Moreover, novel treatment modalities have been directed towards inappropriately activated cell-signaling pathways that may be responsible for the proliferation and/or escape

from apoptosis of leukemic blasts [12]. For this reason, the aim of the present study Stem Cell Compound Library was to evaluate the expression and activity of cell-signaling-related proteins in blasts of children and teenagers affected by high risk haematologic neoplasms, such as AML, T cell ALL and stage IV NHL characterized by bone marrow infiltration. These molecular features have been subsequently correlated to the clinical outcome and to other biological prognostic factors. Materials and methods Patients Seventy-two children with T cell ALL (18 samples), AML (45 samples) and stage IV NHL (9 samples) diagnosed

and treated at the Oncology Pediatric Service of the Second University of Naples were enrolled in this study. The diagnosis was established by cytological examination of bone marrow smears and cytochemical tests included the staining for Periodic Acid Shiff (PAS), Myeloperoxidase (MPO), Alpha-Naphthyl-Acetate Esterase (ANAE) and Acidic Phosphatase (ACP). All samples presented a percentage of blast cells > 90%. The patients with acute leukemias (AL) were sub-classified as ALL or AML according to the French American British (FAB) classification [[24], 25, 26] and NHL patients according Protease Inhibitor Library to the NCI classification according to “”Working Formulation”". All NHL patients were stage IV for bone marrow involvement. The AML patients were treated according to AIEOP-AML protocols (’87, ’92, ’01–’02), ALL and NHL patients according to AIEOP-ALL protocols (’95, ’00) [13]. Immunocytochemistry The bone marrow slides, collected at diagnosis, were fixed in acetone-methanol solution (1:1 dilution) for 30 seconds at 4°C. Mouse anti-human monoclonal antibodies raised Amisulpride against JNK phosphorylated on Serine-63, anti-Caspase8 p20

for p-20 subunit, anti-human Gadd45a (amino acids 1–165) and anti-pErk-1 phosphorylated on Tyrosine-204 were purchased from Santa Cruz Biotecnology (Santa Cruz, CA). All the primary antibodies were used at 1:100 dilution and added to the slides for 30 minutes at 37°C. After three washes in Tris buffer, the Alkaline Phosphatase-conjugated Envision System DAKO was used to visualize the sites of localization of the different proteins expressed in bone marrow cells. This kit is unaffected by endogenous Alkaline Phosphatase activity because includes as blocking reagent levamisole and shows high sensitivity. Fast Red was used as the final chromogen. Cells were counterstained with Mayer’s hematoxylin solution. HL60 cell-line cytocentrifuged slides were used as positive controls. Negative controls for each reaction were performed leaving out the primary antibody. Stained slides were analyzed for percentage of positive cells by two independent investigators. All samples were processed under the same conditions.