C. H. Van Zantwijk

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.

In vitro drug sensitivity of normal peripheral blood lymphocytes and childhood leukaemic cells from bone marrow and peripheral blood.

In vitro drug sensitivity of leukaemic cells might be influenced by the contamination of such a sample with non-malignant cells and the sample source. To study this, sensitivity of normal peripheral blood (PB) lymphocytes to a number of cytostatic drugs was assessed with the MTT assay. We compared this sensitivity with the drug sensitivity of leukaemic cells of 38 children with acute lymphoblastic leukaemia. We also studied a possible differential sensitivity of leukaemic cells from bone marrow (BM) and PB. The following drugs were used: Prednisolone, dexamethasone, 6-mercaptopurine, 6-thioguanine, cytosine arabinoside, vincristine, vindesine, daunorubicin, doxorubicin, mafosfamide (Maf), 4-hydroperoxy-ifosfamide, teniposide, mitoxantrone, L-asparaginase, methotrexate and mustine. Normal PB lymphocytes were significantly more resistant to all drugs tested, except to Maf. Leukaemic BM and PB cells from 38 patients (unpaired samples) showed no significant differences in sensitivity to any of the drugs. Moreover, in 11 of 12 children with acute leukaemia of whom we investigated simultaneously obtained BM and PB (paired samples), their leukaemic BM and PB cells showed comparable drug sensitivity profiles. In one patient the BM cells were more sensitive to most drugs than those from the PB, but the actual differences in sensitivity were small. We conclude that the contamination of a leukaemic sample with normal PB lymphocytes will influence the results of the MTT assay. The source of the leukaemic sample, BM or PB, does not significantly influence the assay results.

MRNA expression levels of methotrexate resistance-related proteins in childhood leukemia as determined by a standardized competitive template-based RT-PCR method

Drug resistance of leukemic blasts is correlated to event-free survival and might be predicted by mRNA expression of drug resistance-related proteins. Methotrexate (MTX) is an important component in the treatment of childhood leukemia. Mechanisms of MTX resistance include (1) decreased transport via the reduced folate carrier (RFC), (2) altered levels of target enzymes, eg dihydrofolate reductase (DHFR) and thymidylate synthase (TS), (3) decreased ratio of folylpolyglutamate synthetase (FPGS)/folylpolyglutamate hydrolase (FPGH). We designed competitive templates for each of these genes to measure mRNA expression by quantitative RT-PCR and normalized the expression to that of β-actin. T-lineage acute lymphoblastic leukemia (T-ALL), relatively MTX resistant compared to common/preB-ALL, displayed higher mRNA levels of DHFR and TS (three- and four-fold higher, respectively; P < 0.001), while FPGS expression was lower (three-fold, P = 0.006) compared to common/preB-ALL. The ratio of (DHFR × FPGH)/(RFC × FPGS) was more discriminating between T-ALL and c/preB-ALL (eight-fold higher; P < 0.001) than either target independently. Acute myeloid leukemia (AML) cells, considered MTX resistant, expressed two-fold lower levels of FPGS mRNA compared to c/preB-ALL (P = 0.04). The ratio of FPGH/FPGS was more discriminating between AML and c/preB-ALL (four-fold higher; P = 0.001) than either target independently. For the total group of 79 leukemic samples, mRNA expression of DHFR varied 549-fold and paralleled TS mRNA expression (r = 0.80; P < 0.001). Although variations in mRNA expression resembled variations in functional activity, no direct correlations were found for RFC (58-fold variation in mRNA expression), FPGS (95-fold) and FPGH (178-fold). In conclusion, differences in mRNA expression of MTX resistance parameters between leukemic subtypes as detected by competitive RT-PCR are in line with known differences in MTX resistance.

Comparison of the Antileukemic Activity In Vitro of Dexamethasone and Prednisolone in Childhood Acute Lymphoblastic Leukemia

It is generally assumed that prednisolone (PRD) and dexamethasone (DXM) have equal glucocorticoid activity of PRD is given at sevenfold higher doses. Results of clinical studies of childhood acute lymphoblastic leukemia (ALL) suggested that DXM is more potent relative to PRD than assumed. The purpose of this study was to determine the relative antileukemic activity of PRD phosphate and DXM phosphate in 133 untreated childhood ALL samples in vitro, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) assay. There was a marked variation in antileukemic activity of both agents among the patient samples. The median LC50 (drug concentration lethal to 50% of the ALL cells) for PRD phosphate was 3.50 microM, for DXM phosphate 0.20 microM. The individually calculated ratios of the LC50 values for PRD and DXM phosphate showed a large range from 0.7 to >500, with a median of 16.2. This 16-fold difference could not be explained by differences between these glucocorticoids in stability, hydrolysis into unesterified drug, adhesion to the wall of the microculture plates, or protein binding. ALL cells were cross-resistant to PRD and DXM phosphate (correlation coefficient = 0.85, P<0.000001). We conclude that the in vitro antileukemic activity of DXM phosphate is median 16-fold higher than that of PRD phosphate, which contrasts to the generally assumed factor of 7. Based on the higher potency of DXM, and its more favorable pharmacokinetics as reported in the literature, DXM may be preferred to PRD as the glucocorticoid in the treatment of ALL.

Clinical and Cell Biological Features Related to Cellular Drug Resistance of Childhood Acute Lymphoblastic Leukemia Cells

Several clinical and cell biological features, such as sex, age, leukemic cell burden, morphologic FAB type, and immunophenotype, have prognostic value in childhood acute lymphoblastic leukemia (ALL). The explanation for their prognostic significance is unclear, but might be related to cellular drug resistance. We prospectively studied the relation between the above mentioned features with resistance to 13 drugs in 144 childhood ALL samples obtained at initial diagnosis. The MTT assay was used for drug resistance testing. The interindividual differences in drug resistance were very large and exceeded those between the several subgroups. There was generally no significant relation between sex, leukemic cell burden, and FAB type with drug resistance. However, subgroups with a worse prognosis as defined by age (< 18 months and > 120 months at diagnosis) or immunophenotype (pro-B ALL and T-ALL) did show relatively resistant drug resistance profiles as compared to the subgroups with a better prognosis (age 18-120 months, common and pre-B ALL). Within the group of common and pre-B ALL and compared to the intermediate age-group, samples of the younger children were significantly more resistant to daunorubicin, mitoxantrone and teniposide, and samples of the older children were significantly more resistant to prednisolone and mercaptopurine. Pro-B ALL samples were significantly more resistant to 1-asparaginase and thioguanine, and T-ALL samples were significantly more resistant to prednisolone, dexamethasone, 1-asparaginase, vincristine, vindesine, daunorubicin, doxorubicin, teniposide, and ifosfamide, than the group of common and pre-B ALL cases. We conclude that the prognostic significance of age and immunophenotype in particular may be explained, at least partly, by its relation with resistance to certain drugs. The results of this study may be useful for future rational improvements of chemotherapeutic regimens in childhood 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.

A possible role for methotrexate in the treatment of childhood acute myeloid leukaemia, in particular for acute monocytic leukaemia

Acute myeloid leukaemia (AML) is thought to be methotrexate (MTX)-resistant. However, a small study suggested that acute monocytic leukemia (AML-M5) is sensitive to MTX. We measured MTX accumulation/polyglutamylation in 20 AML-nonM5, 37 AML-M5 and 83 common/preB-acute lymphoblastic leukaemia (c/preB-ALL) samples. Membrane transport was determined in 11 childhood AMLs (including 3 AML-M5) and in 25 c/preB-ALL samples. MTX sensitivity was determined in 23 AML-nonM5, 15 AML-M5 and 63 common/preB-ALL samples using the thymidylate synthase (TS) inhibition assay. MTX transport was higher in AML samples compared with c/preB-ALL precluding a transport defect in AML. Accumulation of long-chain polyglutamates MTX-Glu(4-6) was 3-fold lower for AML-nonM5 compared with c/preB-ALL cells (median 268 versus 889 pmol MTX-Glu(4-6)/10(9) cells; P < or = 0.001); for AML-M5 samples, median accumulation of MTX-Glu(4-6) was 0 pmol/10(9) cells (P < or = 0.001). After short-term MTX exposure, AML-nonM5 was 6-fold more resistant to MTX compared with c/preB-ALL cells (2.16 versus 0.39 microM; P < 0.001), while AML-M5 was 2-fold more resistant (P = 0.02). In both AML-nonM5 and AML-M5 cells, MTX resistance was circumvented by continuous MTX exposure (median TSI(50) values: 0.052 and 0.041 microM, respectively) compared with a c/preB-ALL value of 0.066 microM. In conclusion, AML-M5 is relatively sensitive to MTX compared with other AML-subtypes even though polyglutamylation of MTX is poor. Using continuous exposure, AML-nonM5 and AML-M5 cells were at least as sensitive to MTX as c/preB-ALL cells. This report suggests that MTX might be an overlooked drug in the treatment of childhood AML.

Infants with acute lymphoblastic leukemia: no evidence for high methotrexate resistance.

The antifolate methotrexate (MTX) has contributed significantly to the great improvement in overall survival and central nervous system prophylaxis in patients with acute lymphoblastic leukemia (ALL) in the past 50 years. After transport into the cell, MTX is polyglutamylated with multiple glutamate residues to MTX-polyglutamates (MTXPGs), which have superior intracellular retention. Cellular resistance to MTX might contribute to treatment failure in childhood ALL. C/preB-lineage ALL ( 1 year) has a favorable prognosis and is in vitro more sensitive to MTX in the TSIA (short exposure) than T-lineage ALL.1 C/preB ALL also have more efficient accumulation of (long chain) MTX-PGs compared to T-ALL and acute myeloid leukemia.2–4 ALL diagnosed in infants less than 1 year of age is closely associated with a number of biological features, especiallyMLL gene (at chromosome 11q23) rearrangements and the proB (CD10-negative precursor B) immunophenotype, and still have a poor outcome.5 So far, little is known of the pharmacodynamics of MTX in infants with ALL, and the relationship thereof with the other biological characteristics frequent in infant ALL. The question then arises whether the poor prognosis of infants with ALL is associated with cellular resistance to MTX. Lymphoblasts isolated from bone marrow or peripheral blood of 47 infants 1 year with newly diagnosed, untreated ALL from the Dutch Childhood Leukemia Study Group (DCLSG), the German COALL study group, the Berlin–Frankfurt–Münster (BFM) Study Group and the Pediatric Oncology Group cell bank (POG) were used for this portion of the study. The distribution of important clinical parameters within the infants showed a high association with the proB immunophenotype and translocations involving the MLL gene (11q23, determined by karyotype, RT-PCR, and/or Southern blotting), and significantly higher white blood cell counts at presentation, features typical of infants with ALL. Since MTX cytotoxicity on primary ALL cells cannot be measured with the MTT assay, we used the thymidylate synthase (TS) inhibition assay (TSIA), which correlates strongly with IC50 values for MTX obtained for cell lines in the MTT assay.1 These data were expressed as the concentration of MTX necessary to inhibit 50% of the TS activity (TSI50), compared to the controls incubated without MTX. A large range of TSI50 values for MTX was observed for both the short (3 h, followed by 18 h drug-free) and continuous (21 h) exposure conditions. Infant ALL cells did not differ in MTX sensitivity from those of a reference group of 109 common(c)/preB ALL patients older than 1 year, with overlapping ranges and similar median TSI50 values (Table 1, Figure 1a and b). The total in vitro accumulation of MTX and the pharmacologically important long-chain polyglutamates (MTX-PG4–6, analyzed by HPLC) did not differ significantly between the infants and the older c/preB ALL group (Figure 1c and d). Hyperdiploid B-lineage ALL cells accumulate higher levels of MTXpolyglutamates.6 Because hyperdiploidy is found in one fifth of the older children but in no case of infant ALL, a comparison was also made of only those patients in both groups known to have a nonhyperdiploid DNA index, as determined by flow cytometry (hyperdiploidy was defined as a DNA index 1.16–1.35). Infants were as sensitive to MTX in the TSIA compared to non-hyperdiploid c/preB ALL patients 1 year (Table 1). In order to examine the influence of proB immunophenotype and MLL gene rearrangements on infant drug sensitivity, the impact of age was investigated within patients with proB immunophenotype and

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

Abstract 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.

Prognostic Significance of Peanut Agglutinin Binding in Childhood T-Cell Acute Lymphoblastic Leukemia

We previously reported the favorable prognosis associated with positive peanut agglutinin (PNA) binding in childhood T-cell acute lymphoblastic leukemia (ALL), and hypothesized that this may be related to glucocorticoid sensitivity. The purposes of this prospective study involving 43 children with newly diagnosed T-cell ALL were to determine the relation between PNA binding and in vitro resistance to prednisolone (PRD) and dexamethasone (DXM), in vivo response to a systemic PRD monotherapy (plus one intrathecal injection with methotrexate) and to multidrug chemotherapy. PNA positivity was found in 28 (65%) out of the 43 cases. PNA-negative samples in vitro were not more resistant to PRD or DXM than PNA-positive samples. However, in 38 evaluable T-cell ALL patients, nine out of 13 PNA-negative cases were poor responders to PRD in vivo, while all 25 PNA-positive cases were good responders to PRD in vivo (p < 0.0001). PNA-positive patients had a 3.4-fold (95% confidence interval, CI, 1.1-10.4, p = 0.03) lower relative risk of any event than PNA-negative patients. The 3-year probability of event-free survival was 0.81 for PNA-positive patients and 0.40 for PNA-negative patients. In conclusion, PNA positivity is a marker for a subgroup of T-cell ALL patients who are very likely to respond well to systemic PRD monotherapy. In addition, PNA positivity is a favorable prognostic factor regarding event-free survival in childhood T-cell ALL.