Summary The methyl-thiazol-tetrazolium (MTU) assay is a drug resistance assay which cannot discriminate between malignant and non-malignant cells. We previously reported that samples with > 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 leukaemia (ALL) samples with > 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 > 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 > 80% leukaemic cells. No such differences were seen between samples with 70-79% and samples with > 80% leukaemic cells after culture. We next studied in another 30 ALL samples whether contaminating mononuclear cells could be removed by using immunomagnetic 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.

Alternative splicing of the primary glucocorticoid receptor (GR) transcript, resulting in glucocorticoid receptor alpha GRa, glucocorticoid receptor beta GRb and glucocorticoid receptor gamma GRc, may influence glucocorticoid (GC) resistance in childhood leukemia. To test this hypothesis, we determined GRa/b protein and GRa/b/c mRNA expression levels in 43 initial acute lymphoblastic leukemia (iALL), 10 initial myeloid leukemia (iAML), 11 relapsed ALL (rALL) samples and one rAML sample. The results were correlated with in vitro GC resistance. GRa mRNA correlated with protein expression (q ¼ 0.39-0.56, Po0.05), but the protein to mRNA ratio was median 2.2-fold lower in rALL than in iALL (Po0.05). GRb mRNA was median 137-fold lower than GRa mRNA and correlated with GRa mRNA expression (q ¼ 0.71, Po0.0001). GRb could not be detected at the protein level. GRc accounted for a median of 2.8% (range 0.95-7.4%) of all GR transcripts. GRa (protein and mRNA) and GRb (mRNA) expressions or GRa/GRb ratios did not correlate with in vitro GC resistance in iALL, but GRc (mRNA) did (q ¼ 0.52, P ¼ 0.007). These results suggest that GRb is not involved in GC resistance in childhood leukemia. The association between GRc expression and in vitro GC resistance in iALL and the decreased protein/mRNA ratio in rALL, a subgroup resistant to GCs, warrants further exploration.

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A preliminary study has linked raised blast glutathione levels with chemoresistance in acute myeloid and lymphoblastic leukemia in adults and children. In this study, therefore, the relationship between leukemic blast glutathione levels and prognosis in childhood acute lymphoblastic leukemia (ALL) was investigated. A total of 77 childhood ALL samples were analyzed, 62 at initial presentation and 15 at relapse. A 20-fold interindividual variation in glutathione levels at presentation (median, 6.54 nmol/mg protein; range, 1.37 to 27.9) was demonstrated. The median level in T-lineage ALL was 2.3-fold higher than in B-lineage ALL (Mann-Whitney test, P < .0001). There was a significant correlation between presenting white cell count (WBC) and glutathione level (Spearman rank correlation coefficient, ‫؍‬ 0.45, P ‫؍‬ .001). A high DNA index correlated with low glutathione levels (MannWhitney test, P ‫؍‬ .013). There was no significant relationship between glutathione levels and in vitro drug sensitivity. Patients with glutathione levels above the median had a significantly greater risk of relapse (log-rank test statistic, 5.55; P ‫؍‬ .018), and the overall survival rate was significantly reduced (logrank test statistic, 4.38; P ‫؍‬ .04). Multivariate analysis demonstrated that glutathione concentration was of independent prognostic value when assessed in conjunction with age, gender, WBC, and immunophenotype. The association of elevated blast glutathione levels with an increased risk of relapse suggests that glutathione-depleting agents may be of therapeutic value in patients who present with a high WBC. (Blood. 2001;97:393-398)