Molecular subtyping of breast cancer by gene expression has proven its significance in females. Immunohistochemical surrogates have been used for this classification, because gene expression profiling is not yet routinely feasible. Male breast cancer is rare and large series are lacking. In this study, we used immunohistochemistry for molecular subtyping of male breast cancer. A total of 134 cases of male breast cancer were immunohistochemically stained on tissue microarrays for estrogen receptor (ER), progesterone receptor (PR), HER2 and epidermal growth factor receptor (EGFR), as well as for CK5/6, CK14, and Ki67. HER2 was also assessed by chromogen in situ hybridization. Cases were classified as luminal A (ER+ and/or PR+, and HER2− and Ki67 low), luminal B (ER+ and/or PR+, and HER2+ or Ki67 high), HER2 driven (ER−, PR−, HER2+), basal-like (ER−, PR−, HER2−, CK5/6+ and/or CK14+ and/or EGFR+), or unclassifiable triple-negative (negative for all six markers). Luminal type A was by far the most encountered type of male breast cancers, representing 75% of the cases. Luminal type B was seen in 21% and the remaining 4% of cases were classified as basal-like (n=4) and unclassifiable triple-negative (n=1). No HER2 driven cases were identified. Patients with basal-like cancer were significantly younger (P=0.034). Luminal B type cancers showed significantly higher histological grade (P<0.001), mitotic index (P<0.001), and PR negativity (P=0.005) compared with luminal type A cancers. In conclusion, most male breast cancers are luminal A and luminal B types, whereas basal-like, unclassifiable triple-negative, and HER2 driven male breast cancers are rare. Luminal type B seem to represent a subtype with an aggressive phenotype. This distribution of molecular subtypes in male breast cancer is clearly different compared with female breast cancers, pointing to possible important differences in carcinogenesis.
Male breast cancer is a relatively uncommon disease accounting for <1% of breast cancer incidence.1 Despite the rarity of this disease, mortality and morbidity are nevertheless significant. Men generally present with higher stage compared with their female counterparts, which is thought to be mainly due to early lymph node metastases formation.2, 3, 4, 5 Overall prognosis has been reported to be poor in male breast cancer, but prognosis of male and female breast cancer seems to be similar when adjusted for stage and age.5, 6 Classification and therapy of male breast cancer has largely been extrapolated from female breast cancer, because large clinical series of male breast cancer are lacking. Several small studies showed, however, differences between female and male breast cancer in hormonal expression,4, 7 expression of oncogenes, tumor suppressor genes,7, 8 and molecular profile.9, 10
In female breast cancer, gene expression profile studies have identified several distinctive breast cancer ‘molecular’ subtypes11, 12, 13 As gene expression analysis by microarray is not (yet) routinely feasible, immunohistochemical surrogates have been used for breast cancer classification.14, 15 Using a panel consisting of estrogen receptor (ER), progesterone receptor (PR), Her2neu, CK5/6, CK14, and epidermal growth factor receptor (EGFR), female breast cancers could be classified as luminal (A or B), HER2 driven or basal-like, with prognostic significance.15 It has been proposed to optimize this algorithm by adding Ki67 for more accurate classification of luminal type B breast cancers.16 These distinctive breast cancers subtypes could reflect specific genetic alterations in the progression from progenitor cells to tumor cells, which give rise to, eg, a basal expression program (EGFR amplification, loss of BRCA1) or a luminal program (16q-losses).17
Only a few published reports on small series18, 19 have tried to classify male breast cancer using immunohistochemistry with conflicting results. In this study, we study the molecular subtypes of a large series of male breast cancer by immunohistochemistry in correlation with clinicopathological features.
Materials and methods
All consecutive cases of surgical breast specimens of invasive male breast cancer from 1986–2010 were collected from four different pathology laboratories in The Netherlands (St Antonius Hospital Nieuwegein, Diakonessenhuis Utrecht, University Medical Center Utrecht, Laboratory for Pathology East Netherlands) and two hospitals in Germany (Paderborn and Koeln). Pathology reports were used to extract age, tumor size, and lymph node status, regarding cases with isolated tumor cells as lymph node positive. In total, 134 cases were included.
Hematoxylin and eosin (HE) slides were reviewed by three experienced observers (PJvD, RK, AHJV-M) to confirm the diagnosis and to characterize the tumor. Histological type (WHO), tubule formation, nuclear grade, mitotic activity index according to the protocol described before,20 and histological grade according to the modified Bloom and Richardson score21 were recorded.
Immunohistochemical stainings were performed using tissue microarray blocks. HE stained slides were used to identify representative tumor areas. From these areas three 0.6-mm punch biopsies from formalin-fixed and paraffin-embedded tissue blocks were obtained and embedded in a recipient paraffin block, using a precision tissue array instrument (Beecher Instruments, Westburg). Sections of 4 μm were cut and immunohistochemistry for ER, PR, HER2, CK5/6, CK14, and Ki67 was performed using a Bond-Max autostainer (Leica Microsystems) with the Bond polymer refine detection kit (Leica Microsystems, DS9800). EGFR staining was done manually (Table 1). Appropriate positive and negative controls were used throughout.
Scoring of the immunohistochemical staining was done by consensus of two experienced observers (RK, PJvD) who were unaware of other tumor characteristics or staining results. Mean staining percentages for available punches were used. ER and PR stainings were considered positive if 10% or more cells showed nuclear staining. In addition, we also evaluated the 1% threshold as recommended in the latest American Society of Clinical Oncology/College of American Pathologists guidelines.22 HER2 staining was interpreted according to the DAKO scoring system. Any cytoplasmic staining for CK5/6 or CK1417 and any membrane staining for EGFR23 was scored positive. Ki67 staining was interpreted as low or high using a 14% threshold.16
For triple-negative (ER−, PR−, and HER2−) tumors not showing reactivity for any of the basal markers (CK5/6, CK14, EGFR), whole tumor tissue sections were cut and stained for CK5/6, CK14, and EGFR in order not to miss focal staining due to the limited sampling for a tissue microarray.
HER2 chromogenic in situ hybridization (CISH) was performed and interpreted using the Spot-light HER2 CISH kit (Invitrogen) according to the manufacturer's instructions as before.24 Cases were also considered to be HER2 positive when they were CISH amplified.
The immunohistochemical stainings were used to classify the breast cancer cases into five different subtypes: luminal type A (ER+ and/or PR+, HER2− and Ki67 low), luminal type B (ER+ and/or PR+, and HER2+ and/or Ki67 high), HER2 driven (HER2+ and ER−/PR−), basal-like (ER−/PR−/HER2−, and CK5/6+ and/or CK14+ and/or EGFR+), and unclassifiable triple-negative (negative for all six markers).
Statistical calculations were performed using SPSS for Windows version 15.0. Differences between breast cancer subtypes regarding clinicopathological characteristics were calculated with ANOVA for continuous variables and with Pearson's χ2 (or Fisher's exact test when appropriate) for categorical variables. Significance level was set at P<0.05.
Patients' age ranged from 32 to 89 years (average: 66 years). Tumor size ranged from 0.4 to 5.5 cm (average: 2.13 cm). Lymph node status was known in 83% of cases by axillary lymph node dissection or sentinel node procedure, 54% of these showing lymph node metastases. During the tissue microarray procedure 4 cases were lost, leaving 130 cases. Table 2 shows the biomarker profile for the 130 cases of male breast cancer.
Molecular Sybtyping Using the 10% ER/PR Threshold
Using the 10% ER/PR threshold, most cases were ER positive (123/130, 95%) and PR positive (88/130, 68%). Only four cases (4/130, 3%) showed HER2 overexpression/amplification (three HER2 3+ and CISH amplified, one HER2 2+ and CISH amplified). Expression of the basal markers CK5/6 (12/130, 9%), CK14 (1/130, 1%), and EGFR (15/130, 12%) was also encountered infrequently.
Characteristics according to the immunohistochemically defined molecular subtypes are presented in Table 3, together with the classical pathological features. The vast majority of cases were classified as luminal type A (98/130, 75%), whereas 27/130 (21%) were luminal type B. No HER2 driven cases were identified. The remaining 4% of cases were basal-like (4/130, 3%) or unclassifiable triple-negative (1/130, 1%).
All 27 luminal type B cases were ER positive and only 4 cases showed HER2 amplification, the rest was considered luminal type B because of high Ki67. PR positivity was seen in only 48% luminal B cases, which was significantly less frequent compared with luminal type A tumors (P=0.005). Luminal type B breast cancers were furthermore characterized by little tubule formation (P=0.008), high nuclear grade (P=0.036), high mitotic activity (P<0.001), and consequently high histological grade (P<0.001) compared with luminal type A cancers. There were no differences in age, tumor size, and the presence of lymph node metastasis between luminal type A and B cancers. EGFR positivity was seen in 5/27 (19%) of luminal type B cancers, which was higher than in luminal type A tumors (8/98, 8%), but not significantly (P=0.120). In three of the four cases with HER2 overexpression/amplification, EGFR overexpression was seen as well (P=0.04).
Three basal-like breast cancers showed CK5/6 positivity and one basal-like breast cancer was identified after staining whole tumor tissue sections for EGFR. In one case, an adenoid cystic carcinoma (considered to be low-grade basal), simultaneous expression of all basal markers was seen (Figure 1). The remaining three cases were grade 2–3 carcinomas. In one case, lymph node metastases were present. Patients with the basal-like cancer subtype had an average age of 54 years, which was significantly younger than patients with luminal type A breast cancers (P=0.034) who had a mean age of 67 years.
There was only one unclassifiable triple-negative case, which did not show any expression of basal markers. This tumor was a moderately differentiated ductal carcinoma. There were no such cases which fulfilled the criteria of the HER2-driven subtype. The cases which showed HER2 overexpression/amplification also showed ER and/or PR positivity.
Molecular Sybtyping Using the 1% ER/PR Threshold
Using the 1% ER/PR threshold, a minor shift of male breast cancer cases toward other molecular groups was seen: two basal-like breast cancers and the unclassifiable triple-negative case were between 1–10% ER/PR positive and (being Ki67 low) moved to the luminal type A group, which now comprised 78% of cases (101/130), whereas 27/130 (21%) were luminal type B. No HER2 driven cases were identified. The remaining 1% of cases were basal-like (2/130) and there were no more unclassifiable triple-negative cases. Statistical analyses revealed similar differences between the groups as was found with a 10% cutoff value for ER and PR. Patients with basal-like breast cancer were significant younger (P=0.007) and luminal type B showed a high malignant phenotype with high nuclear (P=0.038) and histological grade (P<0.001), few tubule formation (P=0.012), and high mitotic count (P<0.001) compared with luminal type A tumors. However, luminal type B tumors were not more often PR negative in case 1% staining was regarded positive.
In female breast cancers, molecular subtyping has extensively been studied and proven its significance.11, 12, 25 In male breast cancer, only a few studies have been conducted in this field, which showed conflicting results, because of small groups and different immunohistochemical definitions.18, 19 The present study, one of the largest series of male breast cancer published until now, demonstrates that luminal A and to a lesser extent luminal B types represent the vast majority of breast cancers in men. HER2 driven, basal-like, and unclassifiable triple-negative breast cancers seem to be very rare in men.
Luminal type A was the dominating subtype of male breast cancer representing 75% of the cases using the 10% ER/PR threshold and even 78% using the 1% ER/PR threshold, and is apparently more often encountered in men compared with female breast cancer.12, 16 In female breast cancer, these tumors are associated with older age and postmenopausal status.15 Like in postmenopausal women, there are only low levels of circulating estrogen in males. Most of the estrogen is synthesized in the peripheral tissue and has local effects in a paracrine or autocrine manner, which is important for the development of hormone-dependent breast cancers26, 27 and probably explains the high incidence in males. Other reports also demonstrated high rates of ER-positive male breast carcinomas.4, 5, 7, 18
None of the 130 cases were classified as HER2 driven, as all HER2 positive cases showed ER positivity and were therefore classified as luminal type B. High rate of EGFR positivity in these tumors has been seen before18 and is in line with previous gene expression studies in women.12, 13 This profile may contribute to the higher malignant phenotype of these tumors also reflected by their poor differentiation, high mitotic activity, and more often PR negativity compared with luminal type A tumors. In females, luminal type B breast cancers are associated with local and regional relapse and bad survival compared with luminal type A tumors;12, 16, 25, 28 in male breast cancer, this has yet to be studied. In the present study, we added Ki67 to the standard biomarker panel for a more accurate classification of luminal type B tumors, as this was shown in previous studies16 to improve the immunohistochemical surrogate molecular classification (only 30% of luminal B cancers are HER2 positive). Nevertheless, there is discussion in the literature on the optimal threshold for Ki67, and the 14% threshold that we chose according to Cheang et al.16 did not have optimal sensitivity and specificity in their study. The Ki67 threshold will therefore likely need to be refined in the future.
The frequency of basal-like breast cancer in female breast cancer is around 16%,15 is associated with high-grade tumors,29, 30 younger age,15, 30 BRCA1 mutations,31, 32 and an overall worse prognosis.14, 15 Our study shows that basal-like breast cancer in men is very rare at 3.0%, in line with previous smaller studies, based on immunohistochemistry18 and high-resolution genomic profiling.9 One of our cases was a low-grade basal-like cancer (an adenoid cystic carcinoma) and three were high-grade basal-like with moderate–high nuclear and histological grade.29, 30 The patients with basal-like breast cancers were significantly younger, which is also a characteristic of basal-like breast cancer in females.15, 30 The low incidence of basal-like breast cancer in men could be associated with their relatively high age at time of diagnosis (66 years) compared with women with breast cancer4, 5 and the low frequency of BRCA1 mutations in men.33, 34, 35, 36 As stated, young age and BRCA1 mutations are associated with basal-like breast cancer in females.15, 30, 31, 32
Seemingly, in contrast with our findings, Ciocca et al.,19 identified four basal-like breast cancers in a small group of male breast cancer (n=28), representing 14% of their studied cases. However, in their study a now outdated definition of basal-like breast cancer was used classifying also ER-positive cases with expression of basal markers as basal-like. Only one of their cases with expression of basal markers had no expression of hormone receptors and in fact, according to our definition, basal-like breast cancer was in their study also rare. In our study, we defined basal-like breast cancer as triple-negative tumors (ER−, PR−, and HER2−) with expression of any basal marker (CK5/6, CK14, and/or EGFR), which is currently probably the most pragmatic approach.14, 15, 37
Similar to in previous studies,14, 38, 39, 40 we used tissue microarrays for defining immunohistochemical profiles, in which focal or heterogeneous staining can be missed. To minimize this sampling error for the basal markers, we stained additional whole tumor tissue sections for CK5/6, CK14, and EGFR in case a tumor was triple-negative (ER−, PR−, and HER2−) and did not show any expression of basal makers in the tissue microarray. In these whole tumor tissue sections, one additional case of basal-like breast cancer was identified.
In conclusion, our study, one of the largest series of male breast cancers published until now, demonstrates that luminal type A is by far the most common breast cancer subtype in males. Luminal type B breast cancer is less common and represents a subgroup of ER-positive tumors with highly malignant phenotype. HER2-driven, basal-like and unclassifiable triple-negative breast cancers in men seem to be very rare. The distribution of breast cancers subtypes in men is different compared with females, pointing to possible important differences in carcinogenesis.
Jemal A, Siegel R, Xu J, et al. Cancer statistics, 2010. CA Cancer J Clin 2010;60:277–300.
Joshi MG, Lee AK, Loda M, et al. Male breast carcinoma: an evaluation of prognostic factors contributing to a poorer outcome. Cancer 1996;77:490–498.
Borgen PI, Wong GY, Vlamis V, et al. Current management of male breast cancer. A review of 104 cases. Ann Surg 1992;215:451–457; discussion 7–9.
Anderson WF, Jatoi I, Tse J, Rosenberg PS . Male breast cancer: a population-based comparison with female breast cancer. J Clin Oncol 2010;28:232–239.
Giordano SH, Cohen DS, Buzdar AU, et al. Breast carcinoma in men: a population-based study. Cancer 2004;101:51–57.
Marchal F, Salou M, Marchal C, et al. Men with breast cancer have same disease-specific and event-free survival as women. Ann Surg Oncol 2009;16:972–978.
Muir D, Kanthan R, Kanthan SC . Male versus female breast cancers. A population-based comparative immunohistochemical analysis. Arch Pathol Lab Med 2003;127:36–41.
Andre S, Pinto AE, Laranjeira C, et al. Male and female breast cancer—differences in DNA ploidy, p21 and p53 expression reinforce the possibility of distinct pathways of oncogenesis. Pathobiology 2007;74:323–327.
Johansson I, Nilsson C, Berglund P, et al. High-resolution genomic profiling of male breast cancer reveals differences hidden behind the similarities with female breast cancer. Breast Cancer Res Treat 2011;129:747–760.
Callari M, Cappelletti V, De Cecco L, et al. Gene expression analysis reveals a different transcriptomic landscape in female and male breast cancer. Breast Cancer Res Treat 2011;127:601–610.
Perou CM, Sorlie T, Eisen MB, et al. Molecular portraits of human breast tumours. Nature 2000;406:747–752.
Sorlie T, Perou CM, Tibshirani R, et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci USA 2001;98:10869–10874.
Sorlie T, Tibshirani R, Parker J, et al. Repeated observation of breast tumor subtypes in independent gene expression data sets. Proc Natl Acad Sci USA 2003;100:8418–8423.
Nielsen TO, Hsu FD, Jensen K, et al. Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma. Clin Cancer Res 2004;10:5367–5374.
Carey LA, Perou CM, Livasy CA, et al. Race, breast cancer subtypes, and survival in the Carolina Breast Cancer Study. JAMA 2006;295:2492–2502.
Cheang MC, Chia SK, Voduc D, et al. Ki67 index, HER2 status, and prognosis of patients with luminal B breast cancer. J Natl Cancer Inst 2009;101:736–750.
Korsching E, Jeffrey SS, Meinerz W, et al. Basal carcinoma of the breast revisited: an old entity with new interpretations. J Clin Pathol 2008;61:553–560.
Ge Y, Sneige N, Eltorky MA, et al. Immunohistochemical characterization of subtypes of male breast carcinoma. Breast Cancer Res 2009;11:R28.
Ciocca V, Bombonati A, Gatalica Z, et al. Cytokeratin profiles of male breast cancers. Histopathology 2006;49:365–370.
van Diest PJ, Baak JP, Matze-Cok P, et al. Reproducibility of mitosis counting in 2,469 breast cancer specimens: results from the Multicenter Morphometric Mammary Carcinoma Project. Hum Pathol 1992;23:603–607.
Elston CW, Ellis IO . Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up. Histopathology 1991;19:403–410.
Hammond ME, Hayes DF, Dowsett M, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer (unabridged version). Arch Pathol Lab Med 2010;134:e48–e72.
van der Groep P, Bouter A, van der Zanden R, et al. Re: Germline BRCA1 mutations and a basal epithelial phenotype in breast cancer. J Natl Cancer Inst 2004;96:712–713; author reply 4.
Moelans CB, Kibbelaar RE, van den Heuvel MC, et al. Validation of a fully automated HER2 staining kit in breast cancer. Cell Oncol 2010;32:149–155.
van ‘t Veer LJ, Dai H, van de Vijver MJ, et al. Gene expression profiling predicts clinical outcome of breast cancer. Nature 2002;415:530–536.
Simpson E, Rubin G, Clyne C, et al. The role of local estrogen biosynthesis in males and females. Trends Endocrinol Metab 2000;11:184–188.
Subramanian A, Salhab M, Mokbel K . Oestrogen producing enzymes and mammary carcinogenesis: a review. Breast Cancer Res Treat 2008;111:191–202.
Voduc KD, Cheang MC, Tyldesley S, et al. Breast cancer subtypes and the risk of local and regional relapse. J Clin Oncol 2010;28:1684–1691.
Livasy CA, Karaca G, Nanda R, et al. Phenotypic evaluation of the basal-like subtype of invasive breast carcinoma. Mod Pathol 2006;19:264–271.
Calza S, Hall P, Auer G, et al. Intrinsic molecular signature of breast cancer in a population-based cohort of 412 patients. Breast Cancer Res 2006;8:R34.
Eisinger F, Jacquemier J, Charpin C, et al. Mutations at BRCA1: the medullary breast carcinoma revisited. Cancer Res 1998;58:1588–1592.
Foulkes WD, Stefansson IM, Chappuis PO, et al. Germline BRCA1 mutations and a basal epithelial phenotype in breast cancer. J Natl Cancer Inst 2003;95:1482–1485.
Friedman LS, Gayther SA, Kurosaki T, et al. Mutation analysis of BRCA1 and BRCA2 in a male breast cancer population. Am J Hum Genet 1997;60:313–319.
Stratton MR, Ford D, Neuhasen S, et al. Familial male breast cancer is not linked to the BRCA1 locus on chromosome 17q. Nat Genet 1994;7:103–107.
Tai YC, Domchek S, Parmigiani G, et al. Breast cancer risk among male BRCA1 and BRCA2 mutation carriers. J Natl Cancer Inst 2007;99:1811–1814.
Wolpert N, Warner E, Seminsky MF, et al. Prevalence of BRCA1 and BRCA2 mutations in male breast cancer patients in Canada. Clin Breast Cancer 2000;1:57–63; discussion 4–5.
Rakha E, Reis-Filho JS . Basal-like breast carcinoma: from expression profiling to routine practice. Arch Pathol Lab Med 2009;133:860–868.
Abd El-Rehim DM, Ball G, Pinder SE, et al. High-throughput protein expression analysis using tissue microarray technology of a large well-characterised series identifies biologically distinct classes of breast cancer confirming recent cDNA expression analyses. Int J Cancer 2005;116:340–350.
Rakha EA, El-Sayed ME, Green AR, et al. Breast carcinoma with basal differentiation: a proposal for pathology definition based on basal cytokeratin expression. Histopathology 2007;50:434–438.
van de Rijn M, Perou CM, Tibshirani R, et al. Expression of cytokeratins 17 and 5 identifies a group of breast carcinomas with poor clinical outcome. Am J Pathol 2002;161:1991–1996.
The authors declare no conflict of interest.
About this article
Cite this article
Kornegoor, R., Verschuur-Maes, A., Buerger, H. et al. Molecular subtyping of male breast cancer by immunohistochemistry. Mod Pathol 25, 398–404 (2012). https://doi.org/10.1038/modpathol.2011.174
This article is cited by
Clinicopathologic characteristics and prognosis for male breast cancer compared to female breast cancer
Scientific Reports (2022)
Breast Cancer Research and Treatment (2021)
Virchows Archiv (2021)
Virchows Archiv (2021)
Breast Cancer (2020)