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ORIGINAL ARTICLE  
Year : 2020  |  Volume : 63  |  Issue : 4  |  Page : 551-558
Associations between the expression of mucins (MUC1, MUC2, MUC5AC and MUC6) and clinicopathologic parameters of human breast carcinomas


1 Department of Pathology, Manavgat State Hospital, Antalya, Turkey
2 Department of Pathology, Ankara Education and Research Hospital, Ankara, Turkey
3 Department of Pathology, Bingol State Hospital, Bingol, Turkey
4 Department of Pathology, Baskent University, Ankara, Turkey
5 Department of Biochemistry, Mustafa Kemal University, Hatay, Turkey

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Date of Submission03-Oct-2018
Date of Decision16-Mar-2020
Date of Acceptance17-Apr-2020
Date of Web Publication28-Oct-2020
 

   Abstract 


Aims: The aim of this study is to evaluate the relationships between the expression of mucins in invasive breast carcinomas and clinicopathologic parameters. Materials and Methods: We examined 150 cases of invasive breast carcinoma, using the 2012 World Health Organization (WHO) classification of the tumors of the breast. We studied the expression of MUC1, MUC2, MUC5AC, and MUC6 by immunohistochemistry. We also evaluated normal breast tissue and ductal carcinoma in situ (DCIS) lesions in nearby invasive tumor areas. Results: In invasive breast carcinomas, MUC1, MUC2, MUC5AC, and MUC6 were expressed in 98.6%, 11.3%, 9.9, and 8.5% of cases, respectively. MUC2, MUC5AC, and MUC6 were overexpressed in invasive tumors and DCIS lesions were compared with normal breast tissue. The apical pattern of MUC1 was correlated with low grade and ER expression. MUC2 was correlated with mucinous carcinoma and an inverse association with invasive ductal carcinoma, not otherwise specified (NOS). MUC6 expression was associated with lymphovascular invasion. Conclusions: Most invasive breast tumors express MUC1 and the apical pattern of MUC1 is correlated with low grade and ER expression. MUC6 expression is associated with indicators of poor prognosis. Further comprehensive studies need to evaluate the role of mucins as a potential biomarker and to be used as a specific therapeutic target against breast cancer.

Keywords: Breast cancer, MUC1, MUC2, MUC5AC, MUC6, prognosis

How to cite this article:
Oral O, Unverdi H, Kumcu E, Turkbey D, Dogan S, Hucumenoglu S. Associations between the expression of mucins (MUC1, MUC2, MUC5AC and MUC6) and clinicopathologic parameters of human breast carcinomas. Indian J Pathol Microbiol 2020;63:551-8

How to cite this URL:
Oral O, Unverdi H, Kumcu E, Turkbey D, Dogan S, Hucumenoglu S. Associations between the expression of mucins (MUC1, MUC2, MUC5AC and MUC6) and clinicopathologic parameters of human breast carcinomas. Indian J Pathol Microbiol [serial online] 2020 [cited 2020 Nov 25];63:551-8. Available from: https://www.ijpmonline.org/text.asp?2020/63/4/551/299318





   Introduction Top


Breast cancer (BC) is the second leading cause of cancer-related deaths in women worldwide after lung cancer.[1] Evidences are emerging that suggest that BC is a heterogeneous disease at the molecular level having a number of distinct entities with specific pathologic features and biologic behaviors. Therefore, much effort is being carried out to identify markers that have biological and therapeutic significance in BC.

Mucins are a large family of glycoproteins attached to serine or threonine residues that are O-glycosylated.[2] They are classified into two main classes: membrane-bound including MUC1, MUC3A, MUC3B, MUC4, MUC11, MUC12, MUC13, MUC15, MUC16, MUC17, MUC20, and MUC 21. Gel forming or secreted include MUC2, MUC5AC, MUC5B, MUC6, MUC19, MUC7, MUC8, and MUC9.[3] Varied expressions of mucins have been reported to be associated with cancer development, influence cellular growth, malignant transformation, dedifferentiation, adhesion, invasion, and immune surveillance.[4] To date, 20 mucin genes have been identified.[5] MUC1 is the mostly studied mucin in BC and normally it is comprised of cell signaling, immunoregulation, and inhibition of cell–cell and cell–matrix adhesion.[6] MUC1 is present in the apical cell membrane of normal breast epithelium but in malign tissues it is detected in variable localization such as cytoplasmic and membrane expressions or both of them.[7] Moreover some studies suggest that the overexpression of MUC1 may contribute in tumor detachment, invasion and metastases, so it is associated with poor prognosis.[8]

MUC2, MUC5AC and MUC6 are gel-forming mucins.[9] MUC2 is an intestinal-type secretory mucin and it is expressed in goblet cells of the intestine and airway epithelium.[10],[11] MUC2 is detected mostly in mucinous carcinomas including colon, breast, pancreas, prostate and it may act as a potential prognostic indicator.[12],[13]

MUC5AC is detected mainly in the surface mucous cells of the cardia, fundus and antrum of the stomach whereas MUC6 is observed in the pyloric gland.[14] Both of them are generally not expressed in normal breast tissue but MUC6 is detected in mucinous carcinomas of the breast.[15]

The aims of this study were to evaluate the expression patterns of various mucins in invasive breast carcinomas and to identify the relationships between mucin expression and clinicopathologic parameters. Thus, we assessed the expression profiles of different mucins and evaluated their prognostic significance in a large series of breast cancer.


   Materials and Methods Top


We studied 150 patients with invasive breast carcinoma including invasive ductal carcinoma not otherwise specified (NOS), lobular carcinoma, mucinous carcinoma, mixed carcinoma, neuroendocrine differentiated carcinoma and apocrine differentiated carcinoma who underwent surgical resection or excisional biopsy without neoadjuvant therapy at Ankara Training and Research Hospital between 2009-2014.

The micropapillary pattern in pure mucinous carcinoma may affect patient survival via their tendency for nodal metastases but we studied only pure mucinous carcinoma cases without a micropapillary pattern.

We evaluated invasive tumor, ductal carcinoma in situ, and normal tissue nearby invasive areas. The median age of patients was 51.4 (range, 26-77 years) years. The follow-up period ranged from less than 4 to 99 months. During this period, 19 patients died. All cases were examined microscopically and re-evaluated histological type, grade (modified Bloom Richardson grade), hormone receptor profile, T and N stage, lymphovascular and perineural invasion.

Immunohistochemistry

All the hematoxyline and eosin-stained slides were revised and the most representative tumor area was selected and marked on the patient's paraffin block. The immunohistochemical stains were performed on 5μm thick sectioned blocks. The antibodies were MUC 1 (1/100, Polyclonal, Thermo Scientific), MUC 2 (1/100, M53, Thermo Scientific), MUC 5AC (1/200, 45M1, Thermo Scientific), and MUC 6 (1/50, CLH5, Thermo Scientific).

Evaluation of immunohistochemistry

The Allred score was used to examine the estrogen receptor (ER) and progesterone receptor (PR) and a score of 3 to 8 was considered positive. HER2 was graded as recommended by the American Society of Clinical Oncology/College of American Pathologist (ASCO/CAP) guidelines and positivity was considered by a score of 2+ or 3+. MUC1 and MUC6 were considered positive if it stained in >30% of malignant cells. Because of their low expression rate, any expression was regarded positive in the cases of MUC2 and MUC5AC.

Data analysis

Data were statistically analyzed using SPSS for Windows version 11.5. The Chi-square test and Fisher's exact tests were used to determine the associations between mucin expression and clinicopathologic parameters. The Kaplan–Meier estimator was used for the survival analysis. Statistically, significance was considered when P value less than 0.05.


   Results Top


In this study, we evaluated 142 cases due to tissue loss. In the assessable cases, in normal breast tissue, MUC1, MUC2, MUC5AC, and MUC6 were expressed in 100%, 1%, 3%, 2% of total cases, respectively. The rates of MUC1, MUC2, MUC5AC, and MUC6 expression were 97.2%, 16.2%, 18.9%, 5.4% in DCIS lesions, respectively. Invasive breast carcinoma demonstrated MUC1, MUC2, MUC5AC, and MUC6 expression in 98.6%, 11.3%, 9.9%, 8.5% of total cases, respectively. In the cases of invasive breast carcinoma and DCIS, the rates of MUC2, MUC5AC, and MUC6 expression were slightly higher than the normal breast tissue. The expression of MUC1 was at similar percentages [Table 1].
Table 1: MUC1, MUC2, MUC5AC and MUC6 expression in invasive carcinoma of breast, ductal carcinoma in situ and normal breast tissue

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Clinicopathological parameters and relationships between mucin expressions and parameters in invasive breast carcinoma

MUC1 was expressed in different sites, such as apical, cytoplasmic, membranous, or a combination of cytoplasmic and membranous [Figure 1]. Other types of mucins were expressed in the cytoplasm [Figure 2]. In the cases of subcellular localization of MUC 1 expression, apical, cytoplasmic, membranous, and combination of cytoplasmic and membranous expression was detected in 12.1%, 58.6%, 12.1%, 17.1% of invasive breast carcinoma cases, respectively [Table 2].
Figure 1: MUC1 expression with cytoplasmic, membranous and apical or combinative location. MUC1 expression shows variable subcellular locations, such as cytoplasmic (a), membranous (b), apical (c), and combinative cytoplasmic and membranous (d) (immunohistochemical staining, ×200, ×400)

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Figure 2: MUC2, MUC5AC, and MUC6 expression. MUC2 (a), MUC5AC (b), and MUC6 (c) show cytoplasm expression (immunohistochemical staining, ×400)

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Table 2: Subcellular localization of MUC 1 in invasive breast carcinomas

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Clinicopathologic parameters comprised histological type, histological grade, lymphovascular invasion, perineural invasion, T stage, N stage, age, triple hormone receptor negativity, Paget' s disease, ER, PR, Cerb B2 status, and the overall survival.

Regardless of the subcellular localization of MUC1, there was no association between MUC1 and clinicopathological parameters. On the other hand, the apical expression of MUC1 was significantly correlated with ER positive-phenotype compared with cytoplasmic expression (P = 0.007). Also, there was an association between subcellular localization of MUC 1 and tumor grade. The apical localization of MUC1 was associated with low grade compared with membranous, combination of cytoplasmic and membranous, and cytoplasmic expressions (P < 0.001).

MUC2 expression demonstrated association with mucinous carcinoma and an inverse association with invasive ductal carcinoma, NOS. (P < 0.001 and P = 0.026, respectively).

MUC5AC expression was not associated with any clinicopathological variables.

MUC6 expression was significantly correlated with lymphovascular invasion (P = 0,029).

The Kaplan–Meier survival analysis revealed no prognostic significance of the overall survival for MUC1, MUC2, MUC5AC, MUC6 expressions and between subcellular localizations of MUC1 (P = 0.556, P = 0.990, P = 0.606, P = 0.130, P > 0.05, respectively) [Figure 3].
Figure 3: Kaplan-Meier survival analysis results for MUC1 (a), MUC2 (b), MUC5AC (c), MUC6 (d) and the subcellular distribution of MUC1 (e)

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Except these findings, no other correlations were found with other parameters [Table 3].
Table 3: Expression of mucins and clinicopathologic parameters of invasive breast carcinomas

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   Discussion Top


In this study, we investigated the expression profiles and prognostic value of different mucins in human breast cancer. To date, twenty mucin genes have been identified. Various previous studies examined especially MUC1, MUC2, MUC5AC, and MUC6 in BC and found different results. We also studied MUC1, MUC2, MUC5AC, and MUC6. In addition to that, we focused on DCIS lesions and normal breast tissue. In the cases of invasive breast carcinoma and DCIS, the rates of MUC2, MUC5AC, and MUC6 expressions were slightly higher than normal breast tissue cases. With future studies, this might be useful in early detection in breast tumors. In all of the normal breast tissue, we observed the apical pattern of MUC1 expression. Previous studies detected similar results in their study but interestingly, Chang et al. showed weak cytoplasmic staining in addition to apical staining in the ducts and lobules in 22 normal breast samples.[7],[15],[16] This different result may depend on the antibody of MUC1 which they used. We detected MUC1 expression in the majority of invasive carcinoma cases (98.6%). Regardless of subcellular localization, there were no associations between MUC1 and clinicopathological parameters.

The presence of apical cellular localization is a marker of an intact MUC1 pathway that is correlated with functional differentiation and a good prognosis. Conversely, the presence of other aberrant patterns is associated with a lack of functional differentiation and worse prognosis.[7] We found membranous, a combination of cytoplasmic and membranous, and cytoplasmic localization of MUC1 was associated with high grade compared with apical expression. Our results correlated with the researches of Rakha et al., Rahn et al., and van der Vegt et al., which showed that apical MUC1 was associated with low-grade tumors.[7],[8],[17] Rakha et al. and van der Vegt et al. also found that the apical pattern was associated with superior outcome compared with the aberrant cytoplasmic and membranous pattern.[8],[17] Rakha et al. showed apical staining in 4.5% of cases. In this study, even so, we observed apical expression in 12.1% of cases, there were no associations between subcellular localization of MUC1 and overall survival. We also detected that the apical expression of MUC1 was significantly correlated with ER positive-phenotype compared with cytoplasmic expression. ER is an extremely useful therapeutic factor and previous studies revealed associations with ER and MUC1 which should be kept in mind.[8],[17],[18]

In BC, the high levels of gel-forming mucin, MUC2, may play the role of a barrier that prevents with the extension of the malignant cells. Previous studies found that MUC2 was expressed with a very high frequency in mucinous carcinomas compared with other tumor types.[8],[15],[19] This may explain the less frequent lymph node metastasis and more favorable prognosis of mucinous carcinoma of the breast. Kim et al. found that MUC2 negativity was correlated to larger tumor size and higher histological grade in mucinous carcinoma. They also showed MUC2 overexpression is related to aggressive tumor behavior in invasive ductal carcinoma, NOS.[20] Conversely, Patel et al. found a significant correlation between the MUC2 expression and histopathological grade, which suggests that MUC2 negative tumors were associated with higher tumor grade. This finding states that negative MUC2 expression in BC is associated with aggressive tumor behavior. Also, Like our study, Rakha EA et al., Do SI et al., found no significant association between MUC2 expression and histologic grade. Further studies are needed to clarify the relationship between MUC2 and prognostic parameters in BC.[21] In our study, MUC2 was expressed in 11.3% of the invasive cancers and MUC2 expression demonstrated the association with mucinous carcinoma and an inverse association with invasive ductal carcinoma, NOS. Nevertheless, in our study, there were no associations with other prognostic factors.

Patel et al. found a high statistically significant correlation between the MUC5AC expression and histopathological grade (P < 0.01) which suggests that MUC5AC negative tumors were associated with higher tumor grade. This finding states that negative MUC5AC expression in BC is associated with aggressive tumor behavior.[21] However, MUC5AC expression was not correlated with any prognostic factors in the current study. MUC5AC's lower expression level of our cases may act role on this result. O'Connell et al. found five-fold increased MUC2 signals and two-fold increased MUC5 signals in mucinous carcinomas compared with the controls by in situ hybridization study.[19] They described MUC5 as a “tracheobronchial mucin'' and MUC5 examined by them may include both MUC5AC and MUC5B. For this reason, a comparative study of both MUC5AC and MUC5B is needed.

In our study, MUC6 expression was detected in 8.5% of invasive breast carcinoma cases and it was associated with poor prognostic factor, such as lymphovascular invasion. Matsukita et al. studied MUC6 expression in mucinous carcinomas and they found that MUC6 is associated with superior outcomes.[15] These authors explained that MUC6 is a gel-forming mucin and in mucinous carcinomas, it may act as a barrier to cancer cell extension like Rakha et al. commented for MUC2.[8] However, they examined MUC6 in a limited number of cases, only 17 cases. Therefore, a large number of studies should be helpful to support this result. Conversely, Do et al. found that MUC 6 was associated with poor prognostic factors such as lymphatic invasion, higher histologic grade, and higher N stage. Similar to us, Do at al. defined positivity as when more than 30% of cancer cells expressed MUC6, whereas the prior studies used a cutoff value of more than 5%.[18] Maybe this different value causes different results. Moreover, in our study, there were only three mucinous carcinoma cases in 150 cases and MUC6 was expressed in one case. For that reason, the role of MUC6 may be different between mucinous carcinomas and other invasive breast carcinomas.


   Conclusions Top


In summary, after the evaluation of the expression of mucins, however, we didn't find any association between mucins and overall survival, we detected some prognostic correlations which can be useful in early detection, predicting prognosis, and therapeutic target of BC patients. Further comprehensive genetic studies are needed to clarify the biological secrets of mucins to be used as a specific therapeutic target against BC.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Adsay NV, Merati K, Nassar H, Shia J, Sarkar F, Pierson CR, et al. Pathogenesis of colloid (pure mucinous) carcinoma of exocrine organs: Coupling of gel-forming mucin (MUC2) production with altered cell polarity and abnormal cell-stroma interaction may be the key factor in the morphogenesis and indolent behavior of colloid carcinoma in the breast and pancreas. Am J Surg Pathol 2003;27:571-8.  Back to cited text no. 12
    
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Correspondence Address:
Onur Oral
Manavgat State Hospital, Department of Pathology, Antalya
Turkey
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/IJPM.IJPM_637_18

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