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.

In the international randomized phase III RATIFY trial, the multi-kinase inhibitor midostaurin significantly improved overall and event-free survival in patients 18-59 years of age with FLT3-mutated acute myeloid leukemia (AML). However, only 59% of patients on the midostaurin arm achieved protocol-specified complete remission (CR) and almost half of patients achieving CR relapsed. To explore underlying mechanisms of resistance, we studied patterns of clonal evolution in patients with FLT3-internal tandem duplications (ITD) positive AML who were entered on the RATIFY or the AMLSG 16-10 trial and received treatment with midostaurin. To this end, paired samples from 54 patients obtained at time of diagnosis and at time of either relapsed or refractory disease were analyzed using conventional Genescan-based testing for FLT3-ITD as well as whole exome sequencing. At the time of disease resistance or progression, almost half of the patients (46%) became FLT3-ITD negative, but acquired mutations in signaling pathways (e.g. MAPK), thereby providing a new proliferative advantage. In cases with FLT3-ITD persistence, the selection of resistant ITD-clones was found in 11% as potential drivers of disease. In 32% of cases, no FLT3-ITD mutational change was observed suggesting either resistance mechanisms bypassing FLT3-inhibition or loss of midostaurin inhibitory activity due to inadequate drug levels. In summary, our study provides novel insights into the clonal evolution and resistance mechanisms of FLT3-ITD mutated AML under treatment with midostaurin in combination with intensive chemotherapy.

In acute myeloid leukemia, there is growing evidence for splicing pattern deregulation, including differential expression of linear splice isoforms of the commonly mutated gene nucleophosmin (NPM1). In this study, we detect circular RNAs of NPM1 and quantify circRNA hsa_circ_0075001 in a cohort of NPM1 wild-type and mutated acute myeloid leukemia (n=46). Hsa_circ_0075001 expression correlates positively with total NPM1 expression, but is independent of the NPM1 mutational status. High versus low hsa_circ_0075001 expression defines patient subgroups characterized by distinct gene expression patterns, such as lower expression of components of the Toll-like receptor signaling pathway in high hsa_circ_0075001 expression cases. Global evaluation of circRNA expression in sorted healthy hematopoietic controls (n=10) and acute myeloid leukemia (n=10) reveals circRNA transcripts for 47.9% of all highly expressed genes. While circRNA expression correlates globally with parental gene expression, we identify hematopoietic differentiation-associated as well as acute myeloid leukemia subgroup-specific circRNA signatures.