Amoureux et al. BMC Cancer 2010, 10:91 http://www.biomedcentral.com/1471-2407/10/91 Page 8 of 12 Figure 4 Olig2 is positively correlated to PSA-NCAM. (A) Immunostaining of GBM9 cells with anti-olig2 (green, Alexa488) and anti-PSA-NCAM (red, CY3) in the presence of EGF + FGF (a), EGF + FGF + endoN (b), no growth factor (c), endoN and no growth factor (d). Cells were counterstained with DAPI (blue); scale bar: 20 μm. (B) Quantification of immunostaining described above from a representative experiment out of 2 performed. Percentage of olig2+ cells is presented as mean +/- standard deviation of the percentage of olig2+ cells grown in presence of EGF and FGF, from at least 5 independent fields of view per condition. Between 174 and 571 cells per condition were counted. (*: p < 0.05, ***: p < 0.001; Fisher’s test). (C) Representative western blot out of 3 independent cultures of C6 and C6-PSA-NCAM with anti-PSA-NCAM, anti-a- tubulin and anti-olig2 antibodies. PSAELISA <10 pg/μg of protein group (4 cases). Never- theless, when confronted with the outcome of patients, the ELISA measurement of these samples seemed to be more predictive. Three out of the 4 samples that had low PSA by ELISA came from patients that had an over- all survival > 20.8 months (median OS in the PSAELISA <10 pg/μg of protein group) and disease free survival > 11.2 months (median DFS in the PSAELISA <10 pg/μg of protein group). Nine out of the 12 samples that had high PSA by ELISA came from patients that had an overall survival < 12.2 months (median OS in the PSAELISA >10 pg/μg of protein group) and disease free survival <6 months (median DFS in the PSAELISA >10 pg/μg of protein group). Importantly, this test presents a number of advantages over currently in use measurement techniques, immuno- histochemical detection and Western blot, which are semi-quantitative. Therefore, once validated on a larger scale, it fulfills the criteria (quantitative test, sensitivity and specificity) that would allow its use in the clinic for GBM and other cancers. Indeed, in addition to the prognosis value of PSA-NCAM in GBM demonstrated here, evidence from our and others’ studies have shown that PSA-NCAM is also a reliable marker in many types of cancers of the CNS and other organs, and that it is involved in the process of metastasis [16,23,26-28]. A correlation could also be established between the level of PSA-NCAM and the severity of the disease and/or