NPM1 Mutated AML Can Relapse with Wildtype NPM1 and Evolve with Novel Mutations: Implications for Pathobiology and MRD Monitoring

Introduction: Minimal residual disease (MRD) monitoring by qPCR is feasible in AML with mutated NPM1 (NPM1 mut ) . NPM1 mut leukemia is associated with a favorable outcome and MRD negativity is associated with an excellent prognosis. Cytogenetic and molecular analysis have proven the concept of clonal evolution in relapsed or refractory AML. Data is conflicting on NPM1 mut AML relapsing with wildtype NPM1 (NPM1 negative). In this study we analyzed 105 relapsed NPM1 mut AML patients including 15/105 patients that relapsed with NPM1 negative AML.

Aims: To analyze in patients with NPM1 mut AML the frequency and mutational landscape of NPM1 negative AML at relapse and provide insight into clonal evolution.

Patients and Methods: Between 2006 und 2017 we investigated a total of 105 intensively treated patients with NPM1 mut AML who attained a complete molecular remission (CMR) and finally relapsed. MRD was monitored by qPCR at 1394 consecutive time points (median=12 per patient, range 3-42). 15/105 patients experienced a NPM1 negative relapse. Unique molecular identifier based sequencing of 63 genes associated with hematological malignancies was performed for patients with NPM1 negative relapse in paired samples at diagnosis and at clinical relapse. The median coverage was 4,900x. Digital error suppression was applied to follow mutations in up to 1/500 molecules (VAF 0.2%).

Results: For 15 patients with an NPM1 negative relapse the median time to relapse was 39 months (range 4-66). In contrast for 90/105 patients with an NPM1 mut relapse the median time to relapse was significantly shorter with 14 months (range 3-77, p=0.005). We analyzed the overall survival (OS) following relapse. For patients with NPM1 negative relapse OS at 2 years was 48% [95% CI, 12-77] versus 68% [52-79] for patients with NPM1 mut relapse (n.s.).

For 61/90 patients with NPM1 mut relapse the diagnostic sample was available for analysis with the 63 gene panel. The mutational landscape of these patients revealed recurrent co-mutations in 14 different genes in 54/61 patients, with the published pattern: DNMT3A, TET2, FLT3- ITD, NRAS, SRSF2, IDH1, IDH2 and FLT3- TKD were affected in more than 10% of cases. A median of 2 genes were co-mutated per patient, no co-occurring mutation was found in only 7/61 patients (11%).

For 12/15 cases with NPM1 negative relapse both diagnostic and relapse samples were available for 63 gene panel sequencing. At diagnosis we identified a total of 33 co-mutations in addition to NPM1 in a total of 12 different genes in 12/12 patients (DNMT3A (n=9 ), TET2 (6), IDH1 (4), SRSF2 (3), PTPN11 (2), IDH2 (1), KRAS (1), NRAS (1), EZH2, (1), STAG2 (1), ASXL1 (1) and FLT3- TKD (1)). All patients had at least one co-mutation with a median of 2 co-mutations per patient at diagnosis (range 1-6). At relapse we identified a total of 37 co-mutations in 13 different genes in 11/12 patients with a median of 2 mutations per patient (range 0-6). Eleven mutations in 9 genes (TET2, STAG2, PTPN11, SRSF2, KRAS, DNMT3A, EZH2, FLT3- TKD and NRAS) ) were lost at relapse and 15 mutations in 10 genes (TP53, RUNX1, NRAS, NF1, IDH2, SRSF2, WT1, IDH1, EZH2, TET2) were gained at relapse. We used digital error correction to reassess these mutations in the diagnostic sample. With a detection limit of up to 0.2% VAF (1/500 molecules) none of these mutations was detectable at diagnosis. DNMT3A was the most prevalent mutation (9/12 at diagnosis, 7/12 at relapse) and compared to the cohort with NPM1 mut relapse the presence of a DNMT3A mutation was associated with NPM1 negative relapse (p=0.024).

One patient (8%) relapsed with the respective ancestral clone from diagnosis (w/o NPM1), 9 patients (75%) showed a relapse with the known ancestral clone plus evolution, and 2 patients (17%) relapsed with a completely unrelated clone.

Conclusion: We identified 14% of NPM 1mut AML patients who relapsed with an NPM1 negative AML following intensive therapy. NPM1 negative relapse occurs significantly later than NPM1 mut relapse and is associated with the ancestral clone in the majority of patients. NPM1 negativity at relapse evades MRD assessment, however mutations in the ancestral clone should be detectable by NGS with digital error correction. This could be included in an extended MRD monitoring strategy, and performed in addition to qPCR for NPM1 at the routine time points.