Abstract B62: Cell cycle alterations in paired diagnosis-relapse childhood acute lymphoblastic leukemia.

Approximately 25% of children with acute lymphoblastic leukemia (ALL) relapse. After relapse, the overall cure rate remains low, despite intensified chemotherapy and stem cell transplantation. The Laboratory of Pediatric Hemato-Oncology of the University of Padua is the AIEOP (Italian Association for Pediatric Hemato-Oncology) referring laboratory and up to 5,000 pediatric leukemia samples are processed every year. For all the ALL diagnosis and relapse samples DNA index is analysed for the risk-treatment stratification of the patients. The analysis evaluates both DNA ploidy and the proliferative state through the determination of the cell population in the S-phase of the cell cycle. Cell cycle analysis of childhood ALL, performed in our laboratory, reveal that S-phase is significant increased in bone marrow from relapsed patients compared to bone marrow of the same patients at the diagnosis. The study of the molecular mechanisms involved in S-phase increase at relapse will help us to improve our knowledge about relapse mechanism and to design new therapeutic strategy exploiting these changes in cell cycle control. We studied 180 patients affected by B-ALL enrolled in AIEOP protocols with more that 60% of blast cells in the bone marrow, both at diagnosis and relapse; extramedullary relapses were excluded. Cell cycles were analysed by flow citometry. To evaluate changes in cell cycle distribution at relapse in respect to diagnosis, for each patients we calculate ΔS as the difference between S-phase fraction at relapse and S-phase fraction at diagnosis. Gene expression profiling were performed using Human Genome U133 Plus 2.0 array (Affymetrix platform). Gene expression analysis, real time PCR and western blot were carry out to compare higher and lower ΔS patients. Preliminary gene expression analysis, on higher ΔS vs lower ΔS samples of relapsed patients, identified 58 differentially expressed probe sets corresponding to genes involved in proliferation, DNA damage and cell migration (LFDR<0.05). Among these genes, we found some E2F1 targets such as MAD2L1, CIP2a and RFC3. Western blot and real time PCR revealed increased cyclinA2 expression, another E2F1 target, in samples from relapsed patients with higher ΔS. We observed that higher [[Unable to Display Character: ∆]]S correlate to very early time to relapse (within 18 month from diagnosis) and these patients are classified as high risk patients that barely respond to treatments after relapse. Very early relapse more frequently happens during maintenance therapy based on methotrexate and 6-mercaptopurine, and methotrexate resistance could be mediated by increased levels of E2F1 transcriptional activity. At the present stage we identified S-phase increase as a molecular determinant of high risk in B-ALL relapse. Further studies of these alterations of cell cycle control and of E2F1 activation may help the development of more specific and less toxic therapies that overcome resistance to currently used chemotherapeutics. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B62. Citation Format: Roberta Bortolozzi, Giampietro Viola, Maddalena Paganin, Silvia Bresolin, Silvia Disaro, Elena Porcu, Valentino Conter, Franco Locatelli, Barbara Buldini, Giuseppe Basso. Cell cycle alterations in paired diagnosis-relapse childhood acute lymphoblastic leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B62.