Gynaecomastia is a benign, non-neoplastic proliferative condition and is the most common lesion in the male breast. Pseudogynaecomastia, or lipomastia, refers to the accumulation of adipose tissue in the male breast without glandular hyperplasia (1). True gynaecomastia can be differentiated from pseudogynaecomastia by the presence of dense subareolar ductal tissue and fibrosis. There are several aetiological factors for true gynaecomastia including physiological causes, such as neonatal, adolescent, and elderly gynaecomastia, resulting from an imbalance between oestrogen stimulation and androgen inhibition (2). Pathological causes of gynaecomastia include Klinefelter’s syndrome, where hypogonadism causes an increased oestradiol-to-testosterone ratio leading to the genesis of gynaecomastia (3). Drugs, obesity, and relative oestrogen excess (e.g., due to liver cirrhosis or prostate cancer) are amongst other causes of gynaecomastia. However, most gynaecomastia cases are regarded as idiopathic with no apparent cause. There is no proven link between gynaecomastia and the development of male breast cancer (4, 5).

The histological features of gynaecomastia are well documented. Microscopically, the early stage of gynaecomastia (florid phase) is characterised by proliferative branching ducts with epithelial and stromal hyperplasia, and the late stage (fibrous phase) is characterised by collagenous stroma, less epithelial proliferation, and decreased vascularity (4).

Little is known about the immunohistochemical profile of male gynaecomastia and how it compares to the female breast due to the lack of large studies to date analysing the expression of putative diagnostic and prognostic markers within this lesion. Therefore, the aim of this study was to analyze the expression of several relevant proteins, including hormone receptors, cytokeratins (luminal and basal types) and proliferation markers in a large, well characterised cohort of surgically resected gynaecomastia specimens.

Ethical approval was sought and granted from the Leeds (East) Research Ethics Committee (reference number 06/Q1205/156). Male patients who had undergone surgical excision for gynaecomastia at the Leeds Teaching Hospitals NHS Trust, during the period between 2000 to early 2011 were identified from the pathology database. Histological sections of those patients were collected and reviewed by two investigators (AB, AMS) to confirm the diagnosis, identify the morphological appearances, classify into early or late stage, and mark representative areas for tissue microarray (TMA) construction. Comprehensive clinical details were collected including patient age, presentation, laterality, history of previous medications, history of previous cancer and family history where available.

Research into male breast lesions has been limited compared with the female breast. Previous, generally small scale, studies on immunohistochemistry of gynaecomastia yielded conflicting results. Here, we detail the immunohistochemical profile of a large series of male breast gynaecomastia lesions represented on TMAs and the association between different protein markers.

Hormones have long been implicated in the pathogenesis of gynaecomastia and endocrine treatment, including androgens, anti-oestrogens, and aromatase inhibitors are currently in use for the medical management of those lesions (14, 15). When gynaecomastia persists after a period of reassurance and observation and/or discontinuation of exposure, Tamoxifen (a selective Estrogen receptor modulator, SERM) is the first medical treatment option (14). Tamoxifen is known to reduce pain and size of gynaecomastia. Examples of lesions that have completely resolved following Tamoxifen treatment have also been reported (16). Danazol, a weak androgen, has been also used but is less effective than Tamoxifen and may indeed exacerbate gynaecomastia due to its conversion to oestrogen and/or the resultant weight gain (17).

We report a predominance of various types of nuclear steroid receptors in gynaecomastia with over two thirds of the lesions expressing androgen receptor. A previous small study reported 80% AR positivity in those lesions (18). An earlier study reported AR positivity in 100% of gynaecomastia samples, compared to 87% positivity in male breast carcinoma (19). A strong correlation between ERα and AR was noted in this study (0.499, p<0.001), which is similar to a previously recorded close association noted between the two markers (p<0.01) (19).

AR immunoreactivity has been shown to range between 38% and 81% in MBC (20) and the presence of it has shown to correlate with better overall survival in MBC (21). The AR expression shown in this study supports the use of non-aromatising androgen therapies, such as dihydrotestosterone, which has shown good response rates in patients with prolonged pubertal gynaecomastia (22).

One intriguing finding in our study is the inverse correlation between ERα expression and age with a decrease in its expression with the increase in patient’s age. To our knowledge, this is a novel finding and is different from the female breast where ERα expression has been shown to increase in mammary epithelium of post-menopausal women, likely a response to decreased circulating levels of oestrogen and increased sensitivity of the receptors (23, 24). The reason for this finding in the male breast awaits further studies but may be due to the relative increase of serum estradiol relative to androgen in elderly men or disturbances in the local breast tissue response to estrogen due to the decreased inhibitory effect of androgen as a result of aging. Table 6 summarises the expression of hormone receptors in non-neoplastic and malignant breast lesions in men and women as reported in the literature.

Within our current study, 99% of samples were deemed ERα positive. High percentages of ERα positivity have been documented in both gynaecomastia and MBC at 100% and 87% expression respectively (30). Cases of MBC are often found to have a high percentage of ERα expression compared to female breast cancer (83% and 68% respectively) (30).

Another novel finding is the high expression of ERß isoforms, ERß1 and ERß2, in all the gynaecomastia lesions examined. In addition to the localisation in the lesional epithelial cells, ERß isoforms were also expressed in the stromal cells suggesting a role of ERß in the pathogenesis of gynaecomastia. Nicoletti et al. confirmed high levels of ERß RNA expression in primary cultured cells from 50 examples of male pubertal gynaecomastia including in stromal cells (31). They concluded that the data support a role of ERß in the pathophysiology of pubertal gynaecomastia. However, that same study noted a significant inverse correlation between AR and ERα (p<0.01) in carcinoma (31). An inverse correlation was also noted between AR and PR (p<0.01) in carcinoma cases. In contrast, our study noted a significant positive correlation between AR and PR (0.281, p=0.005), indicating that the relationship between biomarkers within gynaecomastia is distinctly different from malignant male breast lesions.

Little information is available on the basal and luminal cytokeratin expression in gynaecomastia. Here, we confirm the previously reported tri-layered pattern of expression of outer and inner positive basal cells enclosing a middle luminal layer. Within this tri-layer epithelium of gynaecomastia lesions, CK5/6 and CK14 are commonly expressed, 67% and 21% respectively in the inner luminal layer, 6% and 1% respectively in the intermediate luminal layer, and 84% and 99% respectively in the outer myoepithelial layer². Our study showed similar results of 68.9% expression of CK5/6 and 57% expression of CK14 within glandular epithelium. Basal cytokeratins therefore can be used in the diagnostic setting to confirm hyperplasia. In the female usual ductal hyperplasia, the expression of basal cytokeratins occurs in a heterogeneous/mosaic fashion with positive basal like cells admixed with negative luminal cells (4). Awareness of the different patterns of the immunohistochemical expression of CK5/6 and CK14 in the male and female hyperplastic lesions is important to avoid mistaking the tri-layered pattern in male breast for Pagetoid spread/atypia. It is of note that, in male breast cancer, a basal profile is extremely uncommon (5, 32, 33).

In conclusion, we report on the morphological and immunohistochemical features of a large cohort of male breast gynaecomastia lesions received in a large single institution over 11 year period. The majority of the lesions showed hyperplasia, exhibited high levels of oestrogen and progesterone receptors as well as androgen receptors. We confirm, for the first time, the abundance of ERß isoforms, both in the epithelial and stromal elements, and the negative association between ERα and age. The findings suggest a major role of hormonal factors in the pathogenesis of gynaecomastia and the value of anti-hormonal therapy in the medical management of this common condition. Using hormone receptors basal cytokeratins and ki67 can help differentiate florid examples of hyperplastic male breast lesions from the malignant mimics.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by Leeds (East) Research Ethics Committee (reference number 06/Q1205/156). Written informed consent from the participants’ legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.

PP: analyzed the data and wrote the first draft; AB: performed the laboratory work, collected patient data, analyzed the data and contributed to writing up; VS: ethical approval, contributed to writing up; AS: conceived the idea, supervised experimental work, oversaw writing up. All authors approved the final manuscript.

AS is supported by Birmingham Cancer Research UK Centre grant C17422/A25154.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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