Purpose: Activation of phosphatidylinositol 3′-kinase pathway is implicated in the pathogenesis of mantle cell lymphoma (MCL). The genetic change in phosphatidylinositol 3′-kinase catalytic subunit α (PIK3CA) in MCL has not been identified. Experimental Design: Thirty-five primary MCL cases and 2 MCL cell lines (GRANTA-519 and Rec-1) were used to investigate somatic mutation and gene copy number of PIK3-CA. Gene copy number was determined using quantitative real-time PCR and fluorescence in situ hybridization. We used quantitative real-time reverse transcription-PCR to measure PIK3CA transcription levels. Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and phoshorylated AKT protein levels were analyzed using Western blotting and immunohistochemistry. Flow cytometry was used to assess apoptosis after treatment of MCL cell lines and one control cell line with LY294002, a specific inhibitor of PI3KCA. Results: Fifteen of 22 (68%) MCL cases and the MCL cell lines harbored a gain (≥3) of PIK3CA gene copy number. In addition, cases with increased PIK3CA gene copy number had elevated PIK3CA mRNA levels. Furthermore, amplification of PIK3CA correlated with the status of AKT phosphorylation in 7 of 12 (58%) primary MCL cases. Inhibition of PIK3CA induced increased apoptosis in the MCL cell lines. PTEN protein expression was present in all 14 primary MCL cases and cell lines by Western blotting, whereas 5 of 33 (15%) cases tested by immunohistochemistry had loss of PTEN expression. Conclusions: We conclude that a gain of gene copy number of PIK3CA is frequent genetic alteration that contributes to MCL progression. PIK3CA is a promising therapeutic target in MCL. (Clin Cancer Res 2009;15(18):5724-32) Mantle cell lymphoma (MCL) is now recognized as a distinct clinicopathologic subtype of B-cell non-Hodgkin's lymphoma with well-characterized morphology, a distinct immunophenotypic profile, specific karyotypic abnormality, and even a unique underlying molecular alteration (1). MCL comprises approximately 3% to 10% of non-Hodgkin's lymphoma and is characterized by an aggressive clinical course and poor prognosis with a median survival of 3 to 5 years (2, 3). Although front therapy induces a high rate of complete remission, relapse is inevitable and new treatments are needed for relapsed MCL (4).The genetic hallmark of MCL is the chromosomal translocation t(11;14)(q13;q32), which results in deregulated aberrant expression of cyclin D1 (5-7). However, the concept that MCL represents a distinct clinicopathologic and molecularly defined entity does not preclude the existence of additional distinctive disease subsets that could define disease heterogeneity. The

Introduction. Within the group of core binding factor (CBF) AML, the presence of the t(8;21)(q22;q22) confers a favorable prognosis based on high complete remission rates and high survival probabilities. However within this subgroup the presence of KIT mutations and in some studies specifically mutations at codon 816 in exon 17 have been associated with inferior event free survival, relapse free survival, cumulative incidence of relapse and overall survival. Dasatinib a dual SRC/ABL kinase inhibitor is an active agent already approved for the treatment of imatinib resistant or intolerant chronic myelogenous leukemia which has shown in vitro activity against KIT exon 17 mutations including the D816 imatinib resistant mutation. The aim of the present study was the investigation of the activity of dasatinib on cell proliferation and apoptosis of leukemic cell lines with or without KIT mutations. Materials and methods. The leukemic cell lines ME-1, NB4 and KASUMI were cultured in RPMI. Following RNA extraction RT-PCR was performed for the amplification of the extracellular (exon 8,9), transmembrane/juxtamembrane (exon 10,11) and tyrosine kinase 2 domains (exon 17,18) of c-Kit.Following sequencing only the KASUMI cell line derived from a t(8;21)(q22;q22) AML was found to bear the N822K KIT mutation at exon 17, also described in patients’samples. The KASUMI, the K562 cell line bearing the t(9;22) used as a positive control and the NB4 cell line without KIT mutations used as a negative control, were subsequently cultured under the presence of dasatinib at the concentrations of 1nM, 10nM, 100nM, 500 nM. Cell proliferation, was determined at 24, 48, 72 h using the Cell Proliferation Elisa, BrDU protocol and apoptosis was determined by the method of annexin using flow cytometry at the same time points. Results The BrDU value of K562 cells at 48h without the drug was 1.046 significantly higher compared to those of cells cultured under the presence of Dasatinib at 1nM, 10nM, 100nM, 500 nM (0.6485, 0,5647, 0,4770, 0.4755 respectively) (p<0.001). Similarly the BrDU value of K562 cells without the drug at 72h was 1.320 significantly higher to those under the presence of the drug at 10, 100, 500 nM (0.8137, 0.7292, 0.6637 respectively) (p<0.001). The level of apoptosis was significantly induced by the drug at all concentrations at 24h(p<0.001) and at the concentrations of 10nM, 100nM, 500 nM at 48h (p<0.001) but not at 72h.Ôhere was no effect of the drug on the proliferation and apoptosis of the NB4 cell line. In the KASUMI cells there was a significant reduction of the BrDU values by the presence of dasatinib at the concentrations of 10nM, 100nM, 500nM at 48h (0.9517 vs 0.6462, 0.5653, 0.3467, p=0.038, 0.011, 0.002 respectively). The same was true at the concentrations of 100nM and 500nM at 72h (0.9538 vs 0.2412, 0.1907, p=0.002, 0.004 respectively). Dasatinib significantly increased the level of apoptosis of the KASUMI cells at 24h at 1nM, 10nM, 100nM (2.45 vs 1.41, 1.71, 2.18, p<0.001, <0.001, 0.026 respectively) At 48h dasatinib significantly increased the level of apoptosis at the concentrations of 1nM, 10nM, 100nM (0.84vs 1.03, 1.49, 2.81, p=0.02, p<0.001, p<0.001 respectively). At 72h there was a significant induction of apoptosis by the drug at all concentrations (0.16 vs 1.11, 1.94, 2.93, 1.88 p<0.001) Conclusion. Dasatinib is an effective suppressor of proliferation and inducer of apoptosis of the KASUMI cell line with the t(8;21)(q22;q22) and the N822K KIT mutation. These encouraging results need to be confirmed on patients’ cells with the view to integrate the drug in conventional chemotherapy regimens in future clinical trials.

Myelodysplastic syndromes (MDS) are characterized by genetic instability which is associated with abnormal DNA repair mechanisms. The most lethal type of DNA damage are double strand DNA breaks (DSBs