Downregulation of mTOR and P70S6Kβ2 in Pediatric T-Cell Acute Lymphoblastic Leukemia (T-ALL) Is Correlated with a Poor Prognosis

T-ALL accounts for about 15% of pediatric ALL and more than 20% of children experience a recurrent disease which has a dismal prognosis. Characterization of molecular alterations with prognostic impact may be useful for an early identification of patients at high risk of failure in whom more intensive and/or better tailored treatments, including hematopoietic stem cell transplantation (HSCT) may be considered. PTEN/AKT pathway has been shown to be involved in children with T-ALL (Gutierrez A et al 2009), in glucocorticoid resistance (Beesley A et al 2009) and in NOTCH-1 mutated T-ALL resistant to gamma secretase inhibitors (Palomero T et al. 2009). In the attempt to better characterize the role of the PTEN/AKT/mTOR pathway in pediatric T-ALL, we have evaluated the expression of each protein in this pathway's cascade and investigated its association with outcome.

We enrolled in our study 23 children with T-ALL consecutively diagnosed at our Center in Catania from 1997 to 2009. They were treated by three consecutive AIEOP protocols. We analyzed the mRNA expression of PTEN, using RT-PCR. We evaluate by western blot analysis, the expression of the following proteins: in total (AKT; GSK3β; CK2α; CK2β; PTEN; PDK1; P70S6Kβ2; mTOR; S6K) and phosphorylated [AKT(S473) (T308); GSK3β(S9); PTEN(S380); PDK1(S241); P70S6Kβ2(S371); mTOR(S2448); S6K(Ser235/236)/(Ser240/241)] conformation. The association of these variables with the Event Free Survival (EFS) was assessed using the χ² test. A p value ≤0.05 was considered statistically significant. Furthermore, in order to measure the association level, the Relative Risk (RR) and the corresponding 95% Confidence Interval (95%CI) were calculated. Events were considered dead of complication (DOC), relapse and HSCT.

Seven out of the 23 patients presented an event: 5 relapses, 1 DOC and 1 HSCT. RT-PCR analysis of PTEN expression revealed that only one case did not show any product. Conversely, western blot analysis demonstrated that all patients showed total and phosphorylated PTEN proteins. Interestingly, we observed that total AKT protein was present in all the cases except one; the phosphorylated forms were detected as follows: AKT (T308) in 15 out of the 23 patients (65%), whereas none showed expression of AKT (S473). Surprisingly, we detected a statistically significant downregulation of total and phosphorylated mTOR and P70S6Kβ2 expression in eight, nine, ten and eleven out of 22 analyzed patients respectively. Downregulation or absent expression of both total and phosphorylated P70S6Kβ2 had a statistically significant impact on EFS showing a higher risk of events, when comparing those downregulated with those exhibiting phosphorylated (RR: 2,75; 95%CI: 1,25–6,01) and total protein (RR 3,33; 95%CI: 1,29–8,59) respectively. Moreover, downregulation of mTOR(S2448) confirmed the same pattern of higher risk of events (RR: 2,77; 95%CI: 1,08–7,07) comparing those downregulated with those exhibiting expression of phosphorylated protein.

Our data for the first time have shown that the downregulation or absent expression of mTOR and P70S6Kβ2 is associated with a very poor outcome: 5 cases had very aggressive relapses (3 died for progressive disease); one child died during induction for complication related to aggressive disease (massive splenic hemorrhagic event) and one case underwent a matched-HLA familiar HSCT because of a high risk-MRD pattern. These preliminary findings need to be confirmed in a larger-population based study. Nevertheless our data identify new markers of aggressive and resistant disease, easily available at diagnosis, suggesting that mTOR and P70S6Kβ2, which play a crucial role as negative control in the PI3K/AKT cell signalling pathway, are needed to be evaluated in a future treatment plan design with specifically targeted drugs. Moreover, our data will be confirmed by the use of a reliable and robust method such as flow cytometry which will allow us to perform a sensitive and accurate measurement of single cell characteristics, emphasizing the intracellular signaling pathways of interest.

No relevant conflicts of interest to declare.