Background
Esophageal carcinoma is one of the most common malignancies in China, but primary small cell carcinoma of the esophagus (SCCE) is a relatively rare histopathological type, accounting for only 1-2.8% of esophageal carcinomas [
1,
2]. SCCE is highly aggressive, and characterized by early dissemination and poor prognosis [
3‐
10]. A total of 5,379 cases of esophageal cancer and 2,061 cases of small cell carcinoma were recorded at Sun Yat-sen University Cancer Center from 1994 to 2012, including 93 cases of SCCE. Little is known about the clinicopathological features of SCCE, and it is necessary to identify biomarkers for predicting prognosis and for distinguishing individuals with unfavorable prognoses.
The Wnt signaling pathway is important for adult tissue maintenance. Perturbations in Wnt signaling cause human cancers [
11]. Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) is an orphan G-protein coupled receptor (GPCR) and a stem cell marker first described as a Wnt target gene [
12]. It is also a member of the canonical Wnt-signaling cascade, which forms a signaling gradient in the intestinal crypt, thereby regulating cell proliferation and differentiation [
13]. Lgr5 was also identified as a marker of poor prognosis in colon, ovary and liver cancers [
14,
15], and was considered to be involved in tumorigenesis in Barrett’s esophagus and esophageal adenocarcinoma [
16].
However, the expression and potential clinical significance of Lgr5 in SCCE has not been determined. In this study, we analyzed the expression levels of Lgr5 and their relationships with clinicopathological features in 44 patients with SCCE.
Discussion
SCCE is a highly metastatic disease with a median survival time of less than 1 year [
21]. The recurrence rate after surgery is very high, even in patients with early-stage disease [
22]. However, the aggressive features and poor prognosis of SCCE found in clinical practice have not yet been linked to any specific biomarkers. Recent genetic discoveries based on tumor genome sequencing suggest that the Wnt pathway plays important roles in tumor biology [
23]. Activation of the Wnt pathway releases β-catenin, which interacts with T cell factor family members to activate the transcription of downstream target genes [
24]. R-spondins potently enhance β-catenin signaling and have been implicated in human disease and malignancy [
25,
26]. Lgr5 is a receptor for R-spondin and was shown to activate β-catenin signaling when bound to R-spondins [
25,
27,
28], and may be associated with tumorigenesis via the Wnt pathway.
Iuga et al. [
29] reported that Lgr5 was a novel IHC marker for gastrointestinal neuroendocrine tumors; 88% of primary neuroendocrine tumors and 87% of metastases stained positive for cytoplasmic Lgr5. Moreover, Lgr5 stained positive in most cases expressing CgA and Syn (34/38). These findings were consistent with our study, which also found high Lgr5 expression levels in SCCEs, which were rich in CgA and Syn. von Rahden et al. [
16] reported LgR5 expression in 35 of 41 (85%) patients with esophageal adenocarcinomas with Barrett’s esophagus, and in 16 of 19 (81%) without Barrett’s esophagus. Becker et al. [
30] also detected Lgr5 expression in early esophageal squamous cells. Based on the above findings, we examined Lgr5 expression in SCCE.
Of the 44 patients with SCCE examined by IHC, only 15.8% who presented with high levels of Lgr5 survived for longer than 1 year after diagnosis (data not shown). The median survival in patients with high expression levels of Lgr5 was 7 months, which was 11 months shorter than in patients with lower expression of Lgr5. Less than 20% of patients (18 patients) had survived longer than 2 years at the end of follow-up, none of whom presented with high Lgr5 expression. Our results indicated that high levels of Lgr5 expression were significantly correlated with lymph node metastasis and late stage (stage III/IV), suggesting that high expression of Lgr5 might predict a poor prognosis. Indeed 62% of patients with lymph node metastases failed to survive for longer than 1 year, in accordance with a previous study in which esophageal cancer patients with more than four involved lymph nodes showed similar survival to patients with M1 disease [
31]. In Cox regression analysis, lymph node involvement was an independent prognostic factor, which might explain the association between high expression of Lgr5 and poor prognosis.
SCCE is a systemic disease [
21]. Chemotherapy provides the backbone of SCCE therapy [
21,
32], which was in accordance with the fact that chemotherapy was an independent prognostic factor in the present study. Lgr5 expression may also predict response to chemotherapy in SCCE. High Lgr5 expression was significantly correlated with unfavorable response in this study; only 33% of patients with high expression achieved partial/complete responses during chemotherapy. Additionally, Lgr5 may also predict chemotherapy response in colorectal cancer [
33].
A recent study suggested that GPCRs and their signal transduction pathways may provide promising new therapeutic approaches [
34]. They are involved in the control of blood pressure, maintenance of kidney function, occurrence of neurological diseases and the progression of cancer [
34]. Approximately 36% of currently-marketed drugs target GPCRs [
35]. Lgr5 has been suggested to be involved in cancer progression through regulation of the Wnt signaling pathway [
25,
27,
28]. Lgr5 knockdown was shown to induce cell death [
36], and furthermore, a recently-developed monoclonal antibody (KM4056) was reported to have potent complement-dependent cytotoxicity activity in vitro, and to show strong anti-tumor activity in vivo [
37]
. Lgr5 thus remains a potential for targeted therapy.
Overall, the results of this study suggest that Lgr5 protein expression may represent a possible prognostic marker in SCCE patients. However, these results need to be validated by further studies with larger sample sizes and in randomized patient cohorts before Lgr5 IHC can be used in clinical applications.
Conclusions
In summary, overexpression of Lgr5 was significantly correlated with lymph node metastasis, tumor stage and response to chemotherapy, while high levels of Lgr5 expression were also associated with poor survival in patients with SCCE.
Acknowledgements
We thank Shu-Mei Yan and Mu-Yan Cai for diagnosis confirmation and Yu Chen for editorial assistance. This work was supported by the National High Technology Research and Development Program of China (863 Program), China (No.2012AA02A506); Science and Technology Department of Guangdong Province, China (No. 2012B031800088); Medical Scientific Research Foundation of Guangdong Province, China (No.C2011019).
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
WC, FW carried out the immunohistochemistry staining and drafted the manuscript. WC, FW, DZ, HL, ZW, FW, MQ, CR, XW and WW participated in the design of the study and performed the statistical analysis. WC, FW, YL and RX conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.