Natural killer and lymphokine-activated killer cell functions in chronic myeloid leukemia

Summary The natural killer (NK) and lymphokine-activated killer (LAK) cell activities of peripheral blood lymphocytes from chronic myeloid leukemia (CML) patients in remission and from healthy donors have been studied. Regression analysis to compare both cytotoxic responses in individual donors and the frequency of LAK cell precursors was also carried out. About 42% of CML patients in remission showed low NK activity (less than the mean percentage NK activity of healthy donors — 2 SD) and were categorised as low NK responders. The stage of remission or the drugs used to bring about remission did not influence the NK status. The LAK activity of low NK as well as normal NK responder CML patients was significantly low against the NK-sensitive K562 cell line and the NK-resistant VIP (melanoma) and T-24 (bladder carcinoma) tumor targets, as assessed by linear regression analysis. Allogeneic leukemic cells were more resistant to killing, especially by patients' LAK cells. The frequency analysis of LAK cell precursors revealed a significant reduction in the LAK cell progenitor frequency in CML patients in remission.     

A nine-year cytogenetic follow-up of a patient injected with thorotrast

Summary Chromosomal abnormalities in the peripheral lymphocytes of a Thorotrast case were followed up for almost nine years during which four direct bone marrow observations were made. Chromosomal abnormalities, both Cu type (dicentrics, rings, and acentric fragments) and Cs type (reciprocal translocations, inversions, deletions, duplications, and others), were observed with an average frequency of about 7.5% and 8%, respectively. The frequency of chromosomal abnormalities showed no significant changes during the nine years. Formation of clones of cells with identical chromosomal abnormalities was observed both in bone marrow and peripheral lymphocytes. There were clones common to myeloid and lymphoid cell series, which may be regarded as evidence for the presence of pluripotent stem cells in man. One such common clone had a translocation involving chromosomes 2 and 22. Banding analysis revealed a break in chromosome 22 at the q12 or q12/13 band interface. The frequency of the clone remained fairly constant within 5% for several years and the patient showed no indication of leukemia or any other blood disease. The finding seems to suggest a genetic factor relating to the development of chronic myelocytic leukemia (CML), which generally shows the Ph¹ chromosome resulting from a translocation of chromosome 22 with the break at the q11 or q11/12 band interface. The increase in the number of cells lacking the Y chromosome was observed in our final bone marrow sample, but the phenomenon may be attributed to the age of the patient rather than to the direct effect of radiation. Results of the cytogenetic follow-up study seem to indicate the importance of studies in this direction for a better understanding of radiation effects on human beings.     

Restoration of nonclonal hematopoiesis in chronic myelogenous leukemia with interferon alpha

Studies have shown that recombinant human alpha interferon (rIFN alpha) inhibits the growth of colonies of multipotential stem cells from human bone marrow. This report demonstrates that rIFN alpha inhibits the growth of such colonies from the bone marrow of patients with chronic myelogenous leukemia (CML) to a greater extent than from bone marrow of healthy individuals. It also shows that T lymphocyte colonies subcloned with interleukin 2 (IL-2) from CML mixed colonies were inhibited more by rIFN alpha than were similar colonies subcultured from normal mixed colonies. The report demonstrates that the Ph' chromosome is present in such T cell colonies subcultured from CML mixed colonies. When mixed colonies were grown from CML bone marrow in the presence of rIFN alpha, Ph' negative colonies were observed, whereas no such Ph' negative mixed colonies grew from a similar number of bone marrow cells incubated without rIFN alpha. These observations confirm that T lymphocytes derived from bone marrow stem cells are from the CML clone, and that the inhibition of growth of Ph' positive colonies, by rIFN alpha permits the growth of residual normal stem cells. The disappearance of the Ph-chromosome in subclones of T lymphocytes supports the notion of nonclonal hematopoiesis in patients with CML.     

Detection of B-cell antigen on the blast cells in chronic myeloid leukemia]

The presence of the common antigen on B lymphocytes of healthy donors and myeloblasts of patients with chronic myeloid leukemia in blastic crisis was observed with antimyeloblastic serum in the indirect surface immunofluorescence test. The cytotoxic test showed this antigen in the blastic cells in 27 out of 57 patients with CML BC, in 3 of 11 patients with acute lymphoid leukemia, in 1 of 8 patients with chronic lymphoid leukemia and in 2 of 2 patients with undifferentiated leukemia. The antigen was not found in the peripheral blood cells of healthy donors.     

‘Conservative’ and ‘variable’ clusters of Alu-family DNA repeats in human chromosomes

The distribution of Alu-family DNA repeats (AFRs) in chromosomes of phytohaemagglutinin-stimulated peripheral blood lymphocytes of four normal donors and non-stimulated bone marrow cells of four patients with acute leukemia (ALL and ANLL) was studied by in situ hybridization using DNA of recombinant phage lambda containing multiple inserts of AFR as a probe. Over some chromosome bands (14cen, 16p13, 16cen) from normal donors and from leukemic patients clusters of silver grains were detected. Over other three bands (3q26, 8p11-p12 and 14q24) the clusters were found only in chromosomes from the four acute leukemia patients, and were absent from chromosomes of healthy donors. The results suggest non-random long-range distribution of AFRs in human chromosomes, and somatic variations in the distribution of the repeats.     

Cytotoxic lymphocytes in B-cell chronic lymphocytic leukemia. A flow cytometric study of peripheral blood, lymph nodes and bone marrow.

The occurrence of cytotoxic lymphocyte subpopulations (i.e., CD 16+, CD 57+ and cytotoxic CD 8+) wa studied in the peripheral blood of 18 B-cell chronic lymphocytic leukemia (B-CLL) patients. The absolute numbers of CD 57+, CD 16+ and cytotoxic CD 8+ lymphocytes were increased in the peripheral blood of untreated patients as compared with healthy donors, suggesting a causal relation with the accumulation of malignant B-cells. For 5 B-CLL patients and 5 hematological normal donors, the lymphocyte subpopulations in peripheral blood, lymph nodes and bone marrow were determined. A significant immune response was observed in the lymph nodes of the patients, as reflected by the CD 3+ lymphocytes, which were 1.7-27 times larger in the patients lymph nodes than in their peripheral blood and bone marrow. In contrast, with peripheral blood this was mainly caused by an increase in CD 4+ lymphocytes. The CD 57 lymphocytes in the lymph nodes of the patients had abnormal orthogonal light-scattering signals and an abnormal density of CD 57+ receptors in comparison with their peripheral blood CD 57+ lymphocytes or the CD 57+ lymphocytes in the peripheral blood, bone marrow and tonsils of the hematological normal donors. This study shows that although a significant increase of cytotoxic lymphocytes in the peripheral blood of B-CLL patients is observed, the actual distributions of the non-malignant lymphocytes can be quite different at the actual tumor sites, i.e., bone marrow and lymph nodes.     

The effect of cryopreservation on committed stem cells (CFU-c's) in humans

The effects of different sources of colony-stimulating factor (CSF) and of cryopreservation have been studied on committed stem cells (CFU-c's) in bone marrow aspirates from patients with nonhematological ailments, acute leukemia (AL) in remission, and solid tumors, and in peripheral blood from patients with chronic myeloid leukemia (CML).Results showed significant effects due to the source of CSF and to freezing, colony scores being reduced on human placental-conditioned medium (HPCM) after freezing, whereas they remained the same or were increased on peripheral leukocyte underlayers (PBU). An analysis of the differential morphology of the CFU-c's showed that the ratio of eosinophil to polymor-phonuclear colonies was significantly lower on HPCM and after freezing on both sources of CSF.     

Localisation and distance between ABL and BCR genes in interphase nuclei of bone marrow cells of control donors and patients with chronic myeloid leukaemia

Quantitative measurements of the nuclear localisation of the ABL and BCR genes and the distance between them were performed in randomly oriented bone marrow cells of control donors and patients with chronic myeloid leukaemia (CML). Most ABL and BCR genes (75%) are located at a distance of 20–65% of the local radius from the nuclear centre to the nuclear membrane. A chimeric BCR-ABL gene located on a derivative chromosome 22 resulting from t(9;22)(q34;q11) [the so-called Philadelphia (Ph) chromosome] as well as the intact ABL and BCR genes of patients suffering from chronic myeloid leukaemia are also located mostly in this region, which has a mean thickness of 2 μm in bone marrow cells. We have not found any significant differences in the location of the two genes in the G₁ and G₂ phases of the cell cycle, nor between bone marrow cells and stimulated lymphocytes. Irradiation of lymphocytes with a dose of 5 Gy of γ-rays results in a shift of both genes to the central region of the nucleus (0–20% of the radius distant from the nuclear centre) in about 15% of the cells. The minimum distance between one ABL and one BCR gene is less than 1 μm in 47.5% of bone marrow cells of control donors. Such a small distance is found between homologous ABL and between homologous BCR genes in only 8.1% and 8.4% of cells, respectively. It is possible that the relative closeness of nonhomologous ABL and BCR genes in interphase nuclei of bone marrow cells could facilitate translocation between these genes. In 16.4% of bone marrow cells one ABL and one BCR gene are juxtaposed (the distance between them varies from 0–0.5 μm) and simulate the Ph chromosome. This juxtaposition is the result of the projection of two genes located one above another into a plane, as follows from the probability calculation. Received: 5 September 1996 / Accepted: 15 April 1997     

Isoenzymes of acid phosphatase in blood cells of normal subjects and patients with leukemia (author's transl)]

The activities of acid phosphatases (AP) were measured in leukocytes from patients with chronic myelocytic leukemia (CML), macrophages, granulocytes, in the fractionated mononuclear cells of patients with CML and with hairy-cell-leukemia (HCL) and in the cells from patients with acute leukemia (AL). The lowest activities were found in lymphocytes of normal subjects and of patients with chronic lymphatic leukemia (CLL) and in thrombocytes. Isoenzyme (IsE) 1 was characteristic for thymocytes, IsE 2 for granulocytes, IsE 3 for pathologic blast-cells, lymphocytes and thrombocytes, IsE 4 for macrophages, IsE 5 with components a and b for the mononuclear fraction of patients with HCL. In addition IsE 5 was detected in lymphocytes, macrophages and CLL-cells. In 4 patients with HCL the relative percentage of IsE-5-fraction was slightly greater than the percentage of tartrate resistant cells. In two patients with questionable HCL well marked IsE-5-fractions were recognized but no tartrate resistant cells. In one patient with HCL a relatively high percentage of tartrate resistant hairy-cells and in comparison an inadaquate low IsE-5-fraction was found. These different relations were explained with the more sensitive method of gelelectrophoresis and different affinity of substrates to AP.     

A comparative study of the micronucleus test and chromosome aberrations in PHA-stimulated human lymphocytes

A dose dependence of the number of cells with chromosome aberrations was studied in PHA-stimulated donor's peripheral blood lymphocytes irradiated in vitro with doses of 10-400 cGy. In studying the number of chromosome aberrations and percentage of cells with micronuclei in parallel cultures no correlation was found between these indices within the groups exposed to a similar radiation dose.     

Frequency of acrocentric chromosome associations in a long-term human lymphocyte culture

The frequency of acrocentric chromosome associations (ACA) in the long-term culture of lymphocytes progressively decreases by 25% (on the average) for one mitotic cycle. As a result proliferous lymphocytes after 3-4 divisions contain either no associations or not more than 2 associating acrocentrics. The diversity of the peripheral blood lymphocytes as to the frequency of ACA in the first mitosis is connected with their different proliferative activity in the organism.     

An investigation of Ph1 chromosome in Chronic Myeloid Leukemia patients with different treatment modalities and hematological features

Chronic myeloid leukemia (CML) is characterized by a Ph¹ chromosome that derives through a translocation between chromosomes 9 and 22, i.e., t (9;22). Identifying the Ph¹ chromosome through cytogenetic analysis is an important aspect of CML diagnosis. The aim of this study was to determine the significance of cytogenetic analysis in the diagnosis of CML as well as to find out a relationship between chromosomal abnormalities and CML patients in different stages of treatment. Six CML patients were investigated for this study. The presence of Ph¹ chromosome was detected at different times of treatment using GTG banding on peripheral blood or bone marrow aspirations, and the results were analyzed using cytovision workstation. Hematological features were compared between newly diagnosed patients and patients under treatment. The Ph¹ chromosome was strongly associated with all cases of CML. The regression of Ph¹ chromosomes differed for each patient depending on the treatments and individual response to specific treatments.     

The abl/bcr gene product as a novel leukemia-specific antigen: peptides spanning the fusion region of abl/bcr can be recognized by both CD4<Superscript>+</Superscript> and CD8<Superscript>+</Superscript> T lymphocytes

Chronic myelogenous leukemia (CML) is characterized by a reciprocal translocation leading to the Philadelphia chromosome. Two fusion genes are created by this translocation: bcr/abl and abl/bcr. The fusion regions of both translocation products are unique and strictly limited to leukemia cells, giving rise to potential tumor-specific antigens. Although several studies on the immunogenicity of peptides spanning the bcr/abl fusion region have been reported, little is known about the corresponding reciprocal translocation product abl/bcr. Here we report that synthetic peptides representing the fusion region of the abl/bcr forms a1bb3 and a1bb4 can be specifically recognized by HLA-A2-restricted cytotoxic T lymphocytes from healthy donors. Furthermore, HLA-matched a1bb3-expressing CML cells can be recognized by a1bb3-specific HLA-A2-restricted T cells, indicating natural processing and presentation of abl/bcr protein by leukemia cells. Moreover, a 19-mer peptide encompassing this class I-binding sequence also elicited a1bb3-specific class II-restricted T-cell responses. Thus, both class I- and class II-restricted T-cell responses can be stimulated in healthy donors by abl/bcr peptides in vitro. Because abl/bcr is expressed in the majority of CML patients, it may represent a highly leukemia-specific antigen with potential use in immunotherapy.     

Bone marrow transplantation for leukemia and aplastic anemia: management of ABO incompatibility.

Between February 1971 and October 1980, 34 patients with leukemia or aplastic anemia received bone marrow transplants from HLA-identical siblings whose lymphocytes did not react in a mixed leukocyte culture. The donors of 10 patients were ABO-incompatible, and for five pairs the ABO incompatibility was major. Plasma exchanges followed by a red blood cell exchange transfusion reduced the anti-A titres to 1:4 or less in these patients. The ABO incompatibility had no adverse effect on the results of marrow transplantation. Twenty-two patients, including 16 of the 20 who received their transplant after Jan. 1, 1980, are still living. Seven of the 15 patients with acute leukemia have survived 89 to 466 days, and 4 of the 6 with chronic myelogenous leukemia (CML) have survived 117 to 545 days. Of the 13 patients with aplastic anemia, 11 are alive up to 8 years after transplantation. Marrow transplantation, when possible, is the treatment of choice for young patients with acute leukemia in remission and for patients with aplastic anemia. Marrow transplantation may also prove to be effective in patients with CML.     

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.     

Relationship between chromosome fragility,aneuploidy and severity of the haematological disease in Fanconi anaemia

Fanconi anemia (FA) is a chromosome instability syndrome, characterized by progressive pancytopenia and cancer susceptibility. Other cellular features of FA cells are hypersensitivity to DNA cross-linking agents and accelerated telomere shortening. We have quantified overall genome chromosome fragility and euploidy as well as chromosomes 7 and 8 aneuploidy in peripheral blood lymphocytes from a group of FA patients and age-matched controls that were previously measured for telomere length. The haematology of FA samples were also characterized in terms of whole blood cell, neuthrophil and platelet counts, transfusion dependency, requirement of androgens, cortico-steroids or bone marrow transplantation, and the development of bone marrow clonal cytogenetic abnormalities, myelodysplastic syndrome or acute myeloid leukemia. As expected, a high frequency of spontaneous chromosome breaks was observed in FA patients, especially of chromatid-type. No differences in chromosomes 7 and 8 monosomy, polysomy and non-disjunction were detected between FA patients and controls. The same was true for overall genome haploidy or polyploidy. Interestingly, the spontaneous levels of chromosome fragility but not of numerical abnormalities were correlated to the severity of the haematological disease in FA. None of the variables included in the present investigation (chromosome fragility, chromosome numerical abnormalities and haematological status) were correlated to telomere length.     

Lysis of fresh leukemic blasts by interferon-activated human natural killer cells

In a comprehensive study of 30 leukemia patients, it was found that a measurable fraction of fresh leukemic blasts from 8 of 8 adult patients with chronic myelogenous leukemia (CML) in blast crisis and 10 of 11 pediatric patients with childhood acute lymphocytic leukemia (ALL) were efficiently lysed by human peripheral blood natural killer (NK) cells as measured in 4-hour chromium release assays. The observed lysis of these fresh, noncultured, neoplastic blasts was mediated by a population of interferon-augmentable, FcR-positive, non-adherent large granular lymphoid cells from normal donors, which were also able to kill the 'standard' NK target K562. It was of further interest that all 8 of the patients with blast crisis CML exhibited myeloid type morphology. Furthermore, neoplastic lymphoblasts from 9 of 10 patients with NK-susceptible childhood ALL lacked easily detectable B or T cell markers and were of 'null' cell type. In marked contrast to the lytic susceptibility of fresh leukemic blasts from patients with ALL and CML in blast crisis, fresh neoplastic granulocytes from 5 patients with chronic phase CML (2 of which eventually progressed to myeloid type blast crisis), as well as leukemic blasts from 8 patients with acute myeloid leukemias (AML, AMMoL, and AMoL) were resistant to lysis as mediated by human NK cells from normal donors. The clinical implications of these findings are discussed.     

Clonal Evolution and Blast Crisis Correlate with Enhanced Proteolytic Activity of Separase in BCR-ABL b3a2 Fusion Type CML under Imatinib Therapy

Unbalanced (major route) additional cytogenetic aberrations (ACA) at diagnosis of chronic myeloid leukemia (CML) indicate an increased risk of progression and shorter survival. Moreover, newly arising ACA under imatinib treatment and clonal evolution are considered features of acceleration and define failure of therapy according to the European LeukemiaNet (ELN) recommendations. On the basis of 1151 Philadelphia chromosome positive chronic phase patients of the randomized CML-study IV, we examined the incidence of newly arising ACA under imatinib treatment with regard to the p210BCR-ABL breakpoint variants b2a2 and b3a2. We found a preferential acquisition of unbalanced ACA in patients with b3a2 vs. b2a2 fusion type (ratio: 6.3 vs. 1.6, p = 0.0246) concurring with a faster progress to blast crisis for b3a2 patients (p = 0.0124). ESPL1/Separase, a cysteine endopeptidase, is a key player in chromosomal segregation during mitosis. Separase overexpression and/or hyperactivity has been reported from a wide range of cancers and cause defective mitotic spindles, chromosome missegregation and aneuploidy. We investigated the influence of p210BCR-ABL breakpoint variants and imatinib treatment on expression and proteolytic activity of Separase as measured with a specific fluorogenic assay on CML cell lines (b2a2: KCL-22, BV-173; b3a2: K562, LAMA-84). Despite a drop in Separase protein levels an up to 5.4-fold increase of Separase activity under imatinib treatment was observed exclusively in b3a2 but not in b2a2 cell lines. Mimicking the influence of imatinib on BV-173 and LAMA-84 cells by ESPL1 silencing stimulated Separase proteolytic activity in both b3a2 and b2a2 cell lines. Our data suggest the existence of a fusion type-related feedback mechanism that posttranslationally stimulates Separase proteolytic activity after therapy-induced decreases in Separase protein levels. This could render b3a2 CML cells more prone to aneuploidy and clonal evolution than b2a2 progenitors and may therefore explain the cytogenetic results of CML patients.     

Basic types and vital forms of human peripheral and PHA-stimulated lymphocytes studied in vitro

Natural diversity in peripheral and PHA-stimulated lymphocytes seen in the same donors was studied using digitized streak photo of living cells in observational camera. Cells were monitored for 5-8 h at the superior limit of optical resolution by means of phase-contrast microscopy. Intact lymphocytes were observed in autological blood plasma, and PHA-stimulated lymphocytes were examined in self-conditioned centrifuged growth medium. The majority of intact cells were small- and middle-sized floating lymphocytes with microvilli, and middle-sized caudate lymphocytes capable of stick-slip motion. The lesser part consisted of "spread-eagle" or movable forms of both large granular lymphocytes and middle-sized lymphocytes of several types: narrow-plasm lymphocytes with lamellipodia, wide-plasm lymphocytes without cytoplasmic processes, lymphocytes with single pseudopodia, and lymphocytes with single lobopodia of complex shape. On the contrary, the minor fraction of PHA-stimulated lymphocytes of 3 day old cultures contained floating cells with microvilli or floating cells with microvilli and two pseudopodia, whereas the majority of these lymphocytes were spread-eagle or movable forms of cells of different type. These substrate-adhesive PHA-stimulated lymphocytes had well defined apical and basal cell surfaces, but upon mechanical stress are easily pinched off to become ball-shaped. At least 6 different cell types were distinguished among substrate-adhesive PHA-stimulated lymphocytes, with more than half of these being heavily vacuolated spheroid lymphocytes prone to forming cell clusters. The rest PHA-stimulated lymphocytes were represented by signet-ring lymphocytes with dark or light cytoplasm, narrow-plasm lymphocytes with large prolonged nuclei and lamellipodia, lymphocytes with single lobopodia, and lymphocytes with single spiral structures in the cytoplasm. The spiral structure is 10-11 microns in length and 0.5-0.7 micron in width, being presumably a mitochondrion or a group of butt-joined mitochondria. Since some of the caudate middle-sized lymphocytes also contain this structure, these may be regarded as putative precursors of respective type of PHA-stimulated lymphocytes. Under the conditions of observation, interphase nuclei of all live PHA-stimulated lymphocytes were seen to contain numerous globular or fiber structures of condensed chromatin made of 0.3-0.8 micron beads. These beads are doubtless interphase chromomeres.