INTACT APOPTOSIS SIGNALING IN PEDIATRIC ALL IS ASSOCIATED WITH PROLONGED NOD/SCID ENGRAFTMENT, LOW EXPRESSION OF ANTI-APOPTOTIC MOLECULES AND FAVORABLE PATIENT OUTCOME
M Queudeville, F Seyfried, S Eckhoff, KM Debatin, L Meyer
Background. Defects in cell death signaling have been attributed to be responsible for treatment failure and relapse in acute leukemia. We recently reported the importance of intact apoptosis signaling for good outcome in pediatric ALL and AML in two retrospective studies. Using a NOD/SCID mouse xenotransplant model for pediatric ALL we have also shown that rapid engraftment of primary ALL cells (short time to leukemia/TTLshort) is characteristic for early relapse of the corresponding patient. Aims. In this prospective study the impact of the functional integrity of apoptosis signaling in xenograft ALL samples on NOD/SCID engraftment and patient outcome was investigated. Furthermore, expression of apoptosis regulating molecules was analyzed and correlated to NOD/SCID engraftment, treatment response and apoptosis signaling. Methods. Primary BCP-ALL samples (N=20) obtained at diagnosis were transplanted onto NOD/SCID mice and time to leukemia (TTL) was estimated as weeks from transplant to onset of disease for each sample transplanted. Apoptosis signaling in xenograft leukemia samples was assessed by flowcytometry analyzing two key apoptogenic events, cytochrome c release and caspase-3 activation. Expression of the anti-apoptotic molecules Mcl-1, XIAP, Bcl-2 and Livin was analyzed by qRT-PCR. Results. Of the 20 ALL samples transplanted 6 led to rapid leukemia manifestation in the recipient animals (TTLshort) with an inferior relapse free survival of the corresponding patients in contrast to 14 patients with TTLlong phenotype (log rank P.002). Apoptosis signaling was investigated in xenograft leukemia samples by analysis of caspase-3 activation and cytochrome c release. Both events were closely correlated to each other indicating intact signaling in patients stratified into non-high risk groups, patients showing good response to treatment (remission on day 15, negative MRD on day 33), and patients without relapse. In contrast, no correlation was found in patients with poor outcome. Most importantly, intact apoptosis signaling was also found in TTLlong but not in TTLshort patients strongly indicating that mitochondrial cytochrome c release and consecutive apoptosome formation resulting in activation of downstream effector caspases such as caspase 3 is characteristic for a long engraftment phenotype and prognostic favorable ALL. The functional integrity of this apoptogenic checkpoint is subsumed by the parameter cytochrome c related activation of caspases (CRAC). Patients with positive CRAC values reflecting intact apoptosis signaling showed a significantly superior relapse free survival in contrast to patients with disturbed cytochrome c related caspase activation/negative CRAC (log rank, P<.001). Transcript expression of apoptosis regulating molecules was analyzed. A correlation of cytochrome c release and caspase-3 activation indicating proficient apoptosis signaling was exclusively observed in leukemia samples with low expression of the anti-apoptotic molecules Mcl-1, XIAP, Bcl-2 and Livin. Furthermore, patients samples with low Mcl-1 expression showed a significantly longer time to leukemia (TTL) and lower blast cells on day 8 than samples with high Mcl-1 expression. Conclusions. Thus, the propensity leukemia cells to undergo apoptosis (CRAC positive) leads to prolonged engraftment upon transplant in the NOD/SCID/huALL model (TTLlong), is associated with low expression of anti-apoptotic molecules, and results in favorable treatment response and superior survival of pediatric ALL patients.