CILP2 was overexpressed in CRC.
Aiming at searching for potential novel prognostic markers of CRC, we firstly analyze expression data of the TCGA CRC cohort from Illumina HiSeq 2000 platform, which contains 621 samples and correlating clinical and demographic information. We found that CILP2 was strongly correlated with clinical features of the samples in the TCGA cohort, and CILP2 has not been reported in CRC before. So we focused on CILP2 and furtherly analyzed the association between CILP2 expression and CRC prognosis. To determine the role of CILP2 in CRC, we first analyzed CILP2 gene expression in 50 patients samples with paired adjacent normal tissues in TCGA cohort, and the results suggested that CILP2 gene was overexpressed statistically significant in tumor samples compared to paired adjacent normal tissues (Fig 1A-1B, Fold change=3.412, P<0.001, Table 1). Additionally, CILP2 gene expression was upregulated in the total amount of tumor samples compared with adjacent normal tissue samples in the TCGA cohort (Fig 1C, Fold change=8.6161, P<0.001, Table 1).
To further testify the upregulation of CILP2 in CRC, we detected CILP2 protein expression in a TMA (n=64) by immunohistochemical staining (IHC). We found that 68.75% (44/64) tumor tissues highly expressed CILP2 protein, whereas only 39.06% (25/64) of matched adjacent normal tissues highly expressed CILP2 protein. The staining result in our cohort showed that CILP2 protein expression was statistically significantly more prevalent in tumors than in matched adjacent normal tissues (Fig 1D, P=0.001). The representative images of CILP2 immunostaining were shown in Fig 1E-1F.
Correlations between CILP2 expression and clinicopathological parameters in CRC.
Furthermore, to dissect the role of CILP2 in CRC carcinogenesis, correlations between CILP2 expression and clinicopathological parameters were analyzed based on TCGA cohort (mRNA) and TMA cohort (protein), presenting in Table 2. And Table 3 showed the correlation analysis results of the TCGA cohort using Spearman's test. Among 621 samples from the TCGA cohort, part of clinicopathological data were missed in some cases. Median expression of CILP2 of all samples was chosen as a cutoff to divide samples into the CILP2-high (n=310) group and CILP2-low (n=311) group. We observed that in TCGA cohort tumors of high CILP2 expression were positively associated with the T3/4 stage (T1/2, 36.51%; T3/4, 53.35%; P=0.001, Table 2), and with N1/2/3 stage (N0, 44.89%; N1/2/3, 74.29%; P=0.005, Table 2). Similarly, the percentage of tumors with high CILP2 expression increased with grading of clinical stage (UICC 2010 stage) (stage I, 34.29%; stage II, 49.35%; stage III, 54.19%; stage IV, 60.00%, P<0.001, Table 2), and distant metastasis (M0, 48.69%; M1, 60.23%, P=0.048, Table 2). It was suggested that CILP2 gene expression was strongly correlated with T stage (P=0.001), N stage (P=0.005), M stage (P=0.048), and higher clinical stage (P<0.001), respectively in TCGA cohort (Fig 2). However, there was no significant correlation of CILP2 expression with patients’ age or gender (P>0.05, Table 2). In TMA cohort, tumors of positive CILP2 protein expression was statistically significantly associated with T3/4 stage (T1/2 44.83%; T3/4, 74.29%; P=0.022, Table 2), and higher clinical stage (UICC 2010 stage) (stage I, 35.71%; stage II, 50.00%; stage III, 70.83%; stage IV, 90.00%, P=0.03, Table 2). However, there was no significant correlation of CILP2 protein expression with N stage (P=0.2, Table 2), it may be due to a limited set of data. Although the significant correlation was also not reached in our cohort, there was a tendency that tumors with positive CILP2 protein expression were more likely to distantly metastasize compared with CILP2-negative tumors (P = 0.074, Table 2).
High CILP2 expression is associated with poor outcome of CRC patients.
Kaplan-Meier analysis was performed to investigate the relationship between CILP2 expression and overall survival in the TCGA cohort. There were 609 CRC samples available for prognostic information. Median expression of CILP2 of all samples was chosen as a cutoff to divide samples into the CILP2-high (n=305) group and CILP2-low (n=304) group. As shown in Fig 3, Table 4, CRC patients with high CILP2 expression exhibited a poorer overall survival rate compared with the low‑expression group (P=0.003). Moreover, the univariate Cox regression analysis indicated that high CILP2 expression was strongly associated with a poor prognosis(P=0.003). Other clinical variables, such as age (P<0.0001), T stage (P=0.005), N stage (P<0.0001), M stage (P<0.0001), and clinical stage (UICC 2010 stage) (P<0.0001) were all associated with overall survival (Table 5). Moreover, the multivariate analysis revealed that high CILP2 expression (P=0.034), age (P<0.0001), M stage (P<0.0001), and clinical stage (UICC 2010 stage) (P=0.017) were independently associated with a poor prognosis (Table 5).