Prevalence and Prognostic Impact of FLT3-ITD and NPM1 Mutations: A Cohort of Patients with Acute Myeloid Leukemia from the ABC-SP, Brazil

Several factors determine the prognosis of patients with Acute Myeloid Leukemia (AML) including mutations in FLT3 (15-25%) and NPM1 (35%), which carry a poor and a favorable prognostic value, respectively. In latest LeukemiaNET classification, such mutations are, together, sole factor for classification groups on all 3 risk groups. Few studies are investigating the prevalence and clinical correlation of these mutations in the Brazilian population. Here we show the prevalence and impact of NPM1 and FLT3 mutations in the ABC region of São Paulo.

We prospectively and retrospectively evaluated 38adult AML patients from Hospital Estadual Mario Covas from May 2008 until October 2016.

To investigate the mutations, we first extracted the genomic DNA using DNA Illustrative Blood GenomicPrep Mini Spin® Kit from the collection of 5 mL of peripheral blood and bone marrow from patients previously selected. Second, we performed semi-quantitative PCR to amplify the samples using specific primers for NPM1 and FLT3 mutations. The PCR reaction was done through Taq DNA Polymerase Recombinant® reagent, according to the manufacturer's protocol. The product obtained using primers is approximately 318bp for NPM1 wild-type allele and 322bp for mutant allele of NPM1 and 393bp for wild-type FLT3 alleles and greater than 393bp for mutated alleles of the same gene.

30-50 ng of DNA was used for amplification of the sequence of interest. After PCR reaction, we performed purification of the PCR product with PureLink PCR Micro® Kit.

Afterward, the purified material was diluted with water nuclease-free. FLT3 material was diluted between 1:20 (if weak staining in the gel) and 1:30 (if high staining and DNA drag on the gel). While the dilution of NPM1 material ranges from 1:5 (if weak staining in the gel) to 1:10 (if high staining and DNA drag on the gel).

Finally, we analyzed the PCR product, a mixture of 8.8 μL formamide, 0.2 μL GeneScan™ 600 LIZ® Size Standard v2.0 and 1 μL purified PCR product was made. This mixture was pipetted into a 96-well plate and incubated at 95 °C for 3 minutes, placed on an ice sheet for 5 min and taken to the capillary electrophoresis apparatus (Applied Biosystems 3500) programmed for analysis of long fragments. The injections were pre-analyzed in the Gene Mapper 5 program. As a result of the investigation, we had a good graphic reading of 38 samples for the FLT3 mutation and 34 for NPM1. We found 10 mutated samples for FTLT3 and 7 for NPM1 (26.3 and 20.6%, respectively). We conclude that FLT3 and NPM1 mutations are frequent in the Brazilian population, further studies are needed to confirm our preliminary results.