Effective NPM1 plasmid standards selection for minimal/measurable residual disease monitoring in acute myeloid leukemia.

BACKGROUND: NPM1 plasmid standards are required for absolute quantification of minimal residual disease in acute myeloid leukemia patients. The standards are usually obtained, next to commercially constructed gene fragments, from transgenic bacteria colonies. However, this procedure is laborious and very time consuming. METHODS AND RESULTS: We have developed a PCR method that speeds up, simplifies, and streamlines the process of preparing NPM1 plasmid standards. The method is based on a combination of three primers, two surrounding the usual NPM1 mutation position and one over the mutation site. With this method, we were able to clearly distinguish plasmids with at least 15 different NPM1 mutations from the wild-type NPM1 plasmid. CONCLUSIONS: With the new approach, preparing NPM1 plasmid standards is easier, identifying NPM1-positive colonies is possible in less than a day and moreover, for a lower price than commercially constructed gene fragments.

Prognostic significance of mutation profile at diagnosis and mutation persistence during disease remission in adult acute myeloid leukaemia patients.

In this multi-centre study, we analysed the prognostic impact of mutations in 19 genes associated with myeloid malignancies in 258 newly diagnosed acute myeloid leukaemia patients (aged 19-70 years) undergoing intensive therapy. We identified five patient groups with different prognostic risks and different benefits from allogeneic hematopoietic stem cell transplantation (alloHSCT) within the intermediate cytogenetic risk group patients (n = 184). The most adverse prognosis was observed in patients with DNMT3A and FLT3-ITD co-mutation, whose survival could be significantly improved with alloHSCT. In contrast, the most favourable prognosis without any further benefit from alloHSCT was identified in patients with mutations in NPM1 or CEBPA, after exclusion of the unfavourable prognostic groups defined by mutations in DNMT3A, RUNX1 or genes from chromatin/spliceosome group. An additional analysis of 113 diagnosis-remission paired samples revealed that persistence of non-DNMT3A mutations (above 2% VAF) represented a further negative prognostic factor. The proposed model offers a possible molecular stratification and treatment guidance for intermediate cytogenetic risk group patients.