Introduction. Genomic alterations are frequently associated with acute lymphoblastic leukemia (ALL) prognosis. TCF3-PBX1 fusion is one of these alterations associated with B-cell precursor ALL (BCP-ALL) subtype. Additional genomic aberrations, observed in most pre-leukemic clones, are crucial in both BCP-ALL leukaemogenesis and treatment management. Aims. To explore the role of deletions in genes involved in lymphoid differentiation, cell cycle regulation and cytokine receptors on BCP-ALL TCF3-PBX1 + patients; To evaluate the difference in the gene expression profile of genes involved in cell cycle regulation when comparing patients with and without RB1 deletions (RB1del); To correlate the alterations found with clinical-laboratorial characteristics and with risk stratification variables. Methods. BCP-ALL patients younger than 18 years-old and without previous leukemic treatment were included in the present study. Submicroscopic deletions were identified by multiplex ligation-dependent probe amplification (MLPA) using SALSA MLPA P335 A4/B1 kit. RB1delwill be confirmed by FISH using in-house probes targeting the regions of interest in RB1 . Gene expression of genes involved in cell cycle regulation was assessed using the TaqMan Array 96-well plate - Human cyclins & Cell Cycle Regulation. Univariable analyses of characteristics were performed using the Fisher's exact test or χ2 test for qualitative variables using GraphPad Prism software. Results. Fifty cases harboring the TCF3-PBX1 (6.9% of the overall BCP-ALL series) were identified in the present study. They were predominantly male, aged 1-10 years-old, with WBC count ≤50x109/L and classified as pre-B ALL. Additional alterations in those TCF3-PBX1 + cases were identified by MLPA, with the most frequent alterations affecting genes involved in cell cycle regulation and lymphoid differentiation: CDKN2A/Bdel (40.0%) and PAX5del (37.7%), followed by RB1del (31.1%) and BTG1del (17.7%). The frequency of RB1del in TCF3-PBX1 +patients was markedly different from those with other cytogenetic subgroups of BCP-ALL (P <0.05). RB1del cases were mainly female (68.7% vs 31.3%) and molecular characterized by 9p21 deletions (37.5% CDKN2A/Bdel plus PAX5del). FISH analyses revealed heterogeneity of nuclei harbouring RB1del, varying from 12% to 97% of the interphase nucleus evaluated. Regarding the expression profile, we observed that the presence of RB1del is associated with significantly increased expression of CCND2 (P =0.032) , while the expression of CDKN2D was reduced compared to cases without RB1del (P =0.043). Additionally, we observed that the complete deletion of RB1 (involving all exons) was significantly associated with increased expression of CCNA2, CCNB1 , CDC2 and E2F3 , as a consequence of the loss of critical phosphorylation sites that regulate pRB interactions (P <0.05). On the other hand, RB1 partial deletion (involving exons 17 to 26) was associated with the reduction of CDKN2D expression. Conclusions.RB1 deletions were frequently found as an additional aberration particularly in TCF3-PBX1 + patients. Deletions in 9p21 locus were also frequently found in this cytogenetic subgroup. The loss of critical RB1 phosphorylation sites, as a consequence of RB1del affecting all exons, deregulate the expression of E2F3 , an important transcript factor that interacts directly with pRB and regulate the expression of other genes involved in the cell cycle, such as cyclins A2 and B1 , and CDC2 , essentials for G1/S and G2/M phase transitions. DisclosuresNo relevant conflicts of interest to declare.