Amoureux et al. BMC Cancer 2010, 10:91 http://www.biomedcentral.com/1471-2407/10/91 which lead to altered signaling in core pathways [The cancer Genome Atlas Research network: [3]]. However, to date, the main significant prognosis factors evaluated in large cohorts of patients are O6-methylguanine-DNA methyl transferase promoter methylation status, age, extent of resection, performance status, and mini mental state examination [4]. Another focus of intense investigation has been the existence and the role of cancer initiating cells (CIC) in GBM [reviewed in [5]]. Although early studies have focused on CD133 antigen as the CIC marker in GBM [6], more recent studies have pointed out to the exis- tence of CIC characterized by other cell surface markers such as L1CAM [7] or the ganglioside A2B5 [8,9]. In addition, some studies have shown the critical role of olig2, a transcription factor expressed by normal oligo- dendrocytes. We have reported that olig2 is expressed by all human gliomas whatever their subtype and grade [10], but its level is dramatically increased in highly pro- liferative, tumorigenic GBM cell lines [11]. Furthermore, olig2 seems to be required for gliomagenesis and prolif- eration of neural progenitors [12]. We have postulated that PSA-NCAM could be a powerful biomarker for human GBM. In fact, the spatio- temporal pattern of expression of PSA-NCAM, a critical parameter for proper neural morphogenesis, and its bio- logical functions, are consistent with a possible role in GBM. Indeed, polysialylation of NCAM is a broad feature of growing CNS tissue in development, and it is a marker specifically associated with proliferative and plasticity events in adulthood [13,14]. PSA is a unique polymer of a 2,8-N-acetyl neuraminic acid residues added to the fifth Immunoglobulin domain of the membrane bound NCAM. Importantly, by enabling limited cell-cell and cell-extracellular matrix interactions [15], one hallmark of PSA-NCAM is to favor cell migration, a critical feature of the highly invasive behavior of GBM cells. In order to investigate the expression and the putative role of PSA-NCAM in GBM, we first set up a sensitive ELISA test, which allowed specific detection above 10 pg PSA-NCAM/μg of protein. Results of the ELISA test were correlated with immunohistochemical detection of PSA-NCAM. We studied the expression of PSA-NCAM in a series of 56 GBM samples and found PSA-NCAM expression in 70% of them. Moreover, we report that detectable PSA-NCAM by ELISA test (> 10 pg PSA- NCAM/μg of protein) is an adverse factor of prognosis associated with shorter OS and DFS. We then analyzed PSA-NCAM expression in a human GBM cell line GBM9, previously developed in our laboratory from a patient biopsy and characterized by a highly proliferative behavior and long term sphere formation [9]. We could show that removing PSA-NCAM from these cells down- Page 2 of 12 regulated olig2. Conversely, PSA-NCAM forced expres- sion in the C6 glioma cell line induced olig2 synthesis. Methods Human subjects and clinical parameters Surgically resected samples were obtained from 56 adult patients (age>18 years) from the Departments of Neu- rooncology and Neurosurgery at the Marseille Hospitals (AP-HM), in France from 1986 to 2005. All had primary GBM, histologically proven according to the WHO clas- sification, formalin fixed paraffin embedded tissue. The frozen specimens were stored in the AP-HM tumor bank (Authorization number 2008/70) with written informed consent for tumor banking and study. All patients benefited from surgery followed by radio- therapy and alkylating agents chemotherapy (nitro- sourea), and included long term survivors with a clinical follow up of 8 years. Because the patients were treated before 2005, none of them received the radiotherapy associated to Temozolomide chemotherapy protocol described by Stupp et al. [1]. For all patients, 26 males and 30 females, available clin- ico-pathological data were the age, preoperative Kar- nofsky performance status (KPS), extent of surgical removal, OS and DFS. Mean age at diagnosis was 60.4 +/- 10.7, preoperative KPS was less than 70 in 21 patients and greater than 70 in 35 patients. Total surgical excision based on operative chart protocol was achieved in 39 patients and partial excision in 17 patients. The median OS was 12.5 months [mean confidence interval (CI): 9.7- 15.4]. Of the 56 patients, 48 had relapse. The median DFS was 6.7 months (mean CI: 4.2-9.2). In addition, frozen specimens of 3 medulloblastomas (MB) known to highly express PSA-NCAM [16], and non tumorigenic adult brain tissues taken from a patient biopsy in an area containing only non tumoral tissue were used as controls. For PSA-NCAM immunohisto- chemistry and ELISA analysis, cryostat sections from each patient were checked and consisted of at least 60% tumoral tissue and less than 40% necrotic or non tumoral tissue. PSA-NCAM immunohistochemistry Anti-PSA-NCAM antibody (clone Men B2-2B dilution 1/500, Abcys, Paris, France) was applied on 5 μm cryo- stat sections available for all GBM and control cases. Immunohistochemistry was performed on a Benchmark XT (Ventana, Tucson, AZ, USA). Controls included omission of primary antibody and abolition of the immunostaining after endoneuraminidase N (endo N, 1/ 1000; Abcys) treatment, which specifically cleaves alpha 2,8 linked acetyl neuraminic acid residues added to N glycosylation sites on the fifth immunoglobulin domain