engleski

NPM1 mutations in AML detected by fragment analysis and Sanger sequencing (PP04)

NPM1gene mutations can be detected in approximately 1/3 of all de novo AMLs particularly in cases with normal karyotype and predicts good prognosis. However, associated with FLT3-ITD is of adverse prognosis Aim: We have developed a two-step molecular assay for the detection of NPM1 mutations. Patients and methods: The first part, prescreening for mutation, consists of RT-PCR amplification of exon 12 of the gene with the subsequent gene scan (fragment analysis)by capillary electrophoresis in AB310 Sequencer analyzer. What follows is re- amplification of mutation-positive cases and direct sequencing for the determination of the precise type of mutation. We tested 60 patients with de novo (46) and secondary (14) AMLs from our institution. The karyotype was available for 32% of them and FLT3-ITD analysis was done in all. Results: NMP1 mutations were found in 12 patients with de novo AML (26% of de novo AML ; 20% for all AMLs). None was from the sAML group. Sequencing revealed 8 cases of A-type (TCTG), 2 of D-type (CCTG) and 1 each of H-type (CTTG) and Nm-type (CCAG mutation. Expected frequencies are given in the table. Mutation type N % (95% CI) Expected % A 8 67 (35-90) 70-80 D 2 17 (2-48) approx. 6, < 10 H 1 8 (0-27) approx. 2 Nm 1 8 (0-27) approx. 0, 4, < 1 Σ 12 - - There was also a significant association of NPM1 and FLT3-ITD mutation (H2=6, 322 ; DF=1 ; P=0, 012). Conclusion: In conclusion, the two-step molecular assay for NPM1 mutation detection is rational and acceptable method for the routine diagnostic application. Frequencies of NPM1 mutations in our patients are comparable to the ones reported previously, with type A being the most frequent. However, although in the small number of patients, we have observed increased occurrence of relatively rare molecular types. We can also confirm that there is an increased association of FLT3-ITD with NPM1 mutation.

NPM1 mutations; AML; sequencing

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