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In acute myeloid leukemia (AML), patients may harbor pre-leukemic hematopoietic stem cells (HSCs) containing some, but not all, of the mutations observed in the leukemic cells. These pre-leukemic HSCs may survive induction chemotherapy and contribute to AML relapse by obtaining additional mutations. We report here an acute monoblastic leukemia (AMoL) patient who later developed an unclassifiable myeloproliferative neoplasm (MPN-U). Whole-exome sequencing and cluster analysis demonstrated the presence of three distinct major clones during the clinical course: (1) an AMoL clone with ASXL1, CBL, and NPM1 somatic mutations, likely associated with the pathogenesis, and GATA2, SRSF2, and TET2 mutations, (2) an AMoL remission clone, with mutated GATA2, SRSF2, and TET2 only (possibly the founding clone (pre-leukemic HSC) that survived chemotherapy), (3) a small subclone which had JAK2 mutation during the AMoL remission, appearing at MPN-U manifestation with additional mutations. These findings suggest that pre-leukemic HSCs in AML patients may give rise to non-AML myeloid malignancies. This is the first report to analyze the clonal evolution from AMoL to MPN-U, which may provide new insight into the development of myeloid malignancies.

Purpose: High levels of ROS and ineffective antioxidant systems contribute to oxidative stress, which affects the function of hematopoietic cells in acute myeloid leukemia (AML); however, the mechanisms by which ROS lead to malignant transformation in relapsed AML-M5 are not completely understood. We hypothesized that alterations in intracellular ROS would trigger AML-M5 relapse by activating the intrinsic pathway. Experimental Design: We studied ROS levels and conducted c-Jun activation domain-binding protein-1 ( JAB1/COPS5 ) and thioredoxin ( TRX ) gene expression analyses with blood samples obtained from 60 matched AML-M5 patients at diagnosis and relapse and conducted mechanism studies of Jab1's regulation of Trx in leukemia cell lines. Results: Our data showed that increased production of ROS and a low capacity of antioxidant enzymes were characteristics of AML-M5, both at diagnosis and at relapse. Consistently, increased gene expression levels of TRX and JAB1/COPS5 were associated with low overall survival rates in patients with AML-M5. In addition, stimulating AML-M5 cells with low concentrations of hydrogen peroxide led to increased Jab1 and Trx expression. Consistently, transfection of ectopic Jab1 into leukemia cells increased Trx expression, whereas silencing of Jab1 in leukemia cells reduced Trx expression. Mechanistically, Jab1 interacted with Trx and stabilized Trx protein. Moreover, Jab1 transcriptionally regulated Trx. Furthermore, depletion of Jab1 inhibited leukemia cell growth both in vitro and in vivo Conclusions: We identified a novel Jab1-Trx axis that is a key cellular process in the pathobiologic characteristics of AML-M5. Targeting the ROS/Jab1/Trx pathway could be beneficial in the treatment of AML-M5. Clin Cancer Res; 23(15); 4450-61. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Despite diagnostic criteria are currently available for the distinct subtypes of monocytic-lineage neoplasias, a number of partially overlapping features still remain evident, which may hamper their differential diagnosis. An accurate identification and characterization of monocytic cells is of major relevance for the diagnosis and classification of these neoplasias. In this regard, as compared to other conventional techniques, flow cytometry has shown the highest sensitivity for detection of early monocytic commitment of (normal and neoplastic) bone marrow CD34 + hematopoietic precursors as well as of monocytic aberrations and maturation blockades, which are frequently associated with clonal myeloid disorders. In the present paper we provide basic principles and criteria for multiparameter flow cytometry identification and characterization of bone marrow monocytic cells that contribute to an improved diagnostic and classification of monocytic lineage-associated acute leukemias in clinical settings, particularly when using the EuroFlow antibody panels. © 2015 International Clinical Cytometry Society., (© 2015 International Clinical Cytometry Society.)

Although fatal pulmonary complications frequently occur during the course of acute leukemia, a minor proportion of the complications are due to leukemia itself. Infections, drug reactions and concomitant medical conditions are the major causes of respiratory distress in leukemic patients. We treated four patients with acute myeloid leukemia complicated by leukemic cell lysis pneumopathy (LCLP). All of the patients had leukemia of monocytoid origin and their respiratory function deteriorated soon after chemotherapy initiation. Although two of the patients required mechanical ventilation, all four improved after continued chemotherapy. Our experience indicates that, in cases of LCLP, chemotherapy should be continued with maximal respiratory support.

Background: Automated blood cell analyzers often read leukemic blasts as normal cells. In this study, we evaluated the 5-part differential patterns of blasts using automated analyzers to determine if they can differentiate among blast types., Methods: Blood samples containing 10% or more blasts were collected from patients with acute leukemia (N=175). The 5-part differential count was conducted using DxH 800 (Beckman Coulter, USA) and XE-2100 analyzers (Sysmex Co., Japan), and the results were compared with manual differential counts, which was used as a reference method., Results: The DxH 800 reported the 5-part white blood cell differential count in 98.9% of the cases. The XE-2100 provided an invalid automated differential count in 72% of the cases. Both analyzers counted most lymphoblasts as lymphocytes and most myeloblasts as monocytes. In 11 cases, the DxH 800 reported a 5-part differential count without a blast flag., Conclusions: Some automated analyzers are able to recognize and count blasts according to their characteristic cell types. Therefore, complete blood counts obtained automatically can provide valuable data for making provisional decisions regarding the lineage of leukemia cells before further investigation.

A lineage switch in leukemia, in which the leukemic cell lineage at onset converts to another lineage at a later time, is an uncommon type of hybrid (mixed) leukemia regarded as a variation of bilineage leukemia. We present a case of a 60-year-old female diagnosed with precursor B cell acute lymphoblastic leukemia (ALL), whose markers in flow cytometry shifted from their original status of CD19+, 22+, 79a+, 13+, HLA-DR+, and TdT+. Although her bone marrow achieved remission after induction therapy, there was a small residual population of leukemic cells in the liver. Residual disease was proved by biopsy and pathologically shown to have an immature phenotype of CD5+, CD10-, CD20-, CD79a- and myeloperoxidase negativity. Two weeks after liver biopsy, blast cells progressively appeared in the peripheral blood; these cells had a monocytoid morphology and phenotype (CD13, 14) but were accompanied by myeloid (CD33) and lymphoid (CD2, 4, 20) cells. Markers CD7, 10 and 19 were negative by flow cytometry. This phenotypical conversion from B-ALL to hybrid leukemia featuring monocytoid characteristics is known as a lineage switch. This case suggests that leukemic subclones tend to carry out dedifferentiation, occasionally in extramedullary sites, which serve as a hotbed for the selection of resistant clones.

Acute leukemia is a rare malignancy of pregnancy. When it develops, there are many complications to consider and management becomes exceedingly difficult. We report a case of acute myelogenous leukemia presenting as preeclampsia and fetal demise at 36 weeks of gestation. A 30-year-old multigravida presented with intrauterine fetal demise at 36 weeks' gestation, hypertension, and thrombocytopenia. The patient received platelet and packed red blood cell transfusion, with concurrent prophylactic magnesium sulfate and dexamethasone treatment. Following labor induction, the patient delivered a nonviable female fetus and suffered a stroke postpartum. Peripheral smear and flow cytometry revealed the patient had acute myeloid leukemia with prominent monocytic differentiation. The patient expired on postpartum day six. Acute leukemia during the pregnancy is associated with an unfavorable outcome.

Tumor-associated peptides isolated by acid elution are frequently used for therapeutic immunization against various tumors both in mice and in humans. In acute myeloid leukemia (AML), the frequent accessibility of a large tumor burden allows for extraction of peptides from leukemia cells by using either citrate-phosphate (CP) or trifluoroacetic acid (TFA) buffer. To develop an optimal immunotherapeutic protocol for AML patients, we evaluated both in mice and in humans, the immunogenicity of peptides eluted from leukemia cells with the two acids (TFA or CP). Although ex vivo studies in mice showed that both prophylactic immunizations with mature dendritic cells (DC) loaded with TFA-peptides (DC/TFA), or CP-peptides (DC/CP), were able to stimulate specific antileukemia immune responses, only vaccination with DC/TFA was able to prevent leukemia outgrowth. Moreover, in humans, only DC/TFA generated significant antileukemia CD4(+) and cytotoxic CD8(+) T cell responses in vitro. In summary, these data demonstrate that the choice of the acid elution procedure to isolate immunogenic peptides strongly influences the efficacy of the antileukemia immune responses. These finding raise essential considerations for the development of immunotherapeutic protocols for cancer patients. In our model, our results argue for the use of the TFA elution method to extract immunogenic AML-associated peptides.

Leukemia cutis represents a skin infiltration by leukemic cells. Clinically it can mimic a wide variety of dermatoses. We describe the case of a 64-year-old man with psoriasis who presented with a 4-day history of erythematous, slightly scaly, asymptomatic plaques distributed on the trunk and upper-extremities, and associated asthenia, myalgias, and anorexia. A skin biopsy revealed a leukemic infiltrate. Studies of peripheral blood and bone marrow provided a diagnosis of acute monocytic leukemia. This case report shows the importance of the clinical suspicion for the diagnosis of leukemia.

Hypocholesterolemia is a common finding in patients with acute leukemia (AL). The aim of this study is to investigate if blast myeloid and lynfoid cells take up more high density lipoprotein (HDL) cholesteryl esters than normal cells of the same origin. The HDL-cholesteryl ester uptake followed a kinetic saturation process. Higher maximal velocity rates were found in lymphoblasts and myeloblasts compared to normal cells (Vmax=3.51+/-0.30/3.61+/-0.16 and 2.54+/-0.12/2.28+/-0.12 microg/mg, respectively). High density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) and total cholesterol were significantly lower in AL patients (p<0.05); no differences were observed in triglyceride or VLDL-C levels. In conclusion, low HDL-C levels observed in AL may be related to an overexpression of a selective HDL-cholesteryl ester putative site.

Somatic mutations in PTPN11, the gene encoding the transducer SHP-2, have emerged as a novel class of lesions that upregulate RAS signalling and contribute to leukaemogenesis. In a recent study of 69 children and adolescents with de novo acute myeloid leukaemia (AML), we documented a non-random distribution of PTPN11 mutations among French-American-British (FAB) subtypes. Lesions were restricted to FAB-M5 cases, where they were relatively common (four of 12 cases). Here, we report on the results of a molecular screening performed on 181 additional unselected patients, enrolled in participating institutions of the Associazione Italiana Ematologia Oncologia Pediatrica-AML Study Group, to provide a more accurate picture of the prevalence, spectrum and distribution of PTPN11 mutations in childhood AML and to investigate their clinical relevance. We concluded that PTPN11 defects do not represent a frequent event in this heterogeneous group of malignancies (4.4%), although they recur in a considerable percentage of patients with FAB-M5 (18%). PTPN11 lesions rarely occur in other subtypes. Within the FAB-M5 group no clear association of PTPN11 mutations with any clinical variable was evident. Nearly two third of the patients with this subtype were found to harbour an activating mutation in PTPN11, NRAS, KRAS2 or FLT3.

Objective: To establish a novel human monocytic leukemic cell line and characterize its biological features., Methods: Mononuclear cells isolated from the bone marrow of an acute monocytic leukemia (AML-M(5b)) patient at relapse were inoculated in a liquid culture system. And the biologic features of the established cell line SHI-1 were characterized by morphology, cytochemical staining, flow cytometry, karyotypic analysis, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), fluorescence in situ hybridization (FISH), tumorigenicity in nude mice, quantitative fluorescent polymerase chain reaction, broth medium culture, short tandem repeating sequences-PCR (STR-PCR), multiplex-FISH (M-FISH), and (3)H-thymidine incorporation assay., Results: A human acute monocytic leukemia cell line, SHI-1, was established and has proliferated continuously in vitro for over one year. The cell line presented typical morphology and immuno-profile of monocytic lineage with the original t(6;11)(q27;q23) and del(17)(p11) abnormalities. The MLL-AF6 fusion transcript was detected by RT-PCR. The rearrangement of MLL gene, deletion of p53 gene, and translocation between chromosomes 6 and 11 were revealed by FISH. A point mutation of ATC-->ACC at exon 6 of the p53 gene was found by sequencing of the PCR products. The clonality and the high tumorigenicity of the SHI-1 cell line were confirmed. Infections of EBV and mycoplasma were excluded. A derivative chromosome 7 resulting from a translocation between chromosomes 7 and 13, monosomy 18 and a minute derived from chromosome 8 in addition to t(6;11) and deletion(17)(p11) were detected by M-FISH in SHI-1 cells passaged to March 2003. Cell line authentication by STR-PCR confirmed the identity to the original leukemic cells of the patient. (3)H-thymidine incorporation assay showed that IL-4 and IL-15 had proliferative effects, while IFN-gamma, TNFalpha, IL-2, PDGF, and IL-7 had inhibitory effects on the cell line., Conclusions: SHI-1 is a novel acute monocytic leukemia-derived cell line carrying t(6;11)(q27;q23) and p53 gene alteration and having high tumorigenicity in nude mice. It provides a new useful tool for leukemia research.

Objective: To investigate Wilms' tumor gene (WT1) expression levels in bone marrow (BM) of acute leukemia patients (ALs)., Methods: A real-time quantitative reverse transcriptase polymerase chain reaction (RQ-RT-PCR) method was established for detecting WT1 and internal reference GAPDH expression levels in BM of 108 ALs and 23 non-leukemia controls by Light Cycler., Results: The median expression levels of WT1 in 70 newly diagnosed ALs and 11 relapsed ALs were statistically higher than those in 23 ALs in complete remission (CR) and 23 non-leukemic controls (75.10 and 89.56 vs 2.07 and 1.51 respectively). No statistic differences was found between the CR group and control group, nor between the newly diagnosed group and relapsed group. Of the 70 newly diagnosed ALs, median WT1 expression level of acute granulocytic leukemias was significantly higher than that of acute monocytic leukemias (M(5)), but there was no statistic differences among the M(1), M(2), M(3) and ALL subtypes. Furthermore the WT1 levels were not correlated to peripheral WBC counts, BM blast percentage and multidrug resistant gene (mdr1) expression at presentation, but correlated to chromosome karyotypes. Dynamic analysis of WT1 levels of 2 patients on treatment showed that WT1 expression levels predicted relapse., Conclusion: WT1 expression levels in ALs were strikingly higher than that in non-leukemias. WT1 can be a marker for detecting MRD and evaluating therapy efficacy in leukemias.

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.

A rare case of different leukemias simultaneously occurring in the bone marrow (BM) and the central nervous system (CNS) was encountered. A 4-year-old girl was diagnosed with acute lymphoblastic leukemia and achieved a complete remission with chemotherapy. Two years after diagnosis, she was found to have acute monocytic leukemia at first relapse in the BM. One month after the second course of induction therapy, lymphoblastic leukemia in the BM and monocytic leukemia in the CNS were found simultaneously. Chromosomal analysis of leukemia cells in the BM and CNS showed distinct results. The mechanism of double leukemias occurring was obscure, although a lineage switch, therapy-related, or de novo leukemia could be considered as possibilities. Double leukemias should be kept in mind when leukemias relapse in several sites., (Copyright 2004 Wiley-Liss, Inc.)

The hemoglobin scavenger receptor (HbSR/CD163) is an interleukin-6- and glucocorticoid-regulated macrophage/monocyte receptor for uptake of haptoglobin-hemoglobin complexes. Moreover, there are strong indications that HbSR serves an anti-inflammatory function. Immunoprecipitation and immunoblotting enabled identification of a soluble plasma form of HbSR (sHbSR) having an electrophoretic mobility equal to that of recombinant HbSR consisting of the extracellular domain (scavenger receptor cysteine-rich 1-9). A sandwich enzyme-linked immunosorbent assay was established and used to measure the sHbSR level in 130 healthy subjects (median, 1.87 mg/L; range, 0.73-4.69 mg/L). To evaluate the sHbSR levels in conditions with increased leukocyte stimulation and proliferation, 140 patients admitted to a hematological department were screened. Several patients, with a broad spectrum of diagnoses, had a level of sHbSR above the range of healthy persons. Patients with myelomonocytic leukemias and pneumonia/sepsis exhibited the highest levels (up to 67.3 mg/L). In conclusion, sHbSR is an abundant plasma protein potentially valuable in monitoring patients with infections and myelomonocytic leukemia.

The abnormality in the translocation of chromosomes 4 and 11 (t[4;11]) has been characteristically associated with calla-negative CD15(+) acute lymphoblastic leukemia (ALL) of early pre-B-cell origin. Transformation of a lymphoblastoid to a monoblastoid morphologic structure has rarely been described at relapse in these cases; however, these cases have lacked flow cytometric immunophenotyping (FCI) and genotypic studies (GS) to define the immunophenotype of and the presence of a B-cell gene rearrangement in the monoblastoid component. We report a case of CD15(+), CD10(-) ALL of early pre-B-cell origin defined by morphologic testing and FCI with the t(4;11) abnormality. At relapse, the morphologic testing, enzyme cytochemistry, and FCI data were characteristic of monoblastic leukemia. The t(4;11) abnormality persisted with associated additional chromosomal abnormalities, and the monoblasts contained a B-cell gene rearrangement by GS. These findings support the concept that both processes arose from a multipotential progenitor cell.

In acute myeloid leukemia (AML) close associations are known between cytomorphology and cytogenetics such as in AML M3/M3v showing a t(15;17) and in AML M4eo associated with inv(16)/t(16;16). In AML M5 a heterogenous cytogenetic pattern is observed. We describe the gain of an isochromosome of the short arm of chromosome 5 together with the gain of chromosome 8 as the sole abnormalities in two cases of acute monoblastic leukemia. In a third case of acute monoblastic leukemia we also observed the gain of an isochromosome 5p together with trisomy 8. This patient showed in addition an unbalanced translocation between the long arm of chromosome 1 and the short arm of chromosome 14 leading to a trisomy 1q. So far only two cases of AML with i(5)(p10) have been published. In no other hematological malignancy has an isochromosome 5p been reported up to now. As an isochromosome 5p can be misinterpreted as a deletion 5q, which occurs frequently in AML, fluorescence in situ hybridization with loci specific probes is a helpful method to detect this rare abnormality.

We report two cases of atypical defibrination syndromes in patients with respectively acute monoblastic leukemia (chronic myeloid leukemia initially) and acute lymphoblastic leukemia. Hemostasis studies show low fibrinogen level, elevated D-dimers, decreased alpha 2 antiplasmin and factor V, normal antithrombin III values. Plasminogen is below the normal range in one patient. Soluble complexes, which are an important argument for diagnosis of intravascular coagulation disease, are not detected in both patients. Primary or secondary hyperfibrinolysis seems also excluded since euglobulin clot lysis time was normal. Enzymatic proteolysis of fibrinogen (or fibrin) by the blast cells has been reported by some authors; this mechanism could account for the hemostasis abnormalities observed in these two patients.

Between 1989 and 1996, 21 cases with acute non lymphoblastic leukemia (11 males and 10 females) were diagnosed in our institution. Median age was 9 years (range, 2-15 years). Leukocyte count was more than 50,109/l in 47% of cases. According to the French-American-British (FAB) criteria, 7 cases were classified M1, 10 cases were classified M2, 1 classified M4Eo and 3 classified M5. All patients were treated with "7 + 3" protocol and complete remission was achieved in 17 cases (80%), 2 cases (10%) failed to respond and 2 (10%) died during induction. Relapse was observed in 15 cases. The 3-year survival rate was 20% and the relapse-free-survival rate was 12% confirming the worse prognosis of this leukemia when treated with standard chemotherapy.

The intent of this article is to describe the effect of tumor lysis on automated platelet counts in therapy-related, secondary acute monocytic leukemia. The first patient was a 69-year-old man with large cell carcinoma of the lung who developed acute monocytic leukemia 1(1/2) years after initiation of radiation and chemotherapy for his carcinoma. The second patient was a 72-year-old female with peripheral T-cell lymphoma who developed acute monocytic leukemia 1 year after initiation of chemotherapy for her lymphoma. Platelet counts were determined by the automated Coulter (STKS) counter. Both patients had clinical and laboratory evidences of tumor lysis syndrome and disseminated intravascular coagulation. The peripheral blood smears revealed numerous fragments of leukemic cells and apoptotic cells with pyknotic nuclei. The Coulter machine enumerated these cellular fragments as platelets, resulting in falsely elevated platelet counts. Awareness of this laboratory artifact in secondary acute monocytic leukemia with tumor lysis syndrome is important so that potential life-threatening thrombocytopenia is not overlooked.

The electrochemical voltammetric behavior of bone marrow of leukaemia has been investigated by a self-devised cytosensing system. The two anodic peaks of erythrocytes (red blood cells, RBC) in bone marrow of leukaemia appeared at 0.73 +/- 0.03 and 0.83 +/- 0.02V vs. SCE, respectively, on the first scan. The anodic peak of leukocytes (white blood cells, WBC) appeared at 0.32 +/- 0.03V vs. SCE. The anodic peak of RBC at 0.83V disappeared when the patients were cured. The experimental results show that the voltammetric behavior of erythrocytes is in constant contact with the initial stage of leukaemia. The cyclic voltammetric behavior of 40 cases of leukaemia including acute myeloid leukaemia (AML) and chronic myeloid leukaemia (CML) and 10 cases of healthy volunteer peripheral blood was studied. The cyclic voltammetric behavior of erythrocytes may provide a simple and specific marker for diagnosis of leukaemia.

We describe two cases of acute myeloblastic leukemia, classified as M4 and M5 in the French-American-British nomenclature, with an 11q23 rearrangement at karyotypic analysis. The involvement of the MLL gene with two new partner loci on chromosome 10q22 and 8q24, respectively, was demonstrated by fluorescence in situ hybridization using a YAC clone B22B2L.

Introduction: Cutaneous manifestations of myelodysplastic syndromes are rare and polymorphous. They can be the direct expression of the hematological disease or represent signs of a vasculitis or a neutrophilic syndrome. Myelodysplastic syndromes progress sometimes toward an acute leukemia., Observation: A 53-year-old woman suffering from myelodysplastic syndrome presented for several months a cutaneous vasculitis without any histological specificity. In time, such presentation was complicated by the simultaneous occurrence of a cutaneous Langerhans cell histiocytosis and an acute monoblastic leukemia type LMA 4. The disease was rapidly fatal., Discussion: The complication of a myelodysplastic syndrome by concurrent Langerhans cell histiocytosis and acute monoblastic leukemia suggests a pathogenic relationship between these two latter disorders.

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.

The absolute number of membrane-expressed CD14, the most important endotoxin receptor, on human monocytes and neutrophils shows remarkable variation in the literature. To quantify these numbers two fluorescence methods using fluorescein isothiocyanate (FITC)-labeled monoclonal antibodies (mAb) were applied. A commercially available set of standard beads was used in flow cytometry to quantitate CD14 with eight different mAbs. Independent from their isotype the various mAbs showed minor differences and indicated that peripheral blood monocytes expressed 99,500-134,600 (115,400 +/- 10,600) and neutrophils 1,900-4,400 (3,300 +/- 800) CD14 receptors. There was no significant difference in CD14 expression on leukocytes in unprocessed freshly obtained whole blood and after a Ficoll isolation procedure. However, a short temperature shift resulted in a 1.3- to 1.6-fold up-regulation of CD14. The results obtained with the reference beads were verified with fluorescence Scatchard analysis and spectrofluorometry using mAb 26ic-FITC and showed 109,500 CD14 per monocyte and 6,700 CD14 per neutrophil. For comparison the number of CD14 on the monocytic THP-1 cells and Fc gamma-receptors on human leukocytes were determined using the reference beads and flow cytometry and gave results comparable to published data. Our data indicate that resting human monocytes express roughly 110,000 CD14 molecules on their surface using a simple fluorometric assay. Correct determination of the number of CD14 and other cell surface receptors is of importance in the monitoring of septic patients.

We have employed fluorescence in situ hybridization (FISH) in combination with standard morphology (MGG/FISH) to identify the clonal involvement of different bone marrow cell lineages in 20 AML patients (14 MDS-AML, 6 de novo AML). Even though the number of cells belonging to the abnormal clone varied between individual cases, the percentage of clonal blasts was similar in MDS-AML and de novo AML patients. The erythropoietic cells appeared to be part of the abnormal clone in 13 of 14 patients with MDS-AML, but only in 1 of 6 with de novo AML. Similarly, clonal granulocytes were detected in 13 of 14 patients with MDS-AML, compared to 2 of 6 with de novo AML. Lymphocytes consistently displayed normal, diploid karyotype. The results suggest that it is possible to distinguish between MDS-AML and de novo AML by the use of MGG/FISH; in de novo AML the abnormal chromosomal clone is generally confined to the immature myeloid cells, while in MDS-AML mature granulocytes and erythroid cells are of clonal origin. It is, however, not possible to conclude that MDS-AML is a "multipotent" type of leukaemia, since it cannot be ruled out that the chromosomally aberrant erythroid cells and granulocytes represent surviving cells from the original MDS clone.

Aims: To analyse the forward scatter/side scatter (FSC/SSC) distribution of acute myeloblastic leukaemia (AML) blast cells in order to assess whether it correlates with their morphology, immunophenotype, and clinical and biological disease characteristics., Methods: FSC/SSC patterns were established upon taking into account the localisation of the residual T lymphocytes in the FSC/SSC dot plot as an internal biological standard. One hundred and seventy one newly diagnosed AML patients were analysed and five different FSC/SSC patterns were established. These five patterns could be grouped into two major categories taking into account the FSC/SSC distribution of normal cells in a bone marrow aspirate: immature patterns (1 and 2) and mature patterns (3, 4, and 5). These FSC/SSC patterns were correlated with different clinical and biological characteristics of AML patients., Results: No significant associations were detected in relation to the clinical and haematological disease characteristics and the prognosis of these patients. By contrast there was a significant correlation between the FSC/SSC pattern of the AML blast cells and the FAB classification. An increased reactivity for the antigens associated with myeloid differentiation such as CD13, CD33, CD11b, CD15, CD14, CD4, CD56, and/or CD16 was detected among cases showing a mature FSC/SSC pattern (3, 4, and 5), both in the whole series and even within each of the FAB AML subtypes. By contrast, the reactivity for the CD34 precursor cell associated antigen was higher among those cases displaying an immature FSC/SSC pattern, this being observed even within each FAB subgroup., Conclusions: The FSC/SSC pattern distribution of AML blast cells not only provides an additional objective and reproductible system for the classification of these leukaemias but it may also represent a connection between the FAB morphological groups and the immunophenotypic classification of AML patients.

Tumor necrosis factor (TNF) is a cytokine with pleiotropic biological activity. We have undertaken the present study to evaluate a possible ability of peripheral blood cells of healthy subjects and patients with acute monocytic leukemia to produce TNF alpha. We studied 17 leukemia patients and 8 control subjects. Spontaneous production of TNF alpha induced by LPS and Newcastle disease virus (NDV) were determined in blood cell cultures. Leukemic cells released markedly higher level of TNF alpha than control cells cultured in vitro in contrast to control cells producing TNF spontaneously. It seems that leukemic cells can produce TNF in response to very small amount of LPS; however, we cannot exclude that spontaneous TNF may be involved in autocrine regulation of proliferation and differentiation of bone marrow monocyte precursors.

The level of blast cells in peripheral blood in acute myeloid leukemia (AML) varies in individual patients. The bone marrow egress of hematopoietic cells is an unclear phenomenon in which cell deformability and cytoadhesion to the extracellular matrix are involved. One component of deformability is the membrane fluidity. Using fluorescence polarization, we have studied the fluidity of blast cell membranes from 22 AML patients. This membrane was found to be highly fluid and a statistically significant correlation was found between the increase in membrane fluidity and the number of blast cells in the blood. Studying interaction between blast cells and several components of bone marrow stroma, we found adhesion to fibronectin and fibroblastic extracellular matrix. Adhesion to the extracellular matrix was inversally correlated to the level of blast cells in the blood. The observed increase in membrane fluidity and reduction of adhesion to bone marrow stroma may result in an increase of blast cells egress in AML.

Proteins of the complement system (C2, C3) are synthesized by human monocytes and macrophages, thus providing an important local source of these proteins in vivo which serve as a first-line host defense mechanism. In this study, we investigated the production of complement components C2, C4, and C9 by human monocytes/macrophages and by the pathologic cells of acute monocytic leukemia which represent a source of immature monocytic precursors. Human blood monocytes were collected and purified by cytapheresis and elutriation and leukemic cells by Ficoll gradient. Secretion of complement components was measured by a hemolytic assay. The evaluation of the mRNAs of the various complement components in the cells was performed by polymerase chain reaction (PCR) by adding 32P labeled deoxycytidinetriphosphate (dCTP) to the amplification step. Functional C2 was found to increase during in vitro maturation of macrophages up to the fourth week of culture. C2 mRNA was detected after amplification and increased during the maturation. Interferon-gamma (IFN-gamma) mediated a marked increase of the C2 mRNA. We found a decrease in synthesis of C4 mRNA during in vitro differentiation of human monocytes. The effect of IFN-gamma resulted in an increase in C4 mRNA. C9 mRNA was not detected although it was detected in the HepG2 hepatoma-derived cell line. Functional C2 was not detected by leukemic cells after 24 h of culture but little functional C4 was present in the cell supernatants. As they were by human monocytes and macrophages, C2 and C4 mRNAs were detected after amplification but C9 mRNAs were not detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular cyclic adenosine monophosphate (cAMP) levels were measured in the peripheral mononuclear cells of 25 recipients of ABMT after stimulation with forskolin (10(-4) M), cholera toxin (1 microgram/ml) and prostaglandin E2 (PGE2, 10(-6) M). Significantly increased post-stimulatory cAMP levels were found in cells derived from patients' cells less than 30 days after ABMT (after forskolin stimulation: mean 89.7 +/- 45.4 pmol per 5 x 10(6) cells, p < 0.001; after cholera toxin stimulation: mean 78.6 +/- 37.9 pmol per 5 x 10(6) cells, p = 0.003; after PGE2 stimulation: mean 93.6 +/- 36.6 pmol per 5 x 10(6) cells, p < 0.001). However, at a later time after ABMT (7-60 months), cAMP levels had returned to normal and no significant differences in intracellular cAMP content between cells from patients and normal individuals could be detected. In addition, in six patients cAMP levels after PGE2 stimulation were measured serially pre- or peri-transplantation up to a time when haematologic recovery had occurred: the significantly elevated inducible cAMP levels found early post-ABMT returned to normal with peripheral blood reconstitution. This observation could be of interest in the post-transplantation period, because cAMP, as a 'second messenger', is a modulator of the immune system.

The production of tumor necrosis factor alpha (TNF alpha) of peripheral blood mononuclear cells was investigated in 58 patients with acute leukemia. The results showed that the production of TNF alpha was significantly lower in 41 of the 58 patients than that of normal controls. The TNF alpha activity levels from patients with acute monocytic leukemia and acute myelomonocytic leukemia were higher than those from patients with acute lymphoblastic leukemia and acute granulocytic leukemia (P < 0.05). The TNF alpha activity level changed in various courses of the disease and returned to normal during the period of complete remission. It was noted that patients with higher or normal TNF alpha levels had a higher complete remission rate than those with lower TNF alpha level (P < 0.01). These results indicated that determination of TNF alpha maybe helpful in observing changes of the disease and predicting prognosis.

The authors report two cases of acute myeloid leukemia with prevalent cutaneous lesions. The positivity of granulo-monocytic antibodies and the exclusive cutaneous site of the lesions drove them previously to the diagnosis of histiocytic sarcoma. These cases stress the problem of the immunological identification of cutaneous lymphomas of "histiocytic" type.

The surface expression of the alpha 2-macroblobulin receptor (alpha 2MR), detected by a monoclonal antibody, A2MR alpha-2, was determined on mononuclear blood cells from 90 cases of malignant blood disease. Flow cytometric analyses combined with immunoblotting and ligand binding experiments revealed that alpha 2MR was expressed on malignant cells from patients with acute and chronic myelomonocytic leukemias, while no significant expression was found on malignant cells from acute and chronic lymphatic leukemia, lymphomas, plasma cell leukemias or hairy cell leukemia. In acute myeloid leukemia, alpha 2MR was expressed in 50% of the M4-M5 cases, but only in three of thirty of the morphologically undifferentiated or non-monocytic cases (M1-M3 and M6). In chronic myelomonocytic leukemia five of seven cases were alpha 2MR-positive, while only one of seven cases of chronic myeloid leukemia was positive. The monocytic nature of the hematopoietic cells reacting with A2MR alpha 2 was further confirmed by a close correlation with CD14 surface expression.

Monocytic leukemia (MoL) cells were obtained from the peripheral blood of a patient in whom the leukemic cells infiltrating various lymphoreticular organs exhibited features intermediate between interdigitating reticulum cells (IDC) and ordinary phagocytic macrophages, whereas the leukemic cells in the peripheral blood were essentially monocytic and lacked such features. Peripheral blood CD4+ T-cells were established as an interleukin-2-dependent T-cell line. When the MoL cells were exposed for a few days to conditioned medium from the T-cell line, they extended several dendritic cytoplasmic projections and became intensely positive for HLA-DR antigen, cytoplasmic S-100 beta protein, and CD1 antigen. Functionally, the conditioned medium significantly down-regulated Fc-mediated and Fc-independent phagocytic activities, and the levels of lysosomal enzymes such as lysozyme and nonspecific esterase in the MoL cells. Moreover, the conditioned medium significantly up-regulated the accessory cell function of the MoL cells as measured by the primary allogenic mixed leukocyte reaction (MLR). Furthermore, the conditioned medium significantly down-regulated the expression of CD14 antigen. Biochemical analysis indicated that the factor responsible for these changes is a protein which is distinct from known human cytokines and whose molecular weight is approximately 31 kDa. These findings suggest that IDC are closely related the monocytic lineage and that helper T-cells play an important role in constructing the microenvironment of T-lymphoid tissues which is necessary for the differentiation and maturation of IDC.

THP-1 cells are a monocyte-like cell line derived from a patient with acute monocytic leukemia and unlike other leukemic cell lines has a normal diploid karyotype. We have characterized Fc gamma R expression on this cell line by flow cytometry, radiolabeled IgG1 and monoclonal antibody (mAb) binding assays, and biochemical analysis. Flow cytometric analysis of THP-1 cells with anti-Fc gamma RI, II, and III mAb, and a rabbit anti-Fc gamma RIII F(ab')2 demonstrated that only Fc gamma RI and Fc gamma RII are expressed by these cells. A panel of anti-Fc gamma RIII mAb (anti-CD16) failed to bind to THP-1 cells. Biochemical studies identified polypeptides of 64 to 78 kDa (Fc gamma RI) and of 42 to 53 kDa (Fc gamma RII). Fc gamma R expression was determined by binding of radioiodinated human IgG1 (to detect Fc gamma RI), mAb IV.3 (to detect Fc gamma RII), or rabbit IgG immune complexes. Thirty-five thousand high affinity binding sites (dissociation constant [KD] = 4.22 x 10(-9) M) for IgG1 were found on THP-1 cells. Interferon-gamma (IFN gamma) upregulated Fc gamma RI expression by THP-1 cells 2.8-fold, whereas Fc gamma RI on U937 cells was increased six- to eight-fold by this cytokine. Phorbol myristate acetate (PMA), tumor necrosis factor-alpha (TNF alpha), and vitamin D3 had no effect on IgG1 binding by THP-1 cells. Fifty thousand IgG molecules in immune complexes bound to THP-1 cells. IFN gamma treatment increased this binding by four-fold, PMA treatment resulted in a 50% increase in the number of IgG immune complexes bound, whereas vitamin D3 treated THP-1 cells bound half as many IgG immune complexes as control cells. Binding assays utilizing mAb IV.3 identified 50,000 sites per cell. Treatment of THP-1 cells with IFN gamma, TNF alpha, PMA, or vitamin D3 had no effect on Fc gamma RII expression. That Fc gamma RI plays a predominant role in immune complex binding was demonstrated by inhibition studies. Human IgG1 as well as mouse IgG2a mAb to Fc gamma RII inhibited immune complex binding by 76 to 84%, whereas mouse IgG1 mAb to Fc gamma RII had minimal effect on immune complex binding. Fc gamma R expression may not be linked to differentiation of THP-1 cells since only 1,25 vitamin D3 was able to induce the expression of CD14, a marker of mature monocytic phenotype.

Leukemic cell expression and serum levels of CD4, CD8, and interleukin-2 receptor (IL-2R) were determined at diagnosis for children or adolescents with acute myeloid leukemia (AML). Cellular expression of CD4 was detected in 18 of 62 cases, CD8 in none of 60 cases, and IL-2R in one of 33 cases tested. Myeloblasts of the M4 and M5 subtypes expressed CD4 significantly more frequently than other FAB subtypes (p = 0.0001). Serum levels of the three soluble factors (tested for 91 patients) were positively correlated with each other. Increased serum CD4 levels were significantly associated with cellular CD4 expression, high leukocyte count, M5 leukemia, spleen enlargement, and age less than 1 year. High serum CD8 levels correlated significantly with splenomegaly, extramedullary disease, absence of Auer rods, and high leukocyte count. Cases with high serum IL-2R levels were less likely to have Auer rods and more likely to have splenomegaly and M5 leukemia; serum levels greater than 750 U/ml were associated with a higher probability of treatment failure (p = 0.05), even after adjustment for other potential prognostic factors. Further studies of serum CD4, CD8, and IL-2R levels may help to clarify the immunoregulatory role of T-cells in patients with AML.