Clonal evolution patterns in acute myeloid leukemia with NPM1 mutation

Abstract

Mutations in the nucleophosmin 1 (NPM1) gene are considered founder mutations in the pathogenesis of acute myeloid leukemia (AML). To characterize the genetic composition of NPM1 mutated (NPM1^mut) AML, we assess mutation status of five recurrently mutated oncogenes in 129 paired NPM1^mut samples obtained at diagnosis and relapse. We find a substantial shift in the genetic pattern from diagnosis to relapse including NPM1^mut loss (n = 11). To better understand these NPM1^mut loss cases, we perform whole exome sequencing (WES) and RNA-Seq. At the time of relapse, NPM1^mut loss patients (pts) feature distinct mutational patterns that share almost no somatic mutation with the corresponding diagnosis sample and impact different signaling pathways. In contrast, profiles of pts with persistent NPM1^mut are reflected by a high overlap of mutations between diagnosis and relapse. Our findings confirm that relapse often originates from persistent leukemic clones, though NPM1^mut loss cases suggest a second "de novo" or treatment-associated AML (tAML) as alternative cause of relapse.

Fig. 1

Incidence of mutations in 129 paired (diagnosis/relapse) NPM1^mut pts. Colored bars indicate the presence of a mutation, white bars represent wild-type, data not available is indicated by a gray bar. Light and dark green bars illustrate heterozygous and homozygous FLT3-ITD mutations, respectively. Bars marked by X illustrate different mutation types found in the diagnosis and relapse sample. Stability was calculated by the number of mutations that persisted at relapse divided by all mutations present at diagnosis. D, diagnosis; R, relapse

Fig. 2

CNAs and UPDs identified at diagnosis and relapse by SNP profiling. Bars next to each chromosome indicate genomic gain, loss, and uniparental disomy (UPD). The length of the bar indicates the respective genomic region affected by the aberrations. Bars highlighted by a grey box indicate relapse specific aberrations. Bars marked by a star indicate genomic changes which are lost at relapse

Fig. 3

Mutational map and enriched pathways in diagnosis/relapse pairs of NPM1^mut loss and persistent pts. a Mutational profile of 10 NPM1^mut loss and 10 NPM1^mut persistent pts found by WES. Colors indicate the varying occurrence of mutations during disease progression, including mutations which persist during remission (preleukemic/germline mutations; [green]), mutations which are solely present in the diagnosis sample (blue), relapse specific mutations (red) and mutations which are shared between the diagnosis and relapse sample (black). b Enriched pathways in diagnosis and relapse samples from NPM1^mut loss and NPM1^mut persistent pts based on mutated genes

Fig. 4

Mutation types before and after chemotherapy in NPM1^mut loss and persistent pts. a Frequency of transversions (A to C, A to T, C to A, and C to G) and transitions (A to G and C to T). b Proportion of transversions in NPM1^mut loss diagnosis (rose) and relapse (pink) samples and NPM1^mut persistent diagnosis (light blue) and relapse (dark blue) samples

Fig. 5

Heatmap of differentially expressed genes and GSEA of diagnosis versus relapse NPM1^mut loss samples. a Differentially expressed genes (significant at 0.05 level of the univariate test) from RNA-Seq were assessed using the ClassComparison option from BRB-ArrayTools. Blue indicates upregulation and red downregulation between groups. b Diagnosis samples show enrichment of MYC_UP.V1_UP and BIOCARTA_CELLCYCLE_PATHWAY gene sets. c Relapse samples were enriched in KEGG_MAPK_SIGNALING_PATHWAY, WNT_UP.V1_UP gene sets and genes which are downregulated in NPM1^mut samples (VERHAAK_AML_WITH_NPM1_MUTATED_DN). ES, enrichment score; FDR, false discovery rate; NES, normalized enrichment score

Fig. 6

Flow cytometry of total and active CTNNB1 in diagnosis samples of NPM1^mut loss and NPM1^mut persistent pts. Higher expression of a CTNNB1 total, b CTNNB1 8e7 in 3 NPM1^mut persistent diagnosis samples compared to 4 NPM1^mut loss diagnosis samples, and c mean fluorescent intensity (MFI) including all data points, data is presented as mean ± s.d

Fig. 7

Remission duration of NPM1^mut loss and persistent pts. Pts with NPM1^mut loss (red) had a significantly longer remission duration compared to pts maintaining NPM1^mut (black) at relapse

Fig. 8

Possible mechanisms of relapse in NPM1^mut AML. a Based on our mutation data we postulate different mechanisms of relapse for NPM1^mut loss and NPM1^mut persistent pts. b Variant allele frequency plots of mutated genes from exemplary pts for each group