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J Res Med Sci 2022,  27:20

High claudin-4 antigen expression in triple-negative breast cancer by the immunohistochemistry method

1 Department of Pathology, Isfahan University of Medical Sciences, Isfahan, Iran
2 Poursina Hakim Digestive Diseases Research Center, Isfahan, Iran

Date of Submission27-Dec-2020
Date of Decision17-May-2021
Date of Acceptance18-Jul-2021
Date of Web Publication17-Mar-2022

Correspondence Address:
Dr. Azar Naimi
Department of Pathology, Isfahan University of Medical Sciences, Isfahan
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jrms.jrms_1389_20

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Background: Triple-negative breast cancer is a heterogeneous subtype of breast cancer. Claudin is an epithelial tight junctional protein, and also it is a receptor for clostridium perfringens enterotoxin and shows impairment of expression in several cancers. The chief purpose of this study is to assess the claudin-4 expression in triple-negative breast cancer (TNBC) Iranian patients and evaluate its correlation with some clinicopathological factors. Materials and Methods: In this study, 81 TNBC patients were evaluated for the claudin-4 expression by immunohistochemistry. The slides' staining intensity was examined and scored from 0 to 3. Then, slides were reviewed to assess the percentage of cells with membrane and cytoplasmic staining; the obtaining scores were 1–4. Finally, added the resulting two numbers from two stages, and the final number was a maximum of 7. Final scores of 0–3 were considered the low expression, and 4–7 were considered the high expression. Finally, the collected data were analyzed using the Chi-square test. Results: Eighty-one women with breast cancer and a mean age of 49 ± 12 years participated in the study. In 80% of the patients, there was a high expression of claudin-4 marker, and 20% had low expression. The expression level of the marker was not significantly correlated with age, tumor size, lymph node involvement, tumor grade, disease stage, Ki-67, and metastasis. Conclusion: The present study confirmed the high frequency of claudin-4 antigen expression in TNBC patients, and no significant correlation was observed between the expression of antigen and demographic or clinicopathological factors.

Keywords: Claudin-4, immunohistochemistry, triple-negative breast cancer

How to cite this article:
Naimi A, Zare N, Amjadi E, Soltan M. High claudin-4 antigen expression in triple-negative breast cancer by the immunohistochemistry method. J Res Med Sci 2022;27:20

How to cite this URL:
Naimi A, Zare N, Amjadi E, Soltan M. High claudin-4 antigen expression in triple-negative breast cancer by the immunohistochemistry method. J Res Med Sci [serial online] 2022 [cited 2023 Mar 20];27:20. Available from: https://www.jmsjournal.net/text.asp?2022/27/1/20/339777

  Introduction Top

Breast cancers are categorized into several subgroups based on the expression of hormone receptors. There is a group of breast cancers, none of which expresses progesterone receptors (PR), estrogen receptor (ER), and HER2; they are called triple negative.[1],[2],[3] Triple-negative breast cancer (TNBC) is a heterogeneous group of breast cancers that usually has a poor prognosis and weak response to routine chemotherapy.[4],[5],[6],[7]

Claudin is a tight junctional protein of epithelial cells that makes the epithelial cells close to each other and creates a barrier against the passage of macromolecules. Despite the specific expression of the claudin gene in any other tissue, a tissue may simultaneously appear multiple types of claudin antigens.[8],[9],[10]

Neoplastic cells often show structural and functional defects in tight junctions, which destroys them..[11] Several studies show the impairment of claudin expression in various cancers, including breast cancer,[12],[13] claudin-1 in colon carcinoma,[14] claudin-4 in the colon and gastric cancer,[15] and claudins 1 and 7 in prostate cancer,[16] esophageal cancer,[17] and ovarian cancer.[18]

Furthermore, claudin 4 also has been identified as a receptor for  Clostridium perfringens Scientific Name Search nterotoxin. This enterotoxin can lyse cells rapidly and specifically, so expressing claudins 4 by tumoral cells, could effectively exploit in the treatment of cancers constitutively.[19]

Examining how this antigen is expressed in individuals with TNBC could suggest promising new methods to treat these patients. The present study investigates the frequency of claudin-4 expression in Iranian TNBC patients and the correlation with some prognostic factors.

  Methods Top

This retrospective study included 81 TNBC specimens to evaluate the claudin-4 expression and its association with clinicopathological properties of Iranian TNBC patients. This study included all TNBC patients' primary tumor specimens from January 2012 to December 2017 and paraffin-embedded tumor tissue specimens archived at the Pathology Department of Al-Zahra Hospital and Poursina Hakim Institute, Isfahan, were entered. The patients who received neoadjuvant chemotherapy or were diagnosed with Stage IV of the disease were excluded from the study. The samples which were inconsistent with their clinical reports and data were excluded. There was no medical intervention in this study, so informed consent was not considered.

The 3-5 mm sections were incubated at 60°C (40 min) for deparaffinization. Then, the samples were immersed in xylene and rehydrated in the decreasing ethanol solutions. The samples were incubated in 0.3% hydrogen peroxide to inhibit activation of the endogenous peroxidases. The samples' antigen retrieval was done by heating in an 830-W microwave oven (60°C, 15 min) in 10 mmol/L sodium citrate buffer (pH = 6.0). Subsequently, the slides were incubated with rabbit anti-human claudin-4 monoclonal antibody at 4°C overnight. The primary antibody was replaced with PBS for the negative control. HRP polymer and DAP plus chromogen (Thermo Fisher Scientific, CA) were employed for the detection. Mouse anti-rabbit horseradish peroxidase-conjugated secondary antibody was incubated for 40 min at room temperature. The color was developed using diaminobenzidine as a chromogen. The slides were extensively washed with PBS after each step.

To report claudin-4 expression, first, the intensity was examined using the ophthalmic lens 10 in specific immunohistochemistry staining. Four numbers from 0 to 3 were assigned, which indicated negative, weak, medium, and strong affinity. The slides were then examined using the ophthalmic lens 40 to evaluate the percentage of cells with membrane and cytoplasmic staining, obtained scores 1–4. Number 1 was considered for staining <25%, 2 for 25%–50%, 3 for 50%–75%, and 4 for >75%. Finally, added the resulting two numbers from the first and second stages, and the final number was a maximum of 7. If the final number was 0–3, the antigen expression would consider low [Figure 1] and [Figure 2], and if it was 4–7, it would consider high [Figure 3] and [Figure 4]. Two pathologists reviewed all the specimens by themselves.
Figure 1: Low expression of Claudin 4 in tumoral cells, original magnification×10

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Figure 2: Low expression of Claudin 4 in tumoral cells,original magnification×40

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Figure 3: High expression Claudin 4 in tumoral cells, original magnification×10

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Figure 4: High expression Claudin 4 in tumoral cells, original magnification×40

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Extracted clinicopathological data, including age, tumor size, lymph node involvement, tumor grade, disease progression stage, metastasis, and biomarkers (ER, PR, Her2 neu, and Ki67), were assessed. Biomarkers and Ki67 were scored according to approved guidelines. The Her2 was scored 0+ (negative): nonstaining or mild membranous staining of tumoral cells (≤10%); 1+ (negative): extremely mild and incomplete membranous staining of ≥10% of tumoral cells; 2+ (equivocal): mild-to-moderate incomplete membranous staining of ≥10% of tumoral cells; and 3+ (positive): intense complete membranous staining of ≥10% of tumoral cells. The cutoff for ER and PR was 1%. Ki67 is a proliferative index, and the cutoff for it was 15%.

Finally, the collected data were analyzed using the logistic regression binary and Chi-square tests by SPSS version 23 (IBM Corporation, Somers, NY, USA). P < 0.05 was considered significant.

  Results Top

Eighty-one TNBC patients were investigated in this study. [Table 1] illustrates the clinicopathological characteristics of the patients.
Table 1: Clinicopathological characteristics of the triplenegative breast cancer patients

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The patients were categorized into two groups: low expression (with a total score ≤3) and high expression (with a total score >3), according to scoring for claudin-4 expression. About 80% of patients (65 patients) had high expression, and 20% of patients (16 patients) had low expression. In this categorization, claudin-4 had no significant relationship with the following variables: age, tumor size, lymph node involvement, tumor grade, disease progression stage, Ki67, and metastasis by Chi-square test [Table 2] or logistic regression binary model [Table 3].
Table 2: Correlation between claudin expression and clinicopathological parameters of the triple-negative breast cancer patients

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Table 3: Logistic regression binary model for claudin expression and clinicopathological parameters of the triplenegative breast cancer patients

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  Discussion Top

This study investigated claudin-4 expression and its correlation with clinicopathological features of TNBC. Among 81 TNBC specimens involved in this study, 80% (65 patients) had high expression, and 20% (16 patients) had low expression.

Genomic studies have established four major breast cancer intrinsic subtypes (luminal A, luminal B, HER2-enriched, and basal-like) and a normal breast-like group, showing significant differences in incidence, survival, and response to therapy. However, as gene expression studies evolve, further subclassification of breast tumors into new molecular entities is expected to occur. Prat et al. identified a new molecular subtype, referred to as claudin low. The molecular characterization of the claudin-low intrinsic subtype in tumors and cell lines reveals a breast cancer differentiation hierarchy that resembles the normal epithelial mammary developmental cascade.[20] However, the immunohistochemical expression of claudins in breast cancer has not yet been standardized. The main claudins specifically expressed in human breast tissue are claudins 4 and 7.[21] Prat et al. reported that the majority of triple-negative tumors were either basal-like (39%–54%) or claudin low (25%–39%).[20]

According to Shaulei et al., claudin-7 in the high-grade invasive ductal carcinoma has a low expression, but on the contrary, claudins 3 and 4 have a high expression at both protein and mRNA levels in breast cancer.[12] Abd-Elazeem et al. studied 56 female patients with TNBC and found that about 66% of patients had high expression of claudin-4;[13] their findings were in line with the current study, which indicated 80% high expression. They also demonstrated a significant and negative relationship between claudin-4 and age, tumor size, lymph node involvement, tumor grade, disease progression stage, and Ki-67 inconsistent with the present study.

Some studies contributed claudins to dysregulation in tight junction structures of breast cancers and metastasis. Claudin-4 distribution was seen highly in normal epithelial cells and almost lost in some subtypes of invasive breast cancer and contributed to the metastasis process.[11] Our study revealed that most TNBCs express claudin-4. Although the loss of expression of claudin-4 is attributed to metastasis in breast cancer, we found no relation between claudin-4 loss in TNBCs and metastasis (P = 0.19); it can be related to insufficient metastatic cases in the study or intrinsic traits in the Iranian population.

Logullo et al. reported that claudin-4 exhibited the lowest expression in luminal A and triple-negative subtypes and the highest frequency of expression in HER2-enriched subtypes. However, the majority of the evaluated cases exhibited preserved claudins 4 and 7 expression (62 and 46%, respectively). The distribution of the claudin-low or negative cases did not correspond to the triple-negative molecular profile;[21] however, in our study, most triple-negative tumors were claudin high (80%).

In a study conducted by Kolokytha, in the triple-negative group, the positive expression of claudin-3 and claudin-4 was related to unfavorable and favorable prognostic factors, respectively. Specifically, in triple-negative carcinomas, claudin-4 positivity could probably be considered a biomarker of favorable prognosis.[22] The present study confirmed the high frequency of claudin-4 antigen expression in TNBC patients. It seems that the frequency of claudin 4 expression in the Iranian population may be different from some studies and may explain some differences in clinical presentation, treatment response, and outcome of breast cancer in this population. No significant correlation was observed between the expression of antigen and demographic traits or clinicopathological factors. The logistic regression model also was applied, but no relation is found. According to the high expression of claudin-4 in TNBCs and the expression of this antigen in normal breast tissue, it seems that other members of the claudin family are more appropriate for targeting in therapeutic intervention studies.

However, it may achieve significant relationships in samples with greater size, more expansive geography, and larger time sections. Furthermore, we recommend evaluation of the claudin-4 expression with TNBC patients' survival.


This project was achieved by research project number 399411 and the ethical approval number of IR. MUI. MED. REC.1399.478. We would like to thank Dr. Heidarpour for assisting in the immunohistochemistry part.

Financial support and sponsorship

This study was funded by Vice-Chancellor for Research and Technology of Isfahan University of Medical Sciences.

Conflicts of interest

There are no conflicts of interest.

  References Top

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McNamara KM, Yoda T, Miki Y, Chanplakorn N, Wongwaisayawan S, Incharoen P, et al. Androgenic pathway in triple negative invasive ductal tumors: Its correlation with tumor cell proliferation. Cancer Sci 2013;104:639-46.  Back to cited text no. 3
Ali AM, Ansari JA, El-Aziz NM, Abozeed WN, Warith AM, Alsaleh K, et al. Triple negative breast cancer: A tale of two decades. Anticancer Agents Med Chem 2017;17:491-9.  Back to cited text no. 4
Tang D, Xu S, Zhang Q, Zhao W. The expression and clinical significance of the androgen receptor and E-cadherin in triple-negative breast cancer. Med Oncol 2012;29:526-33.  Back to cited text no. 5
Ieni A, Barresi V, Ricciardi GR, Adamo B, Adamo V, Tuccari G. Prognostic value of androgen receptor expression in triple negative breast carcinomas: Personal experience and comments on a review about “Triple-negative breast cancer: treatment challenges and solutions” by Collignon et al. Breast Cancer (Dove Med Press) 2016;8:157-9.  Back to cited text no. 6
Gasparini P, Fassan M, Cascione L, Guler G, Balci S, Irkkan C, et al. Androgen receptor status is a prognostic marker in non-basal triple negative breast cancers and determines novel therapeutic options. PLoS One 2014;9:e88525.  Back to cited text no. 7
Morin PJ. Claudin protein in human cancer: Promising new targets for diagnosis and therapy. Canser Res 2005;65:9603-6.  Back to cited text no. 8
Mineta K, Yamamoto Y, Yamazaki Y, Tanaka H, Tada Y, Saito K, et al. Predicted expansion of the claudin multigene family. FEBS Lett 2011;585:606-12.  Back to cited text no. 9
Tsukita S, Furuse M. The structure and function of claudins, cell adhesion molecules at tight junctions. Ann N Y Acad Sci 2000;915:129-35.  Back to cited text no. 10
Escudero-Esparza A, Jiang WG, Martin TA. The Claudin family and its role in cancer and metastasis. Front Biosci (Landmark Ed) 2011;16:1069-83.  Back to cited text no. 11
Lu S, Singh K, Mangray S, Tavares R, Noble L, Resnick MB, et al. Claudin expression in high-grade invasive ductal carcinoma of the breast: Correlation with the molecular subtype. Mod Pathol 2013;26:485-95.  Back to cited text no. 12
Abd-Elazeem MA, Abd-Elazeem MA. Claudin 4 expression in triple-negative breast cancer: correlation with androgen receptors and Ki-67 expression. Ann Diagn Pathol 2015;19:37-42.  Back to cited text no. 13
Resnick MB, Konkin T, Routhier J, Sabo E, Pricolo VE. Claudin-1 is a strong prognostic indicator in stage II colonic cancer: A tissue microarray study. Mod Pathol 2005;18:511-8.  Back to cited text no. 14
Resnick MB, Gavilanez M, Newton E, Konkin T, Bhattacharya B, Britt DE, et al. Claudin expression in gastric adenocarcinomas: A tissue microarray study with prognostic correlation. Hum Pathol 2005;36:886-92.  Back to cited text no. 15
Sheehan GM, Kallakury BV, Sheehan CE, Fisher HA, Kaufman RP Jr., Ross JS. Loss of claudins-1 and -7 and expression of claudins-3 and -4 correlate with prognostic variables in prostatic adenocarcinomas. Hum Pathol 2007;38:564-9.  Back to cited text no. 16
Takala H, Saarnio J, Wiik H, Soini Y. Claudins 1, 3, 4, 5 and 7 in esophageal cancer: Loss of claudin 3 and 4 expression is associated with metastatic behavior. APMIS 2007;115:838-47.  Back to cited text no. 17
Rangel LB, Agarwal R, D'Souza T, Pizer ES, Alò PL, Lancaster WD, et al. Tight junction proteins claudin-3 and claudin-4 are frequently overexpressed in ovarian cancer but not in ovarian cystadenomas. Clin Cancer Res 2003;9:2567-75.  Back to cited text no. 18
Kominsky SL, Vali M, Korz D, Gabig TG, Weitzman SA, Argani P, et al. Clostridium perfringens enterotoxin elicits rapid and specific cytolysis of breast carcinoma cells mediated through tight junction proteins claudin 3 and 4. Am J Pathol 2004;164:1627-33.  Back to cited text no. 19
Prat A, Parker JS, Karginova O, Fan C, Livasy C, Herschkowitz JI, et al. Phenotypic and molecular characterization of the claudin-low intrinsic subtype of breast cancer. Breast Cancer Res 2010;12:R68.  Back to cited text no. 20
Logullo AF, Pasini FS, Nonogaki S, Rocha RM., Soares FA, Brentani MM. Immunoexpression of claudins 4 and 7 among invasive breast carcinoma subtypes: A large diagnostic study using tissue microarray. Mol Clin Oncol 2018;9:377-88.  Back to cited text no. 21
Kolokytha P, Yiannou P, Keramopoulos D, Kolokythas A, Nonni A, Patsouris E, et al. Claudin-3 and claudin-4: Distinct prognostic significance in triple-negative and luminal breast cancer. Appl Immunohistochem Mol Morphol 2014;22:125-31.  Back to cited text no. 22


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

  [Table 1], [Table 2], [Table 3]


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