteristics could be coupled to the responsiveness TTA. The effects of TTA were compared to the effects of PA in order to discriminate between general long chain fatty acid properties and specific TTA properties.
Materials and methods
AML blasts from 48 patients with high blasts counts and blast percentages among blood leukocytes were examined. In the first part of the study, the effects of TTA and PA on blast proliferation were investigated in AML blasts derived from 23 of these patients (age 38–87 years, 13 females and 10 males) (Table 1). According to the FAB (French–American–British) classification the distribution was 10 AML-M0/M1, four AML-M2, eight AML-M4/M5 and one AML-M6. In the last part of this work we studied the growth of AML blasts derived from 46 consecutive patients (age 22–87 years, 22 females and 24 males): 16 AML-M0/M1, 13 AML-M2 and 17 AML-M4/M5.
Healthy individuals receiving oral TTA treatment
The safety, tolerance and efficacy of daily oral administration of TTA was assessed in an open, randomized phase I trial. The study was approved by the local Ethic Committee, and the complete results will be published elsewhere. Three groups of healthy adult volunteers (each group including six individuals) received once daily treatment with TTA 200 mg, 600 mg or 1000 mg for 7 consecutive days. TTA was produced in accordance with current Good Manufacturing Practice (GMP; FDA Guidelines; Clauson-Kaas, Farum, Denmark), and administered in capsules. Peripheral blood cell counts and biochemical safety parameters were determined immediately before the first TTA dose, on day 7 after 1 week of TTA treatment, and 7 days after end of treatment.
For cell studies, TTA was prepared at the Department of Chemistry, University of Bergen, as previously described.32 Essential fatty acid-free bovine serum albumin (BSA) and PA was obtained from Sigma Chemical (St Louis, MO, USA). TTA and PA were complexed to BSA as described previously.25 The culture medium consisted of RPMI 1640 with glutamine and Hepes (Gibco, Paisley, UK) supplemented with gentamicin 100 μg/ml and 10% fetal calf serum (FCS; HyClone, Logan, UT, USA). Recombinant human cytokines were used at the following concentrations: 50 ng/ml granulocyte–macrophage colony-stimulating factor (GM-CSF; Sandoz, Basel, Switzerland), 50 ng/ml interleukin-3 (IL-3; PeproTech EC, London, UK), 50 ng/ml stem cell factor (SCF; PeproTech), 50 ng/ml Flt3-ligand (Flt3-L; R&D Systems, Abingdon, UK), and 50 ng/ml interleukin-2 (IL-2; PeproTech). Mononuclear cells were activated by 1 μg/ml phytohemagglutinin (PHA; HA16; Wellcome, Dartford, UK) or monoclonal anti-CD3 (mouse IgE Moab; CLB-T3/4.E; The Central Laboratory of The Netherlands Red Cross Blood Transfusion Services, Amsterdam, The Netherlands) as described in.33
Preparation of AML blasts
Peripheral blood mononuclear cells (PBMC) were isolated by density gradient separation (Ficoll–Hypaque, specific density 1.077; NycoMed, Oslo, Norway).34 The leukemic PBMC contained ≥95% AML blasts, as confirmed by light-microscopy after May–Grünwald-Giemsa staining.
Preparation and culture of normal peripheral blood mononuclear cells
PBMC from healthy donors were isolated by density gradient separation as described above. Normal PBMC were incubated in medium (with 10% FCS) alone or with PHA, anti-CD3, or anti-CD3 + IL-2. Cultures were prepared with TTA complexed to BSA, and with BSA alone. The final volume of medium per microtiter well was 190 μl. After 3 days, cells were pulsed with 3H-thymidine (3H-Thd) for 18 h, followed by harvesting and detection (see below).
Proliferation assay and determination of cytokine secretion
The AML cells were cultured for 6 days in the presence or absence of PA or TTA, before proliferation was measured by 3H-Thd incorporation as described previously.35,36 Cytokine secretion was analyzed by determining cytokine concentrations in the culture supernatants after 48 h of incubation.
The concentrations of secreted TNFα, IL-6 and IL-1β were measured using ELISA assays (Pelikine tool set; Central Laboratory for the Netherlands Red Cross Blood Transfusion Services).
Analysis of enzyme activities and oxidative status
The cells (<20 × 106) were centrifuged (200 g, 5 min) and resuspended in 2.0 ml H-buffer (0.25 M sucrose, 2 mM Hepes and 0.2 mM EGTA, pH7.4). The cell suspension was homogenized using a Dounce homogenizer. Nuclei and cell debris were removed by centrifugation at 1000 g for 10 min. The samples were kept cold (4°C) during the procedure. Protein concentration was measured by using Bio-Rad protein assay (Bio-Rad Laboratories, Richmond, CA, USA). Carnitine palmitoyltransferase II (CPT II) activity was measured as previously described.37 The activity of acyl-CoA oxidase (ACO) was determined by a spectrophotometric assay.38 Glutathione peroxidase activity was measured as described by Flohe and Gunzler39 with t-butyl hydroperoxide as substrate. Determination of the activities of manganese–superoxide dismutase (Mn SOD) and copper–zinc superoxide dismutase (CuZn SOD) were performed by direct spectrophotometry.40,41
For analysis of total (GSH+GSSG) and nonoxidized (GSH)-free glutathione, 75 μl of the suspension was extracted with 75 μl cold 5% sulfosalicylic acid containing 50 μM dithioerythritol, before it was maintained at −80°C. HPLC analysis was performed as previously described.42 Lipid peroxidation was analyzed by measuring (HPLC) the production of malondialdehyd (MDA).43
Presentation of the data
Proliferation and cytokine secretion assays were performed in triplicate and duplicate, respectively. Significant proliferation was defined as 3H-Thd incorporation corresponding to >1000 c.p.m. A significant increase/decrease in proliferation was defined as an alteration exceeding 20%, compared to control. In the statistical analysis of cytokine secretion undetectable levels were given the same value as the corresponding minimal detectable concentration. For statistical comparison the Wilcoxon's test for paired samples was used. Pearson's test for correlation was used in correlation analysis. The level of significance was set at P < 0.05.
Effects of PA and TTA on cytokine-dependent AML blast proliferation
The dose-dependent effects of PA and TTA on AML blast proliferation were investigated for 18 patients (Table 1, patients 1–18), and detectable blast proliferation was observed for all except one patient (patient 7) when the cells were cultured with IL-3+SCF+GM-CSF. Under these conditions, growth inhibition was the most prevalent response to both PA and TTA treatment (Figure 1), and in one case (patient 15) the proliferation was completely blocked by PA and TTA, even at the lowest concentration (50 μM). However, insensitivity was also observed (eg patient 10). Divergent effects were found for certain patients; low doses of TTA and PA enhanced proliferation whereas higher concentrations suppressed proliferation (eg patients 6 and 9). In general, the proliferation was equal to or lower in TTA-treated compared to PA-treated cultures.
Since the effects of PA and TTA reached a plateau at a concentration of 300 μM for most patients, we selected this concentration for the single-dose experiments. In these experiments we reproduced the results for 11 patients who were used in the dose–response experiments described above (Table 1, patients 1, 5, 7–12, 14, 16, 17), and we included five additional patients (Table 1, patients 19–23). The effects of 300 μM TTA or PA for the whole group of 23 AML patients are summarized in Figure 2. Two of these blast populations did not proliferate after cytokine stimulation (patients 7 and 8). AML blasts derived from five patients were unaffected by PA treatment (patients 6, 9, 11, 16 and 21), four were stimulated (patients 10, 14, 19 and 22) and 12 were inhibited. TTA reduced blast proliferation for 15 patients, whereas proliferation was stimulated for only one patient (patient 19) and not affected for five patients (patients 1, 10, 11, 16 and 22). Statistical analysis of the overall results demonstrated that both PA and TTA treatment resulted in significantly reduced proliferation (P < 0.05 and P < 0.005, respectively; Wilcoxon's test for paired samples). To conclude, for the majority of patients both TTA and PA have an inhibitory effect on cytokine-dependent AML blast proliferation, and TTA generally had a stronger inhibitory effect than PA.
Effects of PA and TTA on AML blast proliferation in the presence of single cytokines
We studied effects of PA and TTA on AML blast proliferation in the presence of single cytokines. AML blasts derived from 46 consecutive patients were then cultured with either Flt3-L, GM-CSF or SCF, which are cytokines that commonly stimulate AML blast proliferation.35 Detectable proliferation was observed for a majority of patients in the presence of either Flt3-L (32/46), GM-CSF (35/46) or SCF (35/46). When comparing the overall results, PA had no significant effect on cytokine-dependent proliferation for any of these cytokines (Figure 3). In contrast, TTA significantly reduced GM-CSF- and SCF-stimulated AML blast proliferation (P < 0.05, Wilcoxon's test for paired samples).
The TTA effect was dependent on AML blast differentiation; GM-CSF stimulated proliferation was significantly inhibited by TTA for AML patients classified as AML-M2 (granulocytic differentiation) and AML-M4/M5 (monocytic differentiation) (P < 0.05, Wilcoxon's test for paired samples), whereas no significant inhibition was observed for AML-M0/1 patients (no or minimal blast differentiation). The effects of PA and TTA in individual patients are also summarized in Figure 3. PA had divergent effects on proliferation in the presence of all three cytokines, and similar effects were also observed after TTA treatment of blasts in the AML-M0/M1 subset. However, TTA had an inhibitory effect on blast proliferation for a majority of the AML-M2 and AML-M4/M5 patients.
Effects of PA and TTA on the secretion of TNFα, IL-6 and IL-1β by native AML blasts
AML blasts cultured in vitro usually show constitutive secretion of various cytokines, including TNFα, IL-6 and IL-1β. We therefore investigated the effects of PA and TTA on the release of these three cytokines by native AML blasts derived from 36 consecutive patients (Figure 4). Although there was a wide variation in cytokine levels between patients, PA-treatment increased the release of TNFα and IL-1β (P < 0.005 and P < 0.0005, respectively, Wilcoxon's test for paired samples) in this population. Of these cytokines, TTA only increased the release on TNFα (P < 0.05, Wilcoxon's test for paired samples).
AML blast redox status and fatty acid oxidation capacity
In this study we hypothesized that the intrinsic capacity of metabolites and enzymes involved in cellular redox status, and fatty acid oxidation, could be of importance for the PA- and TTA-mediated effects on AML blast proliferation. In AML blasts derived from 16 patients (Table 1, patients 1–10, 12 and 14–18), the enzyme activities of glutathione peroxidase, MnSOD and CuZn SOD, and the levels of glutathione and lipid peroxides (MDA) varied between the patients (Table 2). There was also significant variation in the activities of key enzymes in mitochondrial (CPT II) and peroxisomal (ACO) β-oxidation. The activity of glutathione peroxidase positively correlated to CuZn SOD activity (Figure 5a). In addition, the activity of ACO correlated inversely to increasing levels of glutathione (GSH+GSSG) (Figure 5b). This correlation indicates a possible coupling between the fatty acid oxidation and cellular redox regulation. A direct correlation between PA or TTA sensitivity and the intrinsic cellular redox status or fatty acid oxidation capacity in the AML blasts was not seen in this analysis.
Effects of TTA on the proliferation of normal mononuclear cells
Proliferation of mitogen activated PBMC derived from eight healthy individuals was determined in order to investigate the influence of TTA on normal blood cells. TTA suppressed the proliferation of mitogen (PHA, anti-CD3) activated T cells (P < 0.05, Wilcoxon's test for paired samples) (Table 3). Antiproliferative effects of TTA were also seen after activation with anti-CD3 in combination with the T cell growth factor IL-2 (P < 0.05, Wilcoxon's test for paired samples). Thus, the growth suppressive effect of TTA does not appear to be caused by decreased release of the autocrine growth factor IL-2.
Effects of TTA therapy on peripheral blood cell counts
The peripheral blood counts of platelets, reticulocytes and white blood cells (WBC, total white blood cells and differential leukocyte counts) were registered for three groups of healthy adults receiving oral treatment with TTA. The individuals received either 200 mg, 600 mg or 1000 mg daily for 7 consecutive days, and the results for those six individuals receiving 1000 mg daily are presented in Table 4. Absorption of TTA from the gastrointestinal tract was documented by detection of TTA in the serum of all individuals after administration (data to be published, Berge et al). Reticulocyte and platelet counts showed a minor increase after 7 days of TTA administration compared with the pretreatment values, but it should be emphasized that all peripheral blood cell counts (reticulocytes, platelets, total WBC, neutrophils) were within the normal ranges after 1 week of treatment (Table 4). All cell counts also remained normal when tested 1 week after the end of treatment, the peripheral blood counts for other leukocyte subsets (monocytes, lymphocytes, eosinophils, basophils) remained within their normal ranges, and normal cell counts were also observed during and after TTA administration for those individuals receiving 200 (n = 6) and 600 mg daily (n = 6) (data not shown). Biochemical analysis showed that TTA administration was not associated with endocrinological disturbances, abnormal blood coagulation, or major changes in serum levels of electrolytes, creatinine, bilirubin, liver enzymes and albumin (data not shown).
In vitro growth potential of AML blasts in short-term culture is reported to be a determinant of prognosis (reviewed in Ref. 35). Hence, compounds that reduce blast proliferation in vitro may also have antiproliferative effects when administered to AML patients. Previous studies have confirmed that the non-β-oxidizable fatty acid TTA retards proliferation in several cancer cell lines. In the present study we examined whether similar effects of TTA could be seen in cultures of AML blasts derived from patients.
The AML blasts investigated in this study originated from patients who were selected because of their high blast percentage and blast count in the blood. From these patients the content of leukemia blasts in the cell population could be enriched to at least 95% by gradient-centrifugation alone.35 With this approach we avoided the risk of alterations in blast functions that may be induced during more extensive cell separation.35,44,45 A high blast count seems to be an adverse prognostic factor, but it is not confirmed that this prognostic impact reflect functional differences between AML cells (reviewed in Ref. 35). Nevertheless, patient selection may make our results representative for only this particular subset of AML patients. The choice of cytokines for studying cytokine-dependent proliferation was based on previous results showing that the combination of IL-3+SCF+GM-CSF, or the presence of Flt3-L, SCF or GM-CSF, separately, initiated strong proliferative response for a majority of patients.35,46 Reproducibility of our proliferation measurements was confirmed in repeated experiments.
In this study we demonstrate that TTA reduces AML blast proliferation in a high proportion of blast populations derived from selected patients. The first part of our study on cytokine-dependent proliferation (GM-CSF+SCF+IL-3) of AML blasts from 23 patients revealed that the effects of PA and TTA were comparable for the majority of the patients; however, TTA caused in general the most pronounced growth reduction (Figures 1 and 2). In addition, the proliferation was less frequently stimulated by TTA compared to PA. These findings were supported in the second part of the study, in which the effects of PA and TTA on the proliferation of blasts from 46 patients were studied in the presence of single cytokines (GM-CSF, SCF or Flt3-L; Figure 3). Under these conditions, TTA significantly reduced blast proliferation in the presence of GM-CSF and SCF, whereas PA had no effect. The TTA responsiveness of the cells seemed to depend on differentiation, as the GM-CSF-dependent proliferation of AML-M2 (minor granulocytic differentiation) and AML-M4/M5 (myelomonocytic differentiation) blasts, but not AML-M0/M1 (minimal differentiation) blasts, were affected.
TNFα is considered as a selective antitumor agent, although it plays pleiotropic roles in inflammation and pathogenesis. On the other hand, IL-1β is known to stimulate proliferation of leukemia cells both through paracrine and autocrine pathways. Thus, altered secretion of both of these cytokines may affect the blast proliferation. In our experiments, both PA and TTA induced secretion of TNFα, whereas only PA induced IL-β release (Figure 4). However, we did not detect any relationship between alterations in TNFα and IL-1β secretion and the influence of PA and TTA on blast proliferation. These data indicate that TTA affects blast proliferation through mechanisms other than autocrine secretion of TNFα and IL-1β.
TTA has metabolic effects that are comparable to those of PUFAs47 which are commonly suggested to induce generation of oxidative stress and lipid peroxides leading to cytotoxicity and decreased growth of cancer cells (reviewed in Ref. 3). We measured intrinsic levels and activities of metabolites and enzymes involved in oxidative status and fatty acid oxidation in AML blasts derived from 16 patients. There were wide variations between the patients, but we found possible correlations between the activities of glutathione peroxidase and CuZn SOD, and the activity of ACO and glutathione content (Figure 5). Our data suggest that AML blasts from different patients are not equally capable of handling reactive oxygen species and metabolizing fatty acids. Although it has been proposed that AML blast populations may be unusually dependent on glutathione-based antioxidant mechanisms,48 we saw no direct correlation between the intrinsic cellular oxidative status and the effects of PA and TTA on proliferation. Hence, the growth regulatory properties of TTA may not be directly linked to the intrinsic antioxidant capacity of the blasts. The effects of TTA on cellular oxidative status were not revealed in the present study due to the restricted availability of native blasts; however, TTA has previously been demonstrated to affect the metabolism of glutathione and increase lipid peroxidation in glioma cells.24
Our present results suggest that TTA can modulate the functional phenotype of native AML cells. Previous studies have also demonstrated that TTA can affect other malignant cells (see below). However, possible adverse effects of TTA on normal cells have to be investigated before the clinical use of TTA can be further considered, especially hematological toxicity that is usually the major problem in intensive AML chemotherapy. Our data demonstrated that TTA had an antiproliferative effect on mitogen (PHA, anti-CD3) activated normal T cells. An inhibition was also observed in the presence of exogenous IL-2, and this last observation suggests that the T cell inhibition is at least partly mediated by mechanisms other than decreased release of this autocrine growth factor. However, TTA does not have a general inhibitory effect on normal cells, because lipopolysaccharide-stimulated activation and cytokine release of normal monocytes is not altered by TTA (submitted, Aukrust P, Wergedahl et al). Effects on normal cells have also been described for drugs that are used in the current AML treatment, eg retinoids.49,50,51,52 Hence, such effects on normal cells may be regarded as acceptable if they are not associated with major clinical toxicity.
Our results from a clinical phase I study showed no evidence for any major toxic effects of TTA, including no hematological toxicity. Furthermore, TTA has been investigated in numerous animal studies and in vitro studies of normal cells (primary hepatocytes) (reviewed in Ref 19, 53, 54, 55). These studies have also reported that TTA seems to be well tolerated without exerting toxic effects on normal cells and tissues. Even megadoses of TTA (up to 2000 mg/kg/day for 2 consecutive days) did not have any detectable effects on hematopoietic cells in out-bred CD-1 mice (data to be published, Berge). Thus, the available results have not demonstrated any major toxic effect of TTA on normal cells.
Through a series of experiments on glioma cells, TTA has been demonstrated to mediate growth regulation mainly through PPAR-independent mechanisms that include intracellular lipid accumulation and induction of apoptosis16 (submitted, Berge et al). In the promyelocytic leukemia cell line IPC-81, we recently found that TTA induced apoptosis through mechanisms that were substantially different from the kinase-driven cell death induced by cAMP (submitted, Tronstad et al). TTA triggered mitochondrial cytochrome c release, caspase-3 activation, depolarization of the mitochondrial membrane potential and selective modulation of the mitochondrial glutathione level. Thus, TTA seems to induce apoptosis via a mitochondrial-mediated pathway. Apoptosis may, however, not be the sole mechanism behind the antiproliferative effects of TTA. Interestingly, it was demonstrated that eicosapentaenoic acid (EPA), which shares various regulatory properties with TTA, inhibited translation initiation, preferentially reducing the synthesis of growth regulatory proteins such as cyclins participating in cell cycle control.5 A similar mechanism might be applicable for TTA-mediated action, and will be studied in future experiments.
In conclusion, TTA reduced cytokine-dependent proliferation of AML blasts derived from the majority of the patients. The blast sensitivity to TTA did not seem to depend on autocrine cytokine secretion or intrinsic oxidative status, indicating that these factors are of minor importance for the antiproliferative effects of TTA. TTA reduced the proliferation of mitogen-activated normal T cells in vitro, but there were no adverse effects on peripheral blood cell counts for healthy individuals after oral administration of TTA.
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The University of Bergen, the Norwegian Research Council and the Norwegian Cancer Society supported the study. We acknowledge Laila Menzoni, Svein Kryger and Kari Williams for excellent technical assistance.
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Tronstad, K., Bruserud, Ø., Berge, K. et al. Antiproliferative effects of a non-β-oxidizable fatty acid, tetradecylthioacetic acid, in native human acute myelogenous leukemia blast cultures. Leukemia 16, 2292–2301 (2002). https://doi.org/10.1038/sj.leu.2402698
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