Acute Myeloid Leukemia (AML) is a hematological disease clonal characterized by proliferation neoplasm cells in the bone marrow and peripheral blood, in consequence genetic alterations at hematopoietic stem cells. Genetic variants, as FLT3-ITD and W288Cfs NPM1 are common in the AML, these variants are important in the diagnosis and treatment of the disease.

In this study, we developed a multiplex PCR method to detection of NPM1 W288Cfs and FLT3-ITD variants in AML patients.

Bone marrow sample was collected from AML patients. The multiplex PCR was performed using specific primers for NPM1 W288Cfs and FLT3-ITD variants, the amplified products were separated by electrophoresis with a 2.5% agarose gel. Clinical, epidemiological, and laboratory data were analyzed from physical and electronic records, with statistical analyses performed to describe and determine the findings. The European LeukemiaNet (ELN) risk stratification was used, considering molecular results of the FLT3-ITD and NPM1 W288Cfs.

The study population was composed of 40 AML patients, female patients were most frequent with 25 (62.5%), the average age was 52 years ± 21.46. The study included 30 (75%) primary AML, 6 (15%) secondary and 4 (10%) relapsed cases, M1 and M2 subtype were prevalent, 30% and 25%, respectively. Hematological data revealed that the patients present anemia 2,728 × 103/mm3 (± 0,572), thrombocytopenia 50,28/mm3 (43,37) and leukocytosis 57,40/mm3 (± 111,2). Curiously, only 4 (10%) patients were NPM1 W288Cfs positive. Accord ELN risk stratification, 3 (7.5%) patients were classified as favorable (NPM1+ e FLT3-ITD-), 30 (75%) adverse (NPM1-) and 7 (17.5%) as intermediary (NPM1+ e FLT3-ITD+ / NPM1- e FLT3-ITD+).

NPM1 W288Cfs variant present low frequency (10%), contrary to other studies, while the risk stratification data are consistent with the ELN. The current World Health Organization classification-2022 highlights the need to implement genetic and molecular tests as routine to evaluate AML patients. Thus, the techniques used had a high sensitivity and specificity in identifying the FLT3-ITD and NPM1 W288Cfs variants, since allowed to characterize AML patients and defining genetic abnormality.

Here, we observed a frequency low of the NPM1 W288Cfs variant in the Amazonas State. The multiplex PCR consist of assay in-house of low cost, with good sensitivity and specificity for FLT3-ITD and NPM1 W288Cfs mutations, allowing to detect the mutations simultaneously.