All Sex Female Male Smoking status Non smokers Smokers Histological type Adenocarcinoma Squamous cell carcinoma Other type Differentiation/Grade Well Moderately Poorly T status T1–T2 T3–T4 N status M status N0 N+ M0 M+ Stage I–II III–IV n 62 8 54 5 57 33 19 10 17 26 19 52 10 38 23 52 10 36 26 (%) 100 13 87 8 92 53 31 16 27 42 31 84 16 62 38 84 16 58 42 Table 1. Clinical and histological features of patients with NSCLC. Other types: include large cell carcinoma, carcinoma, and bronchioalveolar carcinoma. doi:10.1371/journal.pone.0004158.t001 ID2 expression in tumor cells from the 62 NSCLC patients was highly diverse both in terms of intensity and localization (Figure 1). Out of 62 NSCLC patients, a large proportion (98%, 61/62) expressed ID2 protein almost exclusively in tumor cells (median of global staining score = 3; range 0 to 18). Nuclear immunoreactivity was positive in 33 (53.3%) of the NSCLC samples (median of nuclear staining score = 1; range 0 to 11), and cytoplasmic ID2 was detected in 57 (92%) out of 62 tumors (median of cytoplasmic staining score = 2; range 0 to 8). Many poorly differentiated tumors exhibited strong ID2 staining in the nuclei in conjunction with weak or moderate cytoplasmic staining (Figure 1E–J). In contrast, ID2 was not expressed in the nuclei of partially or well- differentiated tumors, while weak to moderate expression was observed in the cytoplasm (Figure 1K–P). Association of ID2 Expression with Clinicopathological Parameters The median of the staining score for either nuclear, cytoplasmic, or global expression of ID2, as described above, was used as a cut off to classify patients in two groups: ‘‘low’’ (i.e., low ID2 expression) and ‘‘high’’ (i.e., high ID2 expression). Multiple component analysis (MCA) was performed on data collected from Expression of ID2 in NSCLC the 62 patients and the graphical representation is presented in Figure 2A. In brief, variable neighborhoods on the graph are potentially associated together. MCA showed that low nuclear expression of ID2 was associated with well-differentiated tumors and lymph node involvement. In contrast, high expression of ID2 in the nucleus was seen more frequently in tumors that were poorly differentiated and had advanced local invasion (T3–T4 status). In association with MCA, DEMOD analysis was performed and demonstrated a significant association between nuclear expression of ID2 and histological differentiation of NSCLC (Figure 2B). Our results showed 15 out of 17 (88%) well- differentiated tumors exhibited low expression levels of ID2. In addition, poorly differentiated tumors had strong nuclear expres- sion of ID2 (p = 0.007) in 12 out of 19 (63%) cases. However, no significant relationship was found between ID2 expression and lymph node involvement or T status, possibly due to the uneven distribution of T status between groups (52 and 10 patients in T1– T2 and T3–T4, respectively). Cytoplasmic expression of ID2 was not associated with the differentiation grade of the NSCLC, but was surprisingly linked to the histological type of tumors. Tumor expression of ID2 was high in 22 out of 33 (67%) cases of adenocarcinoma compared to 5 out of 19 (26%) cases of squamous cell carcinoma (p = 0.016) (Figure 2B). Univariate and Multivariate Survival Analysis To determine whether immunostaining of ID2 had a prognostic value, we examined the association between global, nuclear, and cytoplasmic expression of ID2 with the overall survival of patients. First, we analyzed conventional clinicopathological parameters. We observed a significant impact of tumor stage (p = 0.0001) and tumor size (p = 0.014) on patient overall survival (Figures 3A–C), whereas nodal status (N0 vs. N+) was of borderline significance (p = 0.06). Histological differentiation was not a prognostic marker in our cohort of NSCLC patients (p = 0.48; data not shown). Similarly, we did not observe a significant difference in the overall survival between patients with high expression levels of ID2 and those exhibiting low expression levels of ID2 (Figure 3D–E). However, after stratification of the data according to histological differentiation (Figure 3G–I), poorly differentiated NSCLC patients with high nuclear expression showed a shorter overall survival than patients with low ID2 nuclear expression (median of survival = 24 vs. 41 months, respectively; p = 0.036; Figure 3G). In addition, multivariate analysis using Cox’s regression model demonstrated that nuclear ID2 expression in patients with poorly differentiated tumors was an independent prognostic factor of overall survival (p = 0.034; RR = 13.7; IC 95% = 1.2–161; Table 2). No prognostic value of ID2 nuclear expression was observed in moderate or well-differentiated tumors (data not shown). Discussion The role of ID2 has been studied in several types of cancer (i.e., prostate, breast, colorectal, neuroblastoma, and very recently small cell lung cancer), but to date, nothing is known about the expression of ID2 in non-small cell lung cancer, which affects 80% of patients with lung malignancies. Here, we focused on the expression of ID2 in NSCLC and its relationship with the clinicopathological parameters and prognosis of the disease. Expression of ID2 was evaluated in 62 NSCLC tumors by immunohistochemistry using tissue microarrays. While the majority of the patients analyzed in this study expressed ID2, the patient group was very heterogeneous with regard to both expression level and intracellular localization. However, we have PLoS ONE | www.plosone.org 2 January 2009 | Volume 4 | Issue 1 | e4158