Mutations of the Nucleophosmin (NPM1) Gene Are Common in Adult Acute Myeloid Leukemia and Associated with Favorable Prognosis If Present without FLT3-ITD Mutation.
Authors: Thiede Christian , Koch Sina , Creutzig Eva , Steudel Christine , Illmer Thomas , Schaich Markus , Ehninger Gerhard 
Published: October 2019

Abstract
Nucleophosmin (NPM1) is a nucleo-cytoplasmic shuttling protein with multiple functions, including regulation of the p53-ARF pathway. Rearranged NPM1 has been found in a small subgroup of AML patients with t(5;17). Recently, mutations of NPM1 have been described in patients with acute myeloid leukaemia, especially in patients with normal karyotype (Falini et al, NEJM 2005). These mutations lead to an elongated protein which is retained in the cytoplasm. The precise prevalence as well as the long term clinical impact of this mutation is currently unclear.
Patients and methods: We investigated 1485 samples from adult patients with newly diagnosed AML and advanced MDS for mutations in exon 12 of the NPM1 gene. The majority of these individuals were treated in the AML96 protocol of the DSIL (former SHG). Mutations in NPM1 exon 12 were screened using genomic DNA from the time of diagnosis and Genescan analysis. In a subgroup, mutations were confirmed by sequencing. In addition, confocal laser scanning microscopy was performed to investigate the localization of mutant NPM1 in cases with novel mutations.
Results: A 4-bp insert in the genomic sequence was detected in 408/1485 patients (27.5%). In 229 cases sequenced so far the most common change was the previously reported Type A (80.3%), followed by B (9.2%) and D (3.1) and 13 novel mutations, which showed cytoplasmic localization of NPM1 in all five analyzed cases. The NPM1-mutations were most prevalent in patients with normal karyotype (324/709; 45.7%) compared to 58/686 with karyotype abnormalities (8.5%) (P<.0001). NPM1 mutations were rarely seen in cases with good risk abnormalities [t(8;21): 2/57; inv(16: 2/73; t(15;17): 0/47] but also rarely detected together with high risk cytogenetics [-5/5q-: 3/94;-7/7q-: 5/122; complex: 4/185]. The mutation was found to be associated with high WBC counts, high BM blasts and higher platelet counts (P <.0001). Mutant NPM1 was also more prevalent in females compared to males (33.1 vs 22.9%; P<.00001). Most NPM1 mutations were found in cases with de novo AML, mainly in FAB subgroups M5a, M5b, and M4, and were absent or rare in M3, M0, M6, and M7. Patients with NPM1 alterations had significantly more FLT3-ITD mutations, even if restricted to patients with normal karyotype (NPM1-mut: 43.8% vs. NPM1-wt: 19.9%; p<.0001), whereas a significantly lower rate of MLL-PTD mutations was found (NPM1-mut: 0/207 vs. NPM1-wt: 39/549; P<.0001). To assess the clinical impact, 4 groups were defined based on the NPM1 and FLT3-ITD status for patients with normal karyotype: NPM1-mut/FLT3-ITD neg; NPM1-mut/FLT3-ITD pos; NPM1-wt/FLT3-ITD pos; NPM1-wt/ FLT3-ITD neg. Among these groups, patients having only an NPM1-mutation had significantly better overall survival (OS), disease free survival (DFS) and a lower probability of relapse, compared to all other groups. This was confirmed in a multivariate analysis (HR NPM1-mut/FLT3-ITD neg for OS: 0.76 95% CI: 0.587–.992; DFS: 0.661, 95% CI: 0.449–0.973). In addition, these patients had a significantly lower cumulative incidence of relapse (CIR at 40 months: 37.2%; P = .009).
Conclusion: With more than 27%, NPM1-mutations represent the most frequent genetic abnormality in adult AML identified so far. They characterize a specific subset in patients with normal karyotype. If not associated with FLT3-ITD mutations, NPM1 abnormalities appear to identify a subgroup with improved response towards treatment. The mechanistic background remains to be determined.