Leukemia Research 36 (2012) 401–406 Contents lists available at SciVerse ScienceDirect Leukemia Research jo ur nal homep age: www.elsevier.com/locate/leukres Comparison between multiparameter flow cytometry and WT1-RNA quantification in monitoring minimal residual disease in acute myeloid leukemia without specific molecular targets Giovanni Rossi a,∗ , Maria Marta Minervini a , Angelo Michele Carella a , Chiara de Waure b , Francesco di Nardo b , Lorella Melillo a , Giovanni D’Arena a , Gina Zini c , Nicola Cascavilla a a Department of Hematology and Stem Cell Unit, IRCCS “Casa Sollievo della Sofferenza” Hospital, San Giovanni Rotondo, Italy b Department of Hygiene, Catholic University of Sacred Heart, Rome, Italy c Department of Hematology and Laboratory, Center of Research ReCAMH, Catholic University of Sacred Heart, Rome, Italy a r t i c l e i n f o Article history: Received 6 October 2011 Received in revised form 20 November 2011 Accepted 27 November 2011 Available online 21 December 2011 Keywords: Acute myeloid leukemia Minimal residual disease Flow-cytometry Leukemia-associated immunophenotypes WT1-RNA a b s t r a c t Despite a high remission rate, a significant number of patients with acute myeloid leukemia (AML) relapse. Thus, the evaluation of minimal residual disease (MRD) in AML is an important strategy to better identify high risk patients. Most sensitive methodology to detect MRD is molecular polymerase chain reaction (PCR) but its applicability is restricted to AML with leukemia-specific molecular targets (e.g. AML1-ETO, CBFB-MYH11, MLL, FLT-3). In our study, MRD was monitored at different time points with both MFC and WT1-RNA quantification in 23 AML patients who did not present specific molecular targets. As pre- viously published, we considered values of 10 -3 (0.1%) in MFC and 90 WT1-RNA ×10 4 ABL copies as optimal thresholds. Receiver operating characteristics (ROC) analysis was used to confirm these data. To realize the methodology that better identify high risk patients, an analysis of sensitivity, specificity, pre- dictive values (PV) and likelihood ratio (LR) was provided and similar results were showed. MRD levels ≥10 -3 in MFC as well MRD levels ≥90 WT1-RNA copies in RQ-PCR, identify risk groups of patients with poor prognosis. Therefore, MFC and WT1-RNA quantification showed a comparable capacity in terms of technical performance and clinical significance to identify high risk patients who eventually relapsed. © 2011 Elsevier Ltd. All rights reserved. 1. Introduction Monitoring of the minimal residual disease (MRD) in acute myeloid leukemia (AML) has become essential to predict progno- sis and to select the type of post-remission treatment [1]. Several studies have shown how multiparameter flow-cytometry (MFC) [2,3] polymerase chain reaction (PCR) [4,5] and fluorescence in situ hybridization (FISH) [6] are useful for detection of MRD in AML patients. PCR-based quantification of MRD has a high sensitiv- ity but its applicability is restricted to subgroups of AML with leukemia-specific molecular targets (e.g. AML1-ETO, CBFb-MYH11, MLL, FLT3, NPM1) [7]. These cases comprise only 40–50% of all AML cases [8]. For this reason, several investigators studied WT1 expres- sion as alternative marker of leukemic cells and tested it for MRD detection in AML patients [9–12]. However the results concern- ing its use as follow-up marker during post-remission have been ∗ Corresponding author at: Department of Hematology and Stem Cell Unit, IRCCS “Casa Sollievo della Sofferenza” Hospital, v.le Cappuccini 1, 71013 San Giovanni Rotondo, Italy. Tel.: +39 3922323949; fax: +39 3922323949. E-mail address: giovannirossi.fr@gmail.com (G. Rossi). conflicting. Some groups reported a good association between the clinical course and WT1 levels [13,14] while others failed to show [15,16]. This discrepancy may be due to differences in methodology in measuring WT1 and in patients populations or in the intensity of the regimens used. On the other hand, using an accurate combina- tion of monoclonal antibodies in the MFC allows specific detection of leukemic cells. This approach may be applicable to a percent- age of AML patients that ranges from 80% to 94% [17–20] and shows a sensitivity better than 1 leukemic cell per 10 4 to 10 5 nor- mal cells [8]. However, studies differ in the threshold use for MRD detection and in the timing of testing. A strong association with clinical outcome was found at the presence of both 10 -4 [2,20] and 10 -3 Leukemia-associated immunophenothypes (LAIPs) [3,21–24] positive cells and it is still unclear whether post-induction or post- consolidation have a stronger prognostic power. The majority of previous studies on MRD by immunophenotyping used three-four color MFC but some groups demonstrated that a six color approach improves the identification of LAIP positive cells respect to four- color examination [24]. We provided a six-color MFC analysis for a more sensitive and accurate quantification of MRD in AML. The purpose of the present study was to compare the performance of both MFC and WT1-RNA quantification in detecting MRD in order to 0145-2126/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2011.11.020