Frequency distribution of DNA in blood and bone marrow cells of children with acute non-lymphatic leukemia using impulse cytophotometry

1. As compared with the peripheral blood lymphocytes of healthy subjects the cells in the mononuclear fraction of peripheral blood and in the bone marrow of children with ANLL show a significant higher S- and G2 + M-phase. 2. A high proliferative activity correlate with a bad prognosis or with reaching no haematological remission. 3. Frequently, aneuploid cell populations will occur in the morphological subtypes M 4 and M 5.     

Impulse cytophotometry studies of bone marrow and blood cells in children with acute lymphoblastic leukemia (ALL). 2. Lymphatic cells in blood

The DNA-content of mononuclear cells of the peripheral blood of infantile and juvenile ALL patients was investigated using Pulse Cytophotometry. The fraction of cells in S- and G2 + M-phase is significantly increased in comparison with samples of healthy probands. The fraction of DNA-synthesising cells (S-phase) of both peripheral blood (mononuclear cells) and bone marrow of leukemia patients cannot be significantly distinguished by mathematical methods. On the other hand, the fraction of cells in later phases of cell cycle (G2 + M-phase) is significant enhanced in the bone marrow in comparison with the peripheral blood. A high correlation was found between the number of leukocytes and fraction of G2 + M-phase cells in the peripheral blood of SR- and MR-patients. No correlation was found between the number of leukocytes and S-phase-fraction. The occurrence of aneuploid cell populations in the mononuclear fraction of peripheral blood in the acute state of ALL could be of importance for prognosis and regime of therapy.     

TPEN selectively eliminates lymphoblastic B cells from bone marrow pediatric acute lymphoblastic leukemia patients

BioMetals - B-cell acute lymphoblastic leukemia (B-ALL) is a hematologic disorder characterized by the abnormal proliferation and accumulation of immature B-lymphoblasts arrested at various stages...     

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.     

Characteristics of DNA replication of the chromosomes of the bone marrow cells in patients with acute leukemia]

A study of the late DNA replication pattern in chromosomes of human acute leucaemia cells revealed a significant diffrence from control. Chromosomes, 2,3 and 4-5 of the acute leucaemia cells finish their DNA replication earlier, and chromosomes 1, 13-15 and 16 later, compared to the control chromosomes. The difference in the pattern of DNA replication between analogous chromosomes of acute leucaemia and donor cells was associated with the discovery of large late-replicating chromatin blocks in the pericentromeric regions of leucaemia cell chromosomes. Some relationship is suggested between the pattern of pericentromeric heterochromatin DNA replication and cell differentiation.     

DNA methylation analysis of bone marrow cells at diagnosis of acute lymphoblastic leukemia and at remission

To detect genes with CpG sites that display methylation patterns that are characteristic of acute lymphoblastic leukemia (ALL) cells, we compared the methylation patterns of cells taken at diagnosis from 20 patients with pediatric ALL to the methylation patterns in mononuclear cells from bone marrow of the same patients during remission and in non-leukemic control cells from bone marrow or blood. Using a custom-designed assay, we measured the methylation levels of 1,320 CpG sites in regulatory regions of 413 genes that were analyzed because they display allele-specific gene expression (ASE) in ALL cells. The rationale for our selection of CpG sites was that ASE could be the result of allele-specific methylation in the promoter regions of the genes. We found that the ALL cells had methylation profiles that allowed distinction between ALL cells and control cells. Using stringent criteria for calling differential methylation, we identified 28 CpG sites in 24 genes with recurrent differences in their methylation levels between ALL cells and control cells. Twenty of the differentially methylated genes were hypermethylated in the ALL cells, and as many as nine of them (AMICA1, CPNE7, CR1, DBC1, EYA4, LGALS8, RYR3, UQCRFS1, WDR35) have functions in cell signaling and/or apoptosis. The methylation levels of a subset of the genes were consistent with an inverse relationship with the mRNA expression levels in a large number of ALL cells from published data sets, supporting a potential biological effect of the methylation signatures and their application for diagnostic purposes.     

Similarities in long-term cultures of blood and bone marrow from patients with acute myelogenous leukemia

Dexter-type long-term cultures (LTC) were initiated with peripheral blood (PB) and/or bone marrow cells from 11 patients with acute myelogenous leukemia (AML), and 2 with myelodysplastic syndrome in blastic transformation. Marrow and PB cells from normal subjects served as controls. Assessment of nucleated cells and clonogenic progenitors in the adherent and nonadherent fractions of LTC revealed active hemopoiesis for greater than 5 wks in 4 of 8 cultures of AML blood, and 4 of 7 of AML marrow. The morphology and kinetics of nucleated cells and progenitors with putative normal (granulocyte-macrophage colony-forming units or CFU-gm), and abnormal (blast) phenotype in LTC from AML blood were similar to those from AML marrow, and adherent cells positive for collagen I and III and vimentin were found in both types of LTC. Growth of CFU-gm colonies ceased by wk 5-8 in AML cultures, significantly earlier than in LTC of normal marrow cells (survival of greater than 10 wks), which may indicate derivation of the CFU-gm from a transformed clone or deficiency of stromal function in the leukemic state. In most AML blood and AML marrow LTCs, growth of abnormal (blast) colonies continued until wk 4-6. This study demonstrates certain similarities of morphology and function between LTC of AML blood and AML marrow cells. LTC may provide a useful model for further analysis of circulating primitive hemopoietic progenitor cells in leukemic states.     

Presence of TT virus DNA in bone marrow cells from hematologic patients

Recent observations suggest that TT virus (TTV), in addition to liver, may also infect bone marrow. In this study, bone marrow samples and sera from 33 patients with haematological disorders and sera from 16 healthy controls were investigated for TTV DNA presence. Altogether TTV DNA sequences were demonstrated in bone marrow cells of 84.84% of patients. Moreover TTV DNA was detected in sera from 72.72% of patients and from 93.75% of controls. N22 sequences amplified from bone marrow cells and serum of 3 patients were analysed, after cloning: all these isolates were of type 2c and 2 or 3 variants were present in each isolate. After single strand DNA degradation, replicative forms were detectable in BM cells. This finding, in addition to the detection of variants similar in the BM and in the serum of the same patient could suggest that BM is a site of TTV replication (or one of the sites) from which the virus is spread in blood.     

Factors in the cryopreservation of bone marrow cells from children with acute lymphocytic leukemia.

Bone marrow cells from 42 children with acute lymphocytic leukemia in remission and 19 normal adults were preserved in liquid nitrogen for periods of up to 50 weeks. Many factors in the process of cryopreservation were investigated in an attempt to optimize the recovery of the bone marrow colony forming cells. The effect of cryopreservation on the cells which produce colony stimulating activity also was investigated. With optimization of this technique, it was observed that approximately 100% of the bone marrow nucleated cells were recovered and approximately 50% of the total colony forming cells were viable after 50 weeks in liquid nitrogen.     

Differences in the localization of Alu-repeats in various chromosomes of peripheral blood cells from normal donors and bone marrow cells from patients with acute leukemia

The dispersion of the Alu-family DNA repeats in phytohemagglutinin-stimulated lymphocytes from peripheral blood of normal donors as well as in nonstimulated bone marrow cells of four patients suffering from acute leukemia was studied by hybridization on metaphase chromosomes in situ. DNA of bacteriophage lambda CAR42 clone containing the insertion of at least 8 copies of Alu-family DNA-repeats and labelled with tritium was used as a probe in hybridization. All patients with acute leukemia had the same pattern of changes in hybridization of the bone marrow cells. It consists of silver grains clustering over 3q26, 8p12, 14q24. The pattern may reflect amplification transposition of Alu-family DNA repeats in the human genome connected with cellular differentiation or malignant transformation of blood cells.     

The immunophenotypic and cytogenetic characteristics of the blast cells in subvariants of acute myeloid leukemia

The results of study of morpho-cytochemical peculiarities, antigenic profile and peripheric blood and/or bone marrow blast cell karyotype of 21 patients with acute myeloid leukemia are presented. Hemopoietic cell immunophenotyping was carried out with the use of cytofluorometer FACScan and chromosome cytogenetic analysis with the use of analyzer "Metascan". It has been shown that in the AML M1 blast cell plasmatic membrane carries pan-myeloid CD33 and CD13 antigens, the last having high density of expression, and the CD38 antigen, which is a myeloid cell-precursor marker. In these patients tetraploidy, being the testimony of karyotype change, has been ascertained. It has been found out that the AML M2 blasts, except pan-myeloid antigens, express the cell proliferation CD71 marker. Blast cell karyotype peculiarities typical for this leukemia sub-variant have been revealed. In patients with the AML M4 in 3 of 6 cases an anomalous karyotype has been found. It has been also shown that the CD14 antigen, and rather its percentage in blast total population, is the differential-diagnostic criterion for the AML M4 and M5.     

Impulse cytophotometric studies of bone marrow and blood cells in children with acute lymphoblastic leukemia (ALL). 1. Bone marrow cells

1. Compared with the peripheral blood lymphocytes of healthy children the cell fractions in the S- and G2 + M-phase are significantly higher in the bone-marrow of those children affected with ALL. This increase was proved in the SR- and MR-group irrespective of the cytomorphological subtype and cytochemical reaction. In patients with relapses the percentage of S-phase cells is below 6%. 2. In about 30% of our patients (mainly in the SR-group with L1-morphology) an initial DNA-aneuploidy was identified. As the risk of relapse is higher in children without DNA-aneuploidy, this symptom has a pretherapeutical-prognostic significance. 3. In the phase of hematological full remission, DNA-frequency distribution correlates with the proliferative activity of normal hematopoiesis. It provides no additional information about the pretherapeutical risk.     

Advantages of peripheral blood stem cells from unrelated donors versus bone marrow transplants in outcomes of adult acute myeloid leukemia patients

Background aimsIn allogeneic stem cell transplantation, unrelated donors are chosen in cases where appropriate related donors are not available. Peripheral blood stem cells (PBSCs) are more often selected as a graft source than bone marrow (BM). However, the prognostic benefits of PBSCs versus BM transplants from unrelated donors have not been carefully examined in patients with acute myeloid leukemia (AML). This study compared outcomes of adult AML patients who underwent unrelated PBSC and BM transplantation, evaluating post-transplant complications, including engraftment, graft-versus-host disease (GVHD) and infections, and determined subgroups of patients who are most likely to benefit from unrelated PBSCs compared with BM transplants.MethodsThe authors analyzed 2962 adult AML patients who underwent unrelated PBSC or BM transplants between 2011 and 2018 (221 PBSC and 2741 BM) using the Japanese nationwide registry database, in which graft source selection is not skewed toward PBSCs.ResultsIn 49.7% of patients, disease status at transplantation was first complete remission (CR1). In 57.1% of cases, HLA-matched donors were selected. Myeloablative conditioning was performed in 75.1% of cases, and anti-thymocyte globulin (ATG) was added to conditioning in 10.5%. Multivariate analyses showed a trend toward favorable non-relapse mortality (NRM) in PBSC recipients compared with BM recipients (hazard ratio [HR], 0.731, P = 0.096), whereas overall survival (OS) (HR, 0.959, P = 0.230) and disease-free survival (DFS) (HR, 0.868, P = 0.221) were comparable between PBSC and BM recipients. Although the rate of chronic GVHD (cGVHD) was significantly higher in PBSC patients (HR, 1.367, P = 0.016), NRM was not increased, mainly as a result of significantly reduced risk of bacterial infections (HR, 0.618, P = 0.010), reflecting more prompt engraftments in PBSC recipients. Subgroup analyses revealed that PBSC transplantation was advantageous in patients transplanted at CR1 and in those without ATG use. PBSC recipients experienced significantly better OS and/or DFS compared with BM recipients in this patient group.ConclusionsThe authors' results confirmed the overall safety of unrelated PBSC transplantation for adult AML patients and suggested an advantage of PBSCs, especially for those in CR1. Further optimization of the prophylactic strategy for cGVHD is required to improve the overall outcome in transplantation from unrelated PBSC donors.     

Kinetics of hybridization to human DNA of heterogeneous nuclear RNA isolated from normal human lymphoblasts and acute leukemia blast cells

Heterogeneous nuclear RNA was extracted from normal PHA-stimulated human lymphocytes and acute myeloid leukemia blast cells. Experiments were performed to determine the hybridization kinetics of these RNAs to human DNA. The best least squares solutions indicate in the hybridization reaction of both normal and leukemic RNA two main components. For leukemic cell RNA the rate constants of both components were significantly different from that of normal cell RNA. In particular, the difference between the rate constants of the second slower component suggests that the slowly hybridizing sequences in leukemic cell RNA have a degree of repetition higher than that of the corresponding sequences of normal cell RNA.     

In vitro study of bone marrow from leukemia patients and patients without hematologic disease

The bone-marrow of 26 patients not affected with hematological diseases and 10 patients with untreated leukemia was investigated according to Dexter in long-term cultures. Survival time and cell content of those long-term cultures started with normal bone marrow were not influenced significantly, if reinoculation was made with autologeneic or allogeneic bone marrow. Even without repeated inoculation, leukemic cells grew for a longer time in long-term cultures than normal bone marrow cells. As far as the outcome of the disease is concerned, no conclusion can be drawn from the duration of cultivating leukemic cells growth.     

Scene segmentation techniques for the analysis of routine bone marrow smears from acute lymphoblastic leukemia patients.

Automated analysis of lymphoblast cell morphology is being evaluated as a basis for predicting the response to therapy of patients with acute lymphoblastic leukemia. A new technique of scene segmentation particularly applicable to the "cluttered" images of cells in routine bone marrow smears is described. Morphologic characteristics of lymphoblasts found in bone marrow smears made at time of diagnosis were measured by an automated, interactive image-processing system using the new scene segmentation technique. These characteristics, on a patient by patient basis, are being compared to remission length and survival data to develop and test new prognostic methods.     

A unique antigen expressed on myeloid cells and acute leukemia blast cells defined by a monoclonal antibody

A murine hybridoma-derived monoclonal antibody, PM-81, was obtained from a fusion of cells of the NS-1 myeloma cell line with cells from a mouse immunized with the HL-60 promyelocytic leukemia cell line. This cytotoxic IgM monoclonal antibody was specific for myeloid cells. Employing indirect immunofluorescence and flow cytometry, we determined that this antibody reacts strongly with normal human granulocytes, eosinophils, and monocytes but not lymphocytes (including phytohemagglutinin-activated lymphocytes), null cells, red blood cells, or platelets. Moreover, the PM-81 antibody reacts with leukemia cells from 19 of 22 patients with acute myelocytic leukemia of all FAB subclasses, three of three patients with common acute lymphocytic leukemia, four of four patients with chronic myelocytic leukemia (CML) in myeloid blast crisis (terminal transferase (TdT)-negative) but did not react with cells from two patients with CML in lymphoid blast crisis (TdT-positive) or five patients with chronic lymphocytic leukemia. The myeloid cell lines HL-60, K562, KG-1, and U937 were all reactive with PM-81. The lymphoid lines CCRF-CEM and Daudi did not express PM-81 but HSB-2 was positive. The PM-81 antigen was absent on myeloid and erythroid progenitor cells as determined by their insusceptibility to complement-dependent lysis. In addition, only PM-81-unreactive cells were capable of colony formation. Furthermore, the PM-81 antibody does not appear to induce modulation of the antigen to which it binds. Thus, this monoclonal antibody appears to fulfill several criteria for clinical utility in the diagnosis and treatment of both acute myelocytic and acute lymphocytic leukemia.