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Exosomes are virus-size membrane-bound vesicles of endocytic origin present in all body fluids. Plasma of AML patients is significantly enriched in exosomes, which carry a cargo of immunosuppressive molecules and deliver them to recipient immune cells, suppressing their functions. However, whether these exosomes originate from leukemic blasts or from various normal cells in the bone marrow or other tissues is unknown. In the current study, we developed an AML PDX model in mice and studied the molecular cargo and immune cell effects of the AML PDX exosomes in parallel with the exosomes from plasma of the corresponding AML patients. Fully engrafted AML PDX mice produced exosomes with characteristics similar to those of exosomes isolated from plasma of the AML patients who had donated the cells for engraftment. The engrafted leukemic cells produced exosomes that carried human proteins and leukemia-associated antigens, confirming the human origin of these exosomes. Furthermore, the AML-derived exosomes carried immunosuppressive proteins responsible for immune cell dysfunctions. Our studies of exosomes in AML PDX mice serve as a proof of concept that AML blasts are the source of immunosuppressive exosomes with a molecular profile that mimics the content and functions of the parental cells., (Copyright © 2019 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

The ETS transcription factor ERG has been implicated as a major regulator of both normal and aberrant hematopoiesis. In acute myeloid leukemias harboring t(16;21), ERG function is deregulated due to a fusion with FUS/TLS resulting in the expression of a FUS-ERG oncofusion protein. How this oncofusion protein deregulates the normal ERG transcription program is unclear. Here, we show that FUS-ERG acts in the context of a heptad of proteins (ERG, FLI1, GATA2, LYL1, LMO2, RUNX1 and TAL1) central to proper expression of genes involved in maintaining a stem cell hematopoietic phenotype. Moreover, in t(16;21) FUS-ERG co-occupies genomic regions bound by the nuclear receptor heterodimer RXR:RARA inhibiting target gene expression and interfering with hematopoietic differentiation. All-trans retinoic acid treatment of t(16;21) cells as well as FUS-ERG knockdown alleviate the myeloid-differentiation block. Together, the results suggest that FUS-ERG acts as a transcriptional repressor of the retinoic acid signaling pathway.

Patients with relapsed acute myeloid leukemia (AML) have limited therapeutic options. Vesicular stomatitis virus (VSV)-interferon β (IFNβ)-sodium iodide symporter (NIS) is an oncolytic VSV encoding IFNβ and the NIS reporter. Syngeneic AML C1498 tumors responded to IV therapy with VSV-murine IFNβ (mIFNβ)-NIS in a dose-dependent manner. Imaging for NIS expression showed robust virus infection within the tumors. Virus infection did not increase programmed death ligand 1 (PD-L1) on tumor cells. Combining VSV-mIFNβ-NIS with anti-PD-L1 antibody (Ab) therapy enhanced antitumor activity compared with treatment with virus alone or Ab alone; this enhancement was not significant at higher VSV-mIFNβ-NIS doses. Systemic VSV therapy reduced systemic C1498-green fluorescent protein (GFP) tumor burden in the blood, bone marrow, spleen, and liver of mice with AML. Combination VSV-mIFNβ-NIS and anti-PD-L1 Ab therapy significantly enhanced the survival of these mice with no evidence of toxicity, compared with isotype control, anti-PD-L1, or virus alone. There was an increase in tumor-infiltrating CD4 and CD8 cells. Single-agent VSV-mIFNβ-NIS virotherapy induced both VSV-specific and GFP-specific CD8 T cells as determined by IFN-γ enzyme-linked immunospot, pentamer, and intracellular IFN-γ staining assays. Both of these responses were further enhanced by addition of anti-PD-L1 Ab. Depletion of CD8 or natural killer cells, but not CD4 cells, resulted in loss of antitumor activity in the VSV/anti-PD-L1 group. Clinical samples from chronic myelomonocytic leukemia and acute myelomonocytic leukemia appear to be especially susceptible to VSV. Overall, our studies show that oncolytic virotherapy combined with immune checkpoint blockade is a promising approach to AML therapy., (© 2016 by The American Society of Hematology.)

Adenosine monophosphate-activated protein kinase (AMPK) is a sensor for cellular energy status. When the cellular energy level is decreased, AMPK is activated and functions to suppress energy-consuming processes, including protein synthesis. Recently, AMPK has received attention as an attractive molecular target for cancer therapy. Several studies have revealed that the activation of AMPK by chemical stimulators, such as metformin, induces apoptosis in a variety of hematologic malignant cells. From another perspective, these results suggest that the function of AMPK is impaired in hematologic tumor cells. However, the precise mechanisms by which this impairment occurs are not well understood. In melanoma cells, oncogenic BRAF constitutively activates the extracellular signal-regulated kinase (ERK) pathway and phosphorylates liver kinase B1, an upstream activator of 5' adenosine monophosphate-activated protein kinase (AMPK), resulting in the inactivation of liver kinase B1 and AMPK. In this study, we analyzed whether ERK is involved in the suppression of AMPK activity using established and primary human leukemia cells. We found an inverse correlation between the intensity of ERK activity and the degree of AMPK activation after stimulation with either glucose deprivation or metformin. We also found that the inhibition of ERK activity by U0126 restored AMPK activation after metformin treatment. Furthermore, a combined treatment with metformin and U0126 enhanced the antileukemic activity of metformin. Importantly, metformin induced ERK activation by suppressing the protein levels of dual specificity phosphatase 6, a negative regulator of ERK. This crosstalk between AMPK and ERK could diminish the antileukemic activity of metformin. Taken together, our present observations suggest a novel therapeutic strategy for improving the efficacy of metformin in treating leukemia., (Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

Acute myeloid leukemia (AML) with myelodysplasia-related changes is characterized by the presence of multilineage dysplasia (MLD), frequently related to high-risk cytogenetics and poor outcome. However, the presence of MLD does not modify the favorable prognostic impact of NPM1 mutation. The prognosis of patients with AML presenting marked dysplasia lacking high-risk cytogenetics and NPM1 mutation is uncertain. We evaluated the prognostic impact of MLD in 177 patients with intermediate-risk cytogenetics AML (IR-AML) and wild-type NPM1. Patients were categorized as MLD-WHO (WHO myelodysplasia criteria; n = 43, 24 %), MLD-NRW (significant MLD non-reaching WHO criteria; n = 16, 9 %), absent MLD (n = 80, 45 %), or non-evaluable MLD (n = 38, 22 %). No differences concerning the main characteristics were observed between patients with or without MLD. Outcome of patients with MLD-WHO and MLD-NRW was similar, and significantly worse than patients lacking MLD. The presence of MLD (66 vs. 80 %, p = 0.03; HR, 95 % CI = 2.3, 1.08-4.08) and higher leukocyte count at diagnosis was the only variable associated with lower probability of complete remission after frontline therapy. Concerning survival, age and leukocytes showed an independent prognostic value, whereas MLD showed a trend to a negative impact (p = 0.087, HR, 95 % CI = 1.426, 0.95-2.142). Moreover, after excluding patients receiving an allogeneic stem cell transplantation in first CR, MLD was associated with a shorter survival (HR, 95 % CI = 1.599, 1.026-2.492; p = 0.038). In conclusion, MLD identifies a subgroup of patients with poorer outcome among patients with IR-AML and wild-type NPM1.

The lack of molecular diagnosis in the field of cancer in Iraq has motivated us to perform a genetic analysis of pediatric acute myelogenous leukemia (AML), including class I and II aberrations. Peripheral blood or bone marrow cells were collected from 134 AML children aged ≤15 years. Flinders Technology Associates (FTA) filter paper cards were used to transfer dried blood samples from five Iraqi hospitals to Japan. DNA sequencing was performed to identify class I mutations. Nested RT-PCR was used to detect class II aberrations, except that MLL rearrangement was detected according to long distance inverse-PCR. NPM1 and FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutations were analyzed by GeneScan using DNA template. Among 134 Iraqi pediatric AML samples, the most prevalent FAB subtype was M2 (33.6 %) followed by M3 (17.9 %). Class I mutations: 20 (14.9 %), 8 (6.0 %), and 8 (6.0 %) patients had FLT3-ITD, FLT3-TKD, and KIT mutations, respectively. Class II mutations: 24 (17.9 %), 19 (14.2 %), and 9 (6.7 %) children had PML-RARA, RUNX1-RUNX1T1, and CBFB-MYH11 transcripts, respectively. MLL rearrangements were detected in 25 (18.7 %) patients. NPM1 mutation was detected in seven (5.2 %) cases. Collectively, approximately 30 % of AML children were proved to carry favorable prognostic genetic abnormalities, whereas approximately 10 % had high FLT3-ITD allelic burden and needed a special treatment plan including allogeneic hematopoietic stem cell transplantation. Acute promyelocytic leukemia (APL) was frequent among Iraqi pediatric AML. It is likely that molecular diagnosis using FTA cards in underdeveloped countries could guide doctors towards an appropriate treatment strategy.

Transforming mutations in RAS genes are commonly found in human malignancies, including myeloid leukemias. To investigate the incidence, spectrum, and distribution of activating K- and N-RAS mutations in cytogenetically normal acute myeloid leukemia (CN-AML) patients, 204 CN-AML patients were screened. Activating K- and N-RAS mutations were detected in 3 of 204 (1.5 %) and 22 of 204 (10.8 %) CN-AML samples, respectively. RAS mutated patients presented with a lower percentage of bone marrow blasts (65 vs 80 %, P = 0.022). RAS mutations tended to occur with nucleophosmin-1 (NPM1) mutations (P = 0.079), and all three samples containing K-RAS mutations had concomitant NPM1 mutations. There was no significant overlap between K-RAS mutations and N-RAS, FLT3, CEBPA, IDH1/2, WT1 or MLL mutations. RAS mutation status did not impact relapse-free or overall survival of CN-AML patients. In contrast to reports of noncanonical RAS mutations in other cancers, including some leukemia subtypes, we only observed K- and N-RAS mutations in codons 12, 13, or 61 in CN-AML samples. Our findings suggest that while K-RAS mutations are infrequent in CN-AML, activating K-RAS mutations may cooperate with mutated NPM1 to induce leukemia.

Genetically targeted T cells promise to solve the feasibility and efficacy hurdles of adoptive T-cell therapy for cancer. Selecting a target expressed in multiple-tumor types and that is required for tumor growth would widen disease indications and prevent immune escape caused by the emergence of antigen-loss variants. The adhesive receptor CD44 is broadly expressed in hematologic and epithelial tumors, where it contributes to the cancer stem/initiating phenotype. In this study, silencing of its isoform variant 6 (CD44v6) prevented engraftment of human acute myeloid leukemia (AML) and multiple myeloma (MM) cells in immunocompromised mice. Accordingly, T cells targeted to CD44v6 by means of a chimeric antigen receptor containing a CD28 signaling domain mediated potent antitumor effects against primary AML and MM while sparing normal hematopoietic stem cells and CD44v6-expressing keratinocytes. Importantly, in vitro activation with CD3/CD28 beads and interleukin (IL)-7/IL-15 was required for antitumor efficacy in vivo. Finally, coexpressing a suicide gene enabled fast and efficient pharmacologic ablation of CD44v6-targeted T cells and complete rescue from hyperacute xenogeneic graft-versus-host disease modeling early and generalized toxicity. These results warrant the clinical investigation of suicidal CD44v6-targeted T cells in AML and MM.

Current therapeutic regimens for acute myeloid leukaemia (AML) are still associated with high rates of relapse. Immunotherapy with T-cells genetically modified to express chimeric antigen receptors (CARs) represents an innovative approach. Here we investigated the targeting of the interleukin three receptor alpha (IL3RA; CD123) molecule, which is overexpressed on AML bulk population, CD34(+) leukaemia progenitors, and leukaemia stem cells (LSC) compared to normal haematopoietic stem/progenitor cells (HSPCs), and whose overexpression is associated with poor prognosis. Cytokine-induced killer (CIK) cells were transduced with SFG-retroviral-vector encoding an anti-CD123 CAR. Transduced cells were able to strongly kill CD123(+) cell lines, as well as primary AML blasts. Interestingly, secondary colony experiments demonstrated that anti-CD123.CAR preserved in vitro HSPCs, in contrast to a previously generated anti-CD33.CAR, while keeping an identical cytotoxicity profile towards AML. Furthermore, limited killing of normal monocytes and CD123-low-expressing endothelial cells was noted, thus indicating a low toxicity profile of the anti-CD123.CAR. Taken together, our results indicate that CD123-specific CARs strongly enhance anti-AML CIK functions, while sparing HSPCs and normal low-expressing antigen cells, paving the way to develop novel immunotherapy approaches for AML treatment., (© 2013 Blackwell Publishing Ltd.)

Chemoresistance is a major contributor to the aggressiveness of AML and is often due to insufficient apoptosis. The CFLAR gene is expressed as long and short splice forms encoding the anti-apoptotic proteins c-FLIP(L) and c-FLIP(S) (CFLAR(L) and CFLAR(S) , respectively) that play important roles in drug resistance. In univariate analyses of CFLAR mRNA expression in adult AML patients, those individuals with higher than median mRNA expression of the long splice form CFLAR(L) (but not the short splice form) had significantly lower 3 year overall survival (P = 0·04) compared to those with low expression. In cell line studies, simultaneous down-regulation of c-FLIP(L) and c-FLIP(S) proteins using siRNA induced apoptosis in U937 and NB-4 AML cells, but not K562 or OCI-AML3 cells. However, dual c-FLIP(L/S) downregulation sensitized all four cell lines to apoptosis induced by recombinant tumour necrosis factor-related apoptosis-inducing ligand (rTRAIL). Moreover, specific downregulation of c-FLIP(L) was found to recapitulate the phenotypic effects of dual c-FLIP(L/S) downregulation. The histone deacetylase (HDAC)1/2/3/6 inhibitor Vorinostat was found to potently down-regulate c-FLIP(L) expression by transcriptional and post-transcriptional mechanisms and to sensitize AML cells to rTRAIL. Further analyses using more selective HDAC inhibitors revealed that HDAC6 inhibition was not required for c-FLIP(L) down-regulation. These results suggest that c-FLIP(L) may have clinical relevance both as a prognostic biomarker and potential therapeutic target for HDAC inhibitors in AML although this requires further study., (© 2012 Blackwell Publishing Ltd.)

Activation of the Wnt/beta-catenin pathway has recently been shown to be crucial to the establishment of leukemic stem cells in chronic myeloid leukemia. We sought to determine whether beta-catenin was correlated to clonogenic capacity also in the acute myeloid leukemia (AML) setting. Eighty-two patients were retrospectively evaluated for beta-catenin expression by Western blot. beta-Catenin was expressed (although at various protein levels) in 61% of patients, and was undetectable in the remaining cases. In our cohort, beta-catenin expression was correlated with the clonogenic proliferation of AML-colony forming cells (AML-CFC or CFU-L) in methylcellulose in the presence of 5637-conditioned medium, and more strikingly with self-renewing of leukemic cells, as assessed in vitro by a re-plating assay. In survival analyses, beta-catenin appeared as a new independent prognostic factor predicting poor event-free survival and shortened overall survival (both with P<0.05). Furthermore, variations in beta-catenin protein levels were dependent on post-transcriptional mechanisms involving the Wnt/beta-catenin pathway only in leukemic cells. Indeed, beta-catenin negative leukemic cells were found to increase beta-catenin in response to Wnt3a agonist in contrast to normal counterparts. Altogether, our data pave the way to the evaluation of Wnt pathway inhibition as a new rationale for eradicating the clonogenic pool of AML cells.

To investigate the expression of HoxA(10) mRNA in acute leukemia patients and its significance, HoxA(10) level was detected by reverse transcription polymerase chain reaction (RT-PCR) in 50 patients with acute leukemias, 7 healthy volunteers and 3 patients with ITP (idiopathic thrombocytopenic purpura). The regularity of the expression of HoxA(10) gene in acute leukemia and the relationship between HoxA(10) level and the prognosis of leukemia was explored. The results showed that HoxA(10) was expressed in all types of acute myelogenous leukemia; HoxA(10) message was also observed in acute lymphoblastic leukemia patients and part of control groups. 3 normal donors were found not to express HoxA(10). The level of HoxA(10) mRNA of acute myelogenous leukemia patients was significantly higher than that of acute lymphoblastic leukemia patients and controls (P < 0.01). HoxA(10) gene appeared to be more strongly expressed in AML-M(1) and -M(2) subtypes than in AML-M(4) and -M(5) subtypes, and the gene was notable high expressed in acute promyelocytic leukemia. The number of blast and promyeloid cells in the bone marrow was positive related with the level of HoxA (r = 0.635, P < 0.01). The level of HoxA(10) of 9 non-responsive patients was higher than that of 8 remission patients, but there was no significant difference between them (P = 0.258). HoxA(10) was overexpressed in acute myelogenous leukemia. It is concluded that HoxA(10) is a major transcription factor regulating hematopoiesis and a mark to differentiate lymphoid leukemia and myelogenous leukemia, but not a specific gene of cancer. The level of HoxA(10) is related with load of leukemic cells and curative effect, and can affect occurrence and development of leukemia in combination with many cytokines, HoxA(10) may facilitate the leukemia progression with another cofactors.

To explore CD34(+) antigen expression in new diagnosed acute myeloid leukemia (AML) and analyze the prognosis for CD34(+) AML patients, the expression of antigen CD34 in 238 AML patients was detected by indirect immunofluorescence assay. The results showed that CD34 in 92 out of the 238 patients (38.7%) were positive, there was relationship between the CD34(+) expression and FAB subtypes (M(0), M(1)), and no CD34(+) expression was observed in M(3) subtypes. The complete remission rate of CD34(+) AML patients was 32%, which was lower than that of CD34(-) AML (61%). The lymphoid-associated antigen (CD7) was significantly increased in CD34(+) AML patients, compared with CD34(-) patients (P < 0.05). It is concluded that CD34(+) AML patients show poor prognosis and lower CR rate. The detection of CD34 expression is of some value in predicting prognosis in AML.

Bone marrow monocytic nodules (MNs) can occur in various myeloid disorders. This retrospective review identified 21 patients with myelodysplasia who had unusual and distinct MNs. Eight patients had chronic myelomonocytic leukemia (CMML); 4 had acute myeloid leukemia (AML); and 9 had myelodysplastic/myeloproliferative diseases. In each case, the cells forming MNs expressed strong CD68. MNs appeared to persist even after aggressive chemotherapy, including conventional chemotherapy for 2 AML patients and high-dose chemotherapy preceding allogeneic bone marrow transplantation for 1 CMML patient. Thirteen of 21 patients (62%) died, and acute leukemic transformation was the main cause of death in 3 of 8 patients with CMML. The median survival of the 20 patients with appropriate follow-up was 9.8 months. Our findings demonstrate that MNs are associated with CMML, AML, myelodysplastic syndromes, and myeloproliferative diseases and suggest that MNs are resistant to intensive chemotherapy and patients with bone marrow MNs have a poor prognosis., (Copyright (c) 2005 Wiley-Liss, Inc.)

Immunotherapy of tumor is extensively attentioned as an important part of combined therapy of tumor in recent years. Dendritic cell (DC) is the most powerful antigen presenting cell (APC) by now which not only activates auto-immunity to attack tumor cells, but also does help to enhance antitumor effect for allogenic bodies. To explore the feasibility and safety of clinical therapy application of peripheral blood derived DC cultured ex vivo, and analyze the influence of DC-inducing-immunotherapy upon long-term survival of ANLL patients accepted autologous bone marrow transplantation, peripheral blood mononuclear cells (PBMNC) of 13 ANLL patients after autologous bone marrow transplantation were collected by using CS3000Plus. DC immunotherapy was administered after cultivation of PBMNC ex vivo for 2 weeks, desease-free survival time was observed after therapy for long time follow-up. The results showed that no any severe adverse event associated with DC therapy was observed, the survival analysis of Kaplan-Meier suggested that five year survival rate was 75.52% in DC group while 45.71% in non-DC group. DC group surpassed non-DC group in accumulative survival rate. It is concluded that the ex vivo cultivation and clinical therapy application of DC derived from peripheral blood are feasible and safe, DC immunotherapy in patients with acute non-lymphocytic leukemia after autologous bone marrow transplantation prolongs desease-free survival time and enhances long-term survival rate.

In order to evaluate the effects of FLAG regimen in treatment of refractory and relapsed acute myeloid leukemia (AML), 27 patients with refractory or relapsed acute myeloid leukemia (10 refractory AML patients, 17 relapsed AML patients) were treated with FLAG regimen. The results show that the rate of complete remission was 48.2% (13/27), the rate of partial remission was 14.8% (4/27), and the overall response rate was 63.0%. Main toxicities were gastrointestinal side effectes, myelosupression and neutropenia. It is concluded that FLAG regimen can be employed in treatment of the refractory or relapsed patients who were not respond to other regimen, and the regiment was safe.

AML patients may suffer from a disseminated coagulopathy, which can aggravate a pre-existing bleeding tendency due to thrombocytopenia and platelet dysfunction. The cellular and molecular mechanisms underlying this coagulopathy, however, are not completely understood. Indeed, the broad and increasing therapeutic use of cytotoxic drugs and growth factors is likely to contribute to the complexity of hemostatic abnormalities encountered in this hematologic malignancy. The nature of coagulation activation in AML was therefore investigated in vitro using the human leukemic cell line, HL60. Tissue factor (TF) was almost entirely located on the cell surface and bound factor VIIa, but only 15-25% of this TF was primarily functionally active. Treatment with increasing concentrations of daunorubicin or cytosine-beta-D-arabinofuranoside, two cytotoxic drugs commonly used in AML therapy, induced apoptosis and secondary necrosis of HL60 cells and resulted in marked decryption of TF PCA independent of de novo protein synthesis. This PCA-modulating effect was concomitant with and functionally dependent on the exposure of phosphatidylserine on the outer membrane leaflet. Similar observations were made in analogous ex vivo studies on patient-derived myeloblasts. Incubation of HL60 cells with GM-CSF, a cytokine expressed in the bone marrow microenvironment and used as an adjunct to AML treatment, evoked a cellular response, which included both enhanced TF production and release of VEGF-A and uPA into the culture medium. We conclude that both decryption of pre-formed TF PCA by chemotherapeutic drugs and de novo induction of TF by cytokines such as GM-CSF can regulate the pro-coagulant phenotype of HL60 cells in vitro.

From 1998 to 2001, 5 consecutive cases of AML/TMDS entered our hospital and achieved complete remission (CR) with continuous drip infusion of low-dose etoposide and low-dose Ara-C combined with mitoxantrone (MEtA regimen). The ages of the 5 patients (4 males and 1 female) ranged 32 to 50 years-old, respectively. WBCs were 1,560-45,150/microl, blasts were 12-62%. Bone marrow aspirates revealed trilineage myelodysplasia with various number of blasts. These patients had an acute onset and no preceding hematologic disorders. They were diagnosed M2/TMDS or M4/TMDS. Continuous drip infusion of etoposide (50 mg/body/day) and Ara-C (30 mg/body/day) were given for 11-14 days and a bolus injection of mitoxantrone (8 mg/m2) was added for 2-3 days. Patient 5 was given additional MIT (6.7 mg/m2 on day 6). All cases achieved CR in 21-24 days after the end of the therapy. Toxicities were nausea, vomiting, stomatitis, alopecia and fever due to infection. All were well tolerable, however. Two patients are alive more than 4 years without relapse. MEtA regimen is available for AML/TMDS.

Acute myeloid leukemia (AML) is a disease characterized by a block of maturation. Genes coding for core binding factors are rearranged in a considerable subset of AML cases and result in an altered interaction of core binding factor (CBF) subunits with transcriptional coregulators (NCoR/SMRT). Recruitment of histone deacetylase is also altered in AML, and a subsequent transcriptional repression of target genes involved in myeloid maturation is determined. We determined here the effects of two histone deacetylase inhibitors, sodium butyrate and the stable prodrug xylitol butyrate derivative (D1), on a t(8;21)-positive cell line (Kasumi-1) as well as primary AML blasts. Exposure (24-96 h) to butyrates (1 mM) of Kasumi-1 cells induced histone H4 acetylation, whereas H3 acetylation was unchanged. Induction of morphological and immunophenotypic granulocytic maturation (96 h), also confirmed by an increased expression of CAAT/enhancer binding protein alpha, was observed. Inhibition of proliferation and apoptosis via activation of caspase-9 was also observed. In primary AML blasts, butyrates (0.5 mM) increased histone H4 acetylation of 18 of 19 cases tested. Terminal granulocytic maturation was observed in all cases (5 of 5) characterized by chromosomal translocations involving CBF, whereas in non-CBF cases, maturation was incomplete (4 of 8) or absent (4 of 8). Our data indicate the possibility to effectively remove, in CBF AML cases, the maturation block generated by histone deacetylase stable recruitment, contributing to a possible development of molecularly targeted therapies of AML.

Acute myeloid leukemia (AML) cases with 11q23 abnormalities involving the MLL gene comprise one category of recurring genetic abnormalities in the WHO classification. In an unselected series of 1897 AML cases, 54 patients with an 11q23/MLL rearrangement were identified, resulting in an incidence of 2.8%. The incidence of AML with MLL rearrangement was significantly higher in therapy-related AML (t-AML) than in de novo AML (9.4% vs 2.6%, P <.0001). The frequency of MLL rearrangements was significantly higher in patients younger than 60 years (5.3% vs 0.8%, P <.0001). While the incidence of MLL rearrangements in AML M4, M5a, and M5b was 4.7%, 33.3%, and 15.9%, respectively, it was found in only 0.9% of all other French-American-British (FAB) subtypes (P <.0001). Compared with AML with intermediate karyotype, AML with 11q23/MLL rearrangement had a worse outcome, which was rather comparable with AML with unfavorable karyotype. Compared with t-AML, the median overall survival (OS) of de novo AML with MLL rearrangement was significantly better (2.5 vs 10 months, P =.0143). No significant differences in median OS were observed between cases with t(9;11) compared with all other MLL rearrangements (10.0 vs 8.9 months, P =.36). In conclusion, the category AML with 11q23/MLL abnormalities accounts for 2.8% of unselected AML, is closely associated with monocytic differentiation, and has a dismal prognosis. (

Although many acute myeloid leukemia (AML) colony-forming cells (CFCs) and long-term culture-initiating cells (LTC-ICs) directly isolated from patients are actively cycling, quiescent progenitors are present in most samples. In the current study, (3)H-thymidine ((3)H-Tdr) suicide assays demonstrated that most NOD/SCID mouse leukemia-initiating cells (NOD/SL-ICs) are quiescent in 6 of 7 AML samples. AML cells in G(0), G(1), and S/G(2)+M were isolated from 4 of these samples using Hoechst 33342/pyroninY staining and cell sorting. The progenitor content of each subpopulation was consistent with the (3)H-Tdr suicide results, with NOD/SL-ICs found almost exclusively among G(0) cells while the cycling status of AML CFCs and LTC-ICs was more heterogeneous. Interestingly, after 72 hours in serum-free culture with or without Steel factor (SF), Flt-3 ligand (FL), and interleukin-3 (IL-3), most G(0) AML cells entered active cell cycle (percentage of AML cells remaining in G(0) at 72 hours, 1.2% to 37%, and 0% to 7.6% in cultures without and with growth factors [GFs], respectively) while G(0) cells from normal lineage-depleted bone marrow remained quiescent in the absence of GF. All 4 AML samples showed evidence of autocrine production of 2 or more of SF, FL, IL-3, and granulocyte-macrophage colony-stimulating factor (GM-CSF). In addition, 3 of 4 samples contained an internal tandem duplication of the FLT3 gene. In summary, quiescent leukemic cells, including NOD/SL-ICs, are present in most AML patients. Their spontaneous entry into active cell cycle in short-term culture might be explained by the deregulated GF signaling present in many AMLs.

We report a case of acute myeloid leukemia with tetrasomy 21 as the sole chromosome abnormality in a constitutionally normal patient. Tetrasomy 21 was observed at presentation, disappeared in remission, but reappeared in recurrence of the disease. Fluorescence in situ hybridization analysis using a probe specific for the AML1 gene showed four distinct signals in 82.4% and three signals in 10.8% of interphase nuclei, although conventional G-banding revealed tetrasomy 21 alone in mosaicism with normal karyotype. Spectral karyotyping further confirmed the presence of extra copies of chromosome 21. Tetrasomy 21 as the only anomaly is relatively rare in patients with hematologic disorders other than Down syndrome, and to our knowledge has been reported previously in only seven cases. In a review of the literature, tetrasomy 21 as the only anomaly may be associated with myeloid disorders, although simultaneous numeric abnormalities other than chromosome 21 have been reported in acute lymphoblastic leukemia with hyperdiploid karyotype.

Histone deacetylase (HDAC)-dependent transcriptional repression of the retinoic acid (RA)-signaling pathway underlies the differentiation block of acute promyelocytic leukemia. RA treatment relieves transcriptional repression and triggers differentiation of acute promyelocytic leukemia blasts, leading to disease remission. We report that transcriptional repression of RA signaling is a common mechanism in acute myeloid leukemias (AMLs). HDAC inhibitors restored RA-dependent transcriptional activation and triggered terminal differentiation of primary blasts from 23 AML patients. Accordingly, we show that AML1/ETO, the commonest AML-associated fusion protein, is an HDAC-dependent repressor of RA signaling. These findings relate alteration of the RA pathway to myeloid leukemogenesis and underscore the potential of transcriptional/differentiation therapy in AML.

High levels of telomerase activity and high rates of cell proliferation are associated with a poor prognosis in acute myelogenous leukemia. Furthermore, cytokine production by leukemia cells is believed to play an important role in determining the proliferative characteristics of leukemia. The in vivo effects of two noncytotoxic agents on these parameters were determined in 33 acute myelogenous leukemia patients. Three daily doses of interleukin (IL) 4 or a single dose of amifostine reduced telomerase activity in the leukemia marrow cells in 7 of 9 and 11 of 13 patients, respectively. The administration of a single dose of amifostine resulted in a reduction in tumor necrosis factor alpha and IL-6 transcript levels in the marrow cells of 10 of 13 and 12 of 13 patients in which these transcripts were present. The administration of only three doses of IL-4 or a single dose of amifostine has a significant effect on leukemia cell parameters, which are believed to have a significant impact on the in vivo biology of the disease and on its response to remission induction therapy.

Partial deletion of the long arm of chromosome 5, del(5q), is the cytogenetic hallmark of the 5q-syndrome, a distinct subtype of myelodysplastic syndrome-refractory anemia (MDS-RA). Deletions of 5q also occur in the full spectrum of other de novo and therapy-related MDS and acute myeloid leukemia (AML) types, most often in association with other chromosome abnormalities. However, the loss of genetic material from 5q is believed to be of primary importance in the pathogenesis of all del(5q) disorders. In the present study, we performed fluorescence in situ hybridization (FISH) studies using a chromosome 5-specific whole chromosome painting probe and a 5q subtelomeric probe to determine the incidence of cryptic translocations. We studied archival fixed chromosome suspensions from 36 patients with myeloid disorders (predominantly MDS and AML) and del(5q) as the sole abnormality. In 3 AML patients studied, this identified a translocation of 5q subtelomeric sequences from the del(5q) to the short arm of an apparently normal chromosome 11. FISH with chromosome 11-specific subtelomeric probes confirmed the presence of 11p on the shortened 5q. Further FISH mapping confirmed that the 5q and 11p translocation breakpoints were the same in all 3 cases, between the nucleophosmin (NPM1) and fms-related tyrosine kinase 4 (FLT4) genes on 5q35 and the Harvey ras-1-related gene complex (HRC) and the radixin pseudogene (RDPX1) on 11p15.5. Importantly, all 3 patients with the cryptic t(5;11) were children: a total of 3 of 4 AML children studied. Two were classified as AML-M2 and the third was classified as M4. All 3 responded poorly to treatment and had short survival times, ranging from 10 to 18 months. Although del(5q) is rare in childhood AML, this study indicates that, within this subgroup, the incidence of cryptic t(5;11) may be high. It is significant that none of the 24 MDS patients studied, including 11 confirmed as having 5q-syndrome, had the translocation. Therefore, this appears to be a new nonrandom chromosomal translocation, specifically associated with childhood AML with a differentiated blast cell phenotype and the presence of a del(5q).

Homeobox genes (HOX) may have a regulatory function in the differentiation process of hematopoiesis. We examined the change of HOX B6 and HOX B9 mRNA expressions during the in vitro differentiation of four myeloid leukemia cell lines because HOX B6 may be involved closely in myeloid differentiation. HL-60, NB4, NKM-1 and NOMO-1 were established from acute leukemia of M2, M3, M2 and M5 subtype of the French-American-British classification, respectively. All-trans retinoic acid (ATRA), TPA, and G-CSF were used as differentiation inducers. Each cell line was cultured with each inducer and total RNA was isolated on day 1, 2, 3, or 5. HOX B mRNA was detected by Northern blotting and RT-PCR methods. HOX B6 and HOX B9 mRNAs were constitutively expressed in NB4, NKM-1 and NOMO-1, but were expressed at very low levels in HL-60. HOX B6 and HOX B9 mRNAs were also expressed in fresh acute myelocytic leukemia blasts. HOX B6 mRNA expression in HL-60, NB4, and NKM-1 cultured with ATRA increased on day 3 and decreased on day 5. HOX B6 mRNA expression in NB4 and NKM-1 cultured with TPA decreased on day 3. HOX B9 mRNA expression displayed changes similar to those of HOX B6 mRNA in NB4 and NKM-1. These results indicate that myeloid leukemia cell lines express HOX B6 and HOX B9, and that their respective mRNA expressions in NB4 and HL-60 increase at a mid stage of myeloid differentiation by ATRA induction and then decrease during a late stage. HOX B6 mRNA expression decreased in monocytoid differentiation by TPA induction in NB4, HL-60 and NKM-1. HOX B6 antisense-oligonucleotide inhibited the proliferation of NB4 and NKM-1. These results suggest that HOX B gene expression is related to simultaneous activation of cellular proliferation and differentiation in leukemic cells.

In spite of advances in immunohistochemical techniques, the histological subclassification of acute myeloid leukaemia (AML) in bone marrow biopsy has remained difficult. In particular, the translation of the diagnostic criteria of the French-American-British (FAB) cooperative group as primarily defined by bone marrow cytology into histology poses considerable problems. In this study, we investigated the expression of lactoferrin (LF) in various subtypes of AML and studied the usefulness of its immunohistochemical detection combined with a panel of antibodies directed against myeloperoxidase (MPOX), lysozyme (LYS), CD34 and naphthol-AS-D-chloroacetate esterase (NACE) staining in solving this problem. Trephine biopsies of 52 cases of AML were selected for histological evaluation in comparison to bone marrow aspirates classified according to FAB (M1 n = 10, M2 n = 7, M3 n = 11, M4 n = 13 and M5 n = 11). The results obtained confirmed the specificity of LF as a marker for secondary granules in neutrophilic myeloid cells and as a tool to subclassify AML. Its parallel application with (immuno-)staining for MPOX, LYS and NACE has allowed the identification of M1, M3, M5 cases, where there LF is lacking. Typically M2 is characterized by a subpopulation of LF-positive cells which tend to display a myelocytic differentiation. However, M4 shows a heterogeneous expression pattern of LF: M4a may be defined as more immature variant without LF expression while in M4b a more mature myeloid subpopulation stains positive for LF.

Objective: To investigate whether apoptosis occurs in vivo in patients with acute leukemia during induction chemotherapy and its significance., Methods: Apoptotic effect on peripheral blood of 20 patients with different types of acute leukemia prior to and during routine chemotherapy was studied by cell morphology, DNA electrophoresis and FCM-DNA analysis., Results: After chemotherapy, with falls in WBC and blast count, no apoptotic cell appeared on smears of peripheral blood, nor did ladder pattern of DNA on electrophoresis and subdiploid peak on FCM., Conclusion: Although anticancer drugs have been shown to induce apoptosis in vitro, circulating apoptotic cells are hardly detected probably due to their rapid clearance from the circulation. It is more difficult to study apoptosis in vivo than in vitro.

Nucleoli were studied in circulating myeloblasts of myeloblastic (FAB M1, M2), promyelocytic (FAB M3) and myelomonocytic (FAB M4) acute myeloid leukemias (AMLs) using a cytochemical procedure for the demonstration of RNA. In patients untreated with cytostatic chemotherapy, myeloblasts of myeloblastic acute leukemias possessed less frequently "active large" nucleoli and more frequently "inactive" micronucleoli in comparison with other investigated types of AMLs. When myeloblasts were classified according to the presence of functionally dominant nucleoli, the higher percentage of "terminal" myeloblasts containing only micronucleoli in this type of AML was significantly reduced in patients treated with the cytostatic chemotherapy. In patients suffering from promyelocytic leukemia treated with cytostatic chemotherapy, the decreased percentage of myeloblasts containing functionally dominant active large nucleoli was accompanied by the increased incidence of myeloblasts with functionally dominant "resting" ring shaped nucleoli. In myelomonocytic AML no significant differences were noted between patients untreated or treated with the cytostatic chemotherapy in the incidence of main nucleolar types in myeloblasts and myeloblasts classified according to the presence of functionally dominant nucleoli. Thus a further biological specificity might exist among leukemic blasts in various types of AMLs which should be considered for a rational approach to the therapy of these malignancies. In addition, the cytostatic chemotherapy did not influence incidence of the nucleolar asynchrony in myeloblasts of all investigated types of AML.

Background and Objective: In adult patients with acute myeloid leukemia (AML), central nervous system (CNS) involvement is a rare event and treatment has not yet been defined. Because there are no definitive data as to the most appropriate therapeutic approach to CNS leukemia in AML, we retrospectively analyzed a cohort of AML patients with meningeal leukemia in order to increase our knowledge on this particular matter., Methods: Out of 410 patients with de novo AML observed at our Institute from 1986 to 1995, 9 (2.2%) showed CNS leukemia (CNSL) during the follow-up. CNSL was treated as follows: in a first group of 4 patients we combined systemic HD Ara-C 3 g/m2 (every 12 hours by 3-hour infusion, for 6 doses), cranial radiation therapy and intrathecal (IT) methotrexate (MTX); a second group of 4 patients was treated with HD Ara-C, IT MTX without cranial irradiation; HD Ara-C alone was administered in one patient., Results: All patients of the first group and 2 patients of the second who achieved a complete remission (CR) had a median survival of 10 months (range 5-25+) after CNS involvement, while for the non-remitters it was 2 months (range 1-5). The only patient still living underwent allogeneic bone marrow transplantation., Interpretation and Conclusions: The combination treatment of HD Ara-C, IT MTX and cranial irradiation is well tolerated and seems to be an effective therapy for CNSL, presenting a high incidence of neurologic CR that correlates with a longer survival. As expected, the number of AML patients with CNSL was small, due to the fact that CNS in those patients is a rare complication. However, this study provides further information about the therapeutic possibilities in such restricted subsets of AML patients.

In 141 adult patients with diagnosis of acute myeloid leukemia the overall expression and intensity of expression of CD34 antigen on leukemic cells was investigated. Myeloid blasts were tested by applying direct immunofluorescence staining using anti-CD34 fluorescein monoclonal antibody in flow cytometry. CD34 antigen was found in 73 out of 141 (51%) cases and in particular in M0, M1 and M4 French-American-British (FAB) cytotypes, while M3 and M5 cases were rarely positive. In patients whose blasts expressed CD34 antigen a significantly lower rate of complete remission (CR) was observed as opposed to CD34 negative cases (61% vs 88%) (P = 0.001). Furthermore, a negative correlation between high intensity of CD34 expression, measured as a mean fluorescence index (MFI), and CR rate was observed. In particular, patients with a higher CD34 fluorescence intensity (MFI > 23), showed a further reduction in CR rate (48%). Also, these patients had a significantly lower overall survival (P = 0.03) as compared to patients with no expression of CD34 and patients with CD34 MFI < 23. In conclusion, these findings confirm that CD34 expression is frequently associated with "immature" FAB cytotypes (M0, M1 and M4) and with a reduced probability to achieve CR. Furthermore, a high CD34 intensity of expression should be considered as a reliable poor prognostic factor.

We have identified ten patients with acute myeloid leukemia (AML) and one patient with chronic myeloid leukemia with megakaryocytic crisis who displayed an inv(3)(q21q26). Seven of them had an additional monosomy 7. Most of them had a myelodysplastic syndrome (MDS) preceding AML, normal or increased platelet counts, increased number of megakaryocyte, megakaryocytic dysplasia, and erythroid dysplasia. There was a high incidence of resistance to induction chemotherapy, short remission time, and early relapse. Seven patients were immunologically analyzed. The main immunophenotypes were as follow: CD7+, CD34+, HLA-DR+, CD38+, CD13+, CD33+, CDw65+, CD2-, CD3-, CD4-, CD8-, CD19+, CD20-, CD11b-. Our results suggest that the leukemia with inv(3)(q21q26) represents a new cytogenetic-clinicopathologic subtype, characterized by 1) abnormal megakaryopoiesis and multiple hematopoietic lineage involvement; 2) an antecedent MDS; 3) poor response to conventional chemotherapy; and 4) expression of CD7, CD34, CD38, HLA-DR, CD13, and CD33 antigens. We propose that the malignant transformation in patients with inv(3)(q21q26) occurs in an early stem cell prior to lineage commitment.

On the subject of acute myeloid leukemia (AML), there is little consensus about the target cell within the hematopoietic stem cell hierarchy that is susceptible to leukemic transformation, or about the mechanism that underlies the phenotypic, genotypic and clinical heterogeneity. Here we demonstrate that the cell capable of initiating human AML in non-obese diabetic mice with severe combined immunodeficiency disease (NOD/SCID mice) - termed the SCID leukemia-initiating cell, or SL-IC - possesses the differentiative and proliferative capacities and the potential for self-renewal expected of a leukemic stem cell. The SL-ICs from all subtypes of AML analyzed, regardless of the heterogeneity in maturation characteristics of the leukemic blasts, were exclusively CD34++ CD38-, similar to the cell-surface phenotype of normal SCID-repopulating cells, suggesting that normal primitive cells, rather than committed progenitor cells, are the target for leukemic transformation. The SL-ICs were able to differentiate in vivo into leukemic blasts, indicating that the leukemic clone is organized as a hierarchy.

Previous morphocytochemical, immunological and cytogenetic analyses of blast cells in 174 children and 188 adults admitted to Cancer Research Center have shown that compared to adults in children leukemic precursors belong to earlier stage of differentiation similar to polypotent cell. The analysis covered 2 FAB-variants of ANLL comparable by the number of patients and intensity of the given chemotherapy (M2 and M4 ANLL FAB variants). The study included 65 children (50 with M2 and 15 with M4 FAB variants) and 43 adults (26 with M2 and 17 with M4 FAB variants) given therapy of standard intensity in the regimen 3+7 and 2+5. The children more frequently demonstrated involvement of the liver, spleen and peripheral lymph nodes. The percentage of complete remissions in both groups was not significantly different. 2- and 3-year recurrence-free survival was similar in two age groups with M2 FAB variants of ANLL. However, in M4 variant in adults this survival made up 18% against 0% in children. The differences may arise from lower peroxidase activity in children than in adults. It is suggested that M4 FAB variant in adults may indicate better prognosis than in children. Therefore, therapy in children with M4 FAB ANLL variant should be intensified.

Interleukin-1 beta (IL-1 beta) converting enzyme (ICE) is a cysteine protease that specifically cleaves precursor IL-1 beta to its biologically active form. Recent studies have also implicated ICE in the induction of apoptosis in vertebrate cells. Because IL-1 plays a major role in acute myelogenous leukemia (AML) blast proliferation, we sought to investigate the effect of ICE inhibition on AML progenitors. To do this, we used bocaspartyl (benzyl) chloromethylketone (BACMK) an inhibitor designed to penetrate cells and bind covalently to the active site of ICE. Our preliminary experiments showed that incubation of activated peripheral blood cells with 2.5 mumol/L of BAMCK downregulated production of mature IL-1 beta but had no effect on tumor necrosis factor-alpha. To test the effects of the inhibitor on AML cells, we first used the OCI/AML3 cell line. We found that these cells produce IL-1 beta and bind the biotinylated cytokine and that IL-1 inhibitors, such as IL-1 neutralizing antibodies, IL-1 receptor antagonist, and soluble IL-1 receptors, specifically inhibit OCI/AML3 proliferation, indicating that IL-1 beta is an autocrine growth factor for OCI/AML3 cells. The ICE inhibitor suppressed OCI/AML3 growth in a dose-dependent manner (at 0.4 to 4 mumol/L) and downregulated mature IL-1 beta production, as assessed by Western immunoblotting. Similar results were obtained with marrow aspirates from 16 AML patients. The ICE inhibitor suppressed proliferation of AML precursors (by up to 78%; mean, 44%) in a dose-dependent fashion at concentrations ranging from 0.4 to 5 mumol/L but not proliferation of normal marrow progenitors; the suppressive effect was reversed by IL-1 beta. Furthermore, incubation of AML cells with 4 mumol/L BAMCK downregulated the production of mature IL-1 beta, suggesting that the growth-inhibitory effect is mediated through suppression of the biologically active cytokine. Our data indicate that inhibition of ICE suppresses AML blast proliferation and suggest that ICE inhibitors may have a role in future therapies for AML.

The aim of this study was to compare the thermal sensitivity of normal murine and human hemopoietic progenitors to that of leukemic murine and human clonogenic cells in order to assess the clinical relevance of experimental data. Colony forming units-granulocyte-macrophage (CFU-GM) from normal human bone marrow and from bone marrow of patients with acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), and Hodgkin's disease in complete remission proved to be less sensitive to 42.5 degrees C in vitro hyperthermia (D0: 93.9 min) than murine bone marrow CFU-GM (D0: 49.6 min). Leukemic colony forming cells (CFU-L) from HL-60 suspension culture--when compared to human CFU-GM--showed significantly increased thermal sensitivity (D0: 22.8 min). While the thermal sensitivity of CFU-L from a murine leukemia cell line (WEHI 3-B) was not statistically significant when compared to that of CFU-L from HL-60 (D0 values 17.0 versus 22.8 min), the vertical difference between the parallel regression lines suggested an approximately three-fold greater survival for human CFU-L. Although carefully controlled hyperthermia is an easy purging technique, the relevance of murine data to human clinical practice must be considered critically.

Using serum-free culture system and liquid/semi-solid dual culture technique, we examined the effect of rhSCF on self-renewal of the stem/progenitor cells of human acute myelogenous leukemia (AML-CFU). The results revealed that the rhSCF as a hematopoietic growth factor (HGF) exerted its effect hierarchically earlier than interleukin-3 and granulocyte/macrophage colony-stimulating factor on AML-CFU. rhSCF, whether used alone or in combination with other HGFs, showed potential maintenance of self-renewal of AML-CFU in most AML patients studied. However, considerable discrepancy existed in maintenance among various FAB types of patients or even in patients with the same FAB type. It is suggested that SCF may play an important role in the pathological development of AML.

Background and Methods: Consistent and specific chromosomal aberrations have been observed in an increasing number of neoplasias. In the present report, we describe the cytogenetic findings from 50 cases of de novo ANLL in Argentina, South America, studied at diagnosis. In addition, their relation with the FAB classification is analyzed. Children with Down's syndrome and secondary ANLL were excluded from this analysis., Results and Conclusions: Out of 50 banded cases studied, 11 (22%) had normal karyotype, while the remaining 39 (78%) presented abnormal metaphases with structural alterations in the majority of them. Chromosomes 7 and 22 were most frequently involved in numerical alterations in children, while chromosomes 6, 8, 14 and 16 were the ones most often involved in adults. Consistent chromosome rearrangements were observed and they were linked to specific cytomorphologic subsets. The translocations t(8;21) and t(15;17) were seen only in M2 and M3, respectively. The inversion of chromosome 16, inv(16), was a typical finding in M4, but was not restricted to this subtype. Translocation t(2;3) was observed in three cases, all M4, each with a variable chromosome pattern. These results are in accordance with cytogenetic findings in Western Europe and the USA.

Ginsenosides are the main active component of Panax ginseng. It has been shown that ginsenosides have antineoplastic, antiaging, immunologic function enhancing and other pharmacological actions. In this article, result of experimental studies showed ginsenosides extracted from stem and leaf of Panax ginseng (GSL) has inductive differentiation effect on all types of acute nonlymphocytic leukemia cells in primary culture. The effect on M5, M4 was most potent, followed by M1, M2 and the least, on M3. Through analysis, it was considered that the inductive differentiation effect of ginsenosides might be due to the comprehensive effect of increasing intracellular cAMP and inducing interferon. Since GSL have some other important actions, therefore, if it could be used as a differentiation inducer in clinical practice or combined with other antineoplastic drugs, it would show co-antineoplastic actions in many aspect.

The MTT assay, a colorimetric assay, is found to be suitable for chemosensitivity testing. Recently, it has been suggested that hematopoietic growth factors (HGF) may enhance the effects of cytostatic drugs in acute myeloid leukemia (AML). We therefore studied the effects of granulocyte colony-stimulating factor (G-CSF), interleukin 3 (IL-3), and granulocyte-macrophage colony-stimulating factor (GM-CSF) combined with cytosine arabinoside (Ara-C), daunorubicin (DNR), mitoxantrone (MXT), or etoposide (VP-16) by using the MTT assay. The results were compared with in vitro clonogenic assays. Briefly, AML cells of nine patients were incubated in the presence or absence of G-CSF, IL-3, or GM-CSF under serum-free conditions for 24 hours. Next, for the MTT assay, Ara-C (final dilution range: 0.0024-240 micrograms/ml), DNR (final dilution range: 0.05-3.2 micrograms/ml), MXT (final dilution range: 0.05-3.2 micrograms/ml), or VP-16 (final dilution range: 0.1-100 micrograms/ml) were added and incubated for 48 hours. Cell survival was determined and IC75 values (75% reduction as compared to control cultures) were calculated. For clonogenic assays, the three lowest drug concentrations were used. After 48 hours, the clonogenic response was determined in serum-free, semi-solid cultures with G-CSF, IL-3, or GM-CSF. The results obtained by the MTT assay showed no significant enhancement of cytotoxicity by HGF on cytostatic drug preincubated cells compared to cytostatic drugs alone. The results obtained by the clonogenic assays showed increased cytotoxicity of Ara-C combined with G-CSF, IL-3, or GM-CSF. The median IC75 values of Ara-C decreased from 0.056 to 0.0168 microgram/ml with G-CSF (p = 0.01), from 0.108 to 0.0168 microgram/ml with IL-3 (p = 0.004) and from 0.12 to 0.0204 microgram/ml for GM-CSF (p = 0.02). Only moderate enhanced cytotoxicity was observed when VP-16 was combined with IL-3 (p = 0.036) or GM-CSF (p = 0.036), but not with G-CSF. No enhanced cytotoxicity of DNR and MXT to clonogenic AML cells was found when these agents were combined with HGF stimulation. The results indicate that the MTT assay underestimates HGF enhanced cytotoxicity of Ara-C or VP-16 to clonogenic cells. Therefore, the assay is not useful for accurately detecting differences of clonogenic response due to the proliferative status of cells. In this paper, the potential explanations for the failure of the MTT assay are discussed.

The immunophenotype of 61 de novo AML referring to the Institute of Hematology of Ferrara between January 1988 and September 1992 was studied in order to evaluate the prognostic impact and the biological implications of this approach. In this study we confirm the possible aberrant expression of some antigens and the lack of a strict correlation between FAB classification and immunophenotype. The expression of the CD14 and CD34 was correlated with a worse prognosis, while the CD34+ patients further showed a lower probability to achieve a complete remission.

Bone marrow specimens obtained from 54 patients with de novo AML and 7 patients with AML evolving from MDS were retrospectively examined for the presence of micromegakaryocytes defined as cells of less than 30 microns in diameter with one or two nuclei. At least 25 megakaryocytes were counted in each patient. Micromegakaryocytes were found in 17 cases (31%), M1:1/11, M2:5/18, M3:0/4, M4:5/12, M5:1/4, M6:4/4, M7:1/1. The median age of the patients was higher in de novo AML with micromegakaryocytes (57 years) than in de novo AML without micromegakaryocytes (41 years) (p = 0.014). Chromosomal analysis revealed that deletion of 5 or 5q-, 7 or 7q- was recognized only in the group of de novo AML with micromegakaryocytes and that t(15;17), t(8:21) and inv (16) were not recognized in this group. Micromegakaryocytes were identified in each bone marrow specimen obtained from 9 of 10 patients with de novo AML with trilineage myelodysplasia. The complete remission rate was significantly lower in de novo AML with micromegakaryocytes (33%) than in de novo AML without micromegakaryocytes (86%) (p = 0.001). The duration of survival of the patients with de novo AML with micromegakaryocytes was shown to be shorter than that of the patients with de novo AML without micromegakaryocytes (p = 0.017). Micromegakaryocytes were recognized in all of 7 patients with AML evolving from MDS. The presence of micromegakaryocytes in bone marrow of the patients with AML indicates a subset of AML with poor prognosis that may be closely associated with myelodysplastic syndrome.

Background: Leukemic infiltrates of the esophagus have been described occasionally in autopsy series, but there are no reports of antemortem diagnosis., Methods: Case reports are presented for three patients with acute myeloid leukemia in whom leukemic infiltration of the esophageal mucosa was diagnosed histologically and cytologically by endoscopic examination. Autopsies of patients with leukemia from 1976-1988 were reviewed., Results: The autopsy review of 207 patients with leukemia showed evidence of leukemic infiltration in the esophagus in 7.2% of cases. The only clinical factor identified to be significantly associated with esophageal involvement by leukemic cells was a high initial leukocyte count. Esophageal involvement was associated with leukemic infiltration of other soft tissues and organs., Conclusions: Although the etiology of dysphagia in patients with acute leukemia is usually related to infection, reflux, chemotherapy toxicity, or benign strictures, the frequency of esophageal leukemic infiltration in this autopsy series suggests that this diagnosis must be considered. Esophageal leukemia is usually associated with widely disseminated soft tissue and visceral infiltrates.

The aim of the present study was to compare two different methods for obtaining CD34+ cells from the peripheral blood or the bone marrow of patients with acute non-lymphocytic leukemia (ANLL). Twenty-two samples, obtained from 19 patients, were density cut (Ficoll-Hypaque 1.077) and, after incubation with My10 antibody, separated by panning or by immunomagnetic beads. Immunomagnetic beads provided a significantly better separation than panning, either in terms of concentration of CD34+ cells (85.5 +/- 11.6% vs. 55.7 +/- 25.0%, p = 0.003) or in terms of depletion of CD34+ cells (3.9 +/- 8.0% vs. 30.9 +/- 26.3%, p = 0.008). This was consistent with the virtually complete depletion of colony forming cells (CFC) in the CD34 negative fraction and the recovery of virtually all the CFC in the positive fraction in the samples separated by immunomagnetic beads. In conclusion, separation by immunomagnetic beads can allow collection of nearly pure CD34+ and CD34- cell populations from patients with ANLL, thereby facilitating the study of the biological characteristics of these cell populations. Moreover the method is less time consuming than panning and is not toxic to the CFC.