PAX5 MOLECULAR PROFILE IN PEDIATRIC PATIENTS WITH PRECURSOR B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA

INTRODUCTION AND OBJECTIVES: Precursor B-cell acute lymphoblastic leukemia (pB-ALL) is characterized by cytogenetic-molecular alterations with prognostic value. However, a subgroup known as "B-others" lack such alterations. PAX5 is a transcription factor responsible for B-cell maturation, and it is the most frequently altered gene in "B-others", with somatic genetic alterations observed in about 40% of the cases. These include deletions (27%), intragenic amplifications (2.5%), translocations (2.5%), and point mutations along the gene (7%), with P80R accounting for more than half of them. Recently, the germline variants PAX5 c.547 G>A (p. Gly183Ser) and PAX5 c.113 G>A (p. Arg38His) have been identified in cases with familial aggregation of leukemias. Given this scenario, the main objective of this study is to characterize somatic and germline genetic alterations in PAX5, as well as epigenetic changes and mRNA expression. MATERIAL AND METHODS: This is a retrospective study on a series of 950 pediatric pB-ALL cases (age<18 years) diagnosed between 2015 and 2019. PAX5 copy number alterations (CNAs) are being assessed by Multiplex Ligation-dependent Probe Amplification (MLPA). Gene expression is being quantified by RT-qPCR using TaqMan probes. Somatic mutation analysis of P80R and germline variants c.547 G>A (p. Gly183Ser) and c.113 G>A (p. Arg38His) is being performed by pyrosequencing (PyroMark Q96), along with methylation profiling of PAX5 promoter and enhancer also by pyrosequencing. RESULTS AND CONCLUSION: So far, 126 cases have been evaluated for CNAs, with 19% showing deletions, most frequently affecting exon 8, and 10.3% showing amplifications without a preferred exon. Additionally, 142 cases were sequenced for c.547G>A (p.Gly183Ser), 146 cases for c.113G>A (p.Arg38His), and 136 cases for P80R, and the latter was detected in one case (0.74%). PAX5 gene expression was evaluated in 114 cases according to CNA profile (wild-type; amplified; deleted). In this analysis, the deleted group showed higher PAX5 expression compared to the wild-type group. Next, we will continue CNA analyses; evaluate somatic/germline genetic alterations by whole-genome sequencing, since the frequency of point mutations when assessing hotspots was lower than expected; expand gene expression evaluation using TaqMan probes for the most frequently deleted regions of PAX5; and initiate methylation analyses.