Immunophenotypic profile of biomarkers related to anti-apoptotic and neural development pathways in the ewing's family of tumors (EFT) and their therapeutic implications

Background: Ewing's family of tumors (EFT) comprises a broad spectrum of tumors composed of primitive committed cells with neuroectodermal capacity. The degree of neural differentiation within EFT, as measured with morphological features and expression of neural markers, delimits two members: Ewing's sarcoma (ES) and peripheral primitive neuroectodermal tumor (pPNET). Molecules such as c-kit and its ligand (Stem cell factor, SCF), CD95 (FAS), CD95L (FASL), IGF-IR, protect EFT cells from apoptosis, whereas c-erb-B2, erythropoietin (EPO) and its receptor (EPO- R) participate in the maturation of primitive committed neuroectodermal cells and in the normal embryonal brain development. The aim of the present study was to analyse the expression of these molecules in paraffin-embedded material from a series of EFT. Materials and Methods: Forty-five cases of EFT (23 typical ES, 4 atypical and 18 pPNET) were analysed following the immunohistochemical LSAB method, with antigen retrieval heating using an autoclave, citrate buffer pH 6.0 and the following primary antibodies: FAS (APO-CD 95), FAS-L, c-kit, SCF, IGF-IR and c-erbB2. The expression was evaluated independently by three of the authors and the final score (0 to 3+) was based on the intensity and percentage of positively stained cells. In a second cooperative analysis, tissues from 30 cases of EFT (15 typical, 3 atypical and 12 PNET) were immunostained with EPO and EPO-R. Results: High expression of c-kit/SCF (2+, 3+) was detected in 28/45 cases of EFT (62.2%), whereas FAS-FAS-L and IGF-IR were observed in 16/45 (37.7%) and 9/45 (20%), respectively. Regarding the neuroectodermal pathway, membranous and cytoplasmic expression of c-erb-B2 was observed in 9/45 (20%) EFT, regardless of the morphological and immunohistochemical expression of conventional neural markers. High expression of EPO and EPO-R was observed in 20/30 EFT (66.6%). Conclusion: C-kit/SCF and EPO/EPO-R seem to participate in the pathway of anti-apoptotic and proliferative advantage, while c-erb-B2 does not play an important role in the neuroectodermal differentiation pathway in EFT cells. The Ewing family of tumors (EFT) includes Ewing's sarcoma, the most common malignant bone tumor occurring in children and young adults, and peripheral primitive neuroectodermal tumor which is the second most common soft tissue malignancy in childhood, accounting for 20% of sarcomas. This family of tumors is defined genetically by specific chromosomal translocations resulting in fusion involving the EWS gene with members of the ETS family of transcription factors. From the diagnostic point of view, these tumors express a complex immunophenotype comprised of markers such as CD99 (MIC2, HBA71), Fli 1, NSE, HNK-1 and other neural markers. Despite the use of aggressive multimodal approaches, these tumors present a disappointingly low survival rate, and innovative and more effective treatment methods are needed. The stem cell factor (SCF)/c-kit tyrosine kinase receptor pathway has been considered important for tumor growth and progression in several human neoplasms, furthermore the therapeutic use of tyrosine kinase inhibitors has proven very useful in tumors such as gastrointestinal stromal tumors (GIST) or chronic myelogenous leukemia (1). Moreover, it is known that c-kit/SCF are expressed in EFT and that SCF is capable of protecting tumor cells against apoptosis (2). The c-erb-B2/ HER-2/neu is a key component of a complex signaling network, playing a critical role in the regulation of tissue growth, differentiation and neural development (3). Immunotherapy with trastuzumab, an anti