Ninety-three CRC patients were included who underwent curative surgery in Eastern Hepatobiliary Surgery Hospital, the Second Military Medical University of China, from January 1999 to December 2010. The patients met the following criteria: no anti-cancer treatments were given before surgery; all the visible tumor nodules were resected (including the distant metastatic nodules); patients who died during surgery or from serious surgical complications were excluded; the resected nodules were identified as primary CRC or metastasis of CRC and the surgical margin was free of tumor cells by pathological examination; patients with lymphatic metastasis or/and distant metastasis had received postoperative adjuvant chemotherapy, patients who died from non-CRC diseases or accidents were excluded; and the clinicopathological and follow-up data were available. All the formalin-fixed and paraffin-embedded primary CRC samples were obtained from the Department of Pathology of Eastern Hepatobiliary Surgery Hospital. All patients in this study gave written informed consent.

Patients were followed up until death or until June 15, 2011. All patients were monitored by physical examination, routine blood tests [including serum carcinoembryonic antigen (CEA) concentration], chest X-ray and abdominal ultrasonography every 2 mo in the first year after surgery, and every 3-6 mo thereafter. A computed tomography scan (CT) or magnetic resonance imaging was performed every 6 mo or immediately when a recurrence/metastasis was suspected. If needed, a whole-body fludeoxyglucose positron emission tomography/CT was performed. The follow-up data were recorded during the postoperative examination in our hospital, while patients who were examined in another hospital were followed up by telephone or letter. Recurrence was determined by at least two imaging examination results. Once recurrent tumors were confirmed, further treatment was implemented, such as a second surgery and palliative chemotherapy. Disease-free survival (DFS) was defined as the period from the tumor resection until the tumor recurrence or the last observation. The overall survival (OS) was the interval between the surgery and death or the last follow-up examination.

Immunohistochemstry was carried out as described previously[20]. Representative 4-μm serial sections were prepared from 10% formalin-fixed, paraffin-embedded tissue blocks. To increase the immunoreactivity, microwave antigen retrieval was performed in citrate buffer (pH 6.0) for 5 min, then cooled the sections at room temperature for at least 30 min. Subsequently 3% hydrogen peroxide was used for 10 min to block endogenous peroxidase activity. After nonspecific binding sites were blocked for 30 min with goat serum, a monoclonal antibody against Twist2 (1:300, H00117581-M01, Abnova) and polyclonal antibodies against E-cadherin (1:100, BS1098, Bioworld Technology) were used to incubate the sections in a humid chamber at 4 °C overnight. Next, an EnVision Detection kit (GK500705, Gene Tech, China) was used to visualize tissue antigens. Sections were counterstained with hematoxylin for 5 min. Negative control sections were incubated with phosphate buffered solution instead of the primary antibody.

Two independent pathologists (Dong H and Cong WM), who were blinded to clinical information, assessed the expression of Twist2 and E-cadherin semiquantitatively. Twist2 staining was observed only in the cytoplasm of CRC tumor cells (described in the results); therefore, the nucleolus staining was not evaluated. Cytoplasmic staining of Twist2 was scored according to its extent and intensity (extent + intensity), similar to the methods described previously[21-24]. The extent of staining was graded as follows: 0 for < 15% positive cells, 1 for 15%-30%, 2 for 30%-60% and 3 for more than 60% positive cells. The intensity of staining was scored on the following scale: 0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining. The total score was 0 to 6 when summed (extent + intensity) together. Subsequently, a total score of 0-2 was considered to be a negative/low expression, while a score of 3-6 was considered as positive/high expression. For E-cadherin, the scoring was determined as previous studies[25,26]. Preserved expression of E-cadherin was defined where tumor cells were stained as strongly and homogeneously as normal epithelial cells. Heterogeneous staining, weaker staining or completely negative staining of E-cadherin was considered as reduced expression.

Pearson’s χ² test and Fisher’s exact test (wherever was applicable) were performed to determine the relationship between Twist2 expression and clinicopathological parameters and E-cadherin expression. The prognostic factors for OS and DFS were examined by both univariate and multivariate analyses (Cox’s proportional hazards model). Survival curves were plotted by Kaplan-Meier analysis and by a log rank test. A P value < 0.05 (two-sided) was considered statistically significant. All statistical analysis were performed using SPSS version 19 (SPSS Inc., Chicago, IL, United States).

Although some previous investigations found Twist2 was expressed in both the cytoplasm and the nucleus in several tumors[7,8,11,27], we found Twist2 was mainly expressed in the cytoplasm in CRC, not in the nucleus (Figure 1). A similar expression pattern of Twist2 was found in hepatocellular carcinoma (HCC)[28]. By semiquantitative analysis, 66 (71.0%) of the 93 primary CRC tissue samples were positive for Twist2 expression, while the other 27 (39.0%) were negative. Twist2 expression was generally low in normal colon mucosa compared with the cancer tissues. For E-cadherin, as described previously[29,30], normal epithelial cells were strongly and homogeneously stained in the membrane, while tumor cells were stained mainly in the membrane and occasionally in the cytoplasm (Figure 2). E-cadherin was considered as reduced in 43 (46.2%) patients. The other 50 (53.8%) patients were preserved.

Open in New Tab   Full Size Figure   Download Figure

Figure 1 Immunohistochemical images of Twist2. A: Negative staining in the normal mucosa; B: Negative; C: Weak; D: Moderate; E: Strong cytoplasmic staining in colorectal cancer (200× magnification).

Open in New Tab   Full Size Figure   Download Figure

Figure 2 Immunohistochemical images of E-cadherin. A: Strong and homogeneous staining in normal mucosa; B: Preserved expression in colorectal cancer (CRC); C: Reduced expression in CRC (200× magnification).

As shown in Table 1, we did not find that Twist2 expression correlated with any of the clinicopathological parameters (gender, age, T-stage, N-stage, M-stage, tumor differentiation, vascular invasion, Tumor location and serum CEA level, all P > 0.05). When the relationship between Twist2 and E-cadherin expression was analyzed, we found a significant correlation: Twist2-positive patients showed a higher percentage of reduced E-cadherin than Twist2-negative ones (53.0% vs 29.6%, P = 0.040, Table 2).

For OS on univariate analysis, bad N-stage (lymph node metastasis), bad M-stage (distant metastasis), vascular invasion, serum CEA level (> 5 ng/mL) and Twist2-positive were significantly associated with poor survival (all P < 0.05, Table 3). When adjusted by multivariate analysis by Cox’s proportional hazard model, bad M-stage [hazard ratio (HR) = 7.694, 95%CI: 2.927-20.224, P < 0.001] and Twist2-positive (HR = 5.744, 95%CI: 1.347-24.298, P = 0.018) were considered to independent risk factors for poor OS.

We also analyzed the risk factors for DFS (Table 4). The result of univariate analysis was similar to OS: bad N-stage, bad M-stage, vascular invasion, serum CEA level (> 5 ng/mL) and Twist2-positive were risk factors for poor DFS (all P < 0.05). After adjustment, multivariate analysis revealed bad N-stage (HR = 2.149, 95%CI: 1.226-3.767, P = 0.008), bad M-stage (HR = 10.907, 95%CI: 4.937-24.096, P < 0.001) and Twist2-positive (HR = 3.264, 95%CI: 1.455-7.375, P = 0.004) were independent risk factors for poor DFS, while vascular invasion and serum CEA level were not.

Survival curves plotted according to different expression levels of Twist2 are shown in Figure 3. Significantly, Twist2-negative patients had a higher 5-year OS (86.2% vs 59.6%, P = 0.015, Figure 3A) and 5-year DFS (55.4% vs 24.8%, P = 0.012, Figure 3B) than the Twist2-positive patients. Interestingly, further analysis of the value of Twist2 for CRC patients in different stages showed that for patients in stage I-II (n = 34), there were no differences in OS or DFS (both P > 0.05, Figure 3C and D). For patients in stage III (n = 18) and IV (n = 41), Kaplan-Meier curves showed a clear trend that Twist2-negative patients had a more favorable outcome. Although the differences in OS were not statistically significant (both P > 0.05, Figure 3E and G), we found significant differences in DFS for both stage III and IV (P = 0.033 and P = 0.026 respectively, Figure 3F and H).

Open in New Tab   Full Size Figure   Download Figure

Figure 3 Kaplan-Meier analysis of overall survival and disease-free survival, according to the expression levels of Twist2. A, B: All patients (A, OS, P = 0.015 and B, DFS, P = 0.012); C, D: Patients in stage I-II (C, OS, P = 0.351 and D, DFS, P = 0.652); E, F: Patients in stage III (E, OS, P = 0.178 and F, DFS, P = 0.033); G, H: Patients in stage IV (G, OS, P = 0.101 and H, DFS, P = 0.026). OS: Overall survival; DFS: Disease-free survival.

Colorectal cancer (CRC) is one of the most common malignant tumors and continues to be one of the most common causes of cancer death worldwide. It is important to identify biomarkers to predict patients’ outcomes. Twist2 is a potential prognostic biomarker, but its value for CRC is unknown.

Twist2 is a regulatory factor of epithelial-mesenchymal transition, a well-known progression involved in embryogenesis, tumor invasion, metastatic dissemination and acquisition of therapeutic resistance. Hypoxia, methylation, cancer stem cell self-renewal and drug resistance correlate with Twist2 function. Therefore, Twist2 is a potential prognostic biomarker for tumors, and its prognostic value has also been identified for head and neck squamous cell carcinomas.

This study revealed that Twist2 was overexpressed in CRC at the protein level. Twist2-positive expression correlated with the poor prognosis of CRC, particularly for patients in stage III and IV (tumor-node-metastasis stage).

These results suggest that overexpression of Twist2 can probably serve as a prognostic factor for patients with CRC.

EMT is an important change in cell phenotype, which allows the escape of epithelial cells from the structural constraints imposed by tissue architecture, and was first recognized as a central process in early embryonic morphogenesis. Over recent decades, a series of studies have identified the involvement of EMT in solid tissue epithelial cancers’ invasiveness and metastasis.

This study investigated Twist2 expression in 93 CRC patients and evaluated its value as a prognostic biomarker based on relapse and survival data of patients. The results indicate that Twist2 could be used as an effective prognostic biomarker for CRC. This paper is generally well designed and the result looks reliable.