Significance of EpCAM and TROP2 expression in non-small cell lung cancer

Non-small cell lung carcinoma (NSCLC) is a major cause of cancer-related death in bothmen and women globally [1]. Despite recent advancements in early tumor detection, surgical treatment, radiochemotherapy, and targeted therapy, the NSCLC-related high mortality rate remains a daunting challenge [2]. For example, targeted NSCLC therapy, especially against epidermal growth factor receptor (EGFR), has advanced greatly, yet only approximately 15% of NSCLCs experience therapeutic benefits due to various factors [3]. Since no single marker is sufficient for prediction of prognosis, many studies have focused on the development of new biomarkers.

The tumor-associated calcium signal transducer (TACSTD) gene family consists of two highly conserved and closely related genes, TACSTD1 and TACSTD2, which map to chromosomes 2p21[4] and 1p32[5], respectively. The TACSTD1 gene encodes TROP1, which was originally designated as epithelial cell adhesion molecule (EpCAM). TROP1 is a 39-42 kDa, 314 amino acid, type l transmembrane glycoprotein. EpCAM mediates epithelium-specific, Ca²⁺-independent homotypic cell-cell adhesion and represents the first human tumor associated antigen to be discovered. It has been used as an immunotherapeutic target of carcinoma. Normally, EpCAM is expressed on the basolateral surface of normal simple, pseudostratified, and transitional epithelia. It is also overexpressed in various human carcinomas, including those of the colon and rectum, prostate, liver, esophagus, lung, head and neck, pancreas, and breast [6]. EpCAM participates as a cell adhesion and cell signaling molecule, and its overexpression negatively correlates with survival rates in gallbladder cancer, ampullary carcinoma, and squamous cell carcinoma (SCC) of the head and neck [4, 7]. EpCAM expression is also associated with better survival in clear cell renal cell carcinoma patients [8] and is related to shortened survival in node-positive breast cancer patients [9].

TROP2 is a 35 kDa, 323 amino acid, type 1 transmembrane protein with a single transmembrane domain [10]. TROP2, which was originally identified in human trophoblast and choriocarcinoma cell lines [11], is encoded by TACSTD2. TROP2 is not expressed in normal tissue, but is overexpressed in many carcinomas, including colorectal cancer, gastric cancer, SCC of the oral cavity, and pancreatic cancer [5]. Like EpCAM, TROP2 has been an attractive immunotherapeutic target in cancer treatment. Recently, TROP2 has been actively studied as a prognostic marker for various cancers. TROP2 overexpression has been associated with poor prognosis in colon cancer [10] and oral SCC [11].

Although the roles of EpCAM and TROP2 are not yet fully understood, both proteins are thought to participate in growth and proliferation of carcinoma cells. EpCAM can transduce an intracellular signal through its cleavage of an intracytoplasmic portion [7]. TROP2 is also believed to be a true oncogene involved in initiating signaling mechanisms that can result in increased tumorigenicity, aggressiveness, and metastasis [5]

The present study focused on the differential expression of EpCAM and TROP2 according to SCC or adenocarcinoma (AdC) histology, because certain genetic lesions may play different biological roles depending on the histological subtypes [12]. The study evaluated EpCAM and TROP2 expression in regard to various clinicopathologic factors and survival to investigate their potentials as biomarkers, and also analyzed the prognostic significance of the status.

Although the EpCAM and TROP2 proteins were discovered some time ago, only recently have they become the focus of research in various cancers. While there are increasing reports about the role of EpCAM or TROP2 in various cancers, only a few reports of EpCAM or TROP2 expression in NSCLC have been published [2, 13]. In one recent study, EpCAM expression was investigated in NSCLC, but TROP2 was not involved [2]. A study by Kobayashi et al. has evaluated TROP2 expression in NSCLC [13], and it was limited to small-sized AdC. Hence, the present study is the first comprehensive evaluation of the expression of EpCAM and TROP2 in pulmonary AdC and SCC, simultaneously.

In the present study, we observed different patterns of EpCAM and TROP2 expression between in normal bronchial epithelium and in NSCLC. In normal bronchial epithelium, both proteins showed only infrequent basolateral cytoplasmic membrane positivity. They were not stained in alveolar pneumocytes. As EpCAM is a cell-cell adhesion molecule in epithelial cells, its distribution along the cytoplasmic membrane is not unexpected. On the other hand, EpCAM and TROP2 were overexpressed in NSCLC and their staining along the cytoplasmic membrane was complete along the membrane. This finding corroborates a previous report that the expression of EpCAM increases in a stepwise manner from uninvolved bronchial mucosa, epithelial hyperplasia, to SCC [2]. Cytoplasmic or nuclear expression was not seen in our study. Although Kobayashi et al. advocated that TROP2 is also expressed in cytoplasm in NSCLC [13] and we observed focal cytoplasmic staining, we think that this is non-specific staining regarding that cytoplasmic expression was only focally observed in the cases which show strong membranous TROP2 staining. Both EpCAM and TROP2 were more frequently expressed in SCC in the present study, being consistent with a previous study [6]. But, the pattern of expression was not different to each other. Besides functioning as cell-cell adhesion molecule, EpCAM has been recently known to act as an oncogene, proving to trasduce cell proliferating signal through c-myc, cyclin D, and survivin. In another aspect, the loss of EpCAM has been advocated to promote tumor progression through weakening cell-cell adhesion. When EpCAM is overexpressed, some cancers such as stage I or II gastric cancer, stage II NSCLC, clear cell renal cell carcinoma, and stage II colon cancer show better survival while others such as node-positive breast cancer, epithelial ovarian cancer, gall bladder cancer, cholangiocarcinoma, ampullary pancreas cancer, squamous cell cancer of the esophagus, and squamous cell head and neck cancer do worse. This contradicting result may be determined by which function of cell-cell adhesion or oncogene of EpCAM predominates. Although the function of TROP2 is less defined than that of EpCAM, TROP2 has also been suggested as oncogene, promoting cancer cell proliferation and migration, and cell adhesion molecule [5, 14]. Regarding NSCLC, Kobayashi et al. reported that TROP2 overexpression in small pulmonary AdC was related with worse overall survival [13]. However, in our study, TROP2 overexpression was related with better overall survival and disease free survival in AdC, although SCC showed same tendency but no statistical significance. To our best knowledge, there has not been a study, in which TROP2 overexpression in cancer was associated with better survival than those which do not. This discordance between our study and previous one may be partially explained by that Kobayashi et al. included only AdC which are < 2 cm in size, while we included larger tumors of varying degree of differentiation. In our study, stage II or III AdC with TROP2 overexpression showed better overall survival and disease-free survival while in stage I AdC, TROP2 overexpression did not affect overall survival or disease-free survival. Furthermore, poorly differentiated AdCs expressed TROP2 more frequently. These data imply that poorly differentiated AdCs may show better survival when they express TROP2. Whether TROP2 serves as an adhesion molecule or not has not been fully defined. Some advocated its role as adhesion molecule [15], but early study could not prove it [16]. Recently, it has been discovered that TROP2 binds with Claudin 1 and 7 which are important components of tight junctions, taking part in cell-cell adhesion [17]. Hence, we hypothesized that the biologic role of TROP2 can be different in early and advanced pulmonary AdC. In higher stage pulmonary AdC, the role of TROP2 as an adhesion molecule may be greater than as an oncogene. TROP2 overexpression may strengthen bonds between tumor cells, preventing their shedding and progression. Like EpCAM showed variable results in variable cancers, TROP2 may play different roles in different cancers.

The EpCAM and TROP2 proteins are expressed in NSCLC, and only weakly or not expressed in normal lung tissue. The expression pattern of EpCAM and TROP2 was similar in SCC and AdC. TROP2 protein did confer a significant survival effect in AdC. This finding may reflect that TROP2 is involved in cell-cell adhesion in pulmonary AdC and its loss will promote tumor cell shedding. Hence, our study showed that TROP2 predicts better prognosis in AdC of NSCLC.

Written informed consent was obtained from the patient for publication of this report and any accompanying images.