50 biomarker for prognosis or as a target for molecular ther- apy. In female breast cancer, HER2 is the best described oncogene with frequent ampliﬁcaion. HER2 ampliﬁcation is correlated with poor survival and good response to tar- geted therapy [1, 2]. Other genes, like epidermal growth factor receptor (EGFR), Fibroblast growth factor receptor 1 (FGFR1), topoisomerase IIa (TOP2A) and MYC are also involved in female breast cancer and have prognostic and therapeutic implications [3–6]. Compared to female breast cancer, there is yet little knowledge regarding the genetic makeup of male breast cancer, because male breast cancer is a rare disease and the few available studies are based on small single institutional series [7]. Treatment of male breast cancer has largely been extrapolated from its female counterpart, while there are important differences between male and female breast cancer, with higher ratios of estrogen receptor (ER) and progesterone receptor (PR) positivity in men [8–10]. Also the distribution of molecular subtypes by immunohisto- chemical analysis shows important differences. Luminal type A and B are by far the most frequently encountered subtypes and HER2 driven, basal-like and triple-negative tumors are very rare in men [11, 12]. The few gene expression studies performed recently in men showed that there might be important differences in molecular proﬁle between male and female breast cancer [13–15]. However, the clinical and prognostic signiﬁcance of genetic altera- tions in relevant breast cancer genes still needs to be elu- cidated in male breast cancer. Multiplex ligation-dependent probe ampliﬁcation (MLPA) analysis is a high throughput genomic technique enabling relative quantiﬁcation of copy number or promoter hyper- methylation in a variety of genes in one reaction, based on the simultaneous ampliﬁcation of speciﬁcally hybridized probes on DNA that can be derived from parafﬁn embedded material [16, 17]. We previously showed in female breast cancer that MLPA analysis with a dedicated ‘‘breast cancer kit’’ allows evaluation of copy numbers in 21 important breast cancer genes, providing an overview of the most common ampliﬁ- cations [18]. In the present study, we used MLPA to investi- gate copy number changes of 21 (female) breast cancer related genes in a large group of male breast cancer and correlate these genomic anomalies with clinicopathological features, patients’ outcome, and with previously obtained MLPA data from female breast cancers. Materials and methods Patients: specimens and clinical information All consecutive cases of surgical breast specimens of inva- sive male breast cancer from 1986 to 2010 were collected 123 Breast Cancer Res Treat (2012) 135:49–58 from four different pathology labs in The Netherlands (St. Antonius Hospital Nieuwegein, Diakonessenhuis Utrecht, University Medical Center Utrecht, Laboratory for Pathol- ogy East Netherlands) as described in more detail previously [12]. Hematoxylin and eosin (HE) slides were reviewed by three experienced observers (PJvD, RK, AM) to conﬁrm the diagnosis and to type and grade according to current stan- dards. Pathology reports were used to retrieve information on age, tumor size, and lymph node status. A total of 110 cases from which the parafﬁn blocks contained enough tumor for DNA isolation were included. The age of these patients ranged from 32 to 89 years (average: 66 years). Tumor size ranged from 0.8 to 5.5 cm (average: 2.2 cm). In 86 % lymph node status was known and 55 % of these patients had lymph node metastases. The majority of cases were diagnosed (according to the WHO) as invasive ductal carcinoma (90 %). The remaining cases were lobular (n = 3), mixed type (ductal/lobular) (n = 2), invasive cribriform (n = 1), papillary (n = 1), mucinous (n = 2), invasive micropapillary (n = 1) or adenoid cystic carcino- mas (n = 1). According to the modiﬁed Bloom and Rich- ardson score [19] most tumors were grade 2 (41 %) or grade 3 (36 %). Mitotic activity was assessed as before [20] with a mean mitotic index of 11 per 2 mm2 (range 0–56). For all cases hormone receptor and HER2 status were re-assessed as described previously [12]. Tissue microarray (TMA) slides were used for immunohistochemical stainings for ER, PR and chromogenic in situ hybridization (CISH) for HER2 assessment, the latter showing HER2 ampliﬁcation in only 4/110 cases (4 %). TMA slides were also stained for E-cadherin. Most tumors were ER positive (102/110, 93 %) and PR positivity was also common (71/110; 65 %). Only four cases were E-cadherin negative (three lobular carci- nomas and one ductal carcinoma). DNA extraction and MLPA analysis Representative tumor areas were identiﬁed in HE stained slides and corresponding tumor areas (at least 1 cm2) were dissected with a scalpel from 8 lm parafﬁn slides [21]. DNA was extracted by overnight incubation in proteinase K (10 mg/ml; Roche, Almere, The Netherlands) at 56 C. After boiling for 10 min and centrifugation, 5 ll of this DNA solution was used for MLPA analysis. MLPA was performed according the manufacturers’ instructions (MRC Holland, Amsterdam, The Netherlands), using a Veriti 96-well thermal cycler (Applied Biosystems, Foster City, CA, USA). The P078-B1 kit (MRC Holland), containing 21 breast cancer related genes (Table 1), was used as before [18]. All tests were performed in duplicate. Seven negative references samples (normal breast and blood) were inclu- ded in each MLPA run. The PCR products were separated by electrophoresis on an ABI 3730 capillary sequencer