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ORIGINAL ARTICLE
Year : 2020  |  Volume : 16  |  Issue : 6  |  Page : 1354-1359

Clinicopathologic implications of epithelial cell adhesion molecule expression across molecular subtypes of breast carcinoma


1 Department of Pathology, Sri Ramachandra Institute of Higher Education and Research (SRIHER), Porur; Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai, Tamil Nadu, India
2 Department of Pathology, Sri Ramachandra Institute of Higher Education and Research (SRIHER), Porur, Chennai, Tamil Nadu, India
3 Department of General Surgery, Sri Ramachandra Institute of Higher Education and Research (SRIHER), Porur, Chennai, Tamil Nadu, India
4 Clinical Research Division, Central Research Facility (CRF), Sri Ramachandra Institute of Higher Education and Research (SRIHER), Porur, Chennai, Tamil Nadu, India
5 Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai, Tamil Nadu, India

Date of Submission20-Apr-2020
Date of Decision01-May-2020
Date of Acceptance06-Jul-2020
Date of Web Publication18-Dec-2020

Correspondence Address:
Devarajan Karunagaran
Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai - 600 036, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_490_20

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 > Abstract 


Background: Epithelial cell adhesion molecule (EpCAM), a type I transmembrane protein of the epithelial tissues and known cell adhesion molecule, has been demonstrated to have critical role in carcinogenesis. In breast cancer, EpCAM expression has been associated with poor prognosis. The expression pattern of EpCAM across molecular subtypes of breast carcinoma has been studied in patients reporting to a South Indian multispecialty tertiary care hospital. The prognostic significance of EpCAM expression pattern and probable response to therapy has also been addressed.
Materials and Methods: EpCAM expression was assessed by immunohistochemical studies on 200 breast carcinoma tissue samples of different molecular subtypes, including luminal A, luminal B, Her2Neu, and triple-negative breast cancer (TNBC). The expression was scored using the standard scoring system. A correlation was drawn with detailed clinicopathologic annotation and available outcomes data to analyze the influence of EpCAM on prognosis.
Results: EpCAM expression varied significantly in the different intrinsic subtypes of breast carcinoma. Differential expression was also established with different grades of breast carcinoma with varying levels of differentiation. We observed strong EpCAM expression in TNBC among other subtypes.
Conclusion: The differential expression of EpCAM among intrinsic subtypes of breast cancer and the correlation of EpCAM expression with high-grade breast carcinoma shown in the study have important implications in understanding the role of EpCAM and might form the basis for developing targeted therapies in breast cancer in the future.

Keywords: Breast carcinoma, cell adhesion, epithelial cell adhesion molecule, triple-negative breast cancer


How to cite this article:
Sundaram S, Christian SD, Krishnakumar R, Ramya R, Ramadoss M, Karunagaran D. Clinicopathologic implications of epithelial cell adhesion molecule expression across molecular subtypes of breast carcinoma. J Can Res Ther 2020;16:1354-9

How to cite this URL:
Sundaram S, Christian SD, Krishnakumar R, Ramya R, Ramadoss M, Karunagaran D. Clinicopathologic implications of epithelial cell adhesion molecule expression across molecular subtypes of breast carcinoma. J Can Res Ther [serial online] 2020 [cited 2021 Dec 4];16:1354-9. Available from: https://www.cancerjournal.net/text.asp?2020/16/6/1354/303894




 > Introduction Top


Epithelial cell adhesion molecule (EpCAM) is a transmembrane protein found in the basolateral membrane of the epithelial tissues. EpCAM was initially identified as an antigen prominent in colon carcinoma tissue and later deciphered as a cell adhesion molecule and found to be expressed in a wide variety of cell types. EpCAM, a type I membrane protein (30–40 kDa), located in the intercellular tight junctions, is usually sequestered in normal epithelial cells while in cancer cells homogenously distributed on the cell surface. Although EpCAM has been reported as cancer cell marker and cell adhesion molecule, it has been erstwhile known to be engaged in a variety of roles in cell signaling, cell migration, cellular proliferation, and differentiation.[1]

EpCAM has many documented roles in the process of carcinogenesis[2] and is distinctly expressed in several cancers. Cancers of gastrointestinal origin, genitourinary tract, breast, etc., had higher EpCAM expression, while hepatocellular, renal cell, and urothelial cancers do not show EpCAM expression. Overexpression of EpCAM has been reported in tumor-initiating cells, and hence, EpCAM-targeting therapies play a significant role in the eradication of tumor-initiating cells.[3],[4] Therapeutic agents against EpCAM have been recently shown to work against cancer.[5] Zhang et al. observed that overexpression of EpCAM correlates with the poor prognosis of hepatocellular carcinoma.[6] EpCAM-targeting antibodies such as edrecolomab, adecatumumab, and catumaxomab, as well as conjugated antibodies such as oportuzumab monatox, citatuzumab bogatox, and tucotuzumab, have shown potential anticancer effects, and most of these antibodies are well tolerated in clinical trials.[7]

In breast cancer, EpCAM overexpression has been reported,[8] especially in metastatic breast cancer than in primary breast cancer tissues.[9] EpCAM involved in the metastasis of breast cancer cells to the lymph node.[10] Expression of EpCAM has been associated with tumor aggression[11] and poor prognosis of breast carcinoma.[12] EpCAM over expression has been shown to induce resistance to transforming growth factor beta 1-mediated growth arrest and led to increased proliferation in vitro. In vivo study has also shown hyperplastic growth with EpCAM overexpression, indicating its potential as a prosurvival factor.[13] EpCAM was shown as a clear marker of overall survival and prognosis in basal-like breast cancer.[14] Adecatumumab (MT201), an EpCAM-targeting agent, has shown significant potential against breast cancer in Phase I and Phase II clinical studies,[15] while many other EpCAM-targeting agents are under evaluation. Expression of EpCAM has been used to develop EpCAM-targeting nanodrug delivery system (nanorods) loaded with doxorubicin.[16]

Understanding expression of EpCAM in the molecular subtypes of breast cancer in local population would help in determining prognostic significance and response to anti-EpCAM therapy against breast cancer. Hence, the current study has probed its expression in 200 breast carcinoma patients across molecular subtypes (luminal A, luminal B, Her2Neu, and triple-negative breast cancer [TNBC]) presenting to an urban hospital in a South Indian city.


 > Materials and Methods Top


Tissue samples

A total of 200 breast cancer tissue samples from surgically resected mastectomy specimen were obtained retrospectively from the pathology department of a tertiary care hospital as formalin-fixed, paraffin-embedded blocks after obtaining approval from the institutional ethics committee. Samples were analyzed histologically and categorized into different molecular subtypes based on the immunohistochemical staining for estrogen receptor, progesterone receptor, and Her2Neu. The molecular subtype of breast carcinoma was determined for each sample, and the sample set comprised 56 cases of subtype luminal A, 40 cases of luminal B, 47 cases of Her2Neu, and 57 cases of TNBC [Figure 1]. The clinical and histopathological reports were obtained for all the cases from the hospital network system, and diagnosis was rendered as per the College of American Pathologists protocol.
Figure 1: Molecular subtypes of breast cancer patients included in the study

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Immunohistochemical staining

Immunohistochemical staining method involving streptavidin-biotin immunoperoxidase complex was used. From the tissue samples stored as paraffin blocks, 4-μm thick sections were made. After deparaffinization with xylene and subsequent rehydration with alcohol solutions, endogenous peroxidase was inactivated by the addition of 0.3% hydrogen peroxide in methanol for 30 min. Antigen retrieval by sodium citrate method was performed (0.01 M at pH 6.0 for 10 min). Antigen retrieval with proteinase K was also performed for the detection of EpCAM. Sections were incubated with primary antibodies (EpCAM) for 2 h followed by incubation with biotinylated secondary antibodies at room temperature for 30 min. Chromogenic visualization was achieved using 3,3'-diaminobenzidine, and sections were counterstained with hematoxylin before dehydration and mounting. Positive (kidney tissue) and negative (PBS instead of primary antibody) controls were included in each batch of immunostaining.

Imaging and scoring

Sections were evaluated based on the intensity of staining and proportion of tumor cells stained using Olympus microscope at ×100. EpCAM expression in the tissue sections was evaluated through scoring. Product of positively stained nuclei (fraction of staining: none - 0, <10% - 1, 10%–50% - 2, 51%–75% - 3, >75% - 4) and intensity (intensity of staining: no staining – 0, weak – 1, moderate – 2, strong – 3) represented the total immunostaining Q score ranging between 0 and 12. Total expression of EpCAM, with a Q score above 5, was deemed as overexpression.

Statistical analysis

SPSS Software (IBM Corp. Released 2016. IBM SPSS Statistics for Windows, Version 24.0. Armonk, NY: IBM Corp.) tool was used for analyzing the data. Chi-square and Fisher's exact tests were used to analyze categorical variables. Statistical significance was determined with confidence interval of 95% (P < 0.05).


 > Results Top


Among the study population, the mean age was 53.86 ± 11.492 years ranging from 27 to 84. Eighty-six subjects were found to be in the premenopausal category and 114 in the menopausal category, based on the history [Figure 2]. The clinicopathologic data for all samples obtained for the study are presented in [Table 1].
Figure 2: Age, family history, and menopause status in breast cancer patients

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EpCAM was found to be overexpressed in breast carcinoma when compared to normal breast tissue (P = 0.001)[Figure 3] and [Table 2]. In the 200 breast carcinoma tissue samples analyzed, higher levels of EpCAM expression with a Q score >5 were noticed in 82% of cases, while in the adjacent normal tissue, only 4% of cases showed Q score >5. Further, among the subtypes, triple negatives and Her2Neu were found to show increased expression of EpCAM than the other subtypes luminal A and B [Figure 3], P = 0.001]. Among the luminal A cases, 73.2% scored >5 in expressing the EpCAM, luminal B cases 62.5%, triple-negative cases 96.5%, and Her2Neu cases 91.5%. A significant association of EpCAM expression with increasing grade was analyzed in the study (P = 0.001), and higher tumor grade was seen in TNBC indicating progression [Figure 4]. EpCAM expression is higher in TNBC and Her2Neu type of breast carcinoma when compared to luminal A and luminal B type. More than 90% of the cases in triple negative and Her2Neu were strongly positive for EpCAM compared to the other subtypes. Expression pattern was found to be diffused membranous in tumor cells [Figure 5]. Increased EpCAM expression was observed in ductal carcinoma in situ regions and also in perineural invasion.
Figure 3: Distinct expression of epithelial cell adhesion molecule among molecular subtypes of breast cancer

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Figure 4: Tumor grading in molecular subtypes of breast cancer (Grade 1 = Well differentiated; 2 = Moderately differentiated; 3 = Poorly differentiated)

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Figure 5: Expression pattern of epithelial cell adhesion molecule in breast carcinoma. (a) Epithelial cell adhesion molecule expression in breast carcinoma tissue shown by immunohistochemical staining (brown); (b) higher epithelial cell adhesion molecule expression in the tumor cells (long arrow) when compared to adjacent normal breast acini (short arrow) with faint staining; (c) carcinoma breast showing 1+ epithelial cell adhesion molecule staining intensity; (d) carcinoma breast showing 2+ epithelial cell adhesion molecule staining intensity; (e) ductal carcinoma in situ areas showing 3+ epithelial cell adhesion molecule staining intensity; (f) perineural invasion (long arrow) with 3+ epithelial cell adhesion molecule staining intensity, nerve bundle seen in the center

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Table 2: Comparison of tumor tissue and the adjacent normal tissue with immunoscoring

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Statistical analysis showed association between tumor grade and higher EpCAM expression [Table 3]. Yet, the other tumor characteristics such as tumor staging T, N, M and family history of the subject showed no direct relation with the EpCAM expression. Most of the cases studied were in the age group of >50 years, and among them, 72% were in the menopausal group and 28% were in the premenopausal group. Sixty-seven cases were in the age group <50 years. Age was not found to have any significant association with the tumor characteristics such as subtype, grade, or stage [Table 4].
Table 3: Comparison of tumor characteristics with the immunoscoring

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Table 4: Comparison of tumor characteristics in relation to age

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 > Discussion Top


Our present study showed the expression of EpCAM in 82% of breast carcinoma cases. EpCAM is overexpressed in breast carcinoma when compared to normal breast tissue (P = 0.001). Expression pattern was found to be diffused membranous in tumor cells. EpCAM expression is higher in TNBC and Her2Neu type of breast carcinoma when compared to luminal A and luminal B type. More than 90% of the cases in triple negative and Her2Neu were strongly positive for EpCAM compared to the other subtypes. This indicated the mechanistic role of EpCAM in determining prognosis in triple-negative and Her2Neu tumor subtypes.[17] Some of the studies have reported that no significant association exists between overexpression of EpCAM and the clinicopathologic characteristics.[18] The protein is expressed in other epithelial cancers such as colorectal, gastric, prostrate, ovary, and lung cancers.[17] Our findings relate with the earlier observation by Nehad et al. that higher tumor grade in breast cancer has association with higher expression of EpCAM.[19]

Expression of EpCAM has been known to indicate survival as it promotes the cancer progression and metastasis.[11] Yet, the expression of EpCAM is not higher in all tumor types, and in breast cancer, the expression varies with molecular subtypes.[20] Hence, it is essential to ascertain overexpression of EpCAM in the tumor type to conclude its role as a diagnostic or prognostic marker. EpCAM has been identified as a cancer stem cell marker of tumor cells that associate with progression, differentiation, and upregulation. EpCAM and E-cadherin antagonist are found to be effective in few cancers as it disrupts the linkage between alpha-catenin and F-actin.[16] CD44- and EpCAM-targeted therapy was found to be a potential strategy in epithelial ovarian cancers. On comparing the age with the tumor characteristics, no correlation between EpCAM expression and age and other demographic factors was found. Higher expression was seen in node-positive tumors and in higher grade, high-stage tumors. Many drug-targeted immunotherapeutic approach clinical trials are being conducted in this field. Catumaxomab was approved by the European Commission in 2009 as a treatment for malignant ascites in cancer patients with EpCAM-positive tumor.[15] Bone metastasis showed high EpCAM expression, but no statistical analysis was derived since metastatic cases were limited.

Epithelial tumors undergo transition from epithelium to mesenchymal tissue enabling them to invade blood vessels and survive in bloodstream. This makes most tumors resistant to chemotherapy. The circulating tumor cells (CTCs) located in the peripheral blood of cancer patients are related to invasiveness. CTC enrichment technologies based on EpCAM are now available. Parallel multi-orifice flow fractionation chip for the isolation of CTC from blood is done in cancer models. Immunomagnetic beads coated with EpCAM antibodies are also under trials.[21] Standardized techniques are yet to come. An autopsy cohort study conducted by Hiraga et al., 2016 also showed the involvement of EpCAM in metastatic cases, which is a sign of stem cell signaling.[22]

EpCAM expression has critical and important implications in subsets of breast carcinoma that would benefit from targeted therapies. EpCAM has been found to be overexpressed in breast carcinoma cells when compared to normal epithelial cells of the breast. Triple-negative and Her2Neu molecular subtypes showed strong expression of EpCAM, which possibly accounts for its aggressive clinical course. An association of higher tumor grade (with poor differentiation) with triple-negative and Her2Neu molecular subtypes was also observed. The results point out to the excellent potential of using EpCAM-targeted therapies for specific molecular types of breast cancer.

Correlation with outcome could not be assessed in all cases. A better understanding of the relation between EpCAM expression and prognosis in breast cancer subtypes is important to design and successfully apply as molecular therapy targets. Further studies on larger population to complement and refine our study are needed. This study has not addressed as to the mechanism of EpCAM overexpression in breast cancer subtypes, which could help in the development of EpCAM-targeted therapy in the future. Performing gene expression studies to unravel the pathways involved in EpCAM signaling might be more useful in assessing their role in deciding the prognosis of advanced breast carcinoma. Evaluating the response of anti-EpCAM antibodies by cell culture studies can bring light in this field.

Acknowledgment

We would like to acknowledge Dr. Ravishankar Pitani, Professor of Community Medicine, Sri Ramachandra Medical College and Research Institute, for the statistical analysis.

Financial support and sponsorship

Infrastructure support from the Department of Pathology, Sri Ramachandra Institute of Higher Education and Research, Chennai, and Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology – Madras, Chennai, is acknowledged.

Conflicts of interest

There are no conflicts of interest.



 
 > References Top

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Osta WA, Chen Y, Mikhitarian K, Mitas M, Salem M, Hannun YA, et al. EpCAM is overexpressed in breast cancer and is a potential target for breast cancer gene therapy. Cancer Res 2004;64:5818-24.  Back to cited text no. 5
    
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Zhang J, Qi YP, Ma N, Lu F, Gong WF, Chen B, et al. Overexpression of EpCAM and CD133 correlates with poor prognosis in dual-phenotype hepatocellular carcinoma. J Cancer 2020;11:3400-6.  Back to cited text no. 6
    
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Eyvazi S, Farajnia S, Dastmalchi S, Kanipour F, Zarredar H, Bandehpour M. Antibody based EpCAM targeted therapy of cancer, review and update. Curr Cancer Drug Targets 2018;18:857-68.  Back to cited text no. 7
    
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Sadeghi S, Hojati Z, Tabatabaeian H. Cooverexpression of EpCAM and c-myc genes in malignant breast tumours. J Genet 2017;96:109-18.  Back to cited text no. 8
    
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Cimino A, Halushka M, Illei P, Wu X, Sukumar S, Argani P. Epithelial cell adhesion molecule (EpCAM) is overexpressed in breast cancer metastases. Breast Cancer Res Treat 2010;123:701-8.  Back to cited text no. 9
    
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Gastl G, Spizzo G, Obrist P, Dünser M, Mikuz G. Ep-CAM overexpression in breast cancer as a predictor of survival. Lancet 2000;356:1981-2.  Back to cited text no. 11
    
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de Wit S, Manicone M, Rossi E, Lampignano R, Yang L, Zill B, et al. EpCAM high and EpCAM low circulating tumor cells in metastatic prostate and breast cancer patients. Oncotarget 2018;9:35705-16.  Back to cited text no. 12
    
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Martowicz A, Rainer J, Lelong J, Spizzo G, Gastl G, Untergasser G. EpCAM overexpression prolongs proliferative capacity of primary human breast epithelial cells and supports hyperplastic growth. Mol Cancer 2013;12:56.  Back to cited text no. 13
    
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Agboola AJ, Paish EC, Rakha EA, Powe DG, Macmillan RD, Ellis IO, et al. EpCAM expression is an indicator of recurrence in basal-like breast cancer. Breast Cancer Res Treat 2012;133:575-82.  Back to cited text no. 14
    
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Awada AH Sr., Schmidt M, Scheulen ME, Obrist P, Marschner N, Dittrich DL, et al. Final results from an open-label, randomised, phase II study of adecatumumab (MT201), a fully human anti-EpCAM antibody, in patients with metastatic breast cancer. J Clin Oncol 2007;25:3588.  Back to cited text no. 15
    
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Jenkins SV, Nima ZA, Vang KB, Kannarpady G, Nedosekin DA, Zharov VP, et al. Triple-negative breast cancer targeting and killing by EpCAM-directed, plasmonically active nanodrug systems. NPJ Precis Oncol 2017;1:27.  Back to cited text no. 16
    
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Soysal SD, Muenst S, Barbie T, Fleming T, Gao F, Spizzo G, et al. EpCAM expression varies significantly and is differentially associated with prognosis in the luminal B HER2þ, basal-like, and HER2 intrinsic subtypes of breast cancer. British J Cancer 2013;108:1480-7.  Back to cited text no. 17
    
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Hyun KA, Koo GB, Han H, Sohn J, Choi W, Kim SI, et al. Epithelial-to-mesenchymal transition leads to loss of EpCAM and different physical properties in circulating tumor cells from metastatic breast cancer. Oncotarget 2016;7:24677-87.  Back to cited text no. 18
    
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Spizzo G, Fong D, Wurm M, Ensinger C, Obrist P, Hofer C, et al. EpCAM expression in primary tumour tissues and metastases: An immunohistochemical analysis. J Clin Pathol 2011;64:415-20.  Back to cited text no. 20
    
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Hiraga T, Ito S, Nakamura H. EpCAM expression in breast cancer cells is associated with enhanced bone metastasis formation. Int J Cancer 2016;138:1698-708.  Back to cited text no. 22
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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