Fetal and Perinatal Brain Autopsy: Useful Macroscopic Techniques Including Agar In-situ and Pre-Embedding Methods.

Fragile perinatal and fetal brains are the rule rather than the exception for developmental neuropathologists. Retrieving the fresh brain from the skull and examining early fetal, macerated or severely hydrocephalic brains after fixation can be a challenge. Textbooks on neurodevelopmental pathology mention these challenges to macroscopic examination of the developing central nervous system only in passing, but many perinatal pathologists recognize this diagnostic problem. We reviewed protocols and publications on the removal, fixation, slicing and sampling of these fetal- and perinatal brains. In addition, we describe a technique to facilitate the removal of severely hydrocephalic brains with very thin cerebral walls from the skull by replacing the intraventricular fluid with agar in-situ. Furthermore, we present a method for post-fixation pre-embedding in agar to facilitate slicing, macroscopic examination and sampling of fragile and macerated brains.

Assessment of tumour proliferation by use of the mitotic activity index, and Ki67 and phosphohistone H3 expression, in early-stage luminal breast cancer.

AIMS: Phosphohistone H3 (PhH3) has been proposed as a novel proliferation marker in breast cancer. This study compares the interobserver agreement for assessment of the mitotic activity index (MAI), Ki67 expression, and PhH3 in a cohort of oestrogen receptor (ER)-positive breast cancer patients. METHODS AND RESULTS: Tumour samples of 159 luminal breast cancer patients were collected. MAI and PhH3 scores were assessed by three breast cancer pathologists. Ki67 scores were assessed separately by two of the three pathologists. PhH3-positive cells were counted in an area of 2 mm2 , with a threshold of ≥13 positive cells being used to discriminate between low-proliferative and high-proliferative tumours. Ki67 expression was assessed with the global scoring method. Ki67 percentages of <20% were considered to be low. The intraclass correlation coefficient (ICC) and Cohen's κ statistics were used to evaluate interobserver agreement. The impact on histological grading of replacing the MAI with PhH3 was assessed. Counting PhH3-positive cells was highly reproducible among all three observers (ICC of 0.86). The κ scores for the categorical PhH3 count (κ = 0.78, κ = 0.68, and κ = 0.80) reflected substantial agreement among all observers, whereas agreement for the MAI (κ = 0.38, κ = 0.52, and κ = 0.26) and Ki67 (κ = 0.55) was fair to moderate. When PhH3 was used to determine the histological grade, agreement in grading increased (PhH3, κ = 0.52, κ = 0.48, and κ = 0.52; MAI, κ = 0.43, κ = 0.35, and κ = 0.32), and the proportion of grade III tumours increased (14%, 18%, and 27%). CONCLUSION: PhH3 seems to outperform Ki67 and the MAI as a reproducible means to measure tumour proliferation in luminal-type breast cancer. Variation in the assessment of histological grade might be reduced by using PhH3, but would result in an increase in the proportion of high-grade cancers.

Prognostic models in male breast cancer.

PURPOSE: Breast cancer in men is uncommon; it accounts for 1 % of all patients with primary breast cancer. Its treatment is mostly extrapolated from its female counterpart. Accurate predictions are essential for adjuvant systemic treatment decision-making and informing patients. Several predictive models are available for female breast cancer (FBC) including the Morphometric Prognostic Index (MPI), Nottingham Prognostic Index (NPI), Adjuvant! Online and Predict. The aim of this study was to examine and compare the prognostic performance of these models for male breast cancer (MBC). METHODS: The population of this study consists of 166 MBC patients. The prognostic scores of the patients are categorized by good, (moderate) and poor, defined by the test itself (MPI and NPI) or based on tertiles (Adjuvant! Online and Predict). Survival according to prognostic score was compared by Kaplan-Meier analysis and differences were tested by logRank. The prognostic performances were evaluated with C-statistics. Calibration was done with the aim to estimate to what extent the survival rates predicted by Predict were similar to the observed survival rates. RESULTS: All prediction models were capable of discriminating between good, moderate and poor survivors. P-values were highly significant. Comparison between the models using C-statistics (n = 88) showed equal performance of MPI (0.67), NPI (0.68), Adjuvant! Online (0.69) and Predict (0.69). Calibration of Predict showed overestimation for MBC patients. CONCLUSION: In conclusion, MPI, NPI, Adjuvant! and Predict prognostic models, originally developed and validated for FBC patients, also perform quite well for MBC patients.

Tracing differences between male and female breast cancer: both diseases own a different biology.

AIMS: Male breast cancer (MBC) is a rare and poorly characterized disease. In the present study we used a novel biomathematical model to further characterize MBC and to identify differences between male and female breast cancer (FBC). METHODS AND RESULTS: A total of 134 cases of MBC were stained immunohistochemically for 13 key oncoproteins, and staining percentages were used in a mathematical model to identify dependency patterns between these proteins. The results were compared with a large group of FBC (n = 728). MBC and FBC clearly differed on the molecular level. In detail, the results suggest a different role for progesterone receptor (PR) compared to oestrogen receptor (ER) in MBC, while in FBC ER and PR show a similar pattern. In addition, Androgen receptor (AR) seems to be a more powerful effector in MBC. Grades 1 and 2 tumours were clearly separated from grade 3 tumours, and luminal types A and B tumours also showed a different pattern. CONCLUSIONS: Defined morphological and molecular phenotypes can be identified in MBC, but these seem to be the result of different molecular mechanisms and perhaps multiple genetic pathways, as characterized previously in FBC, emphasizing the rising concept that MBC and FBC should be regarded as different and unique diseases.

Do columnar cell lesions exist in the male breast?

AIMS: In females, columnar cell lesions (CCLs) have been recognized as putative precursor lesions of low-grade breast cancer, but their role in male breast carcinogenesis is as yet unclear. METHODS AND RESULTS: We reviewed surgical resections from males with breast cancer (n = 89), gynaecomastia (n = 20) and normal breast specimens from autopsies (n = 5) for the presence of CCL. In addition, we performed immunohistochemistry for cytokeratin 5/6 (CK5/6), CK14 and oestrogen receptor alpha (ER). In 19 of 89 resections (two DCIS cases and 17 invasive carcinoma), some individual ducts were found to contain cells with snouts on the luminal border but lacking further typical columnar cell lesion features. We mainly found three-layered ductal epithelium, characteristic for gynaecomastia and confirmed by immunohistochemistry. Moreover, we found a few ducts in male breast cancer sections that were clonally negative for basal cytokeratins. CONCLUSION: We found no lesions with convincing CCL morphology at the periphery of invasive male breast cancers, in gynaecomastia or in normal male breast specimens. Although we cannot completely exclude the existence of CCLs in the male breast, these lesions seem to be very uncommon and are therefore unlikely to play a major role in male breast carcinogenesis.

Analysis of copy number changes on chromosome 16q in male breast cancer by multiplex ligation-dependent probe amplification.

Gene copy number changes have an important role in carcinogenesis and could serve as potential biomarkers for prognosis and targets for therapy. Copy number changes mapping to chromosome 16 have been reported to be the most frequent alteration observed in female breast cancer and a loss on 16q has been shown to be associated with low grade and better prognosis. In the present study, we aimed to characterize copy number changes on 16q in a group of 135 male breast cancers using a novel multiplex ligation-dependent probe amplification kit. One hundred and twelve out of 135 (83%) male breast cancer showed copy number changes of at least one gene on chromosome 16, with frequent loss of 16q (71/135; 53%), either partial (66/135; 49%) or whole arm loss (5/135; 4%). Losses on 16q were thereby less often seen in male breast cancer than previously described in female breast cancer. Loss on 16q was significantly correlated with favorable clinicopathological features such as negative lymph node status, small tumor size, and low grade. Copy number gain of almost all genes on the short arm was also significantly correlated with lymph node negative status. A combination of 16q loss and 16p gain correlated even stronger with negative lymph node status (n=112; P=0.012), which was also underlined by unsupervised clustering. In conclusion, copy number loss on 16q is less frequent in male breast cancer than in female breast cancer, providing further evidence that male breast cancer and female breast cancer are genetically different. Gain on 16p and loss of 16q identify a group of male breast cancer with low propensity to develop lymph node metastases.

Promoter hypermethylation in male breast cancer: analysis by multiplex ligation-dependent probe amplification.

INTRODUCTION: Epigenetic events are, along with genetic alteration, important in the development and progression of cancer. Promoter hypermethylation causes gene silencing and is thought to be an early event in carcinogenesis. The role of promoter hypermethylation in male breast cancer has not yet been studied. METHODS: In a group of 108 male breast cancers, the methylation status of 25 genes was studied using methylation-specific multiplex ligation-dependent probe amplification. Methylation of more than 15% was regarded indicative for promoter hypermethylation. Methylation status was correlated with clinicopathological features, with patients' outcome and with 28 female breast cancer cases. RESULTS: Promoter hypermethylation of the genes MSH6, WT1, PAX5, CDH13, GATA5 and PAX6 was seen in more than 50% of the cases, but was uncommon or absent in normal male breast tissue. High overall methylation status was correlated with high grade (P = 0.003) and was an independent predictor of poor survival (P = 0.048; hazard ratio 2.5). ESR1 and GSTP1 hypermethylation were associated with high mitotic count (P = 0.037 and P = 0.002, respectively) and high grade (both P = 0.001). No correlation with survival was seen for individual genes. Compared with female breast cancers (logistic regression), promoter hypermethylation was less common in a variety of genes, particularly ESR1 (P = 0.005), BRCA1 (P = 0.010) and BRCA2 (P < 0.001). The most frequently hypermethylated genes (MSH6, CDH13, PAX5, PAX6 and WT1) were similar for male and female breast cancer. CONCLUSION: Promoter hypermethylation is common in male breast cancer and high methylation status correlates with aggressive phenotype and poor survival. ESR1 and GSTP1 promoter hypermethylation seem to be involved in development and/or progression of high-grade male breast cancer. Although female and male breast cancer share a set of commonly methylated genes, many of the studied genes are less frequently methylated in male breast cancer, pointing towards possible differences between male and female breast carcinogenesis.

Oncogene amplification in male breast cancer: analysis by multiplex ligation-dependent probe amplification.

Gene amplification is an important mechanism for oncogene activation, a crucial step in carcinogenesis. Compared to female breast cancer, little is known on the genetic makeup of male breast cancer, because large series are lacking. Copy number changes of 21 breast cancer related genes were studied in 110 male breast cancers using multiplex ligation-dependent probe amplification. A ratio of >1.3 was regarded indicative for gene copy number gain and a ratio >2.0 for gene amplification. Data were correlated with clinicopathological features, prognosis and 17 genes were compared with a group of female breast cancers. Gene copy number gain of CCND1, TRAF4, CDC6 and MTDH was seen in >40 % of the male breast cancer cases, with also frequent amplification. The number of genes with copy number gain and several single genes were associated with high grade, but only CCND1 amplification was an independent predictor of adverse survival in Cox regression (p = 0.015; hazard ratio 3.0). In unsupervised hierarchical clustering a distinctive group of male breast cancer with poor prognosis (p = 0.009; hazard ratio 3.4) was identified, characterized by frequent CCND1, MTDH, CDC6, ADAM9, TRAF4 and MYC copy number gain. Compared to female breast cancers, EGFR (p = 0.005) and CCND1 (p = 0.041) copy number gain was more often seen in male breast cancer, while copy number gain of EMSY (p = 0.004) and CPD (p = 0.001) and amplification in general was less frequent. In conclusion, several female breast cancer genes also seem to be important in male breast carcinogenesis. However, there are also clear differences in copy number changes between male and female breast cancers, pointing toward differences in carcinogenesis between male and female breast cancer and emphasizing the importance of identifying biomarkers and therapeutic agents based on research in male breast cancer. In addition CCND1 amplification seems to be an independent prognosticator in male breast cancer.

Prognostic value of automatically extracted nuclear morphometric features in whole slide images of male breast cancer.

Numerous studies have shown the prognostic significance of nuclear morphometry in breast cancer patients. Wide acceptance of morphometric methods has, however, been hampered by the tedious and time consuming nature of the manual segmentation of nuclei and the lack of equipment for high throughput digitization of slides. Recently, whole slide imaging became more affordable and widely available, making fully digital pathology archives feasible. In this study, we employ an automatic nuclei segmentation algorithm to extract nuclear morphometry features related to size and we analyze their prognostic value in male breast cancer. The study population comprised 101 male breast cancer patients for whom survival data was available (median follow-up of 5.7 years). Automatic segmentation was performed on digitized tissue microarray slides, and for each patient, the mean nuclear area and the standard deviation of the nuclear area were calculated. In univariate survival analysis, a significant difference was found between patients with low and high mean nuclear area (P=0.022), while nuclear atypia score did not provide prognostic value. In Cox regression, mean nuclear area had independent additional prognostic value (P=0.032) to tumor size and tubule formation. In conclusion, we present an automatic method for nuclear morphometry and its application in male breast cancer prognosis. The automatically extracted mean nuclear area proved to be a significant prognostic indicator. With the increasing availability of slide scanning equipment in pathology labs, these kinds of quantitative approaches can be easily integrated in the workflow of routine pathology practice.

Molecular subtyping of male breast cancer by immunohistochemistry.

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.

Fibrotic focus and hypoxia in male breast cancer.

Fibrotic focus is a scar-like lesion near the center of a carcinoma and has been associated with high-grade, lymph node metastases and poor survival in female breast cancers. Hypoxia is suggested to be the crucial link between fibrotic focus and aggressive tumor phenotype and is also itself a poor prognostic marker. We here set out to study fibrotic focus and hypoxia in male breast cancer for the first time. In a group of 134 male breast cancer patients, the presence and size of a fibrotic focus and the expression of three hypoxia-related immunohistochemical stainings, hypoxia-inducible factor-1α, carbonic anhydrase IX and Glut-1 were studied in correlation with clinicopathological features and prognosis. Fibrotic focus was seen in 25% of the male breast cancer cases and was correlated with hypoxia-inducible factor-1α overexpression (P=0.023), high grade (P=0.005), high mitotic activity (P=0.005) and lymph node metastases (P=0.037). Hypoxia-inducible factor-1α-positive tumors were more often high grade (P=0.003) and HER2 amplified (P=0.005). Glut-1 expression was also more common in grade 3 tumors (P=0.038), but no association between carbonic anhydrase IX and any clinicopathological feature was found. Fibrotic focus >8 mm and hypoxia-inducible factor-1α overexpression were correlated with decreased patients' outcome (P=0.035 and 0.008, respectively). Hypoxia-inducible factor-1α overexpression was an independent and the most powerful predictor of survival in multivariate analysis (P=0.029; hazard ratio 2.5). In conclusion, the presence of a fibrotic focus is associated with hypoxia-inducible factor-1α overexpression, and both are associated with aggressive tumor phenotype and poor survival in male breast cancer. These markers seem to have similar clinical importance as previously reported in female breast cancer.

Immunophenotyping of male breast cancer.

AIMS: Male breast cancer is a rare disease, and knowledge of carcinogenesis is limited. Conflicting results, based on small series, have been reported for clinically relevant biomarkers. METHODS AND RESULTS: One hundred and thirty-four cases of male breast cancer were immunohistochemically stained on tissue microarrays for oestrogen receptor (ER), progesterone receptor (PR), androgen receptor, human epidermal growth factor receptor 2 (HER2), BRST2, cyclin D1, bcl-2, p53, p16, p21, Ki67, cytokeratin (CK) 5/6, CK14, and epidermal growth factor receptor. Data were correlated with clinicopathological features and patient outcome. High mitotic count and high grade were correlated with high Ki67, HER2 amplification/overexpression, p53 accumulation, high p21 expression, low PR expression, and low bcl-2 expression. PR negativity (P=0.009) and p53 accumulation (P=0.042) were correlated with decreased 5-year survival and were independent markers for patient outcome in Cox regression. In unsupervised hierarchical clustering, four groups were identified that were correlated with distinctive clinicopathological features. The hormone negative/ER-positive/high-grade cluster was significantly associated with decreased survival (P=0.011) and was an independent prognostic factor in Cox regression. CONCLUSIONS: Several tissue biomarkers are associated with an aggressive phenotype in male breast cancer. PR and p53 are the most promising individual prognostic markers. On the basis of immunophenotype, four distinctive and prognostically relevant male breast cancer groups were identified, indicating that protein expression profiling may be clinically useful in male breast cancer.

The molecular genetic make-up of male breast cancer.

Male breast cancer (MBC) is extremely rare and accounts for less than 1% of all breast malignancies. Therefore, clinical management of MBC is currently guided by research on the disease in females. In this study, DNA obtained from 45 formalin-fixed paraffin-embedded (FFPE) MBCs with and 90 MBCs (52 FFPE and 38 fresh-frozen) without matched normal tissues was subjected to massively parallel sequencing targeting all exons of 1943 cancer-related genes. The landscape of mutations and copy number alterations was compared to that of publicly available estrogen receptor (ER)-positive female breast cancers (smFBCs) and correlated to prognosis. From the 135 MBCs, 90% showed ductal histology, 96% were ER-positive, 66% were progesterone receptor (PR)-positive, and 2% HER2-positive, resulting in 50, 46 and 4% luminal A-like, luminal B-like and basal-like cases, respectively. Five patients had Klinefelter syndrome (4%) and 11% of patients harbored pathogenic BRCA2 germline mutations. The genomic landscape of MBC to some extent recapitulated that of smFBC, with recurrent PIK3CA (36%) and GATA3 (15%) somatic mutations, and with 40% of the most frequently amplified genes overlapping between both sexes. TP53 (3%) somatic mutations were significantly less frequent in MBC compared to smFBC, whereas somatic mutations in genes regulating chromatin function and homologous recombination deficiency-related signatures were more prevalent. MDM2 amplifications were frequent (13%), correlated with protein overexpression (P = 0.001) and predicted poor outcome (P = 0.007). In conclusion, despite similarities in the genomic landscape between MBC and smFBC, MBC is a molecularly unique and heterogeneous disease requiring its own clinical trials and treatment guidelines.

Chromosome 17 copy number changes in male breast cancer.

BACKGROUND: Overall, HER2-amplified female breast cancer (FBC) is associated with a high grade, an aggressive phenotype and a poor prognosis. In male breast cancer (MBC) amplification of HER2, located on chromosome 17, occurs at a lower frequency than in FBC, where it is part of complex rearrangements. So far, only few studies have addressed the occurrence of chromosome 17 alterations in small MBC cohorts. METHODS: Multiplex ligation-dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH) were used to detect and characterize copy number changes on chromosome 17 in a cohort of 139 MBC. The results obtained were compared to those in FBC, and were correlated with clinicopathological features and patient outcome data. RESULTS: We observed a lower frequency of chromosome 17 copy number changes with less complex rearrangement patterns in MBC compared to FBC. Chromosome 17 changes in MBC included gains of 17q and losses of 17p. Whole chromosome 17 polyploidies were not encountered. Two recurrent chromosome 17 amplicons were detected: on 17q12 (encompassing the NEUROD2, HER2, GRB7 and IKZF3 gens) and on 17q23.1 (encompassing the MIR21 and RPS6KB1 genes). Whole arm copy number gains of 17q were associated with decreased 5 year survival rates (p = 0.010). Amplification of HER2 was associated with a high tumor grade, but did not predict patient survival. Although copy number gains of HER2 and NEUROD2 were associated with a high tumor grade, a high mitotic count and a decreased 5 year survival rate (p = 0.015), only tumor size and NEUROD2 copy number gains emerged as independent prognostic factors. CONCLUSIONS: In MBC chromosome 17 shows less complex rearrangements and fewer copy number changes compared to FBC. Frequent gains of 17q, encompassing two distinct amplicons, and losses of 17p were observed, but no whole chromosome 17 polyploidies. Only NEUROD2 gains seem to have an independent prognostic impact. These results suggest different roles of chromosome 17 aberrations in male versus female breast carcinogenesis.

Differential expression of growth factor receptors and membrane-bound tumor markers for imaging in male and female breast cancer.

INTRODUCTION: Male breast cancer accounts for 0.5-1% of all breast cancers and is generally diagnosed at higher stage than female breast cancers and therefore might benefit from earlier detection and targeted therapy. Except for HER2 and EGFR, little is known about expression of growth factor receptors in male breast cancer. We therefore investigated expression profiles of growth factor receptors and membrane-bound tumor markers in male breast cancer and gynecomastia, in comparison with female breast cancer. METHODS: Tissue microarrays containing 133 male breast cancer and 32 gynecomastia cases were stained by immunohistochemistry for a panel of membrane-bound targets and compared with data on 266 female breast cancers. RESULTS: Growth factor receptors were variably expressed in 4.5% (MET) up to 38.5% (IGF1-R) of male breast cancers. Compared to female breast cancer, IGF1-R and carbonic anhydrase 12 (CAXII) were more frequently and CD44v6, MET and FGFR2 less frequently expressed in male breast cancer. Expression of EGFR, HER2, CAIX, and GLUT1 was not significantly different between male and female breast cancer. Further, 48.1% of male breast cancers expressed at least one and 18.0% expressed multiple growth factor receptors. Since individual membrane receptors are expressed in only half of male breast cancers, a panel of membrane markers will be required for molecular imaging strategies to reach sensitivity. A potential panel of markers for molecular imaging, consisting of EGFR, IGF1-R, FGFR2, CD44v6, CAXII, GLUT1, and CD44v6 was positive in 77% of male breast cancers, comparable to female breast cancers. CONCLUSIONS: Expression patterns of growth factor receptors and hypoxia membrane proteins in male breast cancer are different from female breast cancer. For molecular imaging strategies, a putative panel consisting of markers for EGFR, IGF1-R, FGFR2, GLUT1, CAXII, CD44v6 was positive in 77% of cases and might be considered for development of molecular tracers for male breast cancer.

Automatic nuclei segmentation in H&E stained breast cancer histopathology images.

The introduction of fast digital slide scanners that provide whole slide images has led to a revival of interest in image analysis applications in pathology. Segmentation of cells and nuclei is an important first step towards automatic analysis of digitized microscopy images. We therefore developed an automated nuclei segmentation method that works with hematoxylin and eosin (H&E) stained breast cancer histopathology images, which represent regions of whole digital slides. The procedure can be divided into four main steps: 1) pre-processing with color unmixing and morphological operators, 2) marker-controlled watershed segmentation at multiple scales and with different markers, 3) post-processing for rejection of false regions and 4) merging of the results from multiple scales. The procedure was developed on a set of 21 breast cancer cases (subset A) and tested on a separate validation set of 18 cases (subset B). The evaluation was done in terms of both detection accuracy (sensitivity and positive predictive value) and segmentation accuracy (Dice coefficient). The mean estimated sensitivity for subset A was 0.875 (±0.092) and for subset B 0.853 (±0.077). The mean estimated positive predictive value was 0.904 (±0.075) and 0.886 (±0.069) for subsets A and B, respectively. For both subsets, the distribution of the Dice coefficients had a high peak around 0.9, with the vast majority of segmentations having values larger than 0.8.

The 3-layered ductal epithelium in gynecomastia.

Gynecomastia is the most common abnormality in the male breast and has been associated with male breast cancer, but whether there is an etiological role remains unknown. In the present study we conducted an immunohistochemical investigation to further characterize gynecomastia. A total of 46 cases of gynecomastia were immunohistochemically stained on tissue microarrays for estrogen receptor (ER), progesterone receptor, HER2, androgen receptor, cytokeratins (CK5, CK14, CK7, and CK8/18), p63, E-cadherin, BRST2, cyclin D1, Bcl-2, p53, p16, p21, and Ki67. In addition, 8 cases of male ductal carcinoma in situ and normal breast tissue obtained from autopsies (n=10) and adjacent to male breast cancer (n=5) were studied. Normal ductal male breast epithelial cells were very often ER and Bcl-2 positive (>69%), and progesterone receptor and androgen receptor expression was also common (>39%). Gynecomastia showed a consistent 3-layered pattern: 1 myoepithelial and 2 epithelial cell layers with a distinctive immunohistochemical staining pattern. The intermediate luminal layer, consisting of vertically oriented cuboidal-to-columnar cells, is hormone receptor positive and expresses Bcl-2 and cyclin D1. The inner luminal layer is composed of smaller cells expressing CK5 and often CK14 but is usually negative for hormone receptors and Bcl-2. Male ductal carcinoma in situ was consistently ER positive and CK5/CK14 negative. In conclusion, for the first time we describe the 3-layered ductal epithelium in gynecomastia, which has a distinctive immunohistochemical profile. These results indicate that different cellular compartments exist in gynecomastia, and therefore gynecomastia does not seem to be an obligate precursor lesion of male breast cancer.