Nucleophosmin Gene Mutations Are Predictors of Favourable Prognosis in Acute Myeloid Leukemia with a Normal Kayotype and Can Be Used as a New Marker for Quantitative PCR To Detect Minimal Residual Disease.

Nucleoplasmin (NPM) mutations have recently been described as a new kind of mutation in AML. In the presented work we have analyzed 977 AML cases from different prognostic subgroups. Fifteen different mutations (type A:n=245 (78.3%), B:n=21 (6.7%), D: n=28 (11.4%), I:n=3 (1%), J:n=4 (1.3%), K:n=2, G,H, L, M, N, O, P, Q, R: n=1, each (0.3%) were detected in 313 cases. No mutation was found in inv(16) (n=89), t(8;21) (n=98), t(15;17) (n=17), t(11q23)/MLL (n=8), inv(3) (n=8), other reciprocal translocations (n=3), and complex aberrant karyotypes (n=59). In the normal karyotype group the incidence was 299/603 (49.6%) and in all others 13/90 (14.4%). In the latter cohort cases with NPM+ were: − Y (1 case), +4 alone (1 case), +4 and +8 (2 cases), +4 with multiple trisomies (1 case),+8 (1 case), multiple other trisomies (1 case), del(5q) (1 case), − 7 alone (2 cases), del(9q) (2 cases), unbalanced translocation (1 case).

Data for evaluation of prognosis were available in 401 AML patients with normal karyotype treated within the AMLCG99 study. Results were calculated in relation to MLL-PTD, FLT3-LM, FLT3-TKD, NRAS, KIT, and CEBPA mutations and correlated to further clinical characteristics. NPM-mutated cases were frequently associated with FLT3 mutations but rarely with other mutations. The NPM-mutated group had a higher CR rate (70.5% vs. 54.7%, p=0.003), a trend to longer OS (median 1012 vs. 549 days, p=0.0762), and significantly longer EFS (median 428 vs. 336 days; p=0.0121). There was a clear favourable impact of NPM+/FLT3-LM- on OS and EFS (264/395 cases; 66.8%). This positive effect was lost in the presence of a concomitant FLT3-LM, since survival of the NPM+/FLT3-LM+ double positive pts. was similar to NPM-/FLT3-LM+ cases. We further analysed stability for the NPM mutation at diagnosis and relapse and found stability in 14/15 paired samples. One case with AML M5 lost the NPM mutation at relapse. This case had also a shift from normal karyotype to 46,XX,del(7)(q22),der(7)t(7;21)(p11;q?) and a complete shift of immunophenotype indicating a therapy-related AML. In addition, we have analyzed the applicability of NPM mutations as targets for minimal residual disease (MRD) detection. In total, 82 samples of 22 selected cases were analyzed by use of quantitative real time PCR for the three most common mutation types. Fifteen of these cases had a type A, 3 a type B, and 4 a type D mutation. Samples at diagnosis had an NPM/ABL ratio of 267.6 (range: 54.9 to 3174.7) while each 10 NPM negative cases had a median ratio of 0.03 (assay for A), 0.0001 (assay for B), and 0.002 (assay for D), showing that these assays were nearly specific for the respective mutation. Using limited dilution series of NPM+ in NPM- cDNA a median sensitivity of 1:100.000 could be shown. In both analyzed cases with relapse these were early detectable by increasing NPM levels. Thus, NPM mutations can be used for highly sensitive PCR-based MRD detection in AML, especially if other markers are lacking, i.e. in normal karyotype.

In conclusion, our data show

1. a nearly exclusive association of NPM+ to the normal karyotype,

2. a good prognosis of NPM+/FLT3-LM- in normal karyotype.

3. an intermediate prognosis of NPM+/FLT3-LM+ in normal karyotype

4. NPM as a new marker for PCR-based MRD-detection.