prognostic factor in NSCLC and ENH-mediated translocation of ID2 to the cytoplasm may explain the lack of prognostic value for cytoplasmic ID2. However, the role of ID2 compartmentalization in lung tumor progression, although potentially interesting, would require further evaluation. The mechanism by which ID2 promotes lung tumor progres- sion is not clear yet. However, results obtained in other models suggest that the action of ID2 could depend on the inhibition of Rb tumor suppressor pathway [3,27]. In addition, it has been demonstrated that ID2 also regulates angiogenesis and apoptosis via transcriptional regulation of the VEGF and BCL2 genes, respectively [28,29]. However, action of ID2 could also be tissue specific: in breast cancer cell lines, high expression of ID2 reduced growth potential and invasiveness, while up-regulation of ID2 increased proliferation and invasive potential in prostate tumor cells [11,22,23]. Finally, although our results are preliminary, they suggest that ID2 could be involved in the tumor dedifferentiation processes of NSCLC, and could be used as prognosis marker for patients with poorly differentiated tumors. Interestingly, in NSCLC, poorly differentiated carcinomas are difficult to classify, and as such, the identification of novel prognostic makers for this particular group may help improve the therapeutic outcome of the patients. Materials and Methods Tumor samples We have analyzed tissue samples collected in a prospective group of 62 patients with NSCLC, who had undergone complete surgical resection of the lung tumor as an initial treatment (i.e., without prior radiotherapy or chemotherapy) between January 2002 and August 2004 at the Thoracic Surgery Department of Trousseau Hospital (Tours, France). Surgical specimens were fixed in 10% formalin and paraffin embedded for routine histological examination and immunohistochemistry. Histological types, differentiation, TNM classification, and stage grouping (summa- rized in Table I) were determined in accordance with the World Health Organization (WHO) criteria for lung tumors [30]. Written consent was obtained from all of the patients included in this study as recommended by French legislation. Tissue Microarray Manufacturing and Immunohistochemistry For tissue microarray (TMA) manufacturing, representative areas of the NSCLC samples were identified by a surgical pathologist on hematoxylin-eosin (HE) stained slides. For each tumor, three core biopsies 1 mm in diameter were then taken from selected areas of donor blocks and arrayed into a recipient paraffin block using a manual tissue arrayer (Beecher instruments, Sun Prairie, USA). Sections (4 mm thick) of TMA blocks were cut and immunohistochemistry was performed using a rabbit polyclonal specific antibody raised against ID2 (ZMD.325, Invitrogen, Cergy Pontoise, France). Briefly, the TMA section was deparaffinized, rehydrated, and submitted to heat antigen retrieval in an ethylenediamine tetra acetic acid buffer (MS-Unmasker, pH 7.8, Diapath) for 20 min. ID2 antibody was applied at a 1/50 dilution. Immunostaining was performed with the Lab Vision Autostainer (Lab Vision, Fremont, USA) using a biotin-streptavidin-peroxidase detection system with diaminobenzidine as a chromogen (Chem- MateTM Detection kit, DakoCytomation). Slides were hematoxylin counterstained. The TMA contained core samples of normal bronchiolar mucosa and lung tissue. Specificity of ID2 antibody Expression of ID2 in NSCLC was control by using purified ID2 protein as a negative control to block binding of ID2 antibodies to tumor cells (Figure S1). Two independent observers (J.R., C.B.) evaluated both the localization and intensity of the ID2 staining. This evaluation was blinded to the clinical data. Both nuclear and cytoplasmic staining scores of tumor cells were established by multiplying the staining intensity scaled from 0 to 3 (null = 0, weak = 1, moderate = 2, and strong = 3) by the percentage of positive cells (0 = ,10%; 1 = 10– 25%; 2 = 26–50%; 3 = 51–75%; 4 = .75%). Global expression of ID2 in tumor cells was obtained by the addition of nuclear and cytoplasmic staining scores. Statistical Analysis Multiple component analysis (MCA) was performed on data collected from 62 patients using SPAD software. This method is a descriptive multivariate analysis that allows for rapid identification of statistical relationships between variables. Briefly, the different categories, i.e., ID2 expression and clinicopathological features, were positioned in multidimensional space. In order to observe distances between points, they were projected on a plane of two orthogonal factors, F1 and F2. These ‘first’ factors were chosen to be the most representative of the global variance of the data. Because the position of categories of active variables (nuclear, cytoplasmic, and global expression of ID2) and the position of observations (stage, lymph node status, tumor status, histological types, grade of differentiation, genre, and smoking status) are calculated in the same way, they can be placed on the same graph (observations being replaced by gravity centers). DEMOD method (SPAD software), similar to the chi-square test, was also used to complement the MCA for analyzing the association between the active variables (nuclear, cytoplasmic, and global expression of ID2) and the illustrative variables (clinicopathological features). Overall survival was calculated from the time of histology diagnosis to the date of death due to any cause. The overall survival rate was analyzed according to the Kaplan–Meier method, and the differences in survival rates were assessed by the log-rank test (Medcalc software version 7.3.0.1). Multivariate analysis of prognostic factors was performed using Cox propor- tional hazard model (Medcalc). Supporting Information Figure S1 Immunohistochemical analysis of paraffin-embedded human NSCLC using rabbit polyclonal antibody against ID2. IHC was performed with antibody alone (A and C) or after neutralization of antibody by preincubation with 2 mg/mL of purified ID2 protein (ID2 recombinant protein P01, Abnova, Taiwan), overnight at 4uC, (B and D). Found at: doi:10.1371/journal.pone.0004158.s001 (9.11 MB TIF) Acknowledgments We are grateful to A De Muret (De´partement d’Anatomie Pathologique, CHRU de Tours, France) and Professor P Dumont (De´partement de Chirurgie Thoracique, CHRU de Tours, France) for providing the lung samples. Author Contributions Conceived and designed the experiments: JR XG. Performed the experiments: JR CB. Analyzed the data: JR CB SR AT XG. Contributed reagents/materials/analysis tools: JR CB SR SG XG. Wrote the paper: JR XG. PLoS ONE | www.plosone.org 6 January 2009 | Volume 4 | Issue 1 | e4158