Frequency and spectrum of insertions in the NPM1 gene exon 12 in patients with de novo acute myeloid leukemia living in a large Siberian metropolis

During leukemogenesis, there is a consistent accumulation of driver aberrations leading to the development of acute myeloid leukemia (AML). Since mutations in the NPM1 gene in the absence of a concomitant FLT3-ITD mutation have favorable prognostic value and can serve as independent targets for assessing minimal residual disorder in this disease, their rapid and accurate identification in patients with newly diagnosed AML is required. Currently, the literature contains the results of several domestic studies of the mutation spectrum of NPM1 in an adult cohort of patients with a “narrow” geography. The aim of the work was to study the frequency and spectrum of insertions in the 12th exon of the NPM1 gene in a sample of patients with de novo acute myeloid leukemia living in a large Siberian metropolis.

Material and methods. The study group consisted of 128 primary patients with AML in Novosibirsk. The PCR method with flanking primers was used for screening, and direct Sanger sequencing by capillary electrophoresis was used to establish the sequence and insertion site.

Results and discussion. The frequency of mutations in the 12th exon of the NPM1 gene in the study group was 14.8 %, 84.4 % of the findings were inserts of type A, in one case (5.2 %) an insert of type B. Two new insertions previously undescribed were identified, c.863_864insTGCT and c.868_869insAAGC. The first of them is similar in its functional effect to the changes observed with classical inserts of type A and B: it leads to an elongation of the encoded protein, a shift in the reading frame, and the loss of the nucleolar localization signal motif with the formation of a typical nucleophosmin export signal motif from the nucleus. A distinctive feature of c.868_869insAAGC insertion was the partial preservation of the nucleolar localization signal due to the presence of tryptophan in the 288th position.

Conclusions. Using the developed set of primers, it is possible to screen mutations in NPM1 exon 12 in patients with AML within one working day, as well as their further accurate identification by direct sequencing during the first induction treatment cycle.

1. Vardiman J. Who’s next? The who classification of myeloid neoplasms. XXXVI. Ulusal Hematoloji Kongresi: proc. conf., 3–7 Kasım 2010, Belek, Antalya. 2010. P. 149–154.

2. Falini B., Martelli M.P., Pileri S.A., Mecucci C. Molecular and alternative methods for diagnosis of acute myeloid leukemia with mutated NPM1: flexibility may help. Haematologica. 2010;95(4):529–534. doi: 10.3324/haematol.2009.017822

3. Yuan W., Payton J.E., Holt M.S., Link D.C., Watson M.A., DiPersio J.F., Ley T.J. Commonly dysregulated genes in murine APL cells. Blood. 2007;109(3):961– 970. doi: 10.1182/blood-2006-07-036640

4. Box J.K., Paquet N., Adams M.N., Boucher D., Bolderson E., O’Byrne K.J., Richard D.J. Nucleophosmin: from structure and function to disease development. BMC Mol. Biol. 2016;17(1):19. doi: 10.1186/s12867-016-0073-9

5. Albiero E., Madeo D., Bolli N., Giaretta I., Bona E.D., Martelli M.F., Nicoletti I., Rodeghiero F., Falini B. Identification and functional characterization of a cytoplasmic nucleophosmin leukaemic mutant generated by a novel exon-11 NPM1 mutation. Leukemia. 2007;21(5):1099–1103. doi: 10.1038/sj.leu.2404597

6. Tregnago C., Benetton M., Padrin D., Polato K., Borella G., Da Ros A., Marchetti A., Porcù E., Del Bufalo F., Mecucci C., Locatelli F., Pigazzi M. NPM1 mutational status underlines different biological features in pediatric AML. Cancers (Basel). 2021;13(14):3457. doi: 10.3390/cancers13143457

7. Hindley A., Catherwood M.A., McMullin M.F., Mills K.I. Significance of NPM1 Gene Mutations in AML. Int. J. Mol. Sci. 2021;22(18):10040. doi: 10.3390/ijms221810040

8. Yao Y., Lin X., Wang C., Gu Y., Jin J., Zhu Y., Wang H. Identification of a novel NPM1 mutation in acute myeloid leukemia. Exp. Hematol. Oncol. 2023;12(1):87. doi: 10.1186/s40164-023-00449-4

9. Silin A.E., Kozich Zh.M., Shpudeyko V.K., Tropashko I.B., Silina A.A., Martinkov V.N., Martynenko S.M., Skryabin A.M., Voropayeva A.V. Mutational analysis of FLT3 and NPM1 genes in the group of adult patients with myelodysplastic syndrome and acute nonlymphoblastic leukemia. Problemy zdorov’ya i ekologii = Health and Environment Issues. 2011;(3):85–90. [In Russian]. doi: 10.51523/2708-6011.2011-8-3-17

10. Huet S., Jallades L., Charlot C., Chabane K., Nicolini F.E., Michallet M., Magaud J.P., Hayette S. New quantitative method to identify NPM1 mutations in acute myeloid leukaemia. Leuk. Res. Treatment. 2013;2013:756703. doi: 10.1155/2013/756703

11. Kumar D., Mehta A., Panigrahi M.K., Nath S., Saikia K.K. NPM1 mutation analysis in acute myeloid leukemia: comparison of three techniques – sanger sequencing, pyrosequencing, and real-time polymerase chain reaction. Turk. J. Haematol. 2018;35(1):49–53. doi: 10.4274/tjh.2017.0095

12. Severina N., Sidorova Y., Risinskaya N., Biderman B., Pshenychnyy A., Ryzhikova N., Lukianova I., Kashlakova A., Sudarikov A. PB1792: NGS vs PCR for the detection of NPM1 gene mutations in acute myeloid leukemia. Hemasphere. 2022;6(Suppl.):1672–1673. doi: 10.1097/01.HS9.0000850020

13. Maslyukova I.E., Kurochkin D.V., Martynova E.V., Bakhtina V.I., Subbotina T.N. Comparison of fragment analysis and PCR electrophoresis methods for the detection of FLT3–ITD mutations in patients with acute myeloid leukemia. Onkogematologiya = Oncohematology. 2022;17(4):118–125. [In Russian] doi: 10.17650/1818-8346-2022-17-4-118-125

14. Gebhard C., Glatz D., Schwarzfischer L., Wimmer J., Stasik S., Nuetzel M., Heudobler D., Andreesen R., Ehninger G., Thiede C., Rehli M. Profiling of aberrant DNA methylation in acute myeloid leukemia reveals subclasses of CG-rich regions with epigenetic or genetic association. Leukemia. 2019;33(1):26–36. doi: 10.1038/s41375-018-0165-2

15. El-Gamal R.A.E., Hashem A.E., Habashy D.M., Abou Elwafa M.A.Z., Boshnak N.H. Flow cytometry in detection of Nucleophosmin 1 mutation in acute myeloid leukemia patients: A reproducible tertiary hospital experience. Int. J. Lab. Hematol. 2021;43(1):68–75. doi: 10.1111/ijlh.13317

16. Rastogi P., Naseem S., Varma N., Varma S. Immunohistochemical detection of NPM1 mutation in acute myeloid leukemia and its association with cup-like nuclear morphology of blasts. Appl. Immunohistochem. Mol. Morphol. 2016;24(4):261–267. doi: 10.1097/PAI.0000000000000182

17. Martelli M.P., Manes N., Liso A., Pettirossi V., Verducci Galletti B., Bigerna B., Pucciarini A., de Marco M.F., Pallotta M.T., Bolli N., … Falini B. A western blot assay for detecting mutant nucleophosmin (NPM1) proteins in acute myeloid leukaemia. Leukemia. 2008;22(12): 2285–2288. doi: 10.1038/leu.2008.149

18. Severina N.A., Sidorova Yu.V., Biderman B.V., Risinskaya N.V., Likold E.B., Glinshchikova O.A., Pshenichny A.S., Sudarikov A.B. Comparison of various methods for detecting non-standard NPM1 gene insertion in AML patients. Vestnik gematologii = Bulletin of hematology. 2021;2:75–76. [In Russian].

19. Sekeres M.A., Elson P., Kalaycio M.E., Advani A.S., Copelan E.A., Faderl S., Kantarjian H.M,. Estey E. Time from diagnosis to treatment initiation predicts survival in younger, but not older, acute myeloid leukemia patients. Blood. 2009;113(1):28–36. doi: 10.1182/blood-2008-05-157065

20. Shi Y., Chen X., Jin H., Zhu L., Hong M., Zhu Y., Wu Y., Qiu H., Wang Y., Sun Q., … Qiao C. Clinical prognostic value of different NPM1 mutations in acute myeloid leukemia patients. Ann. Hematol. 2024;103(7):2323– 2335. doi: 10.1007/s00277-024-05786-w

21. Vinogradov A.V., Rezaykin A.V., Salakhov D.R., Ioshchenko S.E., Sergeyev A.G. Comparative analysis of the results of typing molecular damage of the NPM1 gene in acute myeloid leukemia using direct automatic sequencing and immunohistochemical method. Vestnik Ural’skoy meditsinskoy akademicheskoy nauki = Journal of Ural Medical Academic Science. 2013;(4):124– 127. [In Russian].

22. Vinogradov A.V., Rezaykin A.V., Sazonov S.V., Sergeev A.G. Сlinical and pathological features DNMT3A, FLT3, KIT, NPM1, NRAS, TP53 and WT1 genes mutations detection in acute myeloid leukemia patients aged 15–45 years old. Geny i kletki = Genes and Cells. 2018;13(3):70–74. [In Russian]. doi: 10.23868/201811036

23. Vinogradov A.V., Rezaykin A.V., Salakhov D.R., Izotov D.V., Ioschenko S.E., Sergeev A.G. DNMT3A, FLT3, KIT, KRAS, NRAS, NPM1, TP53 and WT1 genes mutations detection in acute myeloid leukemia with normal karyotype. Vestnik Ural’skoy meditsinskoy akademicheskoy nauki = Journal of Ural Medical Academic Science. 2016;(2):89–101. [In Russian]. doi: 10.22138/2500-0918-2016-14-2-89-101

24. Petrova E.V., Martynkevich I.S., Polushkina L.B., Martynenko L.S., Ivanova M.P., Tsybakova N.Yu., Kleina E.V., Shabanova E.S., Chechetkin A.V., Abdulkadyrov K.M. Clinical, hematological and molecular-genetic features of acute myeloid leukemia with mutations in FLT3, CKIT, NRAS and NPM1. Gematologiya i transfuziologiya = Hematology and Transfusiology. 2016;61(2):72–80. [In Russian]. doi: 10.18821/0234-5730-2016-61-2-72-80

25. Belotserkovskaya E.V., Zaykova E.K., PetukhovA.V., Demidov O.N., Levchuk K.A., Budayeva I.G., Zaytsev D.V., Rogovaya Yu.D., Shatilova A.A., Bogdanov K.V., … Girshova L.L. Identification of mutations in IDH1/2, DNMT3A, ASXL1 genes of genome epigenetic regulation and their co-occurrence with FLT3, NPM1, RUNX1 mutations in acute myeloid leukemia. Klinicheskaya onkogematologiya = Clinical Oncohematology. 2021;14(1):13–21. [In Russian]. doi: 10.21320/2500-2139-2021-14-1-13-21

26. Gritsaev S.V., Martynkevich I.S., Chubukina Zh.V., Petrova E.V., Kostroma I.I., Ivanova M.P., Martynenko L.S., Potikhonova N.A., Bubnova L.N., Abdulkadyrov K.M. The specific features of the immunophenotype of blast cells in patients with de novo normal karyotype acute myeloid leukemia and FLT3-ITD mutation. Terapevticheskiy arkhiv = Therapeutic Archive. 2014;86(3):71–77. [In Russian].

27. Sondka Z., Dhir N.B., Carvalho-Silva D., Jupe S., McLaren M.K., Starkey M., Ward S., Wilding J., Ahmed M., Argasinska J., … Teague J. COSMIC: a curated database of somatic variants and clinical data for cancer. Nucleic Acids Research. 2024;52(D1):D1210– D1217. doi: 10.1093/nar/gkad986

28. Heuser M., Freeman S.D., Ossenkoppele G.J., Buccisano F., Hourigan C.S., Ngai L.L., Tettero J.M., Bachas C., Baer C., Béné M.C., … Cloos J. 2021 Update on MRD in acute myeloid leukemia: a consensus document from the European LeukemiaNet MRD Working Party. Blood. 2021;138(26):2753–2767. doi: 10.1182/blood.2021013626

29. Sahasrabudhe K.D., Mims A.S. MRD in AML: who, what, when, where, and how? Blood. 2024;143(4):296–298. doi: 10.1182/blood.2023022226

30. Lachowiez C.A., Loghavi S., Kadia T.M., Daver N., Borthakur G., Pemmaraju N., Naqvi K., Alvarado Y., Yilmaz M., Short N., … DiNardo C.D. Outcomes of older patients with NPM1-mutated AML: current treatments and the promise of venetoclax-based regimens. Blood Adv. 2020;4(7):1311–1320. doi: 10.1182/bloodadvances.2019001267

31. Wei Q., Wang S.A., Loghavi S., Fang H., Medeiros L.J., Wang W. Diagnostic utility of immunohistochemistry in detection of NPM1 mutations in acute myeloid leukemia with a patchy distribution. EJHaem. 2024;5(2):379–382. doi: 10.1002/jha2.866

32. Wessex Regional Genetics Laboratory. NHS England Target Report Turnaround Times. Available at: clck.ru/3N5z3e

33. Gao J., Aksoy B.A., Dogrusoz U., Dresdner G., Gross B., Sumer S.O., Sun Y., Jacobsen A., Sinha R., Larsson E., … Schultz N. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci. Signal. 2013;6(269):pl1. doi: 10.1126/scisignal.2004088

34. Voropaeva E.N., Burundukova M.V., Lyzlova A.A., Chukhontseva I.A., Maksimov V.N., Pospelova T.I. Mutations in the “hot spots” of the FLT3, NPM1, IDH1, IDH2 and DNMT3A genes in acute myeloid leukemia. Sibirskiy onkologicheskiy zhurnal = Siberian Journal of Oncology. 2025;24(1):125–141. [In Russian]. doi: 10.21294/1814-4861-2025-24-1-125-141

35. Clinical guidelines. Acute myeloid leukemia. Available at: clck.ru/3N5ypH [In Russian].