Expression Profile Analysis of Genes Related to Resistance/Sensibility to Prednisolone, Daunorubicin, L-Asparaginase and Vincristine in Childhood Acute Lymphoblastic Leukemia.

Resistance to anti-leukemic agents in childhood ALL has been strongly associated with prognosis. The aim of this study was to analyze the expression profile of genes related to resistance/sensibility of 4 anti-leukemic agents and their correlations with age, diagnosis WBC count, risk group classification, molecular cytogenetics, induction treatment response and event-free survival (EFS) in ALL children. Were analyzed the expression profile of 19 genes related to resistance/sensibility to vincristine ( RPRP2, CD44, TCF5, KCNN1, TIF1 ), prednisolo ne ( F8A, CDK2AP1, BLVRB, CD69 ), daunorubicin ( MAP3K12, SHOCK2, PDCH9, EGF1, KCNN4 ) and L-asparaginase ( GPR56, MAN1, SCGF, IGFBP7, GATA3 ) by real-time quantitative PCR, using SYBR Green approach and melting curve analysis, with GUSB as housekeeping gene. Relative quantification was done using the 2-DDCT method (efficiencies from 1.9 to 2.1 to all the analyzed genes). These genes were chosen according to those described on microarray analysis by Holleman et al. (NEJM, 351:533, 2004). From 154 consecutive children admitted to ALL treatment in the 3 participating institutions, 139 had the gene expression profile analyzed. Sixteen were excluded due to lack of good quality RNA/cDNA. The follow up ranges from 36–60 months. All patients were classified and treated according the Brazilian ALL treatment protocol (GBTLI-99). Association among the variables analyzed and the gene expression levels was evaluated by Mann-Whitney and chi-square tests. EFS analysis was performed using Kaplan-Meier and log-rank test and multivariate analysis by the Cox proportional model to define independent prognostic factors. Correlation between the genes was analyzed by Spearman test. The 4-years EFS was 75,5% to all patients analyzed (81,4% to standard risk and 68,7% to high risk). Statistical correlation was observed among the genes of resistance of daunorrubicin, L-asp and vincristine (P<0,001). There is a significant association (p<0,05) between the levels of expression of the genes TIF1A, BLVRB, CD44, SCFBP1, TCFL5 and risk group; CD44, MAP3K12, SHOCK2 and CD10 antigen and GPR56 , SHOCK2 with favorable event. The overexpression of SHOCK2 and GPR56 genes were also associated to better 4y-EFS in univariate (86,3% x 68,7% (p:0,04) and 86,2% x 72,2% (p:0,03) respectively) and multivariate analysis (p:0,03 and 0,09 respectively). No significant differences were observed in the other variables analyzed. The present data suggest that the expression of some analyzed genes have influence in the resistence/sensibility to the treatment and outcome in childhood ALL; especially over-expression of the SHOCK2 and GPR15 genes were associated with higher sensibility to daunorrubicin and L-asp respectively.