Cell proliferation is related to in vitro drug resistance in childhood acute leukaemia.

Bone marrow and peripheral blood samples from 362 patients with acute lymphoblastic leukaemia (ALL) proliferating cell and 90 patients with acute myeloid leukaemia (AML) were analysed for S-phase fractions, Ki67 antigen, and proliferating cell nuclear antigen expression. The S-phase fractions were correlated with in vitro drug resistance to 15 different anticancer agents. Leukaemia cells isolated from bone marrow had higher S-phase fractions than leukaemia cells isolated from peripheral blood (in initial ALL, median values resp. 6.9 and 2.7%, in initial AML resp. 5.3 and 1.3%; both P<0.01). Relapse ALL samples derived from bone marrow showed increased S-phase fractions (median 9.9%) compared with initial ALL samples (median 6.9%; P<0.01). ALL samples obtained at initial diagnosis showed higher S-phase fractions (median 6.9%) and higher Ki67 expression (median 30%) than initial AML samples (median resp. 5.3 and 14%; both P<0.05). The S-phase fractions were not related to white blood cell count, age, or gender. Within initial ALL, the S-phase fraction correlated significantly but modestly strong (rho=0.3-0.5; P<0.05) with sensitivity to antimetabolites (cytarabine, mercaptopurine, thioguanine), L-asparaginase, teniposide, and vincristine. Similar results were found within subgroups of initial ALL (nonhyperdiploid and common/precursor-B-lineage ALL). In relapsed ALL and AML such correlations were not found. In conclusion, cell proliferation differs between leukaemia subgroups and increased proliferation is associated with increased in vitro sensitivity to several anticancer agents in initial ALL.

BCL-2 expression in childhood leukemia versus spontaneous apoptosis, drug induced apoptosis, and in vitro drug resistance.

The antileukemic activity of cytotoxic drugs is increasingly thought to be the result of induction of apoptosis. Several proto-oncogenes have been related to the regulation of this process. In this study we evaluated the relation between bcl-2 expression, spontaneous and dexamethasone (DXM) induced apoptosis, and in vitro resistance to DXM, prednisolone (PRD) and cytarabine (ARA) determined using the total cell kill colorimetric methyl-thiazol-tetrazolium salt (MTT) assay, in childhood acute lymphoblastic leukemia (ALL). Drug resistance was expressed as the LC50 value, the drug concentration lethal to 50% of the cells. Fourty-six samples taken at initial diagnosis (iALL) and 31 samples taken at relapse (rALL) were incubated in culture medium, with and without DXM. Bcl-2 expression and apoptosis were measured flowcytometrically, the latter using DNA histogram analysis. Bcl-2 expression was 1.4 fold higher in rALL than in iALL (p = 0.008). Both spontaneous and DXM induced apoptosis increased significantly from 0 to 48 hours (in up to 71%, 81% of the cells respectively). Bcl-2 expression was inversely correlated with the extent of spontaneous apoptosis after 24 hours in iALL (r = -0.40, p = 0.05). Relapsed samples, but not samples obtained at presentation, expressing high levels of bcl-2 displayed increased resistance to drug induced apoptosis (r = -0.63, p = 0.02). In iALL high bcl-2 expression appeared to be related to low LC50 values of ARA. No correlations were found for DXM or PRD. In conclusion, DXM excerts its cytotoxic effect at least partly by means of induction of apoptosis. Bcl-2 inhibits drug induced apoptosis in rALL. However in iALL bcl-2 expression is not associated with increased in vitro drug resistance, nor with increased resistance to drug induced apoptosis.

Effects of interleukin 3, interleukin 7, and B-cell growth factor on proliferation and drug resistance in vitro in childhood acute lymphoblastic leukemia.

Growth factors have been reported to enhance the cytotoxicity of anticancer agents. In our study we investigated the capacities of interleukin 3 (IL-3), interleukin 7 (IL-7), low-molecular-weight B-cell growth factor (lmw-BCGF), and IL-3 + 7 to induce proliferation and to modulate the drug resistance of childhood acute lymphoblastic leukemia (ALL) cells. Proliferation was assessed with the methyl-thiazole-tetrazolium (MTT) assay and other parameters. Cellular resistance to cytarabine, thioguanine, and prednisolone was measured using the MTT assay. In 19 samples containing >90% leukemic cells the proliferative response and the modulation of drug resistance was markedly heterogeneous between patient samples and between growth factors. All growth factors were able to stimulate proliferation significantly after 5 days of culture. lmw-BCGF was the most potent growth factor in this respect. Cytotoxicity of cytarabine and thioguanine was significantly increased by IL-7, that of thioguanine by IL-3 as well. IL-7 enhanced the cytotoxicity of thioguanine significantly more than IL-3 and lmw-BCGF and that of cytarabine more than IL-3. Cytotoxicity of prednisolone was not significantly influenced by any growth factor. In individual cases, growth factors reduced the cytotoxicity of the drugs. IL-3 + 7 did not add activity to the most potent single growth factor in both proliferation and drug resistance measurements. This study shows that IL-3, IL-7, and lmw-BCGF generally induce and occasionally inhibit proliferation of ALL cells. Furthermore, they may either increase or decrease cytotoxicity of anticancer drugs. This heterogeneous response to growth factors concerning induction of proliferation and modulation of drug resistance should be taken into account in their clinical use.

Mononuclear cells contaminating acute lymphoblastic leukaemic samples tested for cellular drug resistance using the methyl-thiazol-tetrazolium assay.

The methyl-thiazol-tetrazolium (MTT) assay is a drug resistance assay which cannot discriminate between malignant and non-malignant cells. We previously reported that samples with > or = 80% leukaemic cells at the start of culture give similar results in the MTT assay and the differential staining cytotoxicity assay, in which a discrimination between malignant and non-malignant cells can be made. However, the percentage of leukaemic cells may change during culture, which might affect the results of the MTT assay. We studied 106 untreated childhood acute lymphoblastic leukemia (ALL) samples with > or = 80% leukaemic cells at the start of culture. This percentage decreased below 80% in 28%, and below 70% in 13%, of the samples after 4 days of culture. A decrease below 70% occurred more often in case of 80-89% leukaemic cells (9/29) than in case of > or = 90% leukaemic cells at the start of culture (5/77, P = 0.0009). Samples with < 70% leukaemic cells after culture were significantly more resistant to 6 out of 13 drugs, and showed a trend towards being more resistant to two more drugs, than samples with > or = 80% leukaemic cells. No such differences were seen between samples with 70-79% and samples with > or = 80% leukaemic cells after culture. We next studied in another 30 ALL samples whether contaminating mononuclear cells could be removed by using immunoamagnetic beads. Using a beads to target cell ratio of 10:1, the percentage of leukaemic cells increased from mean 72% (s.d. 9.3%) to mean 87% (s.d. 6.7%), with an absolute increase of 2-35%. The recovery of leukaemic cells was mean 82.1% (range 56-100%, s.d. 14.0%). The procedure itself did not influence the results of the MTT assay in three samples containing only leukaemic cells. We conclude that it is important to determine the percentage of leukaemic cells at the start and at the end of the MTT assay and similar drug resistance assays. Contaminating mononuclear cells can be successfully removed from ALL samples using immunomagnetic beads. This approach may increase the number of leukaemic samples which can be evaluated for cellular drug resistance with the MTT assay or a similar cell culture drug resistance assay.

Expression of 5'-nucleotidase (CD73) related to other differentiation antigens in leukemias of B-cell lineage.

Ecto-5'nucleotidase (5'NT; CD73) expression was studied with a monoclonal antibody (7G2) and a radiochemical assay and compared with the expression of other antigens in B-cell-lineage leukemias on cells from 100 leukemic patients and two cell lines. A B-cell origin was confirmed by the expression of CD19 and HLA-DR. Four stages of B-cell leukemias were defined: stage I (pro-B) as CD10-, cytoplasmic mu- (c mu-), surface Ig- (sIg-); stage II (cALL) as CD10+/c mu-/sIg-; stage III (pre-B) as CD10+ or -/c mu+/sIg-; and stage IV (B) as CD10-/c mu-/sIg+. A linear correlation was found between immunohistochemical and radiochemical determination of 5'NT (r = .86). 5'NT expression was low in T-cell leukemias and stage I, high in stages II and III, and low again in stage IV of B-cell leukemias. 5'NT expression was not related to c mu, CD20, CD21, CD22, CD34, and terminal deoxynucleotidyl transferase (TdT) expression, but was significantly related to CD10 and inversely related to kappa/lambda expression. However, the 5'NT activity in CD10+ leukemias (stages II and III) shows a very wide range. Within the group of CD10+ leukemias no differences were detected between 5'NT+ and 5'NT- cells in their expression of other B-cell antigens. We conclude that the place of 5'NT in leukemias corresponding to early stages of B-cell development has been characterized. 5'NT is expressed in CD10+ stages and decreases before the expression of sIgs. Future studies should make clear whether a high expression of this enzyme in CD10+ stages is a normal maturation phenomenon or a malignant phenomenon.

In vitro drug sensitivity of cells from children with leukemia using the MTT assay with improved culture conditions.

The knowledge about drug resistance in childhood leukemias and acute lymphoblastic leukemia (ALL) in general is limited. This is because of the lack of a suitable in vitro drug sensitivity assay, which is in part due to low in vitro ALL cell survival. We recently adapted the highly efficient 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay to test cells from ALL patients and showed that its results were comparable with those of the DiSC assay, up to now the most valid but laborious assay. In this study, in vitro drug sensitivity was assessed in cells from 82 children with leukemia, 79 of whom had ALL, with the MTT assay. Dose response curves were obtained for 6-mercaptopurine, 6-thioguanine (6-TG), prednisolone (Pred), daunorubicin (DNR), vincristine (VCR), cytosine arabinoside (Ara-C), L-asparaginase (L-Asp), mafosfamide, and mustine. A cytotoxic effect of methotrexate could be detected in only a few cases. Large interindividual differences in drug sensitivity were detected. Compared with leukemia cells from newly diagnosed patients, leukemia cells from relapsed patients were significantly more in vitro resistant to 6-TG, Pred, Ara-C, mafosfamide and mustine but not to DNR, VCR, and L-Asp. Improvements of culture medium and methods to increase MTT reduction were studied. From 10 components tested, addition of insulin and bovine serum albumin to serum-containing medium improved ALL cell survival. Addition of succinate did not increase the amount of MTT reduction. We conclude that the in vitro MTT assay highly facilitates large-scale studies on drug resistance of ALL patients that can lead to rational improvements in existing treatment protocols.

Immunological phenotype of lymphoid cells in regenerating bone marrow of children after treatment for acute lymphoblastic leukemia.

Bone marrow samples from 8 children treated for acute lymphoblastic leukemia (ALL) were investigated at cessation of cytostatic treatment and during 18 months thereafter. The course of the percentage of lymphoid cells and characterization of these cells by means of monoclonal antibodies, peanut agglutinin (PNA) binding and S-phase determination are shown. The percentage of lymphocytes rises in the first 1.5 months, followed by a non-significant decline. The percentage of cells in S-phase is higher at 0 months than at 6, 15 and 18 months. The percentage of T-cells does not change significantly. In the first 1.5 months a sudden rise in the percentage of common-ALL-antigen (cALLA)-positive lymphocytes occurs. The number of B-cells rises to a peak at 6 months. PNA positively increases to a maximum at 3 months and is correlated with positivity for markers of the B-cell lineage. The percentages of B-cells, cALLA-positive, and PNA-positive lymphocytes do not change significantly after they reach their maximum values and are still high at 18 months. Our results show that after cessation of chemotherapy for ALL a lymphoid cell regeneration occurs in the bone marrow consisting of cells of the B-cell lineage; many of these are cALLA-positive, but are discernible from their malignant counterparts by PNA-positivity.

Bone marrow mitotic index: a methodological study.

The mitotic index (MI) of bone marrow specimens is assessed in 3 different ways: method I determines the number of mitotic figures per 1,000 nucleated cells; method II counts the number of mitoses seen per 1,000 nucleated cells belonging to the proliferative pool, and method III is the same as method I, but excludes all lymphoid cells. 30 Giemsa-stained bone marrow smears from 6 children were screened by 3 independent investigators. MI of method II is found to be approximately twice as high as the MI of method I. The results of method III indicate that the size of the lymphoid population introduces a bias, which renders method I less reliable. It is concluded that method II is the method of choice for a reliable assessment of bone marrow MI.