Monitoring of Minimal Residual Disease in NPM1 Mutated Acute Myeloid Leukemia (AML): Results of the AML Study Group (AMLSG)
Authors: Krönke Jan , Schlenk Richard , Jensen Kai-Ole , Eiwen Karina , Habdank Marianne , Späth Daniela , Krauter Jürgen , Ganser Arnold , Döhner Hartmut , Döhner Konstanze 
Published: October 2019

Abstract
Background: Mutations in the nucleophosmin 1 (NPM1) gene represent the most frequent gene mutations in acute myeloid leukemia (AML), with highest frequency (50–60%) in cytogenetic normal (CN)-AML and these mutations can be used as molecular markers for monitoring of minimal residual disease (MRD).
Aims: To examine the applicability and sensitivity of DNA- and RNA-based real-time quantitative polymerase chain reaction (RQ-PCR) assays and to evaluate whether MRD levels are of prognostic relevance in younger (16 to 60 years) patients (pts) with AML harboring NPM1 mutations (NPM1mut ) type A, B or D.
Methods: All pts were treated within the AMLSG 07-04 treatment trial including double induction therapy with ICE (idarubicin, cytarabine, etoposide) followed by three courses of high-dose cytarabine or allogeneic stem cell transplantation from matched related donors as consolidation therapy. Levels of NPM1mut expression ratios, defined as NPM1mut copies/ 104abl copies, were determined by RQ-PCR using TaqMan technology. DNA- and RNA-based RQ-PCR assays were used in parallel.
Results: A total of 537 samples [bone marrow (BM) n=463, peripheral blood (PB) n=74] from 88 pts have been analyzed (mean number of samples per patient, n=6;, range, 1–16) at diagnosis (BM, n=75; PB, n=12), after induction therapy (BM, n=190; PB, n=28), during consolidation (BM, n=115; PB, n=16) and during follow-up (BM, n=83; PB n=18). Sensitivity was 10−6 for the RNA- and 10−4 for the DNA-based assays. Both assays were highly specific as no wildtype (wt) NPM1 could be detected. The lower sensitivity of the DNA-based assay was demonstrated by the finding that the DNA-based RQ-PCR became negative in 102 samples while RNA-based RQ-PCR still showed NPM1mut expression in 68 of these samples (range 2.1–11018.1). In addition, comparison of 63 paired (BM and PB) samples revealed a median 6.8 times higher NPM1mut expression in BM with 13 of 15 samples being negative in PB but positive in BM. Based on these results we subsequently performed RNA-based RQ-PCR analysis in BM samples.
NPM1 mut expression ratios at diagnosis varied between 1.1 × 104 and 6.4 × 106 with a median of 5.6 × 105. Pretreatment transcript levels were not associated with clinical characteristics (e.g., age, WBC counts, BM blasts) and did not impact on relapse-free (RFS) and overall survival (OS). The median decrease of the MRD level ratio after first and second induction therapy normalized to pretreatment levels was 2.24 log10 (−8.57–1.88 log10) and 3.9 log10 (−8.58–0.67 log10), respectively. There was no difference in FLT3-ITD positive and negative pts (p=0.72 and p=0.20, respectively). During consolidation therapy NPM1mut expression levels in the FLT3-ITD negative pts further decreased after each cycle and were significantly lower compared to FLT3-ITD positive patients (p=0.00008, p=0.05, p=0.003). Of note, FLT3-ITD positive pts had rebounding MRD levels during consolidation therapy in comparison to levels achieved after induction therapy. Achievement of RQ-PCR-negativity in at least 1 BM sample (n=20) during consolidation therapy was associated with superior RFS (p=0.004) of 76.8% (95% CI 13%–61%) after 2 years compared to 29.0% (95% CI 59%–100%) for pts with persistent RQ-PCR positivity (n=37). For this analysis pts with allogeneic transplantation were excluded.
Different courses of MRD were identified in 31 relapsed pts. In 19 patients increasing (range 1.0–322.3) or constantly high MRD levels 28 to 518 days (median 66) before occurrence of clinical relapse were detected. For the remaining pts, molecular relapse was not detectable due to loss of the NPM1mut clone in relapse material (n=3), a decrease in MRD levels 71 to 90 days before relapse (n=3) or the lack of samples within 3 months before relapse (n=6).
Conclusions: NPM1 mutations are a sensitive marker for MRD monitoring in AML, in particular when RNA-based RQ-PCR assays on BM samples are used. Achievement of MRD negativity appears to predict favorable clinical outcome.