Differential in Vitro Drug Resistance Profile Between First and Second Relapsed Acute Lymphoblastic Leukemia and Acute Myeloblastic Leukemia in Children

Introduction: In vitro drug resistance profile has so far provided information on chemosensitivity of leukemic cells in acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML). Previous studies have shown that for most tested drugs, patients with relapse had higher IRTR than those with de novo ALL or AML. The objective of this study was the analysis and comparison of the individualized tumor response testing (ITRT) between first and second relapse in children with ALL or AML. 

Patients: A total of 186 pediatric leukemic samples (154 ALL and 32 AML) were tested for ex vivo chemosensitivity for up to 22 drugs. ALL samples included 113 samples obtained at first relapse, and 41 obtained at second relapse. AML samples included 22 samples obtained at first relapse, and 10 obtained at second leukemic relapse. The distribution of patients between first and second relapse groups was comparable. 

Methods: In vitro drug resistance was tested by the MTT assay. 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. The following  drugs were used: prednisolone, dexamethasone, vincristine, idarubicin, daunorubicin, doxorubicin, mitoxantrone, L-asparaginase, cytarabine, fludarabine, cladribine, clofarabine, treosulfan, thiotepa, melphalan, 4-HOO-cyclophosphamide, 4-HOO-ifosfamide, bortezomib, busulfan, 6-mercaptopurine, and 6-thioguanine. For patients with ALL, combined drug resistance profile to prednisolone, vincristine and L-asparaginase (PVA score) was also analyzed. 

Results: ALL: In comparison to first relapse, second relapsed childhood ALL were more resistant to most of tested drugs. Median PVA score in multiple relapsed patients was 8 vs 6 for patients at first relapse (p=0.004). The median relative resistance value between patients with multiple relapse and those with first relapse for all tested drugs was 2.0, indicating higher drug resistance on second relapse. Multiple relapsed ALL samples were more drug resistant to: prednisolone (>1.9-fold), dexamethasone (>1.5-fold), vincristine (3.1-fold), L-asparaginase (5-fold), mitoxantrone (2.4-fold), cytarabine (4.3-fold), mercaptopurine (2.2-fold), thioguanine (4.8-fold), etoposide (2.6-fold) and melphalan (2.7-fold). On the other hand, lymphoblasts at second relapse were comparably resistant to: daunorubicin, doxorubicin, 4-HOO-cyclophosphamide, 4-HOO-ifosfamide, busulfan, treosulfan, fludarabine, clofarabine and bortezomib. No drug showed a trend towards better cellular sensitivity at first versus second relapse. AML: No significant differences between ITRT at first and second relapse of childhood AML were found. Of the all drugs analyzed, no drug was found for which significantly higher resistance of myeloblasts was observed at second 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. 

Conclusion: In comparison to first relapse, leukemic blasts of children at second relapse of ALL are more in vitro resistant to most of tested drugs. Contrary, myeloblasts of children at second relapse of AML show drug resistance comparable to first relapse. (This study was supported by NCN Grant DEC-2011/03/D/NZ5/05749.)