BACKGROUNDDespite the molecular heterogeneity of standard-risk acute myeloid leukemia (AML), treatment decisions are based on a limited number of molecular genetic markers and morphology-based assessment of remission. Sensitive detection of a leukemia-specific marker (e.g., a mutation in the gene encoding nucleophosmin [NPM1]) could improve prognostication by identifying submicroscopic disease during remission. METHODSWe used a reverse-transcriptase quantitative polymerase-chain-reaction assay to detect minimal residual disease in 2569 samples obtained from 346 patients with NPM1-mutated AML who had undergone intensive treatment in the National Cancer Research Institute AML17 trial. We used a custom 51-gene panel to perform targeted sequencing of 223 samples obtained at the time of diagnosis and 49 samples obtained at the time of relapse. Mutations associated with preleukemic clones were tracked by means of digital polymerase chain reaction. RESULTSMolecular profiling highlighted the complexity of NPM1-mutated AML, with segregation of patients into more than 150 subgroups, thus precluding reliable outcome prediction. The determination of minimal-residual-disease status was more informative. Persistence of NPM1-mutated transcripts in blood was present in 15% of the patients after the second chemotherapy cycle and was associated with a greater risk of relapse after 3 years of follow-up than was an absence of such transcripts (82% vs. 30%; hazard ratio, 4.80; 95% confidence interval [CI], 2.95 to 7.80; P<0.001) and a lower rate of survival (24% vs. 75%; hazard ratio for death, 4.38; 95% CI, 2.57 to 7.47; P<0.001). The presence of minimal residual disease was the only independent prognostic factor for death in multivariate analysis (hazard ratio, 4.84; 95% CI, 2.57 to 9.15; P<0.001). These results were validated in an independent cohort. On sequential monitoring of minimal residual disease, relapse was reliably predicted by a rising level of NPM1-mutated transcripts. Although mutations associated with preleukemic clones remained detectable during ongoing remission after chemotherapy, NPM1 mutations were detected in 69 of 70 patients at the time of relapse and provided a better marker of disease status. CONCLUSIONSThe presence of minimal residual disease, as determined by quantitation of NPM1-mutated transcripts, provided powerful prognostic information independent of other risk factors.

SummaryAcute myeloid leukemia (AML) is characterized by recurrent mutations that affect the epigenetic regulatory machinery and signaling molecules, leading to a block in hematopoietic differentiation. Constitutive signaling from mutated growth factor receptors is a major driver of leukemic growth, but how aberrant signaling affects the epigenome in AML is less understood. Furthermore, AML cells undergo extensive clonal evolution, and the mutations in signaling genes are often secondary events. To elucidate how chronic growth factor signaling alters the transcriptional network in AML, we performed a system-wide multi-omics study of primary cells from patients suffering from AML with internal tandem duplications in the FLT3 transmembrane domain (FLT3-ITD). This strategy revealed cooperation between the MAP kinase (MAPK) inducible transcription factor AP-1 and RUNX1 as a major driver of a common, FLT3-ITD-specific gene expression and chromatin signature, demonstrating a major impact of MAPK signaling pathways in shaping the epigenome of FLT3-ITD AML.

SummaryMolecular genetic assays for the detection of the JAK2 V617F (c.1849G>T) and other pathogenetic mutations within JAK2 exon 12 and MPL exon 10 are part of the routine diagnostic workup for patients presenting with erythrocytosis, thrombocytosis or otherwise suspected to have a myeloproliferative neoplasm. A wide choice of techniques are available for the detection of these mutations, leading to potential difficulties for clinical laboratories in deciding upon the most appropriate assay, which can lead to problems with inter-laboratory standardization. Here, we discuss the most important issues for a clinical diagnostic laboratory in choosing a technique, particularly for detection of the JAK2 V617F mutation at diagnosis. The JAK2 V617F detection assay should be both specific and sensitive enough to detect a mutant allele burden as low as 1-3%. Indeed, the use of sensitive assays increases the detection rate of the JAK2 V617F mutation within myeloproliferative neoplasms. Given their diagnostic relevance, it is also beneficial and relatively straightforward to screen JAK2 V617F negative patients for JAK2 exon 12 mutations (in the case of erythrocytosis) or MPL exon 10 mutations (thrombocytosis or myelofibrosis) using appropriate assays. Molecular results should be considered in the context of clinical findings and other haematological or laboratory results.