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ORIGINAL ARTICLE Table of Contents   
Year : 2010  |  Volume : 53  |  Issue : 4  |  Page : 619-623
Immunohistochemical expression and correlation of mammaglobin with the grading system of breast carcinoma


1 Department of Pathology, Institute of Child Health, Lahore, Pakistan
2 Department of Pathology, University of Health Sciences, Lahore, Pakistan

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Date of Web Publication27-Oct-2010
 

   Abstract 

Objectives: In this study it was intended to study mammaglobin expression as a marker for the detection of breast cancer and correlate it with the Bloom-Richardson grading system of breast carcinoma. Materials and Methods: The study was conducted from May 2007 to May 2008. Tissue samples were collected from 50 patients of breast cancer in the various stages of their disease and correlated histologically with the Bloom-Richardson grading system for breast carcinoma. The clinical data of the patients were obtained from their respective files. Results: Positive immunostaining for mammaglobin was seen in 84% of breast carcinoma cases. This immunoreactivity did not correlate with histological and nuclear grades of the tumors, yet it varied according to the histological type of the tumor with ductal carcinoma showing stronger and diffuse staining than other varieties. Conclusion: These results elicit that mammaglobin is overexpressed in carcinoma breast as compared to the normal breast epithelium. This mammaglobin expression can act as a useful tool in the diagnosis of women with breast cancer.

Keywords: Breast cancer, immunohistochemistry, mammaglobin

How to cite this article:
Rehman F, Nagi A H, Hussain M. Immunohistochemical expression and correlation of mammaglobin with the grading system of breast carcinoma. Indian J Pathol Microbiol 2010;53:619-23

How to cite this URL:
Rehman F, Nagi A H, Hussain M. Immunohistochemical expression and correlation of mammaglobin with the grading system of breast carcinoma. Indian J Pathol Microbiol [serial online] 2010 [cited 2019 Dec 14];53:619-23. Available from: http://www.ijpmonline.org/text.asp?2010/53/4/619/72000



   Introduction Top


According to WHO, the age standardized death rate due to breast cancer in Pakistan is 28 per 100,000 deaths per year. [1],[2] The commonest age at presentation of breast cancer in Pakistan is 4 th -5 th decade. The majority of patients present in stage III. [3] Routine hematoxylin and eosin-stained sections are sufficient for the diagnosis of a vast majority of breast lesions. The development of mammography, mass screening, and widespread use of preoperative needle biopsy diagnosis have led to smaller and more preinvasive lesions being detected. [4]

The major applications of diagnostic IHC in breast pathology are the diagnosis of benign breast lesions mimicking malignancy, distinction between simple and atypical ductal hyperplasia or in situ ductal carcinoma, identification of specific histological subtypes, and, diagnosis of intra- and extra-mammary metastases. [5]

The mammaglobin (MGB) gene was identified rather recently by Watson and Fleming in 1996 using a differential screening approach directed at the isolation of human breast carcinoma-associated genes. [6] They isolated a full-length complementary DNA clone which they designated as mammaglobin-1 or MGB-1. Mammaglobin protein is a 93-amino-acid protein with a molecular weight of 10.5 kD. It belongs to a family of epithelial secretory proteins. These proteins are known as secretoglobins due to their structural resemblance to the globins. The mammaglobin gene produces a protein by the same name that is expressed at basal levels in the normal breast epithelium and in 80% of the breast carcinoma but it is not found in normal tissues. This overexpression does not appear to be correlated with tumor grade, tumor stage, or hormone receptor status. However, Nunez-Villar et al.[7] evaluated a series of breast cancer specimens, reporting a correlation between high levels of mammaglobin and expression of estrogen and progesterone receptors, diploid DNA content, low Ki-67 labeling index, low nuclear grade, and the absence of axillary nodal invasion.

The breast tissue-restricted expression of mammaglobin has also generated ideas about mammaglobin-based strategies for the treatment of the breast carcinoma, for example, by targeting breast carcinomas with toxin-conjugated mammaglobin antibodies, mammaglobin-targeted immune therapy, and gene therapy vectors with the mammaglobin promoter driving Bax expression resulting in apoptosis of the breast carcinoma cells.

The aim of this study was to assess mammaglobin expression by means of immunohistochemistry in a consecutive series of 50 breast cancer cases and correlating the expression data with histological grade of the tumors and the available clinical and pathological parameters to clarify the biological role of mammaglobin in breast cancer.


   Materials and Methods Top


Patients

Fifty patients with unilateral, operable breast cancer who underwent resection of their primary tumor from January 2005 to December 2007 (mean age 60 years, age range 20-80 years) were selected for this study. Relevant clinical data including menopausal status, side of breast involved, location of tumor within the breast and the involvement of lymph nodes were collected. All patients gave written informed consent.

Specimen Collection

Formalin-fixed, paraffin-embedded tissues from breast cancer cases and corresponding normal tissue specimens were obtained from the surgical departments of Services Institute of Medical Sciences (SIMS), Sheikh Zayed Hospital, Inmol Hospital, Lahore General Hospital (LGH) and Mayo Hospital. The tumors were histologically confirmed to be breast cancers and classified on the basis of morphology as ductal, lobular, medullary, mucinous, and mixed (ductal and lobular). All the blocks were studied for grading according to the Scarff-Bloom-Richardson grading system of breast carcinoma with Nottingham modification. Five cases of fibroadenoma and three cases of phylloides tumor were also included in this study. Cases of benign reactive lymph nodes were used as a negative control.

Immunohistochemical Staining for Mammaglobin

Formalin-fixed, paraffin-embedded tissue was freshly cut (4 μm) into sections and mounted on poly-l-lysine coated slides for heat-induced epitope retrieval in a citrate buffer. A polyclonal antibody to mammaglobin (clone RP 161-05, DBS, USA) was used. We used the same commercially available streptavidin-biotin-peroxidase kit throughout the whole procedure to ensure the uniformity of the results. Immunopositivity for mammaglobin was scored as follows:

0 - no staining;

1 - weak and sporadic staining in less than 50% of tumor cells;

2 - weak staining in greater than 50% of tumor cells;

3 - strong, diffuse cytoplasmic staining in less than 50% of tumor cells; and

4 - strong, diffuse cytoplasmic staining in greater than 50% of tumor cells.

Sections scoring 3 or 4 were considered as reactive to mammaglobin.

Statistical Analysis

The data were entered and analyzed using SPSS 16.0 (Statistical Package for the Social Sciences). Mean ± SD (standard deviation) were given for quantitative variables. Frequencies and percentages were given for qualitative variables. Pearson's chi-square and Fisher's exact test were applied to observe the associations. Nonparametric Spearman's correlation between quantitative variables was also applied to observe correlation. A P-value of <0.05 was considered as statistically significant.


   Results Top


We studied mammaglobin expression in 50 cases of primary and metastatic breast cancers of various histological grades and types. Among 50 cases of breast cancer, 40 were infiltrating ductal carcinomas and 6 were infiltrating lobular carcinomas whereas remaining 4 were of medullary, mucinous, mixed ductal and lobular, and papillary type. Among them, the infiltrating ductal carcinomas demonstrated strong cytoplasmic staining for mammaglobin protein in 80% (n = 40). The staining was equally frequent in well-differentiated, moderately differentiated, and poorly differentiated carcinomas. This predominant mammaglobin staining pattern was diffuse and cytoplasmic, although some cells demonstrated intense staining particularly around the nuclei. Even the only case of mixed carcinoma containing both ductal and lobular types showed strong, diffuse staining of ductal and a weak, patchy staining of the lobular component. The mucinous and medullary carcinomas were nonreactive for MGB. However, invasive areas of papillary carcinoma were stained for mammaglobin whereas no staining was observed for in situ component.

In nonneoplastic and benign breast lesions including cases of fibroadenoma, phyllode tumors and fibrocystic disease, scattered, weakly positive epithelial cells were seen within the acini of lobules and within the columnar cells of terminal ducts[Figure 1]. However, the overall frequency of mammaglobin immunoreactivity in nonneoplastic ducts and acini was less than 10%. This suggests that although mammaglobin expression is not truly tumor specific, there is quantitative and/or qualitative difference in the level of expression between malignant and nonmalignant breast epithelium. No significant correlation was observed between the histological grade of the tumors and their immunohistochemical scoring of mammaglobin [Table 1] (r = -0.037, P< 0.80). Size distribution and its association with the histological grade of the tumor in 50 cases is shown in [Table 2].
Table 1 :Mammaglobin immunostaining score in 50 cases of breast carcinoma in different grades


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Table 2 :Size distribution and its association with the histological grade of the tumor in 50 cases of breast carcinoma


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Figure 1 :(a) Infiltrating ductal carcinoma, Grade I (H and E, ×100). (b) Mammaglobin positive: cytoplasmic immunohistochemical score of 4+ in infiltrating ductal carcinoma, Grade I (IHC, ×100). (c) Infiltrating ductal carcinoma, Grade III (H and E, ×40). (d) Mammaglobin positive: cytoplasmic immunohistochemical score of 4+ in infiltrating ductal carcinoma with weak, patchy staining in normal acini, Grade III (IHC, ×40)

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Among the 50 cases of breast cancer, 14% of the patients had pre-existing benign breast diseases including adenosis, cystic changes, atypical ductal hyperplasia and periductal mastitis. Only 2% of these lesions were located adjacent to the malignant neoplasm. Their relative percentage is depicted in [Table 3].
Table 3 :Pre-existing benign breast diseases in 50 cases of breast carcinoma


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   Discussion Top


It is due to its tissue specificity that mammaglobin has drawn attention of many workers during the past 5 years. Many have also evaluated its role in the detection of minimal residual disease in breast cancer patients, whereas others have investigated it as a diagnostic and prognostic marker, and its potential role as a therapeutic target. [8]

The frequency reported for mammaglobin expression in breast cancers varies from 20% to 80%; [9] such a broad range might be due to several factors including tumor tissue storage methods (fresh/frozen tissue or paraffin-embedded blocks), the different techniques used for assessing the different expression levels (RT-PCR, quantitative real-time PCR, immunohistochemical staining, and in situ hybridization), tumor heterogeneity, and the specific features of the patient cohorts included in the study. [10],[11]

Mammaglobin has not been extensively studied by immunohistochemical analysis in human tissues because of polymerase chain reaction-based methods. Most of the studies have focused on mRNA to detect mammaglobin overexpression. [12],[13] Most of the previous studies using the immunohistochemical method for mammaglobin expression have demonstrated that mammaglobin is exclusively expressed in about 80% of breast cancers [14] and can be used as a marker gene for the detection of micrometastasis. [12],[13],[14],[15],[16]

Our study is confined to the sensitivity of the MGB as a marker for the detection of breast carcinoma and to correlate its expression with the Bloom Richardson grading system of the breast carcinoma and estrogen receptor status.

Many studies have compared the sensitivity and specificity of mammaglobin with other tumor markers in both primary and metastatic breast carcinomas. Ciampa et al. studied mammaglobin and CrxA-01 expression in the cell block material from malignant pleural effusions. Eighty percent of breast carcinomas were positive for mammaglobin and/or CrxA-01, and none of the extra-mammary carcinomas were positive for mammaglobin[Figure 2]. Han et al. [17] and Bhargava et al. [18] studied mammaglobin and BRST-2 (same as GCDFP-15) expression by immunohistochemical analysis in breast and nonbreast carcinomas. They concluded that mammaglobin has a superior sensitivity to that of BRST-2. Among the nonbreast carcinomas, only 1 of 10 urothelial neoplasms and 1 of 10 thyroid carcinomas showed strong immunoreactivity.
Figure 2 :(a) Infiltrating ductal carcinoma, Grade II (H and E, ×100). (b) Mammaglobin positive: cytoplasmic immunohistochemical score of 4+ in infiltrating ductal carcinoma, Grade II (IHC, ×100). (c and d) Invasive papillary carcinoma with in -situ papillary component, Grade III (H and E, ×100). (e) Mammaglobin positive: cytoplasmic immunohistochemical score of 4+ in infiltrating papillary carcinoma (IHC, ×100). (f) Medullary carcinoma (H and E, ×200)

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Both Watson and Nunez-Villar [7],[19] found no difference in MGB expression in different histological types of breast carcinomas. However, the present study showed that ductal carcinomas are more diffusely and strongly stained with MGB as compared to lobular carcinomas that showed a rather patchy, weak staining pattern. This is in contrast to the study conducted by Bhargava et al. [18] in which infiltrating lobular carcinomas showed strong, diffuse immunostaining for MGB as compared to infiltrating ductal carcinomas (E-cadherin was not used for this study).

Among 50 cases of invasive breast carcinoma, 15 had a variable portion of the in situ component along with the infiltrating tumor. Twelve of these 15 cases showed strong mammaglobin immunoreactivity of the in situ component[Figure 3].
Figure 3 :(a) Mucinous carcinoma (H and E, ×200). (b) Mammaglobin positive: cytoplasmic immunohistochemical score of 3+ in mucinous carcinoma (IHC, ×200). (c) Fibroadenoma: mammaglobin (IHC, ×200). (d) Phyllodes tumor: mammaglobin (IHC, ×200)

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Correlation of mammaglobin immunostaining with Scarff-Bloom-Richardson grading was found out previously. In our study, mammaglobin positivity was seen in 9 out of 11 cases of Grade I, in 11 out of 14 of Grade II, and in 22 out of 25 with Grade III tumors. [Table 1] These findings in various grades of breast cancer are consistent with those of Nunez-Villar et al. [7]

Tumor size and axillary lymph node invasion by the breast tumor cells are the most important pathological variables that predict the occurrence of distant metastasis. Tumors more than 2 cm in size are associated with a greater risk for developing metastatic disease and this is also reflected in an increase in the prevalence of lymph nodes containing invading tumor cells. As observed in this study, no relation was found between mammaglobin expression and tumor size (P = 0.226) [Table 2], as its expression was found equally in both large (T3, T4) and small (T1, T2) invasive breast cancers.

Monica et al.[20] observed high levels of mammaglobin in patients with benign breast disease. It is due to the fact that certain histological types of benign breast diseases, e.g., atypical ductal and lobular hyperplasia and other fibrocystic changes in breast may result in an increased chance of developing breast cancer in certain patients. They also studied the mammaglobin mRNA levels in relation to lactation, menstrual cycle, and menopausal status. In our study, 7 out of 50 patients had preexisting benign breast disease, and of these 7, 1 case was of atypical ductal hyperplasia and apocrine metaplasia each, 2 cases were of periductal mastitis and cystic changes each, and 4 cases of adenosis. [Table 3] The staining in the benign portion of the invasive carcinomas was focal and less intense as compared to the invasive portion of the same slide.

In summary, the current study demonstrates that mammaglobin expression is a promising marker for neoplastic breast epithelial cells and provides sufficient evidence to warrant larger clinical trials and long-term follow-up using mammaglobin as a molecular marker for early detection, staging, and prognosis and/or relapse monitoring of breast cancer.


   Conclusion Top


We conclude that the mammaglobin antibody is a sensitive marker for the detection of tumor cells in patients with breast carcinomas. Furthermore, mammaglobin expression is not altered at the metastatic (lymph node) site. So following points can be noted:

  • Due to the high specificity and overexpression of mammaglobin in the malignant breast tissue, it can be used to confirm a breast origin in a metastatic or undifferentiated tumor with unknown primary.
  • It can also help to differentiate benign, proliferating breast diseases mimicking malignancy on routine histological sections from in situ or infiltrating breast carcinoma.
  • The immunostaining for mammaglobin was almost equally positive in all morphological grades.



   Acknowledgments Top


The authors acknowledge Malik Hussain Mubashar, the vice chancellor of University of Health Sciences, for providing the funds and the working facilities for this project. The authors are also grateful to Mr. Muhammad Waqas for composing and analyzing data with the help of computer software SPSS-11. Miss Bushra and Mr. Naveed are to be especially thanked for giving a very responsible technical assistance.

 
   References Top

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4.Balaton AJ, Guinebretiere JM, Penault-Llorca F. Diagnostic immunohistochemistry of breast epithelial lesions. Ann Pathol 2003;23:570-81.  Back to cited text no. 4
    
5.Bhargava R, Dabbs DJ. Use of immunohistochemistry in diagnosis of breast epithelial lesions. Adv Anat Pathol 2007;14:93-107.  Back to cited text no. 5
    
6.Bernstein JL, Godbold JH, Raptis G, Watson MA, Levinson B, Fleming TP, et al. Identification of mammaglobin as a novel serum marker for breast cancer. Clin Cancer Res 2005;11:18.  Back to cited text no. 6
    
7.Nunez-Villar MJ, Martinez-Arribas F, Pollan M, Lucas AR, Sanchez J, Tejerina A, et al. Elevated mammaglobin (h-MAM) expression in breast cancer is associated with clinical and biological features defining a less aggressive tumour phenotype. Breast Cancer Res 2003;5:65-70.   Back to cited text no. 7
    
8.Zafrakas M, Petschke B, Donner A, Fritzsche F, Kristiansen G, Knuchel R, et al. Expression analysis of mammaglobin A (SCGB2A2) and lipophilin B (SCGB1D2) in more than 300 human tumours and matching normal tissues reveals their co-expression in gynecologic malignancies. BMC Cancer 2006;6:88.   Back to cited text no. 8
    
9.Gargano G, Agnese V, Calo V, Corsale S, Augello C, Bruno L, et al. Detection and quantification of mammaglobin in the blood of breast cancer patients: can it be useful as a potential clinical marker? Preliminary results of a GOIM (Gruppo Oncologica dell′italia Meridionale) prospective study. Ann Oncol 2006;17:vii41-5.   Back to cited text no. 9
    
10.Zach O, Kasparu H, Krieger O, Hehenwarter W, Girschikofsky M, Lutz D. Detection of circulating mammary carcinoma cells in the peripheral blood of breast cancer patients via a nested reverse transcriptase polymerase chain reaction assay for mammaglobin mRNA. J Clin Oncol 1999;17:2015-9.   Back to cited text no. 10
    
11.Grunewald K, Haun M, Feigl M, Urbanek M, Muller-Holzner E, Massoner A, et al. Mammaglobin expression in gynecologic malignancies and malignant effusions detected by nested reverse transcriptase-polymerase chain reaction. Lab Invest 2002;82:1147-53.  Back to cited text no. 11
    
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13.Suchy B, Austrup F, Driesel G, Eder C, Kusiak I, Uciechowski, et al. Detection of mammaglobin expressing cells in blood of breast cancer patients. Cancer Lett 2000;158:171-8.  Back to cited text no. 13
    
14.Watson MA, Dintzis S, Darrow CM, Voss LE, DiPersio J, Jenson R, et al. Mammaglobin expression in primary, metastatic and occult breast cancer. Cancer Res 1999;59:3028-31.  Back to cited text no. 14
    
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19.Watson MA, Fleming TP. Mammaglobin, a mammary-specific member of the uteroglobin gene family, is overexpressed in breast cancer. Cancer Res 1996;56:860-5.  Back to cited text no. 19
    
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Correspondence Address:
Fakeha Rehman
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DOI: 10.4103/0377-4929.72000

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