Abstract
Aim: The analysis of the individualized tumor response testing (ITRT) at first and subsequent relapse in children with acute myeloid leukemia (AML). Patients and Methods: A total of 76 pediatric AML samples underwent ITRT for up to 21 drugs. Results: No significant differences between ITRT at first and subsequent relapse were found, and no drug was found, for which significantly higher resistance of myeloblasts was observed at subsequent relapse, when compared to first relapse of AML. For most tested drugs, patients with relapse had higher IRTR than those with de novo AML. The median relative resistance value between patients with relapse and those with de novo diagnosis for all 21 drugs tested was 1.6. Samples of relapsed AML samples were significantly more resistant to: Idarubicin (1.8-fold), etoposide (5.9-fold), cytarabine (1.7-fold), fludarabine (3.7-fold) and busulfan (4.3-fold). Conclusion: ITRT in relapsed AML is higher in comparison to that at initial diagnosis, while no differences in ITRT between first and subsequent relapse of AML were found.
- Ex vivo drug resistance
- individualized tumor response testing
- acute myeloid leukemia
- children
- relapse
- minimal residual disease
The current cure rate achieves 80% of long-term survival in childhood acute lymphoblastic leukemia (ALL) and 50-60% in acute myeloblastic leukemia (AML) (1-3). Inspite of continuous progress in therapy of acute leukemia, relapses still occur frequently, both in children and adults. The results of therapy, in relapse of AML in childhood, do not exceed 30% and are very poor in subsequent relapses (2, 3). Failure in therapy is dependent on three factors: pharmacokinetic resistance, cellular drug resistance and residual disease (4). Cellular drug resistance can be defined as cellular insensitivity to drugs reaching the cell.
Leukemia cells of children with de novo AML show higher ex vivo resistance (called individual tumor resistance testing, ITRT) to most drugs, when compared to cells of ALL at diagnosis (5, 6). However, little is known regarding drug resistance in relapse of AML in children. There is only a limited number of studies published to date (7, 8). It has been shown that children with relapsed AML were ex vivo, a median of 3-fold more resistant to cytarabine than the initial AML group, however the group of patients was relatively small; in the group of poor responders to chemotherapy, 3-fold higher resistance to cytarabine was observed in comparison to the group of good responders (5). In our study, we aimed to compare ex vivo drug resistance (ITRT) at diagnosis and at first and subsequent relapses in a group of patients with AML.
Patients and Methods
Patient samples. A total number of 76 leukemia samples were included in the study, including 44 samples obtained from children at initial AML diagnosis, 22 at first relapse of leukemia, and 10 obtained at subsequent leukemic relapse. Detailed patient characteristics, with respect to the phase of the disease, are presented in Table I. The distribution of patients between these three groups was comparable.
Individual tumor response testing. ITRT was tested by 3-4,5-dimethylthiazol-2-yl-2,5-difenyl tetrazolium bromide (MTT) assay, as described previously (9). The drug concentration that was inhibitory to 50% of the cells (IC50) was calculated from the dose-response curve and was used as a measure of ex vivo drug resistance for each sample. The relative resistance (RR) between groups analyzed for each drug was calculated as the ratio of the mean values of the IC50 of the respective groups for this drug. Only patients who had a successful MTT assay at-diagnosis were included in the study.
Drugs. The following 21 drugs and concentrations were used: Prednisolone (Fenicort; Jelfa, Jelenia Gora, Poland), tested concentration range 0.007-250 μg/ml; dexamethasone (Dexamethasone; Jelfa), 0.0002-6 μg/ml; vincristine (Vincristine; Eli-Lilly, Indianapolis, IN, USA), 0.019-20 μg/ml; idarubicin (Zavedos; Farmitalia, Milan, Italy), 0.0019-2 μg/ml; daunorubicin (Daunorubicin; Rhone-Poulenc-Rhorer, Paris, France), 0.0019-2 μg/ml; doxorubicin (Doxorubicin; Farmitalia, Milan, Italy), 0.0078-8 μg/ml; mitoxantrone (Mitoxantrone; Jelfa), 0.001-1 μg/ml; etoposide (Vepeside; Bristol–Myers Squibb, Princeton, NJ, USA), 0.048-50 μg/ml; L-asparaginase (Medac; Medac, Hamburg, Germany), 0.0032-10 IU/ml; cytarabine (Cytosar; Pharmacia & Upjohn, Kalamazoo, MI, USA), 0.0097-10 μg/ml; fludarabine (Fludara; Schering, Berlin, Germany), 0.019-20 μg/ml; cladribine (Biodribin; Bioton, Warsaw, Poland), 0.0004-40 μg/ml; treosulfan (Ovastat; Medac, Hamburg, Germany), 0.0005-1 μg/ml; thiotepa (Thiotepa; Lederle, Greifswald, Germany), 0.032-100 μg/ml; melphalan (Alkeran; Glaxo, Parma, Italy), 0.038-40 μg/ml; 4-HOO-cyclophosphamide (Asta Medica; Hamburg, Germany), 0.096-100 μg/ml; 4-HOO-ifosfamide (Asta Medica), 0.096-100 μg/ml; bortezomib (Velcade; Janssen Pharmaceutica N.V., Beerse, Belgium), 19-2000 nM; busulfan (Busilvex; Pierre-Fabre Medicament, Boulogne, France), 1.17-1200 μg/ml; 6-mercaptopurine (Sigma, St. Louis, MO, USA), 15.6-500 μg/ml; 6-Thioguanine (Sigma), 1.56-50 μg/ml.
Statistical methods. Observed differences in proportions were tested for statistical significance using the appropriate chi-square test. For small sample sizes, the Fisher's exact test was used. Differences in the distribution of the IC50 values between two groups were analyzed using the Mann-Whitney U-test. Using the two-tailed test, p<0.05 was considered statistically significant. The study was approved by the Local Bioethical Committee.
Results
No significant differences between ITRT at first and subsequent relapse of childhood AML were found (Table II). Out of the 21 drugs analyzed, no drug was found for which significantly higher resistance of myeloblasts was observed at subsequent relapse, when compared to first relapse of AML. The median RR value between second and first relapse of all tested drugs was 1.0; for 10 drugs the RR was less than 1 (i.e. assumed better sensitivity on subsequent relapse) and for another 11 drugs, the RR value was greater than 1 (i.e. higher drug resistance on subsequent relapse). No drug showed a trend towards better cellular sensitivity at first versus subsequent relapse, as the differences were not significant for each tested drug.
Since the ex vivo drug resistance profile in children with first versus subsequent relapsed AML was comparable, we pooled all patients into one group for further analysis, in order to compare drug resistance between relapse and initially diagnosed AML. For most tested drugs, patients with relapse had higher IC50 in their ITRT profile (Table III). The median RR of all tested drugs was 1.6. For five drugs, RR was significantly higher at relapse: idarubicin (1.8-fold), etoposide (5.9-fold), cytarabine (1.7-fold), fludarabine (3.7-fold) and busulfan (4.3-fold).
Discussion
In the present study, we have shown that drug resistance of myeloblasts in patients with relapsed AML is higher than that of de novo ones. Still, relapse remains a significant problem for all children with AML. In the study of the Dutch-German group, no significant differences in drug resistance were reported in a large cohort of childhood AML samples taken at diagnosis between patients remaining in continuous complete remission versus those with refractory/relapsed AML (10). In general, relapsed AML has a dismal prognosis, mainly related to the time interval between initial diagnosis and relapse, and cellular drug resistance can play a key role in therapy failure of relapsed childhood AML. This is important, as patients with relapse had myeloblasts more resistant to basic drugs used in the therapy of childhood AML, such as: cytarabine, idarubicin, daunorubicin, mitoxantrone and etoposide. Relapsed leukemia blasts were also more resistant to drugs commonly used in therapy of relapsed AML: fludarabine, cytarabine and idarubicin. High ex vivo drug-resistance in childhood AML might partially explain worse clinical results of therapy, when compared to ALL. It is commonly assumed that patients with relapse are more drug resistant than those diagnosed de novo, and this was confirmed in the present analysis of samples of relapsed AML. Patients with relapse were highly-resistant to busulfan, which is a key compound used in the conditioning of patients with AML before hematopoietic stem cell transplantation. On the other hand, no significant differences were found between de novo and relapsed cases for cyclophosphamide and treosulfan. In current therapeutic regimens, based on reduced intensity conditioning, these drugs play an important role.
Unlike ALL, the role of ITRT in childhood AML has not yet been established. Several groups have reported on the possible prognostic value of in vitro drug sensitivity in pediatric AML, showing a good correlation between in vitro drug resistance and short-term clinical outcome after chemotherapy (7, 11-14). These findings have been related mainly to cytarabine (7) and cyclophosphamide (14). Part of these studies included both children and adults. Newer, large studies showed no correlation between in vitro drug resistance to individual drugs and long-term clinical outcome in childhood AML (15-17). As yet, no data exist to support the prognostic value of any in vitro drug resistance profile in childhood AML, while this relationship has been confirmed in adult AML (18). In our previous preliminary report, we showed the possible prognostic value of profile of combined fludarabine, treosulfan and mitoxantrone resistance in children with AML (8). Recently, new compounds were shown to have good antileukemic activity in childhood AML (19, 20). There is still great hope in results obtained in microarray studies (21).
In conclusion, the IC50s for the ex vivo drug resistance profile in relapsed childhood AML are higher in comparison to those at initial diagnosis, however, we did not find differences in ex vivo drug resistance between first and subsequent relapse of AML. It seems that development of subsequent relapses in pediatric AML is also related to factors other than drug resistance.
Acknowledgements
The study was supported by grant MNiSW N407 541339. The Authors thank Beata Kolodziej, Beata Kuryl o-Rafinska and Malgorzata Kubicka for technical assistance.
Footnotes
-
↵* These Authors contributed equally to this study.
- Received January 8, 2013.
- Revision received February 3, 2013.
- Accepted February 4, 2013.
- Copyright© 2013 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved