Indian Journal of Pathology and Microbiology

: 2011  |  Volume : 54  |  Issue : 2  |  Page : 284--289

Expression of human mammaglobin and clinicopathologic correlations in breast cancer: The findings in Malaysia

Fawwaz S Al-Joudi1, Fahme A. K. Kaid1, Ismarulyusda Ishak1, Norhazilah Mohamed1, Kairol Osman1, Iskandar Zulkarnain Alias2,  
1 Department of Biomedical Sciences, Faculty of Allied Health Sciences, National University of Malaysia (Universiti Kebangsaan Malaysia), Jalan Raja Muda Abdul-Aziz, Kuala Lumpur, Malaysia
2 Department of Chemical Pathology, School of Medical Sciences, University of Science of Malaysia (Universiti Sains Malaysia), Kota Bharu, Malaysia

Correspondence Address:
Fawwaz S Al-Joudi
Department of Biomedical Science, Faculty of Allied Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul-Aziz, Kuala Lumpur 50300


Background: Human mammaglobin (hMAG) is a secreted protein which has been detected in breast epithelial cells of mammary glands and has been used as a specific marker for breast cancer. Objectives: This study aims at studying the hMAG expression and identifying the significant predictors of hMAG expression in breast cancer tissues. Materials and Methods: The tissue samples were obtained from two major teaching hospitals in the country. They were examined by immunohistochemistry (IHC) and the hMAG expression was evaluated using an established scoring system. Results: Out of 84 breast cancer tissue samples, hMAG was expressed in 50 samples (59.6%). The expression of hMAG was found to be increased with cancer grade. The output of logistic regression model showed that hMAG was overexpressed in breast cancer samples from the first hospital (P = 0.014), but not with those from the second hospital. Conclusions: It can be concluded that hMAG may serve in the diagnosis and the assessment of progression with the increased cancer grade. The dominance in hMAG expression in samples from HUSM may correlate with ethnic, environmental or genetic factors.

How to cite this article:
Al-Joudi FS, Kaid FA, Ishak I, Mohamed N, Osman K, Alias IZ. Expression of human mammaglobin and clinicopathologic correlations in breast cancer: The findings in Malaysia.Indian J Pathol Microbiol 2011;54:284-289

How to cite this URL:
Al-Joudi FS, Kaid FA, Ishak I, Mohamed N, Osman K, Alias IZ. Expression of human mammaglobin and clinicopathologic correlations in breast cancer: The findings in Malaysia. Indian J Pathol Microbiol [serial online] 2011 [cited 2019 Nov 17 ];54:284-289
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Full Text


Breast cancer is the second most fatal cancer in women after lung cancer. It is estimated that more than 1 million new cases of breast cancer are diagnosed worldwide every year, with a death rate of approximately 410,000 people per year. [1] It is also the most common female malignancy in Malaysia: 1 in 20 women has the chance of developing breast cancer. [2] There are no systematic data for the incidence of breast cancer in Malaysia because of the lack of a cancer registry. [3] However, there are few published reports about the breast cancer prevalence in specific states of the country. The breast cancer tissue markers known and used are not sensitive and not even tumor-specific, hence are not sufficient to be ideally suited for diagnosis or for screening the general population. [4] Various biomarkers have been used in the detection of breast cancer in tissues from biopsies, peripheral blood and/or bone marrow, such as survivin, human mammaglobin (hMAG), carcinoembryonic antigen (CEA), cytokeratins (CK19 and CK20), polymorphic epithelial mucin (MUC-1), epidermal growth factor receptor (EGFR), maspin, estrogen receptor (ER) and progesterone receptor (PR). [4],[5],[6],[7],[8] Correlations between these markers with each other and with clinicopathologic features of breast cancer are not fully understood and still await further investigations.

hMAG is a 93-amino acid protein belonging to the uteroglobin/Clara cell protein family of small epithelial secretory proteins, the secretoglobins. [9],[10] It is expressed in normal breast epithelial cells and is specifically overexpressed in breast cancer. By using immunohistochemistry (IHC), hMAG was reported to be present in 81% of the breast cancer cases. [11],[12] Thus, hMAG is promising for its potential diagnostic value as well as its prognostic indications in breast cancer, especially as it can be detected almost exclusively in breast cancer, and occasionally, endometrial tumors. [13],[14],[15],[16],[17] Furthermore, the detection of hMAG in the blood in circulating tumor cells has been correlated with poor prognoses. [18],[19]

The present study was carried out to evaluate the hMAG expression in breast cancer tissue samples by IHC. The association of hMAG expression with clinicopathologic and demographic factors of the patients was also investigated.

 Materials and Methods

A total of 84 formalin-fixed, paraffin-embedded tissue samples of breast cancer tissues were obtained from the operating theaters and the pathology departments of the Medical Centre of the National University of Malaysia (MCUKM) in Kuala Lumpur and the Hospital of the University of Science of Malaysia (HUSM), Kota Bharu, Kelantan. Biodata and clinical information were obtained from the records and the histopathology reports, which included age, race, marital status, menopausal status, breast feeding, family history, histological grade, ER, PR and HER-2. The protocol of this study was approved by the Research and Ethical Committee of the MCUKM.

The IHC technique was performed as described previously. [12] Tissue blocks were trimmed and sectioned with a microtome to obtain 4 μm tissue sections. The sections were floated in a 38 o C water bath, fished and mounted on poly-l-lysine slides and dried overnight at 37°C. The slides were deparaffinized and rehydrated by dipping into containers containing 400 ml each of xylene, and graded concentrations of ethanol and distilled water. For antigen retrieval, the slides were boiled in sodium citrate buffer at pH 6.0 for 30 minutes and cooled down at room temperature (RT) for 20 minutes. The slides were then blocked against endogenous enzyme activity by dipping in 3% hydrogen peroxide for 20 minutes. For the immunohistochemical analysis, a diagnostic kit, the Chemicon IHC select, Immunoperoxidase Secondary Detection System (Chemicon International Inc., Temecula California, USA) was used as instructed by the manufacturer. The sections were preincubated with normal goat serum in phosphate buffered saline (PBS) for 20 minutes. Following this, a polyclonal rabbit anti-mammaglobin serum (abcam ab53134-100, Abcam, Cambridge, USA) was added at 1:50 dilution and left for 30 minutes at room temperature. After washing in the rinsing buffer, the sections were incubated with biotinylated goat anti-rabbit IgG (1:100) for 30 minutes at RT. The secondary antibody solution was washed away and the tissues were then incubated in streptavidin horse radish peroxidase and diluted in tris buffered saline (TBS) for 20 minutes. Then, the slides were washed, followed by adding the chromogen solution containing 3, 3 diaminobenzidine diluted in TBS and hydrogen peroxide diluted in TBS for 20 minutes. Finally, the tissue sections were washed and counterstained with hematoxylin solution for 2 minutes. After dehydration with graded alcohol and xylene, the slides were finally mounted under cover slips.

The expression of hMAG was examined microscopically using an established scoring system. [8] In brief, at least five fields in each slide were examined for both number of cells with specific staining and the intensity of staining. The number of cells stained was counted per 1000 cells in each field and then the total percentage was obtained. The rate of stained cells was scored as follows: 0 = <5% (less than 5% of breast cancer cells were stained), 1 (between 5 and 25%), 2 (between 26 and 50%), 3 (between 51 and 75%) and 4 (more than 75%). Intensity of staining was scored as weak (1+), moderate (2+) or intense (3+). The sum was obtained by multiplying the percentage by the intensity. The total score for the hMAG expression was considered negative when the score was <1 and positive with score ≥1. The negative control consisted of tissue section with a negative primary antibody.

The statistical analysis of the data was performed using Statistical Package for Social Sciences for Windows (SPSS version 13, 2002). Proportions were used to present the percentages of the variables. Some data were not traceable from the records particularly for the breast feeding practice, family history of breast cancer and marital status. Such data were recorded as "missing" and statistical analysis was done using only the available data. The associations of hMAG expression as a dichotomous variable (positive/negative) with the demographic and clinicopathologic factors were examined using Chi-squared test. The logistic regression model "ENTER" was used to identify the predictors of positive of hMAG expression. All the categorical variables were coded as "0" and "1," and code "1" was given for the respondent. To ensure correct output for the logistic regression model, dummy variables were created for the grade of cancer variable. The dependent variable was hMAG expression, whereas the independent variables included age, ethnicity (race), marital status, menopausal status, breast feeding practice, family history of breast cancer, histological grade of breast cancer, ER, PR and HER-2 expression.


The mean ± SD age of the study subjects was 52.3 ± 13.0 years. The minimum and maximum ages were 26 and 82 years, respectively. According to age, the subjects were categorized into two groups: those with age ≤50 years (43 subjects, 51.2%), and those of >50 years of age (41 subjects, 48.8%). The basic characteristics of the subjects involved in this study are shown in [Table 1]. {Table 1}

Expression of hMAG

The breast antigen hMAG was positively detected in 50 (59.5%) tissue samples [Figure 1]. Among these hMAG-positive samples, 15 (30%) showed high immunostaining intensity and 28 (56%) showed a moderate intensity, and the remaining 7 samples (14%) showed weak intensity [Figure 2], [Figure 3] and [Figure 4]. {Figure 1}{Figure 2}{Figure 3}{Figure 4}

Associations of hMAG Expression with the Demographic and Clinicopathologic Factors

The relationship between positive hMAG expression and patients' demographic and clinicopathologic data were examined using Chi-square. Patients with positive hMAG expression from hospital 1 were more than those from hospital 2 and the difference was statistically significant (P < 0.01) [Table 2]. The expression of hMAG showed a tendency to increase with the grade of cancer [Figure 5] although statistically this relationship was not significant (P > 0.05). In addition, the distributions of hMAG expression according to the age group, menopausal status, breast feeding practice, family history of breast cancer, ER, PR and HER-2 were all not significant (P > 0.05). {Figure 5}{Table 2}

Multivariate analysis

Logistic regression model was developed using the "ENTER" method and the output of the model showed that only the location (hospital 1) variable has been retained as a significant predictor (P < 0.001) for positive hMAG expression [Table 3]. In this model, the possibility of a breast cancer patient from HUSM expressing hMAG is 34.5%. {Table 3}


The present study showed that the positive reactions for hMAG were detected in more than half of the examined samples. The reported figures of hMAG expression in breast cancer tissues have ranged from 20 to 95%. [4],[12] This broad range may be attributed to several factors, including environmental and genetic factors, in addition to the effects of the preservation methods of tissues, since these may be fresh tissues obtained, or can be preserved frozen, or otherwise paraffin-embedded tissues. Furthermore, the variability in the detection techniques used for assessing the hMAG expression, such as reverse transcriptase-polymerase chain reaction (RT-PCR), IHC or in situ hybridization may be reflected in the results obtained, due to variable sensitivities and specificities. [20],[21],[22]

The mean age of the patients was 52.3 years, similar to findings from the neighboring countries of Indonesia and Thailand. [23] However, breast cancer in western countries is usually diagnosed at a mean age of 60 years. [2] Nevertheless, age did not show a significant association with hMAG expression either in this study or in earlier reports, in spite of the increased risk of development of breast cancer with the longer exposure of a woman to sex hormones. [24],[25],[26],[27],[28] Furthermore, it was found that almost two-thirds of the women had advanced breast cancer (grades II and III) at first diagnosis. Similar findings were reported previously in the state of Kelantan [8],[29] in Malaysia, especially among Malay patients. [3],[30] Breast cancer in western countries is usually diagnosed at early stages. [31] The Malay community comprises more than half of the total population in Malaysia, and this could be the reason for the predominance of Malays in both locations ((MCUKM & HUSM)). The expression of hMAG was more predominant in samples from Malay women (63.2%) than in other ethnic groups (51.9 among Chinese and Indian patients) although this association was statistically not significant. The relatively high hMAG expression among Malay women could be related with genetic, environmental, or even cultural factors. Some unique characteristics are that Malay women have a high fertility rate and there are more Malay women in the younger age groups compared to the other ethnic groups. [32] Although the available data regarding the genetics of breast cancer in Malaysian women are still limited, more focus is shed on environmental and cultural factors in the light of the knowledge that Malay women in Singapore have a lower incidence of breast cancer compared to Chinese and Indian women. [33] This may be the result of social and cultural factors such as the reference to traditional medicine, the negative perception of the disease, low socioeconomic status and poor education, coupled with fear and denial. Health promotion and education provided by the authorities and societies may increase the awareness of women toward the disease. Similarly, and mostly for cultural and religious reasons, breast feeding is practised by Malay women, which may help reduce the incidence of breast cancer. [34] Nevertheless, its relationship with hMAG expression has not been significant, in agreement with the previous findings. [35]

It has been shown previously that women with a family history of breast cancer are at increased risk of the disease. [36],[37],[38] However, the findings in the current study showed that the majority of cases (56.6%) had negative family history of breast cancer. The best model to explain this is that of the multifactorial nature of the disease. [39] However, it is not unusual that some cancer patients would not report their illnesses due to social and cultural influences. [7],[29]

The correlations of hMAG expression with the clinicopathologic factors were generally not remarkable, except for the tendency to increase with the tumor grade, although this particular association has been described with contradictory reports. In these reports, hMAG expression coincided with high tumor grade, [40] with low tumor grade, [25] with no obvious correlation, [4] or even a description of a correlation between the absence of hMAG and high tumor grades. [41] Such contradictions in the findings may point at a possible existence of genetic differences that regulate the expression of hMAG in different populations. A better explanation would rely on the multifactorial nature of breast cancer. [39] This view is further supported by the lack of a solid and agreed upon correlation between hMAG expression and the hormone receptors' status and HER-2 receptors, either in this work or in previous works. [12],[17],[24],[41],[42],[43] Other than that, hMAG remains an issue for study because of its closeness to breast biology, and also because uteroglobin, one of the members of the secretoglobin family, plays a role in the regulation of progesterone. These steroid hormones have been implicated in the development of breast cancer. [14],[44],[45] Hence, the question of the role of the secretoglobin hMAG in breast cancer development remains unanswered and requires more research.

In conclusion , hMAG is a promising marker of breast cancers, as its expression was detected in a substantial number of the examined breast cancer tissues samples. The findings suggest that hMAG has the potential to be used as a diagnostic breast tumor marker, which may, at the same time, serve as a marker of progression of tumors. Moreover, it may serve in the post-therapeutic follow-up. The remark on the differential expression among samples from different locations may imply, at least theoretically, that hMAG expression may be influenced by environmental, genetic or ethnicity-related factors. This work may require further expansion through acquiring a bigger sample size and a proper long-term follow-up of breast cancer patients, to further assess its prognostic value.


This work was supported by a research grant from the Universiti Kebangsaan Malaysia (National University of Malaysia (Research Grant No. 02-01-02-SF0122).


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