Wilms’ Tumor 1 Gene Mutations in Childhood Acute Myeloid Leukemia: A New Prognostic factor with Implications for MRD Detection
Authors: Hollink Iris H.I.M. , van den Heuvel-Eibrink Marry M. , Zimmermann Martin , Balgobind Brian V. , Arentsen-Peters Susan T.C.J.M. , Alders Mariel , Willasch Andre , Kaspers Gertjan J.L. , Trka Jan , Baruchel Andre , de Graaf Siebold S.N. , Creutzig Ursula , Pieters Rob , Reinhardt Dirk , Zwaan C. Michel 
Published: April 2020

Abstract
In an array-CGH screening study of cytogenetically normal AML (CN-AML), we detected a cryptic 11p13-deletion including the WT1 gene in one childhood AML sample. The remaining WT1 allele in this sample carried a nonsense mutation. WT1 gene mutations have recently been identified in approximately 10% of adult CN-AML. Of interest, WT1 mutations were found to be a new independent poor prognostic factor in adult CN-AML (Virappane et al. JCO2008, Paschka et al. JCO2008). WT1 mutations have also been reported in childhood AML; however, their clinical relevance in childhood AML is not known. In this study, we investigated the frequency, clinical characteristics and prognostic value of WT1 mutations (exons 7–10) in a large, well-characterized cohort of childhood AML samples (n=298). Additionally, a subset of these samples was screened for mutations in exons 1–6 (n=68), and for micro-deletions in the WT1 gene (n=24). Survival analysis was restricted to the subset of patients with de novo AML who were treated using uniform DCOG and BFM treatment protocols (n=232). Fifty-three pathogenic WT1 mutations were detected in 35/298 (12%) samples taken at diagnosis. Mutations were mainly located in exon 7 (n=43), but also in exons 1 (n=2), 2 (n=1), 3 (n=2), 8 (n=1) and 9 (n=4). Predominantly frame-shift mutations were found (n=41), next to nonsense mutations (n=6) and missense mutations (n=6); the former two resulting in a truncated WT1 protein. In 19/35 (54%) of the WT1-mutated samples, we detected more than one WT1 aberration. This included either a different WT1 mutation (n=15), a homozygous WT1 mutation (n=2), or a deletion of the other WT1 allele (n=2). WT1 mutations clustered significantly in the CN-AML subgroup (21/94=22%; p&lt;0.001). NPM1 and WT1 mutations were mutually exclusive, but WT1-mutated samples were more likely to carry FLT3/ITD (43% vs. 17%; p&lt;0.001) and CEBPα mutations (26% vs. 9%; p=0.007). Mutations in patients below the age of 3 years were only found sporadically (1/60=2%). The highest frequency was found in the age category 3–10 years (17/76=18%), and decreased above the age of 10 years (17/128=12%; p=0.008). WT1-mutated AML was correlated with a higher white blood cell count at diagnosis (WBC) (57.2×109/l vs. 34.1×109/l; p=0.007); no correlation was found with sex or FAB-classification. WT1-mutated AML patients had a significantly worse outcome when compared with patients with WT1 wild-type AML (5-year overall survival (pOS) 35% vs. 66%; p=0.002; 5-year event-free survival (pEFS) 22% vs. 46%; p&lt;0.001; and 5-year cumulative incidence of relapses (CIR) 70% vs. 44%, respectively; pGray&lt;0.001). Moreover, using multivariate analysis including age, WBC, cytogenetics, FLT3/ITD and stem cell transplantation, WT1 mutations were identified as an independent poor prognostic factor for pOS (HR1.79; p=0.04), pEFS (HR2.05; p=0.005) and relapse-free survival (pRFS) (HR2.44; p=0.001). We identified patients carrying both a WT1 mutation as well as a FLT3/ITD as a very poor prognostic subgroup (5-year pOS 21%). The mutational hotspots in the WT1 gene were located within areas of primer-probe combinations used for WT1-based minimal residual disease (MRD) detection. Furthermore, in 4/28 (14%) wild-type diagnostic-relapse pairs a mutation was gained at relapse, which may also effect MRD detection. In conclusion, WT1 mutations are present in 12% of childhood AML at diagnosis and in 22% of patients with CN-AML, and are a novel independent poor prognostic marker in childhood AML. Furthermore, their presence may have implications for current WT1-based MRD detection.