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Immunoexpression of claudin-4 and correlation with estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2-neu in breast cancer


1 Department of Surgery, Tata Main Hospital, Jamshedpur, Jharkhand, India
2 Department of Surgery, VMMC and Safdarjung Hospital, New Delhi, India
3 Department of Pathology, National institute of pathology ICMR, Safdarjung Hospital, New Delhi, India

Date of Submission31-Dec-2020
Date of Acceptance29-Jan-2021
Date of Web Publication18-Aug-2021

Correspondence Address:
Niranjan Kumar,
Department of Surgery, Tata Main Hospital, C Road, West Northern Town, Bistupur, Jamshedpur - 831 001, Jharkhand
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.jcrt_1909_20

 > Abstract 


Background: Claudins are important transmembrane proteins in tight junction. The role of intercellular tight junctions in breast epithelial cells is traditionally thought to be in maintaining polarity and barrier function. However, claudin-4, a tight junction protein, is overexpressed in breast tumor cells compared to normal epithelial cells, which generally corresponds to loss in polarity and can provide valuable information about biology of the tumor. A prospective clinical study was conducted to assess the expression claudin-4 in patients with breast cancer and its correlation with hormone receptors – estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2)-neu.
Materials and Methods: The study included 102 biopsy-proven breast carcinoma patients. The biopsy samples were evaluated immunohistochemistry for expression of ER, PR, HER2-neu, and claudin-4. The expression of claudin-4 was correlated with ER, PR, and HER2-neu.
Results: In the study, we found that out of 26 cases of high claudin-4, 25 cases (96.15%) were ER negative and P < 0.001, which was significant. Similar results were found with PR-negative cases. Whereas, out of 76 cases with low claudin-4, 54 cases (71.05%) were HER2-neu negative and P = 0.022, which was significant.
Conclusions: Claqudin-4 expression has a negative correlation with ER and PR and has a positive correlation with HER2-neu. Hence, it can be effectively utilized as a prognostic and therapeutic marker in breast cancer in the future.

Keywords: Breast, carcinoma, claudin-4, estrogen receptor, human epidermal growth factor receptor 2-neu, progesterone receptor



How to cite this URL:
Kumar N, Tandon M, Chintamani C M, Saxena S. Immunoexpression of claudin-4 and correlation with estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2-neu in breast cancer. J Can Res Ther [Epub ahead of print] [cited 2021 Dec 5]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=324034




 > Introduction Top


Breast cancer is the most common type of malignancy and second most common cause of death among females after lung cancer.[1] It represents 14% of cancer-related death in women.[2] There are various biomarkers, and a study of these biomarkers in breast cancer has gained such widespread importance in the management. Earlier, estrogen receptors (ERs) were thought to be the only receptors expressed by breast cancer cells; nowadays, a number of other receptors have also been found to be present, for example, progesterone receptors (PRs), androgen receptors, apoptotic markers (bcl-2/bax ratio), human epidermal growth factor receptor 2 (HER2)-neu, cytokines, E-cadherin, p53, P-glycoprotein, Ki-67, and stem cell markers. However, none of them has been found satisfactory to predict the response[3],[4],[5],[6] of tumor, and the search for an ideal predictor is still on. Harrell et al. reported that claudin protein expression is an important prognostic factor in tumor known as claudins low, which comes from progenitor cells.[7] There are a minimum of 24 different known claudins found in humans, which are tissue specific. Claudin-3, claudin-4, and claudin-7 are often present in breast tumors and are occasionally expressed at elevated levels.[8],[9],[10] In our study, we correlated the expression pattern of claudin-4 with established biomarkers (ER, PR, and HER2-neu).


 > Materials and Methods Top


The study was conducted in VMMC and Safdarjung Hospital in the Department of General Surgery from May 2013 to May 2018. For this, we enrolled 102 biopsy-proven breast carcinoma patients who underwent surgery (modified radical mastectomy or breast conservative surgery). A detailed history of the present illness along with past, family, menstrual, and obstetric history was recorded; triple assessment (clinical examination of both breasts, ultrasonography/mammography, and core-needle biopsy) and metastatic workup of all patients were done after taking “informed consent.” The mastectomy/lumpectomy specimen and axillary nodes were subjected to histopathological examination for margin assessment, claudin-4, and ER/PR/HER2-neu expression using immunohistochemistry (IHC). After that, we compare the expression of claudin-4 with the established biomarkers ER, PR, and HER2-neu.

After formalin fixation, paraffin embedding, and staining with hematoxylin and eosin [Figure 1]a, histopathological features were determined, before immunohistochemical examination. Histopathological grade was assessed using Bloom–Richardson method, modified by Elston and Ellis (1991). Tumor tissue was routinely fixed overnight (12 h.) in 10% buffered formalin. Representative sections were taken the next day, processed, and embedded.
Figure 1: (a) Hematoxylin and eosin-stained section showing invasive ductal carcinoma of the breast, (b) Immunohistochemical staining showing positivity for human epidermal growth factor receptor 2-neu

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Four-micrometer thickness sections were mounted onto poly-L-lysine-coated slides and left to dry overnight at 37°C. Sections obtained on poly-l-lysine-coated slides were subjected to immunohistochemical staining using mono/polyclonal antibodies by avidin–biotin complex technique.

Slides were deparaffinized, rehydrated, and underwent antigen retrieval (citrate buffer, pH 7.1) by microwaving for 20 min and HER2-neu for 4 min. After cooling down to room temperature, the sections were incubated for 20 min with 3% hydrogen peroxide to block any endogenous peroxidase activity, washed with Tris-buffered saline (TBS), and incubated with (power block) for 15 min to block any nonspecific staining. They were then washed with TBS and incubated with primary antibodies (ER clone ID5, PR clone PR88, and HER2 clone EP1045Y, BioGenex, and claudin-4 clone 3E2C1) for 1 h to identify tumor markers by antigen–antibody reaction. The sections were then washed with TBS and incubated with secondary link antibody (supersensitive poly-horseradish peroxidase) for 30 min. Then, the sections were washed with TBS and incubated for 10 min with 3,3′-diaminobenzidine chromogen. Counterstain with hematoxylin for contrast was done. Both internal and external controls were used for interpretation. Both positive and negative external control slides were used in each staining. Normal breast tissue was used as a positive internal control for ER and PR. Level of ER is expressed as a product of the percentage of epithelial cells stained and intensity of staining through IHC. According to the American Society of Clinical Oncology (ASCO) (2010), ER [Figure 2]a or PR [Figure 2]b was considered positive if finding of more than 1% of tumor cell nuclei is immunoreactive. ER or PR was considered negative if finding of <1% of tumor cell nuclei is immunoreactive (Hammond et al., 2010, and David J. Dabbs, 2010). Reporting of HER2-neu was also done as per the standard criteria: 0 (negative) – no immunoreactivity or immunoreactivity in <10% of tumor cells, 1+ (negative) – faint weak immunoreactivity in >10% of tumor cells but only a portion of the membrane is positive, 2+ (equivocal) – weak-to-moderate complete membrane immunoreactivity in >10% of tumor cells, and 3+ (positive) – more than 10% of tumor cells must show circumferential intense and uniform membrane staining, with homogeneous chicken-wire pattern. For HER2 status, tumors were considered positive [Figure 1]b if scored as 3+ according to the guidelines of the ASCO/College of American Pathologists (Wolff A. C. et al., 2007), and fluorescent in situ hybridization with an amplification ratio of 2.2 or more was used to segregate immunohistochemically equivocal (2+) results.
Figure 2: Immunohistochemical staining showing (a) positivity for estrogen receptor, (b) Positivity of progesterone receptor

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Scoring of claudin-4 was done at ×20 magnification. For manual scoring, tumor samples were evaluated by two independent observers, including one pathologist, and scored for claudin-4 expression on a scale of 0–3. Claudin immunoreactivity was assessed based on a combined score of the extent and intensity of staining. Scores 0–3 were assigned according to the percentage of positive tumor cells (0 = 0%, 1≤25%, 2 = 25%–50%, and 3≥51%) and the intensity of staining in tumor (0 = 0, 1 = 1+, 2 = 2+, and 3 = 3+).[11]

The two scores were multiplied to give an overall score of 0–9, of which 0 was considered negative, 1–2 was considered weak, 3–6 moderate, and 9 strong staining.[12] Negative and weak expression was considered as low, whereas moderate and strong as high. Tumors with low expression of all five claudins were defined as claudin low. Any discordant scores were reviewed together by both scorers to obtain a consensus score. Zero (0) is negative, 1 is weakly positive, 2 is medium positive, and 3 is strongly positive. Only membranous staining was classified as positive. After scoring, the mean value of both scores for each patient was used for statistical analysis. Accordingly, the patients were classified into two groups – high and low claudin-4 [Figure 3] and [Figure 4]:
Figure 3: High claudin-4

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Figure 4: Low claudin-4

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  1. High claudin-4: Tumors with a staining intensity of ≥2
  2. Low claudin-4: Tumors with a median intensity of <2.


Statistical analysis

Categorical variables were presented in number and percentage (%) and continuous variables were presented as mean ± standard deviation. Normality of data was tested by Kolmogorov–Smirnov test. If the normality was rejected, then nonparametric test was used.

Statistical tests were applied as follows: (a) quantitative variables were compared using Wilcoxon rank-sum test to compare pre- and postdata, (b) qualitative variables were compared using Chi-square/Fisher's exact test, and (c) P < 0.05 was considered statistically significant.

The data were entered in MS Excel spreadsheet, and analysis was done using the Statistical Package for Social Sciences version 21.0 Manufacture details- IBM, Chicago, USA. Chi-square/Fisher's exact test was used for calculating the statistical significance, and at a confidence limit of 95% (P < 0.05), a response in terms of the change in tumor size was observed and it was found to be statistically significant.


 > Results Top


In our study, we found that out of 26 cases of high claudin-4, 25 cases (96.15%) were ER negative and 1 case (3.85%) ER positive [Table 1], and P = 0.0001, which was significant. It was also found that high claudin expression (25 out of 26 cases), that was correlated with PR-negative status of the tumor and P = 0.018, was significant [Table 2]. The study showed that ER and PR were inversely related to claudin-4.
Table 1: Correlation of claudin-4 expression with estrogen receptor (ER)

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Table 2: Correlation of claudin expression with progesterone receptor (PR)

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It was also seen that out of 76 cases with low expression of claudin-4, 54 (71.05%) were HER2-neu negative. Out of the remaining cases, i.e., 26 of high claudin-4 expression, 14 cases (53.855%) were HER2-neu positive and 12 were HER2-neu negative. This showed that there was a direct correlation of HER2-neu with claudin-4 (P = 0.022) which was significant [Table 3]. When we compare triple-negative cases with claudin-4, it was seen that out of 22 cases of triple negative, 12 cases were high claudin-4 [Table 4] and [Figure 5].
Table 3: Correlation of claudin-4 expression with human epidermal growth factor receptor (HER-2neu)

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Table 4: Correlation of claudin-4 expression with triple negative breast cancer (TNBC)

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Figure 5: Graphical representation of triple negative with claudin-4

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 > Discussion Top


During the progression of breast cancer, the tissue becomes increasingly disorganized, with associated loss of defined ductal structure and reduced ability of mammary epithelial cells to polarize. These alterations are often accompanied by modified cell-cell and cell-matrix adhesions. One of the first adhesion proteins found to play a role in cancer progression was E-cadherin (the most transmembrane protein of the adherens cell-cell junctions, which is usually lost during cancer progression). Tight junctions, the other main cell-cell adhesion complexes, are apically located and act to maintain cell polarity and control paracellular permeability, also creating a barrier between the apical and basolateral compartments of the cell wall.[13] Integrals to these junctions are members of the claudin family of transmembrane proteins. These proteins have four transmembrane domains and are considered to be the backbone of tight junctions. The extracellular loops of every claudin molecule bind to a claudin molecule on an adjoining cell at the so-called membrane “kissing points.” These structures, together with other integral proteins, such as occludin and junctional adhesion molecule, and a variety of peripheral proteins, form the tight junction macromolecular complex.[14] Tight junctions can vary in composition, permeability, and ion specificity depending on the tissue and therefore the state of differentiation. Hence, breast cancers are heterogeneous aggressive tumors implicating a difficult challenge in their management. Nowadays, available therapy of breast cancer is based on morphological and genetic characteristics of tumor.[15],[16] In clinical practice ER, PR, HER-2neu are established biomarker and are used to determine breast cancer subtype. These molecular subtypes are luminal A (positive for ER and PR, respectively, and HER2 negative, with low levels of Ki-67 expression), luminal B (positive for ER and PR, but negative or positive for HER2, and with high levels of Ki-67), triple-negative (or basal-like) tumors (ER and PR negative, also as HER2 negative), and HER2-enriched tumors (ER and PR negative, but HER2 positive).[17],[18],[19] These subtypes are useful in clinical management because they have distinct prognoses and predictive response of target therapy.[20],[21],[22],[23] Tumors that are ER positive have better prognosis[11],[20] and respond well to hormonal therapy, whereas triple-negative tumors have poor prognosis.[22] In the present study, we investigate claudin-4 protein expression and its relationship with a known clinical marker of breast cancer. Claudins are the transmembrane proteins[24] that were first named in 1998 by Japanese researchers Mikio Furuse and Shoichiro Tsukita at Kyoto University. The name comes from the Latin word Claudine (“to close”) suggesting the barrier role of these proteins. It forms a family of 27 related members. Among which, claudin-4 expression is usually tissue specific and also seen in epithelial malignant tumors.[25] It has been also observed that claudin-4 is overexpressed in the majority of the breast tumors tested and that expression is increased 2–3 folds compared to normal breast tissue.[26] Claudin determination may give additional predictive information on the behavior of tumor tissue and prognosis. Claudin-4 is an interesting marker originally described in the Perou's classification when considering the claudin-low molecular patterns. These proteins are major components of tight junction, and loss of their expression has been associated with malignancies.[24],[27]

Fiona Lanigan et al. studies suggest a negative correlation between claudin expression and ER status.[9] It was also suggested a negative correlation between claudin-4 expression and ER/PR status.[20],[24] It was also reported in triple-negative breast cancer; a combination of high claudin-4 with low claudin-7 was identified as worse prognosis.[9] In view of the limited studies available in the literature, it is an endeavor to continue with the study to arrive at a significant conclusion in Indian patients. In our study, there were inverse relationships between claudin-4 and ER and PR expression. P-value < 0.001 (which was significant). There was a positive correlation between claudin-4 and HER2-neu expression, P = 0.022 (which was significant).

Szasz et al. were the first to demonstrate the relation between loss of claudin-4 and poor prognosis of breast cancer. They also showed that there was more lymph node metastasis in claudin-4-negative breast cancer and poor prognosis than positive claudin-4 tumors.[10],[28],[29] Kolokytha et al. demonstrated the favorable prognosis of triple-negative carcinoma expressing claudin-4.[30] Our study also had similar observations.


 > Conclusions Top


Claudin-4 was inversely associated with ER, PR, and directly with HER2-neu. It was also observed that triple-negative tumors had high claudin-4. Hence, single biomarker claudin-4 can be effectively utilized instead of ER, PR, and HER2-neu as a prognosis of breast cancer. Claudin low subtype is heterogeneous and can be seen in triple negative, luminal A and B, and HER2 enriched type. Claudin low tumors are aggressive tumors and warrant further study to better understand this mysterious subtype with more cases in the future. Therefore, an intense search for markers that may be crucial in the course of disease, especially those with prognostic and therapeutic purposes, will be needed to develop targeted and personalized treatment.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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

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