Comparison of the Performance of Multiparameter Flow-Cytometry and wt1-RNA Levels in Detecting Minimal Residual Disease and Predicting Relapse in Patients with Acute Myeloid Leukemia

Despite a high remission rate, a significant number of patients with acute myeloid leukemia (AML) relapse (Lowenberg et al. 2003). Thus, the evaluation of minimal residual disease (MRD) in AML is an important strategy to better identify high risk patients. Most sensitive is the molecular polymerase chain reaction (PCR) methodology but its applicability is restricted to subgroups of AML with leukemia-specific molecular targets (e.g. aml1-eto, cbfb-myh11, mll, flt3). However, these cases comprise only 40–50% of all AML cases (Kern et al. 2004). Detection of aberrant phenotypes by multiparameter flow cytometry (MFC)(San Miguel et al. 2001, Al Mawali et al. 2009) and quantification of wt1-RNA levels (Cilloni et al.2008) provide excellent options for MRD monitoring in AML. In the present study we investigated patients with AML withouth leukemia- specific genetic alterations. In this subgroup, in order to realize the methodology that better identify high risk patients, the performance of both MFC and wt1 expression (RQ-PCR) was evaluated. Then, if threshold values defining a presence of MRD for both of methodologies are predictors of poor prognosis were also evaluated. Patients and methods Fresh bone marrow samples from 74 AML patients were obtained at diagnosis between January 2008 and May 2011. CR was achieved from 59 patients. Of these patients, 23 did not show genetic alterations and were studied for MRD with both MFC and wt1 expression (RQ-PCR) at different time points: after induction therapy (T1), after consolidation therapy (T2) and every 3 months during follow-up. The immunophenotypic analysis was performed using two panels of markers in a six-color combinations in order to correctly identify leukemia –associated immunophenotypes (LAIP). The follow MoAb CD45/CD34/CD117/CD15 and CD45/CD34/CD64/14 were combined with specific myeloid and lymphoid markers respectively. RQ- PCR to test wt1 expression was made according to the standardized and quality-controlled method (Willasch et al. 2009). The obtained wt1 copy numbers were normalized with respect to the ABL transcripts. Results An analysis of sensitivity, specificity, predictive values(PV), likelihood ratio(LR), Receiver Operating characteristic Curve (ROC) and the Area Under the Curve (AUC) for both MCF and RQ-PCR of wt1 were performed. MFC showed higher sensitivity than RQ-PCR at each time point (80% vs 70% at T1) but specificity of RQ-PCR was always superior to MFC, particularly at T2 (76,9% vs 53,8%). Although both methodologies showed comparable LR+ values at each time point, a better LR− for MFC analysis was found (LR+: 1,73 vs 1,82; LR−:0,37 vs 0,48 at T1).However both methodologies showed low positive PV (57,1 vs 58,3 at T1). AUC and 95% confidence intervals demonstrated a moderate accuracy for both MFC and RQ-PCR [0,715 (0,499–0,932) vs 0,713 (0,506–0,940) at T1]. Analyzing the performance of combined methodologies a lower sensitivity and a progressive higher specificity were evidenced at each examined time. Excluding allograft patients better values of sensitivity, specificity, PV (MCF PPV/NPV:87,5%/83,3% vs RQ-PCR PPV/NPV:100%/75% at T1), LR (MFC LR+/LR−:5,25/0,15 vs RQ-PCR LR+/LR−: +∞/0,25 at T1) were obtained as well as a higher AUC. Combining both methodologies any advantage was achieved. A cut-off value of 10⁻³ in MFC and 90 wt1-RNA × 10⁴ ABL copies was selected as most significant in terms of risk of relapse and survival (DFS and OS). The median OS of the whole sample was 20,37 months while the DFS was 14,03. As far as DFS is concerned, 57,1% of patients with positive MRD, according to MCF analysis, relapsed with a relapse median time of 11,50 months compared to 22,2% of patients with negative MRD with relapse median time of 29,24 months (p< 0.05).On the other hand, 58,3% patients of high MRD, according to the wt1 expression, relapsed after a median time of 10,97 months compared to 27,3% patients with negative MRD who relapsed after a median time of 29,24 months (p<0,05). Conclusion According to this study, MFC and wt1-RNA copies number are comparable methodologies to detect MRD and to predict relapse. Combination of both does not provide any improvement in the result analysis. At the time of induction therapy we observed the best performance by both methodologies in detecting MRD. In AML MRD levels ≥10⁻³ in MFC as well as MRD levels ≥ 90 wt1-RNA copies in RQ-PCR, identify two risk groups of patients with different prognosis.

No relevant conflicts of interest to declare.