ROR1 expression as a biomarker for predicting prognosis in patients with colorectal cancer

Abstract

There is a lack of reliable prognosis biomarker in the current treatment of colorectal cancer. The receptor-tyrosine-kinase-like orphan receptor 1 (ROR1) is overexpressed and associated with poor prognosis in certain tumors. This study aimed to explore the prognostic significance of ROR1 in colorectal cancer. Western blot analysis and immunohistochemistry showed that the expression of ROR1 in colorectal cancer was significantly higher than that in the adjacent normal tissues. ROR1 expression was positively associated with the clinical stage and lymph-node metastasis (p < 0.01). Kaplan-Meier survival analysis revealed that patients with higher ROR1 expression had a significantly shorter overall survival (p < 0.01). Multivariate Cox regression analysis confirmed that ROR1 is an independent prognostic marker in colorectal cancer (p = 0.002, HR = 2.08, 95% CI: 1.314-3.292). Thus, our study demonstrated that ROR1 expression is correlated with malignant attributes and may serve as a novel prognostic marker and therapeutic target for colorectal cancer.

Keywords: colorectal cancer; immunohistochemistry; prognostic factor; receptor-tyrosine-kinase-like orphan receptor 1; tissue microarray.

Figure 1. ROR1 expression in CRC tissues and CRC cells

(A) Western blot of ROR1 expression in human CRC tissues (T) and their adjacent normal tissues (N). Total proteins were used as loading control. (B, C) The intensity of each lane was calculated by Image J software and analyzed by paired t-test. Statistics results showed that P<0.001. (D) Flow cytometry of ROR1 expression in the immortalized normal colon cells and CRC cells.

Figure 2. ROR1 expression on CRC tissue microarray by immunohistochemistry staining

(A) The IHC staining of ROR1 expression in breast tumor (1) and its adjacent normal tissue (2). (B) Detection of ROR1 expression in CRC tissues. Positive ROR1 staining was shown in brown color and the nucleus counterstained with hematoxylin was shown in blue color. The magnification was ×200 in B1, ×400 in B2. (C) Different levels of ROR1 expression detected by TMA-IHC analysis. (1) Score 0 indicates that none or little cells exhibit ROR1 expression; (2) score 1 indicates that more than 25% of tumor cells exhibit weak ROR1 expression; (3) score 2 indicates more than 50% of tumor cells have weak expression or more than 25% of tumor cells have moderate ROR1 expression; (4) score 3 indicates more than 75% of tumor cells have moderate expression or more than 50% of tumor cells have strong ROR1 expression. (D) The proportion of negative (score 0), weak (score 1), moderate (score 2) and strong (score 3) staining for ROR1 protein in CRC patients. (E) The scores of ROR1 in CRC tissues at different stages were analyzed by two tail t-test (P < 0.001). (F) The scores of ROR1 in CRC tissues with different status of lymph node metastasis were analyzed by two tail t-test (P < 0.001). LN, lymph node.

Figure 3. Correlation of ROR1 expression with overall survival in CRC patients

(A) Kaplan-Meier survival analysis of ROR1 expression in CRC patients. The low ROR1 expression group had longer OS than the high ROR1 expression group. (B) Multivariate Cox regression survival analysis in CRC patients. ROR1 expression could serve as an independent prognostic factor.