Reg IV expression and clinicopathologic features of gallbladder carcinoma

Summary

Regenerating islet-derived family, member 4 (Reg IV) has been shown to be associated with colorectal carcinogenesis and gastric carcinogenesis through intestinal metaplasia. In this study, we examined Reg IV expression in the gallbladder and gallbladder carcinoma, and measured Reg IV levels in sera from patients with gallbladder carcinoma. Quantitative reverse transcription–polymerase chain reaction revealed that high Reg IV levels were identified in 17 of 31 gallbladder carcinomas, whereas there was no apparent amplification in normal gallbladders. Immunohistochemically, although only a small part of the epithelium with intestinal metaplasia in 2 of 4 cases with adenomyomatosis showed Reg IV expression, Reg IV was negative in all cases with normal gallbladder (n = 15) and cholelithiasis (n = 13). In contrast, 34 (56%) of 61 gallbladder carcinomas were positive. Expression was more frequently observed in well to moderately differentiated than in poorly differentiated adenocarcinomas and significantly correlated with expression of caudal-related homeobox transcription factor (a candidate for involvement in the induction of intestinal metaplasia). Multivariate analysis revealed negative Reg IV expression, as well as hepatic parenchymal invasion, to be independently associated with a poor prognosis in patients with advanced gallbladder carcinoma. Before surgical resection, 4 (33%) of 12 patients with gallbladder carcinoma had high serum Reg IV levels, whereas Reg IV was never elevated in 12 patients with benign diseases. The serum levels of Reg IV decreased after surgical resection of the tumors. These results suggest that Reg IV is involved in gallbladder carcinoma carcinogenesis through intestinal metaplasia and is associated with relatively favorable prognosis in patients after surgery. The serum level of Reg IV may be of use or indicative of neoplasia.

Introduction

The regenerating islet-derived family, member 4 (Reg IV) gene was originally identified by high-throughput sequencing of a complementary DNA library derived from patients with inflammatory bowel disease [1] and encodes a secreted Reg IV protein. Reg IV is known to be expressed in various normal tissues such as the stomach, colon, small intestine, and pancreas [2], [3]. In cancers, although lung and breast cancers do not express Reg IV, gastric, colorectal, pancreatic, and prostate cancers have been reported to express Reg IV, and the levels of expression in these cancers are much higher than in normal tissues [2], [3], [4].

The biological function of Reg IV in cancers, however, has been poorly understood, although several possible functions have been reported. Among these, several studies have shown that Reg IV may be involved in early carcinogenesis in certain cancers. Zhang et al [5] reported that overexpression of Reg IV may be an early event in colorectal carcinogenesis, on the basis of their observations in which Reg IV mRNA expression was upregulated in colorectal adenomas and adenocarcinomas when compared to paired normal mucosa, and the staining of Reg IV by in situ hybridization was stronger in adenomas with more severe dysplasia. Oue et al [2] showed that Reg IV protein was expressed in the goblet cells of intestinal metaplasia of the stomach and goblet-like cell vesicles of gastric carcinoma, suggesting the association of Reg IV protein with intestinal differentiation of the stomach and tumorigenesis of intestinal-type gastric carcinoma.

Gallbladder carcinoma (GBC) is a highly malignant neoplasm. Despite the improvement of diagnostic modalities, most cases with GBC are diagnosed at advanced stages, resulting in poor prognosis even after surgical resection [6], [7], [8]. Although the mechanisms of carcinogenesis of GBC have not been fully evaluated, dysplasia is considered to be a precursor of invasive GBC, because of their close topographical relationship [9], [10]. In addition, several reports have shown that the most common epithelial change associated with dysplasia is intestinal metaplasia [9], [10], [11], suggesting that dysplasia arises from metaplasia, which is possibly attributable to chronic inflammation in the mucosa of the gallbladder [12], [13]. Furthermore, Mukhopadhyay and Landas [14] proposed a sequence of gastric (antral) metaplasia–intestinal metaplasia–dysplasia, as evidenced by an age gradient from antral metaplasia to dysplasia, and significant statistical associations between antral metaplasia and intestinal metaplasia and intestinal metaplasia and dysplasia. This sequence is similar to that in the gastric carcinogenic pathway: chronic inflammation and regeneration lead to intestinal metaplasia in the gastric mucosa, followed by dysplasia, and, finally, intestinal-type gastric carcinoma [15]. These observations led us to the idea that Reg IV may also play an important role in gallbladder carcinogenesis through intestinal metaplasia.

On the other hand, it has been reported that intestinal metaplasia and caudal-related homeobox transcription factor (Cdx2) in the gallbladder are related [12], [16]. Cdx2 plays a crucial role in the regulation of intestinal epithelial differentiation and the maintenance of its phenotype [17]. Besides the expression in the fetal and adult epithelial nuclei of normal intestines, Cdx2 is ectopically expressed in the nuclei of the epithelium with intestinal metaplasia in some organs and some intestinal-type neoplasms. Although in intraductal papillary mucinous neoplasms of the pancreas Cdx2 expression is shown to be significantly correlated with Reg IV expression [18], the relationship between Reg IV and Cdx2 expressions remains unknown in GBC.

In this study, we investigated the expression of Reg IV mRNA and its protein in the gallbladder and GBC using molecular and immunohistochemical techniques. The relationship between Reg IV and Cdx2 protein expressions in GBC was also examined. In addition, because Reg IV is a secreted protein, we measured Reg IV levels in sera from patients with GBC by ELISA to assess the possibility of Reg IV as a serum biomarker for GBC.