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

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

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 ( 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 I-asparaginase and thioguanine, and T-ALL samples were significantly more resistant to prednisolone, dexamethasone, I-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

Favorable prognosis of hyperdiploid common acute lymphoblastic leukemia may be explained by sensitivity to antimetabolites and other drugs: Results of an in vitro study

DNA hyperdiploidy is a favorable prognostic factor in childhood acute lymphoblastic leukemia (ALL). The explanation for this prognostic significance is largely unknown. We have studied whether DNA ploidy was related to cellular resistance to 12 drugs, assessed with the methyl-thiazol- tetrazolium assay, in samples of 74 children with common (CD10+ precursor B- cell) ALL. Sixteen patients had hyperdiploid ALL cells and 58 patients had nonhyperdiploid ALL cells. Hyperdiploid ALL cells were more sensitive to mercaptopurine (median, 9.0-fold; P = .000003), to thioguanine (1.4-fold; P = .023), to cytarabine (1.8-fold; P = .016), and to I-asparaginase (19.5-fold; P = .022) than were nonhyperdiploid ALL cells. In contrast, these two ploidy groups did not differ significantly in resistance to prednisolone, dexamethasone, vincristine, vindesine, daunorubicin, doxorubicin, mitoxantrone, and teniposide. The percentage of S-phase cells was higher (P = .05) in the hyperdiploid ALL samples (median, 8.5%) than in the nonhyperdiploid ALL samples (median, 5.7%). However, the percentage of cells in S-phase was not significantly related to in vitro drug resistance. We conclude that the favorable prognosis associated with DNA hyperdiploidy in childhood common ALL may be explained by a relative sensitivity of hyperdiploid common ALL cells to antimetabolites, especially to mercaptopurine and to I-asparaginase

Prednisolone resistance in childhood acute lymphoblastic leukemia: Vitro-vivo correlations and cross-resistance to other drugs

As an important determinant of response to chemotherapy, accurate measurement of cellular drug resistance may provide clinically relevant information. Our objectives in this study were to determine the relationship between in vitro resistance to prednisolone (PRD) measured with the colorimetric methyl-thiazol-tetrazolium (MTT) assay, and (1) short-term clinical response to systemic PRD monotherapy, (2) long-term clinical outcome after combination chemotherapy within all patients and within the subgroups of clinical good and poor responders to PRD, and (3) in vitro resistance to 12 other drugs in 166 children with newly diagnosed acute lymphoblastic leukemia (ALL). The 12 clinical poor PRD responders had ALL cells that were median 88-fold more in vitro resistant to PRD than 131 good responders (P = .013). Within all patients, increased in vitro resistance to PRD predicted a significantly worse long-term clinical outcome, at analyses with and without stratification for clinical PRD response, and at multivariate analysis (P ≤ .001). Within both the clinical good and poor responder subgroups, increased in vitro resistance to PRD was associated with e worse outcome, which was significant within the group of clinical good responders (P < .001). LC50 values, ie, lethal concentrations to 50% of ALL cells, for PRD and each other drug correlated significantly with those of all other 12 drugs, with an average correlation coefficient of 0.44 (standard deviation 0.05). The highest correlations were found between structurally related drugs. In conclusion, in vitro resistance to PRD was significantly related to the short-term and long-term clinical response to chemotherapy, the latter also within the subgroup of clinical good responders to PRD. There was a more general in vitro cross-resistance between anticancer drugs in childhood ALL, although drug-specific activities were recognized

In vitro cellular drug resistance and prognosis in newly diagnosed childhood acute lymphoblastic leukemia

As an important determinant of the response to chemotherapy, measurements of cellular drug resistance may provide prognostically significant information, which could be useful for optimal risk-group stratification. The objective of this report is to determine the relation between in vitro resistance to 12 drugs, measured with the colorimetric methyl-thiazol-tetrazolium (MTT) assay, and long-term clinical response to chemotherapy in 152 children with newly diagnosed acute lymphoblastic leukemia. At risk-group stratified analyses, in vitro resistance to prednisolone, L-asparaginase, and vincristine were each significantly (P < .01) related to the probability of disease-free survival (pDFS) after combination chemotherapy. The combination of data for prednisolone, L- asparaginase, and vincristine provided a drug-resistance profile with prognostic independent significance superior to that of any single drug or any other factor. The 3-years pDFS was 100% for the group with the most sensitive profile, 20% of all patients, 84% (SE 6%) for the group with an intermediately sensitive profile, 40% of all patients, and 43% (SE 8%) for the remaining group with the most resistant profile (P < .001). In conclusion, the extent of in vitro cellular resistance to prednisolone, L- asparaginase, and vincristine, measured using the MTT assay, was significantly related to the clinical response to combination chemotherapy. Treatment failure in newly diagnosed childhood ALL can be predicted based on cellular drug resistance data

Different cellular drug resistance profiles in childhood lymphoblastic and non-lymphoblastic leukemia: A preliminary report

The better prognosis of acute lymphoblastic leukemia (ALL) than of acute non-lymphoblastic leukemia (ANLL) in children, and the often observed better prognosis of myeloid-antigen (MyAg) negative ALL than of MyAg-positive ALL, may be related to differences in cellular drug resistance. We therefore compared the resistance to 12 drugs of 125 ALL and 28 ANLL samples with the MTT assay. ALL samples were median > 75-fold more sensitive to the glucocorticoids prednisolone and dexamethasone (p < 0.00001), and 2-fold more sensitive to vincristine (p = 0.05) than ANLL samples. Differences for the other drugs were not significant. MyAg-negative ALL samples were more sensitive to glucocorticoids than MyAg-positive ALL-samples (p ≤ 0.04). Prednisolone, and dexamethasone if tested, had a stimulatory effect on leukemic cell survival in 36% of ANLL, but in only 2% of ALL samples (p < 0.0001). Vincristine, and vindesine if tested, had a similar effect in 11% of ANLL, and in 4% of ALL samples (p = 0.11). We conclude that the more favorable response of ALL against ANLL to combination chemotherapy in children may be explained by the higher antileukemic activity of glucocorticoids and of vincristine in ALL, while none of the drugs was more active in ANLL. Similarly, the better prognosis of MyAg-negative ALL than of MyAg-positive ALL may be explained by a relative sensitivity to glucocorticoids. Glucocorticoids and vinca-alkaloids induced leukemia cell proliferation in part of the samples, most frequently in ANLL. The findings may be useful in the design of new chemotherapeutic regimens for ALL and ANLL

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 (PRO) 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

Prognostic significance of peanut agglutinin binding in childhood 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 202 children with newly diagnosed ALL were to determine the relationship between PNA binding and (1) immunophenotype; (2) in vitro resistance to prednisolone (PRD) and dexamethasone and other drugs; (3) clinical response to a systemic PRD monotherapy (plus one intrathecal injection with methotrexate); and (4) multidrug chemotherapy. PNA positivity was more frequent in T cell ALL (65% of 43 cases) than in pro-B (0% of seven cases), common (17% of 106 cases) and pre-B (16% of 45 cases) ALL (P < 0.001). PNA binding was not associated with in vitro resistance to PRD or dexamethasone. However, in 38 evaluable T cell ALL patients, nine of 13 PNA-negative cases were clinically poor responders to PRD, while all 25 PNA-positive cases were good responders to PRD clinically (P < 0.0001). The four clinically poor PRD responders with B cell precursor (BCP)-ALL were also PNA negative. Within T cell ALL, PNA-positive patients had a 3.4-fold (95% CI, 1.1-10.4, P = 0.03) lower relative risk of any event, than PNA-negative patients. Within BCP-ALL, PNA binding was not of prognostic significance. In conclusion, PNA positivity, especially frequent in T cell ALL, is a marker for a subgroup of childhood ALL patients who are very likely to respond well to systemic PRD 'monotherapy'. In addition, PNA positivity is a favorable prognostic factor in T cell ALL

Ex vivo activity of methotrexate versus novel antifolate inhibitors of dihydrofolate reductase and thymidylate synthase against childhood leukemia cells

Leukemic cells of 27 children [14 patients with initial acute lymphoblastic leukemia (iALL), 8 patients with relapsed ALL (rALL), and 5 patients with acute nonlymphoblastic leukemia (ANLL)] were evaluated for their sensitivity to methotrexate (MTX) and five novel antifolate drugs, which have the potential to circumvent MTX resistance. The novel antifolates include a polyglutamatable [edatrexate (EDX)] and a lipophilic (trimetrexate) inhibitor of dihydrofolate reductase and two polyglutamatable inhibitors (ZD1694 and GW1843U89) and one lipophilic inhibitor (AG337) of thymidylate synthase (TS). Drug activity was assessed via the determination of in situ inhibition of TS activity after exposing leukemic cells to antifolate drugs for: (a) 3 h, followed by a 15-h drug-free period; and (b) 18 h of continuous exposure. For human CEM leukemia cell lines with well-defined mechanisms of resistance to MTX, in situ TS inhibition correlated with the growth-inhibitory effects of MTX and the novel antifolates (r = 0.86-0.93; P <0.01). Although a wide interpatient variability in MTX sensitivity was observed within the three leukemia groups, the median drug concentration required to inhibit TS activity to 50% of untreated controls (TSI50) for a 3-h exposure to MTX was similar for iALL and rALL cells but was up to 9-fold higher in ANLL cells. After a 3-h exposure, EDX, ZD1694, and GW1843U89 displayed a markedly (10-150-fold) increased potency over MTX in all leukemia groups with comparable TSI50 values for ANLL and iALL cells. Compared with a 3-h MTX exposure, continuous exposure resulted in lower TSI50 values for iALL (14-fold), rALL (14-fold), and ANLL cells (85-fold). In comparison to MTX, the TSI50 values in these groups were also lower for EDX (1.6-3.5-fold), ZD1694 (2.1-4.3-fold), and GW1843U89 (15-35-fold). On short-term exposure, the lipophilic drugs trimetrexate and AG337 displayed markedly less potency as compared with that of long-term exposure. In conclusion, the efficacy of novel antifolates against childhood leukemia cells can be tested with the in situ TS inhibition assay. These novel antifolates displayed a greater efficacy than MTX against childhood leukemia cells and may have potential for the circumvention of MTX resistance in ANLL cell