FLT3 is a receptor tyrosine kinase involved in the proliferation and differentiation of hematopoietic stem cells. FLT3 internal tandem duplications (FLT3/ITDs) are reported in acute myeloid leukemia (AML) and predict poor clinical outcome. We found FLT3/ITDs in 11.5% of 234 children with de novo AML. FLT3/ITD-positive patients were significantly older and had higher percentages of normal cytogenetic findings or French-American-British (FAB) classification M1/M2 and lower percentages of 11q23 abnormalities or FAB M5. FLT3/ITD-positive patients had lower remission induction rates (70% vs 88%; P ‫؍‬ .01) and lower 5-year probability rates of event-free survival (pEF) (29% vs 46%; P ‫؍‬ .0046) and overall survival (32% vs 58%; P ‫؍‬ .037). Patients with high ratios (higher than the median) between mutant and wild-type FLT3 had significantly worse 2-year EFS rates than FLT3/ITD-negative patients (pEFS 20% vs 61%; P ‫؍‬ .037), whereas patients with ratios lower than the median did not (pEFS 44% vs 61%; P ‫؍‬ .26). FLT3/ITD was the strongest independent predictor for pEFS, with an increase in relative risk for an event of 1.92 (P ‫؍‬ .01). Using an MTT (methyl-thiazol-tetrazolium)-based assay, we studied cellular drug resistance on 15 FLT3/ITD-positive and 125 FLT3/ITD-negative AML samples, but we found no differences in cellular drug resistance that could explain the poor outcomes in FLT3/ ITD-positive patients. We conclude that FLT3/ITD is less common in pediatric than in adult AML. FLT3/ITD is a strong and independent adverse prognostic factor, and high ratios between mutant and WT-FLT3 further compromise prognosis. However, poor outcomes in FLT3/ITDpositive patients could not be attributed to increased in vitro cellular drug resistance. (Blood. 2003;102:2387-2394)

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...

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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.

Cellular drug resistance is thought to be an important cause of the poor prognosis for children with relapsed or refractory acute lymphoblastic leukemia (ALL), but it is unknown when, to which drugs, and to what extent resistance is present. We determined in vitro resistance to 13 drugs with the MlT assay. Compared with 141 children with initial ALL, cells from 137 children with relapsed ALL were significantly more resistant to glucocorticoids, L-asparaginase, anthracyclines, and thiopurines, but not to vinca-alkaloids, cytarabine, ifosfamide, and epipodophyllotoxins. Relapsed ALL cells expressed the highest level of resistance to glucocorticoids, with a median level 357-and >24-fold more resistant to prednisolone and dexamethasone, respectively, than ini-OWADAYS, using intensive front-line multiagent chemotherapy along with improved supportive care, more than 95% of children with acute lymphoblastic leukemia (ALL) can achieve a complete remission (CR), of whom 70% will remain in continuous CR and be considered cured.'-3 These results show the tremendous improvement in the development of more effective chemotherapy regimens for childhood ALL, which was once a fatal d i~e a s e .~ Nevertheless, current protocols fail in the remaining 30% of the children with newly diagnosed ALL, with bone marrow relapses representing the most common treatment failures.'-3 Second remissions can be induced with intensified chemotherapy in more than 90% of the children with ALL whose disease relapsed while they were on modem protocols, but their long-term prognosis is p~o r . ' .~.~ Long-lasting second hematologic remissions can be expected in about 10% of the children with early relapses and in up to 30% of those with late relapses, despite even more intensive second-line therapy that includes the effective front-line drugs used in an alternative Knowledge about the nature of relapsed ALL is limited, but it is assumed that regrowth of drug-resistant leukemic cells plays an important role.6 It is unknown to which drugs and to what extent relapsed leukemic cells express resistance, mainly because a suitable drug-resistance assay was lacking until recently. The poor growth capacity of ALL cells in vitro, limiting the use of long-term clonogenic assays, could be circumvented with the introduction of so-called shortterm cell culture drug-resistance assay^.^ The 3-[4,5-dimethyl-thiazol-2,5-diphenyl] tetrazolium bromide (MTT) assay, first described by Black and Speer in 1954' and revised by Mosmann in 1983: has been adapted by us for testing ALL cells."." The 4-day semiautomated MTT assay is an efficient tool for large-scale drug-resistance testing and results showed a good correlation with the prognosis in childhood We present here the results of in vitro drugresistance testing on samples from 141 children with relapsed or refractory ALL.