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.

Central European Institute of Technology Masaryk University Brno Czech Republic

Department of Internal Medicine Hematology and Oncology Faculty of Medicine Masaryk University Brno Czech Republic

Department of Internal Medicine Hematology and Oncology University Hospital Brno Cernopolni 9 613 00 Brno Czech Republic

See more in PubMed

Papaemmanuil E et al (2016) “Genomic Classification and Prognosis in Acute Myeloid Leukemia,“ (in eng), N Engl J Med, vol. 374, no. 23, pp. 2209–2221, Jun 9 2016, doi: https://doi.org/10.1056/NEJMoa1516192

Federici L, Falini B (2013) “Nucleophosmin mutations in acute myeloid leukemia: a tale of protein unfolding and mislocalization,“ (in eng), Protein Sci, vol. 22, no. 5, pp. 545 – 56, May doi: https://doi.org/10.1002/pro.2240

La Manna S et al (2021) “Type F mutation of nucleophosmin 1 Acute Myeloid Leukemia: A tale of disorder and aggregation,“ (in eng),Int J Biol Macromol, vol. 188, pp.207–214, Oct 01 2021, doi: https://doi.org/10.1016/j.ijbiomac.2021.08.023

La Manna S et al (2021) doi: https://doi.org/10.1016/j.bioorg.2021.104997

Dohner H et al (2017) “Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel,“ (in eng), Blood, vol. 129, no. 4, pp. 424–447, Jan 26 2017, doi: https://doi.org/10.1182/blood-2016-08-733196

Schuurhuis GJ et al (2018) “Minimal/measurable residual disease in AML: a consensus document from the European LeukemiaNet MRD Working Party,“ (in eng), Blood, vol. 131, no. 12, pp. 1275–1291, 03 doi: https://doi.org/10.1182/blood-2017-09-801498

Arber DA et al (2016) “The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia,“ (in eng), Blood, vol. 127, no. 20, pp. 2391 – 405, 05 doi: https://doi.org/10.1182/blood-2016-03-643544