NPM1 Mutated AML Is Characterized By Pre-Leukemic Mutations and the Persistence and Acquisition of Co-Mutations in Molecular Remission Leads to Inferior Prognosis
Authors: Cappelli Luca Vincenzo , Meggendorfer Manja , Baer Constance , Nadarajah Niroshan , Kern Wolfgang , Haferlach Torsten , Haferlach Claudia , Höllein Alexander 
Published: October 2019

Abstract
Background: In AML patients (pts), pre-leukemic mutations in DNMT3A, TET2 and ASXL1 (DTA) were shown to persist in remission, which was not associated with survival (Jongen-Lavrencic et al. NEJM 2018). On the other hand Abelson et al. (Nature 2018) recently identified a specific pre-leukemic mutational spectrum in pts that eventually develop AML. NPM1 mutation (NPM1mut) identifies a WHO AML entity which accounts for about 30% of all AML. The absence of NPM1 transcripts following treatment defines complete molecular remission (CMR). We aimed to dissect the clonal hierarchy of co-mutations at diagnosis of NPM1mut AML and analyze the role of persistent mutations in this well-defined CMR setting.
Methods: We investigated 136 pts with diagnosis of NPM1mut AML (from 2006 to 2016) who attained a CMR (absence of NPM1 transcripts, sensitivity 0.001%) after intensive treatment. The mean follow-up was 40 months (2-96). Next-generation sequencing of 39 genes associated with myeloid malignancies was performed for all pts in paired samples at diagnosis and at CMR. The median coverage was 5200x. FLT3-ITD was analyzed by gene scan.
Results: At diagnosis, a total of 375 mutations were detected, with an average of 2.8/pt (range 1-6); at least one mutation other than NPM1 was present in 123/136 pts (90%). DTA-genes DNMT3A (39% of pts) and TET2 (15%) were among the most frequently mutated, while no ASXL1 mutation was observed. Mutated non-DTA genes included: FLT3-ITD (35%), IDH2 (27%), IDH1 (21%), NRAS (18%), FLT3-TKD (14%) and PTPN11 (13%). The mean variant allele frequency (VAF) was 36% (2.7-94), and was higher for DTA than non-DTA mutations (44% vs 34%, p<0.0001). We established the clonal hierarchy of NPM1mut AML at diagnosis by analyzing the VAF of co-mutations in relation to NPM1 ±5%). In the majority of pts (75/123: 61%) a higher VAF was detected for co-mutations than for NPM1. Specific mutations with higher VAF were DNMT3A (35/52, 67%), IDH1 (21/28: 75%), IDH2 (31/36: 86%), SRSF2 (9/11: 82%) and TET2 (14/21: 67%). Mutations in those genes therefore represent first hits and occur at an earlier phase of AML development. This phenomenon was more frequent in pts ≥60 years (yrs) vs pts <60 yrs (72% vs 52%, p=0.021, OR: 2.4). On the contrary, mutations in FLT3-TKD (10/18: 56%), NRAS (14/23: 61%) and WT1 (4/6: 67%) had a significantly lower VAF than NPM1mut (p<0.0001) indicating that these are second hit mutations. We detected a significant higher rate of persistent mutations in CMR in pts with NMP1mut as 2nd hit vs cases with NPM1 as 1st hit (55% vs 33%, p=0.026, OR=2.4). No difference in OS was detected for pts with NPM1 as 1st or 2nd hit mutation. At CMR, a total of 84 mutations were detected, with an average of 0.6/pt (range 0-3). DTA mutations in DNMT3A and TET2 persisted with the highest frequency (59% and 46%). The most frequently persisting non-DTA mutations were: SRSF2 (42%), IDH2 (28%) and IDH1 (21%). The VAF at CMR was significantly lower than VAF at diagnosis (mean: 16% vs 36%, p<0.0001). In line with previous reports, we confirm in NPM1mut AML that the detection of persistent non-DTA mutations (n=20 pts) at CMR was associated with shorter OS, while this was not true for DTA mutations (5-yrs OS: 46% vs 86% for no persisting and 84% for DTA persisting, p=0.001). Moreover, 15/136 pts (11%) acquired novel mutations at CMR (among which: 2 BCOR, 1 RUNX1, 1 SF3B1 and 2 TP53 mutations) and showed worse prognosis (5-yrs OS: 52% vs 84%, p=0.036; 5-yrs EFS: 17% vs 65%, p=0.04) than those who did not (121/136, 89%). By multivariate analysis including known risk factors such as: age, bone marrow blast levels and karyotype, we found that both persistence (HR: 2.8, CI=1.3-6, p=0.007) and acquisition (HR: 3.3, CI=1-11, p=0.05) of non-DTA mutations are independent predictors of outcome.
Conclusions. Here we challenge the notion that in NPM1mut AML the NPM1 mutation is generally the first hit. We show that especially in older patients NPM1 is a frequent 2nd hit mutation and that the corresponding 1st hit mutations are more likely to persist in CMR. We also show for the first time in NPM1mut AML that the persistence of non-DTA gene mutations and the acquisition of further hits at CMR impacts prognosis. These findings suggest that the longitudinal assessment of pts at CMR will potentially aid prognostic stratification of NPM1mut AML pts and eventually lead to therapeutic strategies aimed at eradicating a pre-leukemic/relapse state in this subset of patients.

Disclosures
Cappelli: MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Baer:MLL Munich Leukemia Laboratory: Employment. Nadarajah: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. Höllein:MLL Munich Leukemia Laboratory: Employment.