Children with Down syndrome (DS) have an increased risk for leukemia. The prognosis for DS acute myeloid leukemia (AML) is better than for non-DS AML, but the clinical outcome of DS acute lymphoblastic leukemia (ALL) is equal to that of non-DS ALL. Differences in prognosis may reflect differences in cellular drug resistance. In vitro drug resistance profiles were successfully investigated on leukemic cells from 13 patients with DS AML and 9 patients with DS ALL and were compared with reference data from 151 non-DS AML and 430 non-DS B-cell precursor (BCP) ALL. DS AML cells were significantly more sensitive to cytarabine (median, 12-fold), the anthracyclines (2-7-fold), mitoxantrone (9-fold), amsacrine (16-fold), etoposide (20-fold), 6-thioguanine (3-fold), busulfan (5-fold), vincristine (23-fold), and prednisolone (more than 1.1-fold), than non-DS AML cells. Compared with DS ALL, DS AML cells were significantly more sensitive to cytarabine only (21-fold). After short-term exposure to methotrexate, DS AML cells were 21-fold more resistant than non-DS AML cells, but no difference was observed after continuous exposure. DS ALL cells and non-DS BCP-ALL cells were equally sensitive to all drugs, including methotrexate. Normal peripheral blood mononuclear cells from DS and non-DS children without leukemia showed highly resistant drug profiles. It was concluded that the better prognosis of DS AML might, at least partially, be explained by a specific, relatively sensitive drug-resistance profile, reflecting the unique biology of this disease. IntroductionChildren with Down syndrome (DS) have an elevated risk for acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). 1 Before the age of 5 years, the risk for AML is 4 times higher than the risk for ALL. 1 When treated with chemotherapy, children with DS also experience more side effects (such as infections and mucositis) from treatment than children without DS. [2][3][4][5][6] Remarkably, children with DS AML have a better prognosis than children with non-DS AML. 2,[7][8][9] In addition, their clinical characteristics differ from those of non-DS AML children: younger age, lower white blood cell count (WBC), and high incidence of French-American-British (FAB) M7. 2,[8][9][10][11] Children with DS ALL have a similar clinical outcome when compared with children with non-DS ALL. 4,5,12,13 Some studies report a lower frequency of T-ALL in DS; others do not confirm this. [3][4][5] Differences in prognosis may reflect differences in cellular drug resistance, pharmacokinetics, or regrowth potential of residual disease. We have shown previously that drug-resistance testing provides clinically relevant information on resistance profiles from specific subgroups of leukemia patients. [14][15][16] In addition, cellular resistance has been shown to predict treatment outcome, independent of other prognostic factors. [17][18][19] In the current study, we analyzed differences in cellular drug resistance between samples from children with DS AML and DS ALL, and we compared t...

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.

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.