Concomitant monitoring of <i>WT1</i> and <i>FLT3‐ITD</i> expression in <i>FLT3‐ITD</i> acute myeloid leukemia patients: which should we trust as a minimal residual disease marker?
Authors: Federico De Marchi, Anna Candoni, Maria Elena Zannier, Lisa Haley, Bonnie Wing Yin Lau, Renato Fanin
Journal: American Journal of Hematology (2017)

FLT3-ITD mutations (FMS-like tyrosine kinase 3) are the most common molecular alterations observed in Acute Myeloid Leukemia (AML). The presence of these mutations has an unfavorable prognostic significance and is the target of specific FLT3-ITD inhibitor drugs, currently in advanced stage clinical trials.1, 2 In recent years minimal residual disease (MRD) detection in AML has taken a very important prognostic role, becoming essential for the choice of the most appropriate consolidation strategy.1-4 A recent study by Gaballa et al has shown how FLT3-ITD molecular status at the time of transplant is a key predictor of disease relapse and survival in patients with FLT3-ITD AML.3 Similarly, a study by our group has demonstrated the high predictive value of MRD assessment by the Wilms' tumor (WT1) gene expression at the time of stem cell transplant in this subset of patients.4 However, to date, there are no studies comparing the effectiveness of FLT3-ITD and WT1 detection on clinical MRD monitoring in FLT3-ITD AML. The reliability of the FLT3-ITD mutation as a MRD marker has not yet been clarified and the use of polymerase chain reaction (PCR) techniques for its detection do not seem to guarantee high sensitivity (“PCR bias”).1, 2 Therefore, to verify the usefulness of serial FLT3-ITD tests along the course of the disease, we compared its expression level with the quantitative analysis of WT1, a known pan leukemic marker available for MRD assessment in AML.5 We analyzed 24 consecutive FLT3-ITD positive AML patients (diagnosed at the University of Udine-Hematology Department), all with WT1 overexpression at diagnosis. The bone marrow samples were collected at three specific time-points for each AML case: at diagnosis, postinduction, and finally either post consolidation therapy (in cases in cytological remission), post salvage therapy (in primary refractory cases) or at cytological relapse (in cases of relapse after a previous remission). Of note, none of these patients received therapy with FLT3-ITD inhibitors. The expression level of FLT3-ITD mutation was determined by PCR (Applied Biosystem, Foster City, CA), followed by capillary gel electrophoresis to determine allelic burden.6 Concomitantly, at the same time points, we performed quantitative PCR for WT1 gene expression, in accordance with the LeukemiaNet guidelines and the detection of WT1 copies > 250/104 copies of the control gene Abelson (Abl) was defined as WT1 overexpression (Ipsogen WT1 ProfileQuant Kit, Qiagen, EU).5 At diagnosis, the median bone marrow blast cells (BC) count was 90% (range 20%-100%), with a 37% median FLT3-ITD allelic burden (range 3%-83%) and 9970 copies of WT1 (range 481-36423). As reported in Table 1, patients were divided into three groups according to the cytologic remission status of their AML after induction therapy: (1) patients with disease refractory (RES) to induction chemotherapy (AML-RES, 8 patients, median bone marrow BC after chemotherapy of 30%, range 15%-80%); (2) patients in cytologic Complete Remission (CR) post Induction therapy (AML-CR, 10 patients, BC < 5%); (3) patients in relapse (REL) post CR (AML-REL, six patients, median bone marrow BC at relapse of 35%, range 20%-100%). We found that in those patients who achieved and maintained a cCR after induction (10 AML-CR, group 1), both WT1 and FLT3-ITD became negative in a concordant manner: 8/10 cases (80%) were double negative after induction and 2/10 (20%) were double positive (concordance 100%); after consolidation 10/10 cases (100%) were double negative. In cases with resistant AML (8 AML-RES, group 2) or at relapse (6 AML-REL, group 3) post chemotherapy, WT1 was overexpressed in the totality of cases (14/14, 100%), while FLT3-ITD was positive only in 9/14 cases (64%), being negative in 5/14 (36%) cases, even in the presence of cytologically evident bone marrow disease. We have therefore observed a discrepancy between WT1 and FLT3-ITD expression levels in relapsed (AML-REL) and disease-resistant (AML-RES) patients, with WT1 showing greater stability as a marker of MRD and a stronger concordance with cytology. FLT3 positive (allelic burden-%) 10/10 48,5 (29–76) 2/10 (20%)a (3; 6) WT1 positive (number of copies/104 Abl) 10/10 11488 (4164–26393) 2/10 (20%)a (1154; 438) FLT3 positive (allelic burden-%) 8/8 38 (3–83) 6/8 (75%) 30,5 (3,4–89) 6 /7° ( 85%) 44,5 (16–98) WT1 positive (number of copies/104 Abl) 8/8 10733 (481–36423) 8/8 (100%) 5266 (752–17047) 7/7° (100%) 6081 (757–28371) FLT3 positive (allelic burden-%) 6/6 34 (4,7–39) 2/6 (33%)b (5; 10,5) 3/6 (50%) (43; 37; 48,4) WT1 positive (number of copies/104 Abl) 6/6 7497 (1328–13719) 2/6 (33%)b (1451; 447) 6/6 (100%) 11613 (651–21589) These data, within the limitations of a small series, suggests that WT1 could be a reliable MRD marker and highlight the instability of FLT3-ITD, with possible switch from FLT3-ITD positivity at diagnosis to FLT3-ITD negativity at the time of cytological relapse or during postchemotherapy resistance. This probably reflects the fact that the AML is constituted by leukemic subclones heterogeneous for FLT3 expression and that a FLT3-ITD negative subclone may be responsible for the recurrence or resistance of AML.1, 2 Conversely, overexpression of the pan leukemic marker WT1 seems to be more stable along the course of the disease and more homogeneous in the different leukemic subclones, making this an appropriate and reliable marker for MRD detection; furthermore, overexpression of WT1 appears to have a high degree of concordance with cytology (100% in this series). In summary, these data suggest that FLT3-ITD is not, by itself, an optimal MRD marker and that the current available technique for its detection has lower sensitivity compared to the one in use for WT1. More sensitive molecular methods, such as new PCR based tests or next-generation sequencing (NGS) assays, could increase FLT3-ITD sensitivity and its use as MRD marker.2, 7 However, even with the current technique, two patients in our series were negative for WT1 and positive for FLT3-ITD after induction and later relapsed. It might therefore be useful, until more efficient methods are routinely available, to test both WT1 and FLT3-ITD markers at diagnosis and throughout the AML course to better evaluate the depth of remission and to guide MRD driven therapy in FLT3-ITD positive AML.