Significance of CD133 expression in esophageal squamous cell carcinoma

Background CD133 was recently reported to be a cancer stem cell marker and a prognostic marker for several tumors. However, few studies have investigated CD133 expression in esophageal squamous cell carcinoma (ESCC). Therefore, we examined whether CD133 could serve as a prognostic marker of ESCC and investigated the correlation between CD133 expression and the clinicopathological findings of ESCC patients and several markers. Methods We studied 86 ESCC patients who underwent curative surgery without neoadjuvant treatment at Tohoku University Hospital (Sendai, Japan) between January 2000 and December 2005. We analyzed tissue specimens by immunohistochemical staining for CD133, p53, p16, p27, murine double minute 2 (MDM2), Ki-67, and epidermal growth factor receptor (EGFR). Results Pathological tumor depth and tumor stage were significantly more advanced among CD133-negative patients than among CD133-positive patients. A log-rank test showed that CD133 immunoreactivity was significantly correlated with the overall survival of the patients (P = 0.049). However, multivariate analysis showed that it was not significantly correlated (P = 0.078). Moreover, CD133 was significantly positively correlated with p27 immunoreactivity (P = 0.0013) and tended to be positively correlated with p16 immunoreactivity (P = 0.057). In addition, p16 immunoreactivity was correlated with smoking history (P = 0.018), pathological lymph node status (P = 0.033), and lymphatic invasion (P = 0.018). Conclusions This study indicated that CD133 immunoreactivity is a good predictor of prognosis in ESCC patients. In addition, CD133 may play a role in the regulation of tumor cell cycle through p27 and p16 in ESCC. At present, it thus remains controversial whether CD133 expression is a valid prognostic marker for ESCC. To elucidate this relationship, further investigations are required.


Background
Prognosis or clinical outcome of esophageal squamous cell carcinoma (ESCC) has markedly improved over the last several decades, owing to advancements in medical treatment. However, in Japan, 11,867 people succumbed to this disease in 2010, and esophageal cancer was the seventh most common cause of cancer mortality in men (3.4% of the total cancer deaths in Japan) [1]. Various prognostic markers have recently been evaluated, including the stem cell marker CD133 (Prominin-1), which was reported to be a cancer stem cell marker for cancers of the brain [2], colon [3,4], prostate [5], liver [6,7], lung [8], kidney [9], ovaries [10], and skin [11,12]. It was also reported to be a marker of poor prognosis for cancers of the brain and spinal cord [13], colon [14], rectum [15], pancreas [16], breast [17], and stomach [18]. In addition, a potent cytotoxic drug, monomethyl auristatin F, which acts as an anti-CD133 antibody-drug conjugate for hepatocellular and gastric cancer cells, may be utilized to treat CD133-positive tumors [19]. To date, there have been limited studies of CD133 in esophageal cancer, and thus, the significance of CD133 in this form of cancer remains unclear. Therefore, we examined whether CD133 could serve as a prognostic marker of ESCC. In addition, we explored the correlation between CD133 expression and the clinicopathological findings of ESCC patients and the correlation between CD133 expression and the immunolocalization of several markers, such as p53, p16, p27, murine double minute 2 (MDM2), Ki-67, and epidermal growth factor receptor (EGFR), which are known as prognostic markers or tumor proliferation factors in ESCC [20][21][22][23][24][25][26][27].

Patients and tissue samples
A total of 86 consecutive ESCC patients, who underwent curative surgery without neoadjuvant treatment at Tohoku University Hospital (Sendai, Japan) between January 2000 and December 2005, were selected. All patients underwent thoracoscopic esophagectomy with twoor three-field node dissection, except for four patients who underwent pharyngo-laryngo-esophagectomy with one-field node dissection, six patients who underwent transhiatal esophagectomy with one-field node dissection, and eight patients who underwent esophagectomy by right thoracotomy.
The resected specimens and lymph nodes were fixed in 10% formalin, and representative sections were embedded in paraffin wax. The sections were histologically examined according to the Union for International Cancer Control (UICC) TNM (tumor, node, metastasis) classification (7th edition) system [28]. Patient survival time was determined from the date of surgery until death, recurrence, or the last follow-up examination. This study was approved by the ethical committee of the Tohoku University Hospital (Accession number 2011-596).

Statistical analyses
Statistical analyses were performed using JMP Pro Version 9.0.2 (SAS Institute Inc., Cary, NC, USA). The correlation of factors was evaluated by the chi-square test, Fisher's exact test, or Wilcoxon test, as appropriate. Survival curves were determined by the Kaplan-Meier method, and differences in survival between groups were compared by the log-rank test. The Cox proportional hazard model was used for multivariate analysis. A P value of <0.05 was considered statistically significant.

Results
Correlation between CD133 and clinicopathological findings of patients Table 1 summarizes the clinicopathological findings of the patients examined. The median follow-up time was 69.0 months (range, 1 to 149 months). The patients included 73 men and 13 women with a median age of 64 years (range, 37 to 81 years). The number of patients in each pathological stage was as follows: 20, pStageI; 28, pStageII; 33, pStageIII; and 5, pStageIV. There were five patients with M1 lymph nodes. Of the 86 patients, 38 (44.2%) were immunohistochemically positive for CD133 ( Figure 1). pT and pStage were significantly more advanced among CD133-negative patients compared with CD133-positive patients (Table 1). Table 2 summarizes the correlation between expression of CD133 and expression of other molecular markers examined. CD133 and p27 expression were positively correlated (P = 0.0013), and CD133 and p16 expression tended to be positively correlated (P = 0.057) but did not reach statistical significance. No significant correlations were detected between expression of CD133 and expression of any other marker.

Correlations for other molecular markers
In terms of correlations between the other molecular markers and clinicopathological findings, p16 expression was correlated with smoking history (P = 0.018), pathological lymph node status (P = 0.033), and lymphatic invasion (P = 0.018) (Additional file 1). With regard to correlations among other molecular markers, p53 expression was positively correlated positively with Ki-67 expression (P = 0.0030) (Additional file 2).

Survival analysis
The 3-and 5-year survival rates of all patients examined were 65.0% and 61.5%, respectively. Results of univariate analysis of postoperative overall survival (OS) and disease-free survival (DFS) are summarized in Table 3.  Overall survival was significantly correlated with pT, pN, pStage, and CD133 status, and was significantly longer in CD133-positive patients than in CD133negative patients (P = 0.049) (Figure 2). No significant correlation between OS and the other markers was observed (Figure 3). Multivariate analysis demonstrated that pStage was a significant prognostic factor for OS and that pStage and tumor location were significant prognostic factors for DFS. Correlation between CD133 expression and patient survival did not reach statistical significance by multivariate analysis (Table 4).

Discussion
CD133 was originally identified as a transmembrane glycoprotein in normal hematopoietic stem and progenitor cells [31] that participated in proliferation, selfrenewal, and multilineage differentiation [32]. Furthermore, CD133 was recently used to identify putative cancer stem cells of several tumors [33]. According to several studies, CD133 was associated with tumor differentiation in several organs [16,[34][35][36]. For example, Jiang et al. [36] reported that CD133 expression was increased in diffuse-type gastric cancers compared with intestinal-type cancers and was increased more so in poorly differentiated than in moderately or well differentiated gastric cancers. In addition, Feng et al. [35] reported that CD133 was negatively correlated with the cellular differentiation status of colon cancer cells. Finally, Fan et al. [34] reported that CD133 expression was correlated with well differentiated or moderately differentiated cholangiocarcinomas and that subcellular CD133 localization was correlated with the tumor differentiation status. In terms of ESCC, Hang et al. reported that CD133 expression was increased in well differentiated and moderately differentiated ESCCs compared with poorly differentiated ESCCs [30]. However, in our study, no correlation was detected between CD133 expression and tumor differentiation of carcinoma cells. We think that this was because there is a difference among pathologists or facilities regarding the histological evaluation of tumor differentiation status, and in addition, our study was small. On the other hand, CD133 expression correlated with p27 expression (P = 0.0013) and tended to correlate with the status of p16 immunoreactivity (P = 0.057). The relationship between CD133 and cell cycle regulators has remained unclear in esophageal cancer. There may be a correlation between CD133 and cell cycle pathways associated with the INK4 family or the  CIP/KIP family of cyclin-dependent kinase inhibitors [37], but this possibility requires further investigation.
To the best of our knowledge, there are few reports that have investigated the effect of CD133 expression on survival of ESCC patients. Nakajima et al. [38] reported that CD133 expression in resected ESCC specimens following neoadjuvant chemoradiotherapy tended to be correlated with poor prognosis, but multivariate analysis did not produce a significant correlation. In contrast, CD133 expression was significantly correlated with poor response to neoadjuvant chemoradiotherapy. Hang et al. [30] reported that CD133 expression in resected ESCC specimens without preoperative treatment was not significantly correlated with prognosis. Our study revealed that OS was significantly longer in CD133-positive patients than in CD133-negative patients, as determined by log-rank test. One reason for this was that the tumors were significantly more advanced (according to their pStage classification) in CD133-negative patients than in CD133-positive patients. Although correlation between CD133 expression and patient survival did not reach statistical significance, as determined by multivariate analysis, CD133 immunoreactivity may have the potential to be a good predictor of prognosis in ESCC patients. With regard to other tumors, CD133 expression in non-small cell lung cancers [39,40], hepatocellular carcinomas [18], and pancreatic cancers [41] was not correlated with patient survival. Moreover, CD133negative expression in cholangiocarcinomas was correlated with poor prognosis [34], which is similar to that revealed in our study. Further studies are needed to clarify this issue.

Conclusions
In conclusion, this study demonstrated that CD133 immunoreactivity may have the potential to be a good predictor of prognosis in ESCC patients, and that CD133 may play a role in the regulation of tumor cell cycle through p27 and p16 in ESCC. At present, whether CD133 expression is a valid prognostic marker for ESCC remains controversial. To elucidate this relationship, further investigations are required, including verification of an evaluation method for CD133 immunoreactivity in ESCC.

Consent
Written informed consent concerning the procedure of this study was obtained from all patients prior to study enrollment.