Research Paper
Expression and Prognostic Significance of EpCAM Philip Went1, Stephan Dirnhofer, Daniel Schöpf, Holger Moch, and Gilbert Spizzo Institute of Pathology, University of Basel, Switzerland [P. Went, S. Dirnhofer]; Department of Radiology [D. Schöpf] and Division of Haematology & Oncology [G. Spizzo], Innsbruck Medical University, Austria; Institute of Surgical Pathology, University of Zurich, Switzerland [H. Moch]
AIM: EpCAM (CD326) is a transmembrane glycoprotein on epithelial cells. To assess its expression, an increasing number of commercially available antibodies combined with multiple staining protocols with varying sensitivities and specificities are in use. There is no consensus about definition of positivity. Systematic and reliable comparison of results is therefore hampered. To maximise the reproducibility of the results, we investigated EpCAM expression on tissue microarrays. METHODS: The murine monoclonal antibody ESA (clone VU-1D9) was used at a dilution of 1:800 after antigen retrieval by microwave at 80°C for 30 min. Tissue microarrays containing gastrointestinal carcinoids (n = 91) and endometrial (n = 316) and esophageal (n = 54) carcinomas were analysed and the results were compared with clinicopathological features. RESULTS: In gastrointestinal carcinoids, 87% of cases had strong EpCAM expression, and less than 2% were completely EpCAM-negative.
This EpCAM expression
was not associated with tumor localization within the gastrointestinal tract. All endometrial and esophageal carcinomas expressed EpCAM to some extent, but there was no correlation of EpCAM expression with histological grade, tumor stage or patient survival in the uterus and esophageal cancers. CONCLUSION: EpCAM was frequently expressed in adenocarcinomas of the uterus, esophageal squamous cell carcinomas and gastrointestinal carcinoids. This high frequency makes these tumors as potential targets of anti-EpCAM therapies and may render individual testing before application of the therapy unnecessary.
Keywords: EpCAM tissue microarray squamous cell carcinoma carcinoid
Journal of Cancer Molecules 3(6): 169-174, 2008.
Introduction EpCAM (epithelial cell adhesion molecule; CD326) is a transmembrane protein able to interact with many other proteins, such as EpCAM itself, claudin-7, tetraspanins, CD44v and cathepsin [1-5]. The official symbol for the gene, which is localised to the chromosome 2p21, is TACSTD-1 (tumor-associated calcium signal transducer 1), though recently epcam1 was proposed as a new name [6]. Its extracellular domain recently has been suggested to contain an EGF-like domain and a TY domain [6]. As the mentioned spectrum of interacting proteins suggests, the function of EpCAM is multifaceted and varies in time and during cell biology. Whereas its adhesion function was discovered early, its role as potent signalling molecule acting during morpho- and carcinogenesis has been demonstrated only recently. Though an increasing number of commercially available antibodies combined with multiple staining protocols with varying extents of sensitivity and specificity are in use and there is no consensus about definition of positivity, EpCAM is accepted to be widely distributed in human normal tissues Received 12/10/07; Revised 1/03/08; Accepted 1/06/08. 1 Correspondence: Dr. Philip Went, Institute of Pathology, City Hospital, Birmensdorferstrasse 497, 8063 Zurich, Switzerland. Phone: 41-44-4661384. Fax: 41-44-4662138. E-mail:
[email protected] 2 Abbreviations: SCC, squamous cell carcinoma; GIT, gastrointestinal tract; TMA, tissue microarray.
2008 MedUnion Press − http://www.mupnet.com
[7]. Additionally, it has been detected in many tumor tissues [8]. Interestingly, whereas the deregulation of most molecules, as for example HER2/Neu, is unidirectional in most cancers, i.e. overexpressed or lost, EpCAM seems to have different regulatory pathways in different tumors. EpCAM can be (i) overexpressed, as for example in most colon cancers [9], it can be (ii) lost, as for example in some poorly differentiated colon cancers [8], or it can be (iii) acquired, as for example in squamous cell carcinoma (SCC2) of the esophagus [10], because it is negative or weakly positive in normal squamous cell epithelium but strongly positive in SCC. Accordingly, the prognostic value EpCAM is discussed contradictorily. As EpCAM is detected on carcinoma cells, it is also a possible therapeutic target. Effectively, the immunization of mice with EpCAM-positive colon cancer cell lines followed by the search of tumor specific antibodies led to the discovery of EpCAM [11]. The first anti-EpCAM antibody, Edrecolomab, was used in phase I to III trials on colon carcinoma patients and showed contradictory results [12,13]. Other therapeutic modalities have been added. The actual spectrum of clinical trials targeting EpCAM includes passive immunotherapy, administered locally or systemic, and active immunotherapy. EpCAM had been conjugated with exotoxin A and used as an effective locally administered immunotoxin in head and neck cancers [14]. Experimental approaches now aim at controlling EpCAM gene promoter [15] or at silencing EpCAM expression with short interfering RNA [16].
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Table 1: Frequency of EpCAM in gastrointestinal carcinoids Weak/ Tumor location (case no.) Negative Strong moderate All (91) Stomach (11) Duodenum (8) Ileum (11) Appendix (9) Colon, right-sided (7) Rectum (7) Pancreas, nonfunctioning (38)
2% 9% 13% 0% 0% 0% 14% 5%
11% 18% 13% 0% 0% 14% 0% 3%
87% 73% 74% 100% 100% 86% 86% 92%
In this paper, we add results on EpCAM expression in cancers of the uterus, esophagus and gastrointestinal carcinoids, and review the distribution of EpCAM in human tumors. The knowledge of its distribution clarifies the broad application potential of anti-EpCAM target therapies. The relation of EpCAM expression to prognosis is highlighted with special attention to the contradictory effects and the antidromic characteristics of EpCAM expression in normal tissues and corresponding malignant tumors.
Materials and Methods Tumor specimens The tumor tissues of 336 uterine carcinoma patients were retrieved from the archives of the Institute of Pathology in Basel (Switzerland) including the years 1985-1998. Sixty esophagus cancer tissues were collected from the archive of the Institute of Pathology in Innsbruck (Austria) encompassing the years 1994-1999. Tissue retrieval for 104 carcinoids comprised the years 1982-2000 from the archive of the Institute of Pathology in Varese (Italy). These carcinoids were all localised to the gastrointestinal tract (GIT). Additional clinical data were obtained by directly contacting the primary care physicians and hospitals, which included age, sex, stage, grade, administered therapy, date of diagnosis, date of last follow-up, and survival status (alive or deceased). Retrieval of tissues and clinical data was performed according to the regulations of the local institutional review board and data safety laws. Tissue microarray (TMA) Tissue microarrays were constructed as described previously [17]. The staining procedure and slide analysis were done identical to our previous studies [8,9,18]. In brief, the antibody ESA (clone VU-1D9, Novocastra, Hamburg, Germany) was diluted at 1:800 and the tissues were heated by microwave at 80°C for 30 min before staining. A case was considered strongly positive when the antibody detected ≥ 70% positive tumor cells, otherwise weakly positive, or negative if no cells were stained. The staining intensity was recorded but not used for correlation with clinical findings, because it could vary depending on tissue fixation. To validate the TMA results, we compared the results of the esophagus cancer TMA with conventional sections. Statistical analysis Statistical analysis including data description was performed using the Statistical Package of Social Sciences version 13 (SPSS, Chicago, IL, USA) for Mac OS X.
Results In all three TMAs derived from different primary organs, there was a tendency of EpCAM to show a bipolar expres-
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Figure 1: EpCAM expression in gastrointestinal carcinoids and uterine and esophagus carcinomas. A, The extent of the tumor infiltration of a gastrointestinal carcinoid in the appendix is highlighted by EpCAM immunohistochemistry (×10). B, Uterine carcinoma with a solid growth pattern and a diffuse positivity in all tumor cells with a partial slight decrease in staining intensity from the centre to peripheral parts of the tumor (×20). C, SCC of the esophagus with the diffuse expression of EpCAM (×20).
sion pattern: tumor cells expressed EpCAM with strong staining intensity in a high frequency, whereas a minority of tumors were EpCAM-negative or showed weak EpCAM expression.
EpCAM expression in carcinoids From 104 carcinoid specimens, 91 cases in TMA format could be analyzed. All originated from the GIT (Table 1). The results showed that 87% of cases had strong EpCAM expression, whereas less than 2% were completely EpCAMnegative (Figure 1A and Table 1). There was no correlation Print ISSN 1816-0735; Online ISSN 1817-4256
EpCAM in Human Tumors
Table 2: EpCAM expression in uterine carcinomas Weak/ Negative moderate All (n = 316) 0% 17% Histology 0% 20% Non-endometrioid (n = 206) Endometrioid (n = 110) 0% 12%
Strong 83% 80% 88%
Table 3: EpCAM in carcinomas of the esophagus Weak/ Negative moderate All (n = 54) 0% 54% Histology 0% 33% Adenocarcinoma (n = 6) 0% 56% SCC ( n = 48) Normal epithelium (n = 52) 33% 19%
Strong 46% 67% 44% 0%
Grade 1 (n = 136) 2 (n = 89) 3 (n = 89)
0% 0% 0%
15% 19% 20%
85% 81% 80%
Stage (FIGO) Not available (n = 46) 1 (n = 159) 2 (n = 52) 3 (n = 30) 4 (n = 13)
Grade 1 (n = 9) 2 (n = 18) 3 (n = 18)
0% 0% 0%
44% 67% 39%
56% 33% 61%
0% 0% 0% 0% 0%
17% 18% 17% 17% 8%
83% 82% 83% 83% 92%
Tumor stage pTis (n = 4) pT1 (n = 3) pT2 (n = 5) pT3 (n = 6) pT4 (n = 4)
0% 0% 0% 0% 0%
50% 67% 60% 17% 75%
50% 33% 40% 83% 25%
0% 0% 0%
57% 44% 50%
43% 56% 50%
0% 0%
22% 5%
14% 8%
Nodal stage pN0 (n = 14) pN1 (n = 18) pN2 (n = 6) Metastasis pM0 (n = 36) pM1 (n = 13)
Figure 2: Kaplan-Meier analysis of survival in patients with endometrioid adenocarcinomas of the uterus corpus (n = 195). Blue: patients with low/moderate EpCAM-expressing tumors; green: patients with strong EpCAM-expressing tumors.
between EpCAM expression and tumor localization within the GIT. No additional patient data were available for correlation analyses.
EpCAM expression in uterine carcinomas On the TMA of endometrial carcinomas, samples from 316 cancers were analyzable. All tumors expressed EpCAM at least weakly. Mean age of the patients was 68 years (ranging from 31 to 92). EpCAM showed a tendency to be expressed more frequently in the endometrioid subtype when compared to non-endometrioid carcinomas (Figure 1B and Table 2). Still, there was no correlation of EpCAM expression with grade or stage. Likewise, EpCAM was not associated with survival (Figure 2), neither in a subgroup analysis of the different histological subtypes separately. Mean survival of the patients (n = 195) was 60 months (ranging from 0 to 173). EpCAM expression in esophagus cancers In regard to esophagus cancers, a TMA was used to successfully feature 54 tumors. Mean age of the patients was 62 years (ranging from 30 to 84). EpCAM was expressed in all tumors, and only three tumors (5.6%) showed a weak EpCAM expression (Table 3). In some larger sections, there was a 2008 MedUnion Press − http://www.mupnet.com
tendency of the tumors to show EpCAM at the periphery of tumor cells in a higher staining intensity (Figure 1C). The TMA results were in concordance with those of the conventional sections with corresponding tumors. For further analysis, only SCC cases were considered, because these tumors showed a differentially regulated EpCAM expression if compared with adenocarcinomas. SCC specimens were significantly more frequent to express EpCAM if compared with normal squamous epithelium (P < 0.001). Still, there was no correlation of EpCAM expression with grade, stage or disease-specific survival (Table 3).
Discussion EpCAM was detected as antigen in mice immunized with human colon cancer cell lines more than 25 years ago [11]. Since then, EpCAM has been detected on the majority of human carcinomas. Interestingly, the EpCAM expression as detected by immunohistochemistry approximates a dual expression pattern with strongly and homogeneously positive or completely negative tumors. Technical and biological reasons could contribute to this phenomenon: (i) Upregulation of EpCAM has been shown in colon cancers. (ii) Pretreatment procedures can be optimized until the highest possible contrast in the expected cell number is achieved; therefore, the method tends to produce a bipolar result with the smallest possible grey zone. Irrespective of these caveats, immunohistochemistry represents the most widespread, cost-effective and fast method to detect in situ protein expression. In normal tissues, EpCAM is in general not expressed in mesenchymal tissues, although exceptions exist, e.g. erythroid precursors in the bone marrow have been found to be EpCAM positive [19]. Additionally, elements of the spermatogenesis have been found to express EpCAM weakly [8]. As a general rule, the normal squamous epithelium does express EpCAM weakly in the basal cells, whereas the cylindric epithelium shows high EpCAM expression. For example in the GIT, EpCAM expression can be found in
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Table 4: EpCAM expression and its correlation with prognosis Tumor Colon (G2) Stomach (stage I/II) Esophagus: adenocarcinoma Esophagus: SCC Prostate Lung: adenocarcinoma Lung: SCC Mamma: pN+ Gall bladder Ovarian carcinoma Kidney: clear cell carcinoma
EpCAM expression
Prognosis
Positive association with G3 Not significant Not significant
increasing intensity and frequency from the esophagus to the colon, in the latter all normal tissue samples were found to express EpCAM [8]. In endometrium at various stages of the female cycle, all normal tissue samples were found to be EpCAM positive [8]. In a multitumor TMA investigation, 85% of 963 adenocarcinomas of different origins were positive, which is in contrary to 72% of 423 squamous cell carcinomas [8]. This expression pattern reflects the expression pattern in normal tissues. In breast cancers, 90% of 1715 tumor samples showed a minimal EpCAM positivity at least [20-23]. Still, lobular carcinomas were significantly less frequent to overexpress EpCAM than ductal carcinomas or other types of breast cancer (18% vs. 44% vs. 49%, P = 0.001). Additionally, EpCAM overexpression exhibited a positive correlation of with increasing tumor grade and a negative correlation with increasing lymph node metastasis. In ovarian cancer, EpCAM expression varies according to the histological subtype and is highest in endometrioid carcinoma and lowest in mucinous carcinomas, whereas serous carcinomas are in between. Similar to breast cancer, EpCAM expression correlates positively with increasing grade of ovarian cancer [24]. In the kidney, EpCAM expression is highest in collecting duct (71%) and chromophobe carcinomas (75-100%) and decreases in papillary (49-55%) and is lowest in clear cell renal carcinomas (18-20%)[18,25], reflecting the expression pattern found in the normal cellular counterparts of these tumors. In the prostate, 87-100% of cancers express EpCAM [9,26-28]. Discrepancies exist regarding the association of EpCAM expression and clinico-pathological parameters in prostate cancer. Whereas one study found a significantly differential EpCAM expression in hormone-refractory and untreated prostate cancers [8], this was not confirmed by another study looking at hormone-refractory cancers, localised cancers and metastases [26]. Likewise, two studies found no difference in the EpCAM expression and the Gleason score or the outcome of patients [9,28], but one found a significant association of the Gleason grade with EpCAM [26]. In the colon, EpCAM is detected in 98% of all cancers [9], and its expression decreases from 100% in well differentiated to 92% in poorly differentiated colon cancers. In the lung, EpCAM expression depends on the histological cancer subtype: SCCs show the lowest level of EpCAM expression (53-98%)[9,29], whereas adenocarcinomas show the highest probability of EpCAM expression with 91% of all tumors. In SCC of the lung, EpCAM expression is higher in advanced and high-grade tumors [29]. On the other hand, mesenchymal tumors are EpCAM negative, which are analogous to the lack of EpCAM expression in mesenchymal tissues. Mixed mesenchymal-epithelial tumors, e.g. synovial
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Other
Inverse association with hormone resistance
Trend: Trend:
Positive association with G3 and higher stage
sarcomas or phyllode tumors of the breast, show EpCAM expression in the epithelial tumor component [8]. Adenomas of adrenal gland represented the only epithelial tumor found to be EpCAM negative [8]. There are few notoriously difficult, but for the patient relevant situations in which EpCAM expression can be used as a part of the diagnostic panel to finalize a diagnosis: (i) EpCAM was suggested to be a part of the diagnostic panel in malignant mesotheliomas [30,31]. EpCAM expression decreases in malignant mesotheliomas from the epithelioid to sarcomatoid type. Several anti-EpCAM antibodies were included in the panels to discriminate mesothelioma from primary lung cancer, such as MOC-31 or BerEP4, and acknowledged its usefulness. The use of these different antibodies with different specificities and sensitivities could also explain the variation of 2% [32] to 42% [8] of EpCAMexpressing mesotheliomas. (ii) In renal tumors, we showed that EpCAM positivity is an additional useful tool to differentiate renal oncocytoma from chromophobe RCC [18]. (iii) Esthesioneuroblastomas were also found to be EpCAM positive [8,31]. When positivity for neuroendocrine markers could raise the possibility of small cell carcinoma [31], EpCAM expression together with the lack of cytokeratin could point at esthesioneuroblastoma. The correlations of EpCAM with the prognoses of cancers were revealed by only recently significant results (Table 4). Presumably, the dual expression pattern with the predominance of a strong heterogeneous positivity contributed to this delay. Additionally, the technology to analyse a very large scale of tumor specimens simultaneously under standardized conditions, i.e. the TMA technology, has been developed only a few years ago. The most puzzling finding of EpCAM and tumor prognosis is that in some cancers, e.g. node-positive breast cancer, high EpCAM expression denotes a poor prognosis [20], whereas in other tumors, e.g. clear cell carcinoma of the kidney, it is associated with an improved prognosis [25]. In the lung, high EpCAM expression is discussed controversially. One study found a trend of strong EpCAM expression and an improved survival for adenocarcinomas of the lung, which was inversed in SCCs of the lung [9]; however, no association of EpCAM expression and outcome was found by Piyathilake et al. [29]. There has been a finding that is more as expected, i.e. the loss of EpCAM expression is associated with a poor prognosis. Some poorly differentiated colon cancers, which lost EpCAM expression during cell dedifferentiation, represent this category. The association of EpCAM loss with an improved survival has to our knowledge so far not been described. In this study, we could not find any association of EpCAM expression with the survival or the grade or stage in esophagus SCC, although higher EpCAM expression is
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EpCAM in Human Tumors
significantly more frequent in tumors than in normal tissues. This result is most probably attributed by a too small patient cohort, as in the case of colon cancers where more than 95% of cancers were EpCAM-expressing and the prognostic significance was demonstrated in a cohort comprising > 1000 patients. In analogy to SCC of the lung, EpCAMexpressing SCCs of the esophagus are expected to have a poorer prognosis. Indeed, Stoecklein et al. [10], who did not find a correlation with histopathological parameters such as pT but with the clinical follow-up data, where high EpCAM expression indicated decreased relapse-free and overall survivals, confirmed this assumption. Interestingly, Kimura et al. [33] found a correlation of high EpCAM expression with advanced local tumor extension and growth pattern, additionally, the survival rates were on the contrary higher in patients with high EpCAM expression. All three studies, ours included, looked predominantly at SCCs, but differed in the EpCAM scoring and the used anti-EpCAM antibody. The prognostic impact of EpCAM expression in esophageal cancers therefore may only be weak and remains to be defined. In summary, EpCAM is expressed in many human carcinomas but not mesenchymal tumors. The study of larger patient cohorts showed that EpCAM expression could be of some usefulness in the differential diagnosis of selected tumors. Additionally, it is of some use as a prognostic factor, although its relevance depends on the tumor type investigated. The fact that higher expression of EpCAM is frequent in human carcinomas will develop its most important impact if future anti-EpCAM targeted therapies can be successful.
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Acknowledgments We are grateful to Prof. L. Terracciano (Institute of Pathology, Basel, Switzerland) and Prof. C. Capella (Department of Pathology, Ospedale di Circolo, University of Insubria, Varese, Italy) for the collection and the use of the tissue microarrays containing the carcinoid tumors. Additionally, we would like to thank M. Mirlacher, B. Stalder and H. Weisskopf for their assistance in the construction and staining of all TMAs.
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