Granulopoietic studies in acute lymphocytic leukemia of children.

Studies have been carried out on the levels of serum and urine colony stimulating activity (CSA) and peripheral blood and bone marrow colony forming cell numbers in children with acute lymphocytic leukemia (ALL) during various phases of their disease. These studies have suggested that serum and urine levels of colony stimulating factor are reduced during the inital or relapse phase of the disease compared to levels found during remission. It has also been found that the number of bone marrow colony forming cells is reduced in relapse or before treatment and elevated during remission while the number of peripheral blood colony forming cells is increased during relapse or before treatment and normal during remission. It has also been shown that mixing of serum or leukemic cells with normal human bone marrow cells inhibits colony formation.     

Evaluation of rearrangement of immunoglobulin genes and T cell receptors in determining low numbers of leukemic in children with acute lymphoblastic leukemia]

The samples from blood and/or bone marrow were analysed in 7 children at the time of initial diagnosis or relapse and in 9 children with clinical and hematologic remission of ALL. Molecular genetic methods were more sensitive than morphologic ones in leukemic cells detection: 5 children in remission of ALL demonstrated gene rearrangements. Clinical significance of the presence of low numbers of leukemic cells in remissions is still unknown.     

Pre CFU-F in bone marrow cultures from children with hematopoietic disease including acute leukemia

Stromal precursor cells from bone marrow aspirates of children have been studied in culture. In 7 day liquid cultures normal individuals and patients with acute leukemia in remission grew 110 +/- 50 CFU-F and 100 +/- 40 CFU-F (colony forming unit--fibroblasts) respectively, per 6 X 10(5) buffy coat mononuclear cells. Staining with monoclonal antibodies suggests that stromal cells from CFU-F colonies are fibroblasts. CFU-F colony growth from the bone marrow of patients with active leukemia was low. After cultivation periods of more than 21 days, we observed, in addition, still more immature, clonogenic fibroblast precursor cells, "pre CFU-F", and round cells attached to stromal cells from pre CFU-F colonies. From the round cells, we have passaged pre CFU-F and CFU-GM (colony forming unit--granulocytic, monocytic) in secondary cultures. Our observations are in agreement with the concept that the bone marrow stromal cell matrix serves as a sanctuary for reversibly attached clonogenic cells of both the hematopoietic and fibroblast lineages.     

CFU-gm colony formation of peripheral blood and bone marrow in adult acute leukemia at presentation, during remission, and at relapse

Granulocyte/macrophage colony-forming unit (CFU-gm) formation was studied simultaneously in bone marrow and peripheral blood of 52 previously untreated adult patients with acute non-lymphocytic (ANLL) and 36 with acute lymphoblastic leukemia (ALL). They were followed during induction therapy at monthly intervals while in remission and in 19 ANLL and 22 ALL cases, until relapse. Patients showing a decreased colony number in the marrow but normal or increased colony numbers in the peripheral blood had a high probability of entering remission. Non-responding patients displayed an opposite pattern. The higher the degree of marrow repopulation with granulocytic progenitor cells after induction treatment, the longer remission duration and survival for ANLL patients and the longer survival for ALL patients. CFU-gm formation returned to normal in the early stages of complete remission, but then declined progressively. At ANLL and ALL relapse, colony growth was reduced markedly while cluster formation remained normal. The number of marrow colonies and clusters in ANLL were significantly higher at first and second relapse compared to the growth pattern at first presentation. A similar trend had been observed in ALL, suggesting a selection advantage.     

Ex vivo enrichment of mesenchymal cell progenitors by fibroblast growth factor 2

Bone marrow stromal cells, obtained from postnatal bone marrow, contain progenitors able to differentiate into several mesenchymal lineages. Their use in gene and cell therapy requires their in vitro expansion and calls for the investigation of the culture conditions required to preserve these cells as a stem compartment with high differentiative potential during their life span. Here we report that fibroblast growth factor 2 (FGF-2)-supplemented bone marrow stromal cell primary cultures display an early increase in telomere size followed by a gradual decrease, whereas in control cultures telomere length steadily decreases with increasing population doublings. Together with clonogenic culture conditions, FGF-2 supplementation prolongs the life span of bone marrow stromal cells to more than 70 doublings and maintains their differentiation potential until 50 doublings. These results suggest that FGF-2 in vitro selects for the survival of a particular subset of cells enriched in pluripotent mesenchymal precursors and is useful in obtaining a large number of cells with preserved differentiation potential for mesenchymal tissue repair.     

Characterization of early B lymphocyte precursors present in long-term bone marrow cultures

Lymphoid precursor cells are present in long-term bone marrow cultures (LTBMC), but their differentiation into mature lymphocytes is blocked. A quantitative assay for B cell precursors in LTBMC, which gives a linear relationship between the number of grafted LTBMC cells and the frequency of B cell colony forming units (CFU-B) in the spleen and bone marrow of immunodeficient CBA/N mice 19 days after reconstitution, is described. Characterization of the B cell precursor indicates that this assay is detecting a very early precursor and not a B lymphocyte or a late pre-B cell. This conclusion is based on the observations that a) pre-B cells transformable by Abelson murine leukemia virus are not present in LTBMC by 3 days postrecharge and CFU-B are absent by 6 days postrecharge; b) late B cell progenitors capable of rapid repopulation of irradiated CBA/N mice are not present in LTBMC, since a lag in the kinetics of B cell reconstitution in animals grafted with LTBMC cells is observed compared with fresh bone marrow cells; c) the B cell precursors in LTBMC have high proliferative potential, since they can stably repopulate recipient mice for at least 8 wk postreconstitution and through two serial passages in irradiated CBA/N recipients; and d) the B cell precursors are large, rapidly sedimenting cells as determined by velocity sedimentation. The serial transplantation experiment further shows that a split is often observed between lymphoid and myeloid reconstituting ability of LTBMC cells. The LTBMC B cell precursor may be a pluripotent stem cell or a lymphoid stem cell, although its differentiative potential remains to be determined.     

In vitro proliferation of normal and leukaemic human leukocytes controlled by an inhibitory endopeptide

GI-3, an endogenous inhibitory fraction isolated from leukocytes, selectively inhibits the proliferation of granuloid precursor cells in a non-toxic manner. Its active principle was determined as an acidic chlor-tolidine positive decapeptide [ 3 ]. The in vitro effect on normal and acute leukaemic human bone marrow and blood cells was examined. A dose dependent inhibition by GI-3 of 3H-TdR incorporation into myeloid cells of normal bone marrow was found, the sensitivity of human cells being higher than that of rat cells. The proliferation of the target leukaemic bone marrow and blood cells (AML, AMMoL) was also decreased by the endogenous inhibitor in a dose dependent manner in untreated subjects as well as in patients in remission or relapse. The rate of inhibition of leukaemic of well-known cytostatics (adriamycin hydrochloride, dianhydrogalactitol) applied for comparison. Beyond its direct cytostatic effect, GI-3 could be used in the differential diagnosis of blastic leukaemias, complementing the routine cytochemical methods.     

Compensatory adjustment of hemopoietic stem cell pool of CBA mice after acute intake of 90Sr

It was investigated the functional status of stem cell pool (CFUs) of bone marrow, spleen and peripheral blood in mice (CBA) in early (1-30 days) and late (180-360 days) period after acute intake of 90Sr (29.6 kBq/g). Cumulative dose in red bone marrow due to incorporated 90Sr was 0.98-87.7 Gy. The kinetics, proliferative and differentiative potential of stem hemopoietic cells (CFUs) and productivity of hemopoietic tissues were significantly influenced by dose rate, absorbed dose and degree of suppresssion of bone marrow functions.The obtained results indicated that the sarcomogenous doses of 90Sr (29.6 kBq/g) resulted in realization of compensatory reactions in hemopoietic stem cell pool to support the life ability of irradiated animals: higher proliferative potential of CFUs and its repopulation, redistribution of cell subpopulations during differentiation and activation of spleens hemopoiesis.     

Osteoclast precursors in bone marrow and peritoneal cavity

Osteoclasts differentiate from cells that share some phenotypes with mature macrophages and monocytes, but early precursors for osteoclasts still remain obscure. To characterize osteoclast precursors, using monoclonal anti-c-Fms and anti-c-Kit antibodies, bone marrow cells were separated and the frequency of clonogenic progenitors were measured. Osteoclast precursors in the bone marrow mainly expressed c-Kit and diminished in frequency when they expressed c-Fms. In contrast to bone marrow, the precursors in the peritoneal cavity were enriched with a population of c-Fms⁺. Injection of these antibodies into mice demonstrated that peritoneal osteoclast precursors were sensitive to anti-c-Fms but not to anti-c-Kit antibodies, whereas those in bone marrow only declined in the presence of both antibodies. Meanwhile, c-Fms as opposed to c-Kit played an essential role in the generation of osteoclasts in cultures. We also compared osteoclast precursors with colony forming cells (CFU-M) by a macrophage colony stimulating factor. CFU-M in bone marrow decreased when anti-c-Kit antibody was administered and no CFU-M was detected in peritoneum. In this study, we show differences between proliferative potential osteoclast precursors maintained in bone marrow and peritoneum and between CFU-M and osteoclast precursors. J. Cell. Physiol. 170:241–247, 1997. © 1997 Wiley-Liss, Inc.     

Growth of hemopoietic cells after incubation with VP 16-213

We examined the effect of various concentrations of VP 16-213 (25-125 microM/l, 2-h incubation on normal and complete remission bone marrow from patients with acute leukaemia and on leukaemic blasts. The maximal tolerated dose of the drug for normal bone marrow GM-CFC was between 75 and 100 microM/l whereas that for complete remission bone marrow was distinctly lower. More early stem cells measured by aid of LTBMC were more resistant in normal, but not in every remission bone marrow. We have to examine if these LTBMC results are influenced by a damaged microenvironment by using 2 stage LTBMC. Spontaneous leukaemic cells showed a different, sometimes lower sensitivity to VP 16-213 doses maximally tolerated by normal hemopoietic cells so that the VP 16-213 incubation must not be effective for every leukaemia.     

Characterization of cytokine, growth factor receptor, costimulatory and adhesion molecule expression patterns of bone marrow blasts in relapsed childhood B cell precursor all

Relapse of childhood acute lymphoblastic leukaemia (ALL) comprises a leading challenge of investigation. Characterization of leukaemic cells regarding their potency to express growth factors and surface molecules can provide insight into their aberrant biology. Thus, we analyzed bone marrow blasts from 10 children with relapsed B cell precursor ALL. The gene and protein expression of essential haematopoietic growth factors (IL-2, IL-4, IL-7, IL-10, IL-15, IFN-gamma, G-CSFR), their corresponding receptors as well as the expression pattern of adhesion molecules (ICAM-1, CD58) and costimulatory proteins (CD40, CD40L, B7.1, B7.2, CD28, MHC-I and II) was analyzed by RT-PCR and flow cytometry. Constitutive gene expression was found for IL-7, IL-10, IL-15 and IFN-gamma and their corresponding receptors. Flow-cytometric analysis showed that IL-10R, IL-7Ralpha, IL-4Ralpha and the gamma(c)chain are constitutively expressed, and that some cells bear the G-CSFR. IL-10 and IL-15 protein-producing leukaemic cells were easily detectable. The neoplastic cells mainly lack B7.1, and ICAM-1 is mostly decreased. Furthermore, high CD40, and, surprisingly, CD40L expression could be found. These studies show that ALL cells are likely to be sensitive to many growth factors and some factors are produced by the neoplastic cell itself. The secretion of IL-10 by leukaemic cells, and the absence or downregulation of conventional adhesion and costimulatory molecules might represent an effective mechanism of escape of immune surveillance in relapsed ALL.     

Binding of lectins to human leukaemic cells

The interactions of five different lectins: peanut (PNA), lentil (LEN), wheat germ (WGA), soybean (SBA), Asparagus pea (FBP) with leukaemic cells obtained from 31 children: 25 with acute lymphoblastic leukaemia (ALL) and 6 with acute myeloblastic leukaemia (AML) were examined in this study. The relationship of lectin-binding ability to cells cytomorphological, cytochemical and immunological features and its potential clinical application were investigated. It has been shown that PNA and LEN receptors were found in the majority of blast cells. The SBA reacting cells were found only in few patients and FBP binding was not found in studied ALL and AML cells. There was a clear difference in the WGA binding capacity in ALL cells with L1 and L2 characteristics respectively. No differences were found in PNA. WGA and LEN reactivity between PAS negative and PAS positive leukaemic cells. Only PNA of all studied lectins seemed to differentiate T- from B-ALL blast cells. Only WGA binding of ALL cells showed the positive correlation to the risk index value.     

Acute leukemia in adults: assessment of remission induction with combination chemotherapy by clinical and cell-culture criteria.

Remission induction was assessed by clinical and cell-culture criteria for 65 patients with acute myelogenous leukemia (AML), 11 patients with chronic myelogenous leukemia (CML) in blast crisis and 19 patients with acute lymphoblastic leukemia (ALL). Cyclophosphamide, cytosine arabinoside and vincristine (CAV) therapy resulted in complete remission in 23 of 50 previously untreated patients with AML and in 3 of the 11 patients with CML. Fourteen patients with ALL responded to vincristine-prednisone induction therapy and two to induction therapy with CAV. The median duration of survival of the responding patients was 2.2 years, compared with 4 months for the patients who did not respond to treatment. Granulopoietic colony formation, assessed by assay of colony-forming units dependent on colony-stimulating activity in culture (CFU-C), was abnormal in 37 of 42 bone marrow aspirates from patients with AML before treatement. CFU-C concentration increased when leukocyte-conditioned medium (LCM) was added to the cultures; 13 cultures had normal or elevated CFU-C concentration with LCM. Marrow cells of patients with ALL or CML in blast crisis demonstrated a similar pattern. Serial studies of marrow CFU-C concentration of 31 patients with AML demonstrated a change to a normal pattern with successful remission induction. Results of this study suggest that administration of purified LCM to leukemic patients might increase granulocyte production from potential but unstimulated granulopoietic precursors. This therapy would lessen the probability of death from infection during remission induction.     

Acute lymphoblastic leukemia (ALL) antigens detected with antisera to E rosette-froming and non-E rosette-forming ALL blasts.

Based on the presence or absence of erythrocyte receptors(E) a T cell marker, acute lymphocytic leukemia (ALL), can be divided into E+ALL and E-ALL. We studied cell surface antigens on blasts from 12 children with untreated ALL: eight with E-ALL and four with E+ALL. Heterologous antisera were raised against thymus cells, E+ and E-ALL blasts, appropriately absorbed and tested by immunofluorescence and a radiolabeled antibody assay with normal and leukemic lymphoid cells. By both methods, anti-thymus and anti-E+ALL sera reacted with human thymocytes. Specific binding of anti-E+ALL serum to T antigens was indicated by the fact that a single absorption with thymocytes abolished its binding to allogenic thymocytes, and the reactivity of anti-E+ALL serum with thymus, blood and bone marrow lymphocytes was similar to that of anti-thymus serum. After exhaustive absorption with blood leukocytes, anti-E+ALL and E-ALL sera were negative against normal lymphocytes and bone marrow cells from children with ALL in remission. Anti-thymus and anti-E+ALL sera reacted with blasts from patients with E+ALL, but not with E-ALL. In contrast, anti-E+ALL serum reacted with 40 to 96% of blasts from all children with E-ALL, whereas of the four patients with E+ALL, two were negative and two had the lowest percentage of immunofluorescent cells (10 to 22%). These results were confirmed with the radiolabeled antibody assay. Patients with active E-ALL had cells bearing E-ALL antigen(s) in the peripheral blood and bone marrow, but the number of immunofluorescent cells was lower in blood. Cells reactive with anti-E-ALL serum did not react with thymus cells, blood lymphocytes, remission bone marrow cells, Raji cells, PWM and PHA-induced blasts and CLL cells bearing mIg (uk). These data suggest that the antigen detected on E-ALL blasts by anti-E-ALL serum is neither a HLA-related nor a cell differentiation antigen. Thus, by using antiserum to E+ALL blasts, we have confirmed the presence of a T cell-specific antigen(s) on E+ALL cells. This antiserum did not recognize other leukemia-associated antigens common to E+ and E-ALL. We have also demonstrated an antigen(s) which is regularly expressed on E-ALL blasts and is either not detectable or is present in a lower proportion of E+ALL blasts.     

Immunological detection of residual leukaemic disease in the bone marrow of children with acute lymphoblastic leukaemia

Peripheral blood lymphocytes incubated with tumour cells or extracts may undergo blastogenesis. This is the basis of a technique studied in children with acute lymphoblastic leukaemia (ALL) in childhood in an attempt to predict relapse. Samples of peripheral blood and bone marrow from 82 children with varying degrees of ALL were analysed. Cultures were prepared by incubating a lymphocyte suspension with an autologous bone-marrow suspension. Final ratios of lymphocytes to bone-marrow cells (L: BM) were 1: 1 and 2: 1. Control wells received bone-marrow or lymphocyte suspension only. Cultures were incubated for 72, 96, and 120 hours. All were pulse-labelled with ³H-TdR and radioactivity was measured by scintillation counting. Results were expressed as the stimulation index, calculated by dividing the mean counts per minute (cpm) of wells containing both lymphocytes and bone-marrow cells by the sum of the mean cpm for control wells. If the stimulation index exceeded 1 at 72, 96, or 120 hours at either L: BM ratio a positive response was recorded.Seventy-six children were in clinical remission at the time of testing (group A) and six were in clinical relapse (group B). In group A 24 patients showed stimulation and relapsed later at a mean time of 3·8 months (21 with marrow disease, two with testicular infiltration, and one with lung infiltration). Sixteen patients showed stimulation and had up to 4% blasts in their bone marrow but remained in remission. Nineteen other patients showed a positive response and several factors may have contributed to this: two underwent a “rebound” lymphocytosis after stopping treatment, nine had current or intercurrent infections, two had persistent unexplained bone-marrow lymphocytosis, but six had no causative symptoms and thus their responses were “true false-positives.” Seventeen patients from group A showed no response and remained in remission for a mean of 22·9 months after testing. None of the six children in group B responded, and at testing had 17-85% blasts in their bone marrow.During the study no patient relapsed who had not shown a positive response. The technique merits further study as a guide to the presence of leukaemic cells.     

Effect of thymic humoral factor in vitro on human bone marrow and blood cells from B-, T- and null-cell acute lymphatic leukemia.

The nature of null-cell acute lymphatic leukemia (ALL) was investigated with the aid of a thymic humoral factor (THF), bone marrow cells, and a local xenogeneic graft-versus-host reaction (GVHR). Lymphocytes obtained from the blood and bone marrow of six children with T-cell ALL, five with null-cell ALL, one with perinatal B-cell ALL, one with acute myelocytic leukemia, and one with erythroleukemia were tested for membrane surface markers (E, EAC, and SM Ig); functional activity of T cells was tested by a local GVHR. All of the specimens obtained at the initial presentation showed a lack of functional activity of the lymphocytes. Incubation of null cell and acute myelocytic leukemia (AML) bone marrow with THF led to the acquisition of the characteristics of functional, immunocompetent T cells. No such effect was seen when the bone marrow of T-cell ALL and peripheral blood lymphocytes of B-cell perinatal ALL were incubated with THF. This study demonstrates that the null cell in ALL bone marrow can be differentiated into a T cell whereas the stem cell in AML bone marrow constitutes a pluripotential undifferentiated cell which also can mature into a T cell.     

Proliferative and differentiation potentials of skeletogenic bone marrow colony-forming cells

The clonal nature of bone marrow fibroblast colonies derived from clonogenic bone marrow osteogenic cells (CFUf) was proved by the chromosome analysis. During subsequent passages of multi-colony derived bone marrow fibroblast strains there occurs a pronounced increase in the cell number and in the number of osteogenic units (tested by transplantation in diffusion chambers). Single colony-derived strains are capable of forming bone and cartilage simultaneously. It follows that CFUf or part of them are clonogenic cells with high proliferative potentials and are common precursors for bone and cartilage tissue. Thus, CFUf may be regarded as osteogenic stem cells.     

Autologous responses to human leukaemic cells in mixed leucocyte culture

Summary Cryopreserved leukaemic blasts and remission non-T cells from 22 patients with acute leukaemia (15 lymphocytic, 7 non-lymphocytic) were tested as stimulators of autologous remission T cells and normal allogeneic T cells in primary and secondary MLC. In most cases the autologous response elicited by leukaemic cells was less than or equal to that elicited by remission non-T cells. However, T cells from 2 patients in long-standing first remission from ANLL displayed greater proliferation in response to leukaemic blasts than to remission non-T cells in both primary and secondary MLC. The results are suggestive of sensitization of these 2 patients to leukaemia-specific antigens, but other possible explanations are discussed. Abbreviations used: MLC, mixed leucocyte culture; ANLL, acute non-lymphocytic leukaemia; ALL, acute lymphoblastic leukaemia; AMLR, autologous mixed lymphocyte reaction; NK cells, natural killer cells; MNC, mononuclear cells     

Prognostic significance of neoplastic cell proliferation parameters in human haematological malignancies.

High percentage of neoplastic cells in S, G2 and M phases of cell cycle is unfavourable prognostic sign in human haematological malignancies. In chronic leukaemias (CML and CLL) it is true for peripheral blood leukaemic cells, in non-Hodgkin lymphomas--for lymph node cells, in multiple myeloma--for bone marrow plasma cells. In acute leukaemia results are controversial: some authors found a correlation between proliferation parameters of bone marrow blast cells while others did not. These parameters correlate positively with the rate of complete remission and negatively with its duration. It is concluded that proliferation parameters of neoplastic cells may be used for individual prognosis in patients with haematological tumours especially in combination with other biological and clinical prognostic markers.     

Growth of normal versus leukemic bone marrow cells in long term culture from acute lymphoblastic and myeloblastic leukemias

The ability of the in vitro long-term bone marrow culture (LTBMC) system to impair the survival of leukemic cells and to enhance the growth of normal progenitors has been studied. Bone marrow cells from 19 acute lymphoblastic leukemia (ALL) and 30 acute myeloid leukemia (AML) patients at diagnosis were grown in LTBMC for 4-10 weeks. In half of the cases the leukemic population declined down to undetectable levels and was replaced by putative normal hemopoietic precursors, both in ALL and in AML. In the remaining cases, leukemic cells persisted throughout the culture time and few if any normal hemopoietic cells were detected. These data led us to extend to the lymphoid compartment the previous observation of decreasing leukemic myeloid blasts in LTBMC. The potential of such cultures as an in vitro purging system for autologous bone marrow transplantation in selected poor-prognosis lymphoid malignancies should be explored, as has been done for acute and chronic myeloid leukemias.