IDENTIFICATION OF GENE FUSIONS IN ACUTE LYMPHOBLASTIC LEUKEMIA

INTRODUCTION AND OBJECTIVES: Acute lymphoblastic leukemia may originate from lymphoid precursors of B or T lineages. However, 80-85% of ALL cases are associated with B cell lineage (B-ALL), which comprises multiple subtypes defined by genetic alterations that are also important for risk stratification. Chromosomal translocations can theoretically occur between any chromosome loci. In B-ALL, most of them are reciprocal translocations promoting gene fusions which deregulate lymphopoiesis, leading to formation of leukemia phenotype. This study aimed to standardize the methods for detecting the main gene fusions associated with B-ALL – BCR-ABL1, ETV6-RUNX1 and KMT2A-AFF1 – in order to establish novel studies on B-ALL in the state of Bahia. MATERIAL AND METHODS: The bone marrow or peripheral blood samples from patients diagnosed with B-ALL and commercial cell lines: K562, REH and RS4;11 were used for the standardization of methods. The cell lines presenting abovementioned gene fusions were cultured at 37°C in RPMI medium supplemented with fetal bovine serum. Subsequently, we proceeded with RNA extraction, and reverse transcription (RT) followed by polymerase chain reaction (PCR) assays for the amplification of the gene fusions. PCR products were visualized on agarose gel electrophoresis stained with ethidium bromide. RESULTS AND CONCLUSION: The first results of the standardization of the RT-PCR assays revealed the presence of the BCR-ABL1 fusion transcript in the K562 cell line, which was confirmed by visualizing the PCR product band in the agarose gel (expected band size was 210 bp). After standardization of RT-PCR reactions, we could also detect the gene fusions ETV6-RUNX1 and KMT2A-AFF1, which are present in REH and RS4;11 cell lines, respectively. Since the breakpoint distribution of KMT2A-AFF1 fusions is heterogeneous, more than one primer pair was used in the amplification reaction. Thus, a multiplex PCR was established to cover most of predicted KMT2A rearrangements and led to the formation of more than one amplicon for the same target. Therefore, in this study it was possible to establish the RT-PCR assays for detecting recurrent primary alterations in B-ALL, which allowed us to establish the first laboratory in the state of Bahia to provide molecular biology tests for patients with leukemia in the public system.