Is there a relationship between in vitro drug resistance and level of minimal residual disease as detected by polymerase chain reaction at the end of induction therapy in childhood acute lymphoblastic leukaemia? (2002)

Both measurement of drug resistance by the methyl thiazol tetrazolium (MTT) assay (Kaspers et al, 1997) and detection of minimal residual disease (MRD) at the end of induction therapy as determined by quantitative polymerase chain reaction (PCR) (Van Dongen et al, 1998) have shown a significant value in the prediction of relapse in childhood acute lymphoblastic leukaemia (ALL) and can thus be used to modify treatment. However, it is not known how the results from both tests correlate and whether both tests recognize the same low- and high-risk patients. Therefore, bone marrow samples of 20 ALL patients were analysed in this retrospective study, using the MTT assay as well as the PCR-based MRD detection to determine the correlation between their results (Table I). In vitro resistance to three drugs (prednisolone, vincristine and l-Asparaginase, PVA) was determined as described earlier (Kaspers et al, 1997). The drug concentration lethal to 50% of ALL cells (LC50) was used to express the level of drug resistance. Results for each drug were also combined in a PVA score. Quantification of MRD by real-time quantitative PCR (RQ-PCR, n = 12) or limiting dilution assay (LDA, n = 8) was performed using a patient-specific forward primer (Ig and TCR gene rearrangements) and a consensus probe as described previously (Verhagen et al, 2000; de Haas et al, 2001). Correlation of the continuous variables was studied using the non-parametric Spearman's test, while differences in the MRD levels between subgroups with a different PVA score were analysed by the Mann–Whitney U test. For the group of 16 identically treated patients (treated during induction therapy with prednisolone, vincristine, l-asparaginase and daunorubicin), both the combined drug resistance, as defined in the PVA score (P = 0·48), and the drug sensitivity to prednisolone (rho 0·45; P = 0·08), vincristine (P = 0·8) and l-asparaginase (P = 0·5) as single drugs were not significantly correlated with MRD at the end of induction. If we included the four differently treated patients (in which prednisolone was replaced by dexamethasone and daunorubicin was not included during induction therapy) in the analysis, a significant correlation (rho 0·49, P = 0·02) was found for prednisolone (PRD) as a single agent and MRD level. Previously, a poor in vivo clinical response to initial PRD monotherapy was shown to be associated with poor prognosis (Schrappe et al, 2000) and also with antileukaemic activity of PRD in vitro (Kaspers et al, 1998). We hypothesize that in early treatment, a patient group can be characterized by PRD resistance in vitro and in vivo, and by high MRD levels, which have a high relapse risk. This was recently demonstrated in a small study reported by Schmiegelow et al (2001). It is important to stress that although both techniques are influenced by drug sensitivity, they are fundamentally different, as they take hold of two different mechanisms. Whereas the MTT assay reflects the in vitro drug resistance of a subset of leukaemic cells that survive during a 4 d culture period, the number of leukaemic cells, as detected by PCR, is an in vivo measurement of kinetics of all leukaemic cells and is dependent on multiple factors, such as pharmacokinetics, dosage and administration of drugs, and proliferation of vital leukaemic cells. These differences between the two techniques might explain the lack of a strong correlation between the two tests. Because both tests have been shown to predict relapse risk in patients treated with this induction regimen, our results suggest that a combination of both tests will give an even better correlation with disease outcome. Prospective studies on a large number of equally treated patients, in which both techniques are performed and evaluated with regard to clinical outcome, are warranted. Such studies are being performed in the Netherlands [Dutch Childhood Leukaemia Study Group (DCLSG) ALL-9], in Germany [Cooperative for Acute Lymphoblastic Leukaemia (COALL)-97] and by the Nordic Society for Paediatric Haematology and Oncology (NOPHO). We thank the technicians of the Research Laboratory of Paediatric Oncology of the VU University Medical Center who performed the MTT assays and Mrs J.M. Wijkhuys and Mrs D. Jacobs (Department of Immunology, Erasmus University, Rotterdam) for performing the MRD analysis with the RQ-PCR technique. We also acknowledge the members of the DCLSG protocol ALL-8.