Identification of Clinically Relevant Predictive MRD Checkpoints in AML Patients with NPM1 Mutations: A Study of the AML Study Group (AMLSG).

Mutations in the nucleophosmin 1 (NPM1) gene represent the most frequent gene mutations in acute myeloid leukemia (AML), with highest frequency (50-60%) in cytogenetically normal (CN)-AML. Several studies have shown the applicability and prognostic value of an NPM1 mutation (NPM1^mut)-based assay for detection of minimal residual disease (MRD). So far, there are no studies evaluating the prognostic value of NPM1^mut MRD levels in a large controlled cohort of AML patients (pts) enrolled on prospective clinical trials.

To evaluate the prognostic value of NPM1^mut MRD levels in younger (16 to 60 years) AML pts harbouring NPM1 mutations type A, B or D, and to assess the influence of concurrent FLT3 internal tandem duplications (ITD).

All pts were enrolled in the prospective AMLSG 07-04 and AML HD98A treatment trials. Treatment comprised double induction therapy with ICE (idarubicin, cytarabine, etoposide) followed by high-dose cytarabine-based consolidation, autologous or allogeneic stem cell transplantation. Levels of NPM1^mut expression ratios, defined as NPM1^mut copies per 10⁴ABL copies, were determined by RQ-PCR using TaqMan technology. Dilution series showed a maximum sensitivity of 10^-6 and high specificity as no wildtype NPM1 could be detected.

A total of 1079 samples, [bone marrow (BM), n=1062; peripheral blood, n= 17) from 212 pts were analyzed at diagnosis, after each treatment cycle, during follow-up and at relapse (median number of samples per pt, n=4; range, 1-16). NPM1^mut expression ratios at diagnosis varied between 1.1×10⁴ and 10.4×10⁶ (median, 6.9×10⁵). Pretreatment transcript levels were not associated with clinical characteristics (e.g., age, white cell counts, BM blasts) and did not impact on relapse-free (RFS) and overall survival (OS). Following the first induction cycle, the median decrease of the MRD level ratio normalized to pretreatment levels was 4.21×10^-3, independent of the presence of concurrent FLT3-ITD (p=0.39)_. After the 2nd induction cycle, the median reduction of MRD levels was significantly stronger in the FLT3-ITD^neg group (6.75×10^-5) compared with the FLT3-ITD^pos group (4.19×10^-4) (p=0.003) and this differential effect was observed throughout consolidation therapy.

For evaluation of the prognostic impact of NPM1^mut MRD levels, we compared patients achieving PCR-negativity with those with positive values at different checkpoints. The first reliable checkpoint was after double-induction therapy: the cumulative incidence of relapse (CIR) at 4 years of PCR-negative patients (n=27) was 0% compared with 48% (SE, 4.4%, p<0.00001) for PCR-positive patients (n=105). This translated into a significant better OS (p=0.0005). The second checkpoint was after completion of consolidation therapy (first measurement during follow-up period). Again, 4-year CIR was significantly (p<.00001) lower in the PCR-negative group with 11% (SE, 6.5%) compared with 51% (SE, 4.8%) in PCR-positive patients, again translating in a significantly better OS (p<.00001). In addition, the level of NPM1^mut expression ratio at any time point examined after completion of therapy correlated with the risk of relapse, since 20 of 22 pts with a value above 1000 NPM1^mut/10⁴ABL copies relapsed after a median interval of 90 days (range, 11-709 days). The remaining 2 pts had increasing levels at last follow-up but are still in continuous complete remission (CR). In a few cases relapse prediction appeared to be limited due to inadequate increase of NPM1^mut expression levels or to loss of NPM1 mutation at the time of relapse (n=5). On the other hand, we observed a number of pts (n=17) in continuous CR who had intermittent low (<1000 NPM1^mut/10⁴ABL copies) NPM1^mut expression ratios.

The levels of NPM1^mut expression at two distinct checkpoints, after double induction therapy and after completion of consolidation therapy, can be used as a prognostic factor in NPM1^mut AML pts. The adverse outcome of pts carrying a concurrent FLT3-ITD is reflected by a significant lower reduction of tumor burden.

Göhring:Celgene Corp.:. Schlegelberger:Celgene Corp.:.