WT1 Mutations Are Secondary Events In AML and Show Varying Frequencies Within Genetic Subgroups and Different Impact On Prognosis
Authors: Schnittger Susanne , Eder Christiane , Alpermann Tamara , Dicker Frank , Ulke Madlen , Kohlmann Alexander , Kuznia Sabrina , Kern Wolfgang , Haferlach Torsten , Haferlach Claudia 
Published: October 2019

Abstract

Background
Mutations (mut) in the WT1 gene belong to the first genetic aberrations described in AML. In contrast to recurrent fusion genes or NPM1mut WT1mut do not seem to be disease defining. Also in contrast to other mutations in AML, for most of which a certain prognostic value has been established, the impact of WT1mut still is discussed controversially.

Aim
Analyze the frequency and prognostic impact of WT1 mutations in comparison to other genetic aberrations.

Patients and Methods
3,157 unselected AML patients (pts) were analyzed (de novo: n=2,699, s-AML: n=234, t-AML: n=224). 1,708 pts were male and 1,449 female. Median age was 67.1 years (y) (range: 17.8-100.4 y) with 1,108 pts <60 y and 2,049 ≥60 y. The mutational hot spot regions of WT1 (exons 7 and 9) were analyzed by direct Sanger sequencing with a sensitivity of ∼10%. Karyotype and WT1 mutation status was available in all cases. Other mutations were assessed in subsets: ASXL1 (n=1,951), CEBPA (n=2,670), DNMT3A (n=1,293), FLT3-ITD (n=3,149), FLT3-TKD (n=3,004), IDH1R132 (n=2,431), IDH2R140 (n=2,380), IDH2R172 (n=2,412), KRAS (n=1,409), NRAS (n=1,780), NPM1 (n=3,003), MLL-PTD (n=2,961), RUNX1 (n=2,390), TET2 (n=1,016) and TP53 (n=1,215).

Results
A total of 189 WT1 mutations were detected (exon 7: n=151, exon 9: n=38). The total frequency of WT1mut pts was 175/3,157 (5.5%). 11 pts were double to quadruple mutated. The frequency was heterogeneous with respect to AML subtypes. Compared to all others, significantly higher frequencies were detected in biallelic CEBPAmut (15/110; 13.6%; p=0.001), followed by t(15;17)/PML-RARA (18/164; 11.0%, p=0.004), and FLT3-ITD (58/682; 8.5%, p<0.001). Lower frequencies were observed in DNMT3Amut (18/412; 4.3%, p=0.014, ASXL1mut (6/355; 1.7%, p<0.001), IDH2R140 (5/286; 1.7%, p=0.001), and IDH1R132 (2/222; 0.9%, p<0.001). WT1mut were never detected in pts with complex karyotypes (0/175; p=0.047) or those with IDH2R172 (0/68; p=0.020).
Further, WT1mut were more frequent in females (95/1,449, 6.6%) than in males (80/1,708, 4.7%) (p=0.014) and in younger pts (<60 y: 102/1,108, 9.2% vs ≥ 60 y: 73/2,049, 3.6%; p<0.001). Median age of pts with WT1mut was 55.5 y compared to 63.6 in WT1wt (p<0.001). Further, WT1mut were associated with lower platelet count (58.4 vs 84.7 x109/L; p<0.001) and lower hemoglobin level (8.8 vs 9.3 g/dL, p=0.001). There was no association to the history of the disease or white blood cell count.
Stability of WT1mut was analyzed in 35 paired diagnostic and relapse samples (median time of relapse after diagnoses: 11.1 months (m); range: 2.6-60.6 m). In 23 cases (65.7%) the WT1mut was retained at relapse and in 12 cases (34.3%) it was lost. In 5 cases a sample at 2nd relapse was available (median time from 1st relapse: 8.5 m, range: 6.0-18.0 m). 3 of these cases retained and 2 lost the WT1mut.
Analysis of prognostic impact was restricted to intensively treated pts (n=1,936, WT1mut: n=132, 6.8%). In the total cohort, there was no impact of WT1mut on prognosis. In pts ≥60 y there was a trend to shorter event free survival (EFS) for WT1mut (9.3 vs 12.3 m, p=0.052). In the two prognostically favorable groups with high WT1mut incidences (biallelic CEBPAmut and PML-RARA) no effect on outcome was seen. When restricting the analysis to normal karyotype AML (WT1mut: n=85, WT1wt: n=1,093) WT1mut pts had shorter EFS (10.8 vs 17.9 m, p=0.008). This was true for the younger (12.2 vs 29.0 m, p=0.007) as well as for the older pts (9.3 vs 13.9 m, p=0.016). In a multivariate analysis all parameters with significant impact on EFS in univariate analysis were included: age (p<0.001, HR: 1.24), ASXL1mut (p<0.001, HR: 1.36), FLT3-ITD (p<0.001, HR: 1.55), NPM1mut/FLT3-ITD wild-type (p<0.001, HR:1.55), RUNX1 (p=0.019, HR: 1.23, and WT1mut (p=0.009, HR: 1.64). In multivariate analysis WT1mut was found to have independent adverse impact on EFS (p=0.002, HR: 1.64) besides FLT3-ITD status (p<0.001, HR: 1.71) and age (p<0.001, HR: 1.28).

Conclusions
WT1 mutations are 1) more frequent in females and younger AML, 2) more frequent in t(15;17)/PML-RARA, biallelic CEBPAmut, FLT3-ITD mutated AML, and nearly mutually exclusive of ASXL1, IDH1, IDH2 and complex karyotype. 3) The distribution pattern in different genetic subtypes and the instability during follow-up as shown by paired sample analyses clearly emphasize a secondary character of this mutation. 4) For AML with normal karyotype an independent adverse impact of WT1mut on EFS was shown.

Disclosures:
Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Eder:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Ulke:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kuznia:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.