Leukocyte mean corpuscular volume in the differential diagnosis of acute leukemias

The leukocyte mean corpuscular volume was measured in 61 cases of acute leukemia. The volume of blast cells allows a clear distinction between lymphoblastic and other varieties of acute leukemia. There seems to be good correlation between patients'' age, leukocyte corpuscular volume, cytological and cytochemical findings. Furthermore, myeloblastic leukemias.Leukemic cells can be characterized by their volume, which is close to that of normal cells of the same line. This observation supports the clonal theory of their origin and concurs with the classical nomenclature which separates them into lymphoblastic, myeloblastic and monoblastic types.     

Expression of the surface membrane receptors of neutrophils in myeloproliferative diseases

For 147 patients with myeloproliferative diseases, a study was made of the expression of Fc-receptors to immunoglobulins IgC, IgA, and that of FcH-receptor and receptors to C3-components of the complement in peripheral blood neutrophils. The data obtained show the lower level of neutrophils having membrane receptors in patients with acute myeloblastic leukemia and chronic granulocytic leukemia at the stage of blast transformation. A decrease in expression of membrane receptors of neutrophils was shown in patients with chronic granulocytic leukemia and benign subleukemic myelosis. The finding of a higher level of neutrophils having surface receptors revealed in patients with real polycythemia is close to the data obtained in the study of expression of membrane receptors in patients with chronic myeloproliferative diseases and healthy persons.     

The differentiation factor in the bone marrow and blood serum of healthy subjects and in acute leukemia

The factor of differentiation--i.e. systemic morphogen of connective tissue (SMCT)--have been discovered in bone marrow and blood serum of healthy humans. SMCT calls forth the differentiation of mesodermal cell types in early embryonic amphibian cells. These cell types are the following: notochords, muscles, mesothelium, blood cells, mesenchyme. Under the influence of the punctates of bone marrow the frequency of muscle and blood cell appearance is not constant, which might be the consequence of the individual variability of SMCT. Under the influence of bone marrow and blood serum in patients with acute lymphoblastic, monoblastic and myelomonoblastic leukemia the embryonic cells differentiate only into atypical epidermis, which proves the absence of the SMCT activity in the sources used. In some cases under the influence of bone marrow from patients having the same disease the early embryonic cells differentiate into mesodermal cell types, which normally appear under the low concentration of SMCT. This was observed however only in those cases when bone marrow or blood serum have been taken from patients in the state of remission. In patients with remission the correlation is observed between the activity of factor of differentiation in bone marrow and that of blood serum.     

Disturbances of desmofibrinogenesis in pateints suffering from acute myeloblastic leukemia

Significantly decreased levels of blood plasma clotting factor XIII (FSF) were found in the blood of 20 patients with acute myeloblastic leukemia as compared to the control values. It was found that after administration of cytostatic drugs (Cerubidyne and Cytosar) FSF deficiency was higher. This effect was associated with a proteolytic activity detectable in plasma which destroys FSF in vitro. This proteolytic activity was neither inhibited by EACA nor by Trasylol. These results indicate that in patients with acute myeloblastic leukemia beside DIC the treatment with cytostatic drugs as well as the presence of proteases from leukemic cells in the plasma will cause an impairment of transformation of soluble fibrin polymer into insoluble desmofibrin.     

Cholesterol, phospholipids, and fatty acids of normal immature neutrophils: comparison with acute myeloblastic leukemia cells and normal neutrophils.

The lipid composition of immature myeloid cells from the bone marrow of normal persons and myeloblasts from patients with acute myeloblastic leukemia was studied and compared with the lipid composition of normal mature human neutrophils. Total cholesterol, phospholipid, and fatty acid composition was determined on each cell type. The leukemic cells showed decreased total cholesterol and cholesterol-to-phospholipid ratio, increase phosphatidylcholine and phosphatidylinositol, decreased phosphatidylethanolamine, and an increased percentage of unsaturated fatty acids when compared to normal mature neutrophils. A nearly identical pattern was seen in the normal immature myeloid precursors from normal bone marrow. We conclude that the altered lipid composition of acute myeloblastic leukemia cells is related to unexplained factors related to cell age and not to malignancy per se.     

Monoclonal antibodies to the antigen of myeloid cells

ICO-G-2 hybridoma clone was obtained after fusion of spleen cells from BALB/c mice immunized with cells from a patient with acute myelomonoblastic leukemia (AMML) and P3 X 63 Ag8.653 cells, using 50% polyethyleneglycol, molecular weight 1500 KD. The antigen with a molecular weight of 100 KD was present only on polymorphonuclear neutrophils and eosinophils of the peripheral blood. The antigen expression was also found on the majority of myeloid precursors and some nuclear erythroid cells. CFU-GM did not express the antigen. Monoclonal antibodies ICO-G-2 reacted with blast cells of some patients with AML, AMML and CML. The antibodies did not react with cells from patients with AMonL, CMonL, ALL, CLL and LSA. Such pattern of reactivity makes these monoclonal antibodies useful for the differential diagnosis of acute nonlymphocytic leukemias and CML in blast crisis.     

Direct versus cultured preparation of bone marrow cells from 22 patients with acute myeloid leukemia

Summary The 24-h culture of bone marrow from patients with acute myeloblastic leukemia (AML) and acute promyelocytic leukemia (APL) gave more analyzable metaphase cells and improved chromosome morphology compared with direct preparations. Culture increased the proportion of cytogenetically abnormal cells, and in six bone marrows where the direct preparation failed, a result was obtained from the cultured preparation. The culture of bone marrow from patients with APL led to the detection of clones carrying the t(15;17) that were not found in direct preparations. Such sequestered clones were not found in AML and acute myelomonocytic leukemia (AMMoL). Cultured preparations were no better than direct preparations from AMMoL.     

Disturbances of coagulation and fibrinolysis in patients with acute leukemia]

In 30 patients with acute leukemia--18 with myeloblastic acute leukemia, 1 with promyelocytic acute leukemia, 4 with myelo-monocytic acute leukemia, 4 with chronic myelocytic leukemia exacerbation--coagulation and fibrinolysis tests were performed in different stage of the disease. Most of the disorders were noted in the III period of the disease (significant levels of the factors II, IX decrease, clot contractility weakness and platelets count decrease). I in patients with manifestation of haemorrhagic diathesis and in patients without them disturbances in examined tests were similar, but platelets count in patients with bleeding was always significantly reduced. The main reasons of the bleeding in acute leukemias are thrombocytopenia together with the in coagulation factors.     

Comparative ultrastructural morphometrical studies in acute leukemia.

Four hundred paramyeloblasts (from meyloblastic, promyelocytic, monoblastic and lymphoblastic types), isolated from peripheral blood of untreated leukemia patients, were studied by planimetric ultrastructural morphometry. The data of 19 parameters for these four paramyeloblastic types were compared with statistical methods. More central "scattered" heterochromatin was found from this morphometric investigation, i.e. early prophases in the lymphoblastic type of acute leukemia (these cases are more sensitive to therapy). The absolute mean values of the areas of whole cells, areas of the nucleus and nucleolus, areas of the heterochromatin and other indices show that different cell clones will undergo leukaemic transformation.     

Results of randomized trials of immunotherapy for acute leukemia

Summary For more than a decade clinical trials have attempted to define the role of immunotherapy in the treatment of patients with acute leukemia. Based on animal studies which indicated that non-specific immune stimulation had an antitumor effect if the tumor burden was small, the use of immunotherapy during remission in patients with acute leukemia seemed appropriate following the initial report of the success of bacillus Calmette-Guerin (BCG) in prolonging remission duration and survival in acute lymphoblastic leukemia. Therefore a series of randomized clinical trials was initiated to confirm these original observations. In four studies comparing BCG inoculations, with or without allogeneic leukemia cells, and chemotherapy or no therapy, no advantage of immunotherapy was noted. Immunotherapy appeared to be equally as good as chemotherapy. A combination of BCG and chemotherapy showed some advantage in one study, but no advantage was noted in two other studies.In acute myeloblastic leukemia several randomized trials suggested that BCG or one of its derivatives when given alone, in combination with allogeneic cells, or with chemotherapy had a marginal effect in prolonging remission duration and survival when compared to chemotherapy or rno therapy.In conclusion, immunotherapy during remission has marginal activity in acute leukemia.     

Localization of the cellular retinoic acid binding protein (CRABP) gene relative to the acute promyelocytic leukemia-associated breakpoint on human chromosome 15

Summary A human genomic fragment comprising the cellular retinoic acid binding protein (CRABP) gene was isolated. By using a panel of somatic cell hybrids, this gene could be assigned to human chromosome 15. Subsequently, a possible involvement of the CRABP gene in translocation (15;17) (q22;q11) positive acute promyelocytic leukemia (APL) was investigated. Although transposition of the CRABP gene could be demonstrated, we did not observe any gross CRABP rearrangement in a series of primary APL patients, nor in the acute myeloblastic leukemia cell line HL-60. Thus, the observed lack of CRABP expression in these leukemic cells may not be caused by disruption of its gene. CRABP maps to the region 15q22-qter.     

The detection of Fc-IgA receptors on T and non-T,non-B acute leukemic lymphoblasts

Fc-IgA receptors have been described on purified human T-lymphocyte populations from normal individuals. More recently, non-T-cell subpopulations bearing Fc-IgA receptors have also been described. In this study, receptors for the Fc portion of IgA were detected on both T and non-T, non-B leukemic lymphoblasts from 15 patients with acute lymphoblastic leukemia (ALL). From 1.6 to 6.8% of the T lymphoblasts of 7 patients expressed Fc-IgA receptors and 0.5 to 3.9% of the non-T, non-B lymphoblasts of the remaining 8 patients expressed Fc-IgA receptors. The expression of IgA receptors on these cells may reflect the maturational state of these leukemic lymphoblasts. The detection of Fc-IgA receptors on leukemic lymphoblasts suggests that expression of these receptors on lymphoid cells represents an early maturational event.     

Cholesterol regulates VEGFR-1 (FLT-1) expression and signaling in acute leukemia cells

VEGF receptors 1 (FLT-1) and 2 (KDR) are expressed on subsets of acute myeloid leukemia (AML) and acute lymphoid leukemia cells, in which they induce cell survival, proliferation, and migration. However, little is known about possible cofactors that regulate VEGF receptor expression and activation on leukemia cells. Here we show that cholesterol accumulates in leukemia-rich sites within bone marrow of xenotransplanted severe combined immunodeficient (SCID) mice. Therefore, we hypothesized that cholesterol-rich domains might regulate FLT-1 signaling and chemotaxis of acute leukemias. We then showed that FLT-1 accumulates in discrete cholesterol-rich membrane domains where it associates with caveolin-1 and that placenta growth factor (PlGF)/VEGF stimulation promotes FLT-1 localization in such cholesterol-rich domains. Accordingly, FLT-1 localization and its phosphorylation are abrogated by methyl-β-cyclodextrin (MβCD), which removes cellular cholesterol, and by nystatin, an inhibitor of lipid-raft endocytosis. Mechanistically, cholesterol increases FLT-1 expression and promotes PlGF/VEGF-induced leukemia cells viability and also induces VEGF production by the leukemia cells in vitro. Taken together, we conclude that cholesterol regulates VEGF:VEGFR-1 signaling on subsets of acute leukemias, modulating cell migration, and viability, which may be crucial for disease progression. Finally, we provide evidence obtained from human AML samples that primary leukemia cells accumulate significantly more cholesterol than do normal cells and that cholesterol accumulation correlates with disease aggressiveness.     

Vitamin B12 Content of Circulating Leukocytes as an Aid in the Differentiation of the Acute Leukemias

From 25 patients with acute leukemia 116 specimens of leukocytes were assayed microbiologically for total vitamin B₁₂ to determine if variation in vitamin B₁₂ content would help in differentiating the acute leukemias. The mean cell vitamin B₁₂ levels (μμg./10⁸ cells) in the different types of leukemia were: lymphoblastic 464, myeloblastic 1058 and monocytic 200. Cell vitamin B₁₂ levels above the normal range (100-800 μμg./10⁸ cells) are suggestive of myeloblastic leukemia. The only elevated cell vitamin B₁₂ levels comparable to those found in myeloblastic leukemia were in reticulum cell leukemia, and this type of leukemia was not difficult to diagnose morphologically. Blast cells contained more vitamin B₁₂ than mature cells of the same series; there was a significant positive correlation between the percentage of blast cells and cell levels of total vitamin B₁₂ in both lymphoblastic and myeloblastic leukemia.     

Hydrocortisone in culture protects the blast cells in acute myeloblastic leukemia from the lethal effects of cytosine arabinoside

The blast cells in acute myeloblastic leukemia (AML) respond to many of the same regulatory mechanisms that control normal hemopoiesis. These include the growth factors that bind to membrane receptors and steroid hormones or vitamins that have intracellular receptors. We report the effects in culture of the steroid glucocorticoid hydrocortisone on freshly explanted AML blasts from patients and on two continuous AML cell lines. Only small changes in clonogenic cell numbers in suspension cultures were seen in the presence of hydrocortisone. The most striking effect of the hormone was on the sensitivity of blasts cells to cytosine arabinoside (ara-C). In contrast to the response of AML blast cells to retinoic acid, a ligand for intracellular steroid receptors that sensitizes some blast populations to ara-C, hydrocortisone reduced the toxic effects of the drug. The protective action of hydrocortisone was not mediated through the cell cycle since exposure of blasts to hydrocortisone did not affect the percentage of cells in DNA synthesis as measured with the tritiated thymidine (3HTdR) "suicide" technique. The hydrocortisone effect could be demonstrated using a pulse (20 min) exposure protocol. Blasts pulsed with increasing specific activities of 3HTdR showed the usual response pattern with an initial loss in plating efficiency to about 50% of control, followed by a plateau, regardless of whether the cells had been exposed to hydrocortisone. Control blasts exposed to increasing ara-C concentrations gave very similar dose-response curves; in striking contrast, blast cells cultured in hydrocortisone, then pulsed with ara-C did not lose colony-forming ability even though the same population was sensitive to 3HTdR. The hydrocortisone effect was dose and time related; protection from ara-C increased from 10(-8) to 10(-5) M and was seen after 4 hr exposure but required 8 hr to reach a maximum. We conclude that hydrocortisone can protect blasts from the lethal effects of ara-C even while the cells are in active DNA synthesis.     

CD26/DPPIV cell membrane expression and DPPIV activity in plasma of patients with acute leukemia

CD26/DPPIV (dipeptidil peptidase IV) displays an array of diverse functional properties, with a role in the development of several human cancers. This enzyme is found mainly anchored in the membrane of cells although it also has an enzymatically active plasma isoform. The regulation of biological activities of cytokines by DPP IV activity has a potential role in the homeostatic regulation of hematopoiesis. In this study, we analyzed the CD26 antigen cell membrane expression by flow cytometry and the DPPIV activity in plasma of patients of acute leukemia. The results showed that the plasma DPPIV activity is significantly higher in leukemia patients and could be 100% inhibited by Januvia? (Merck Sharp & Dohme) a selective DPPIV inhibitor. Although CD26 expression on immune cells were not leukemia-dependent the analysis of the correlation between CD26 expression and the DPPIV plasma activity were statistically significant (p < 0.01) in acute lymphoid leukemia (B-ALL and T-ALL).     

Stimulant of antibody producers isolated from the supernatants of cell cultures of normal human bone marrow and in various diseases

The biological activity of a stimulant of antibody producers (SAP) isolated from normal human bone marrow was studied and compared with that of a stimulant of antibody producers from the bone marrow of patients with acute myeloblastic leukemia, acute lymphoblastic leukemia, lymphosarcoma, and multiple myeloma. The activity of the SAP from human bone marrow in health was similar to that of analogous transmitters from the bone marrow of other species of the mammals and birds. The activity of the SAP in patients with multiple myeloma was elevated, whereas in patients with acute myeloblastic leukemia, it was lowered.     

Recurrent DNMT3A R882 mutations in Chinese patients with acute myeloid leukemia and myelodysplastic syndrome

Somatic mutations of DNMT3A gene have recently been reported in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). We examined the entire coding sequences of DNMT3A gene by high-resolution melting analysis and sequencing in Chinese patients with myeloid malignancies. R882 mutations were found in 12/182 AML and in 4/51 MDS, but not in either 79 chronic myeloid leukemia (CML), or 57 myeloproliferative neoplasms (MPNs), or 4 chronic monomyelocytic leukemia. No other DNMT3A mutations were detected in all patients. R882 mutations were associated with old age and more frequently present in monoblastic leukemia (M4 and M5, 7/52) compared to other subtypes (5/130). Furthermore, 14/16 (86.6%) R882 mutations were observed in patients with normal karyotypes. The overall survival of mutated MDS patients was shorter than those without mutation (median 9 and 25 months, respectively). We conclude that DNMT3A R882 mutations are recurrent molecular aberrations in AML and MDS, and may be an adverse prognostic event in MDS.