54 Fig. 2 Comparison of frequency of copy number gain ([1.3, upper graph) and ampliﬁcation ([2.0, lower graph) of 17 genes between luminal type male and female breast cancer. MBC Male breast cancer, FBC Female breast cancer, Amp ampliﬁcation. Genes signiﬁcantly more affected in men, *genes signiﬁcantly more affected in women B from unsupervised hierarchical clustering had adverse patients’ outcome (p = 0.004). Using a Cox regression analysis, CCND1 ampliﬁcation appeared to be the only single gene which was a predictor of survival aside from grade, mitotic count, and tumor size (p = 0.015; hazard ratio 3.0). When chemotherapy and hormone therapy were included in Cox regression, CCND1 was retained as an independent prognosticator. However, hormone therapy was an independent prognostic factor as well and was correlated with a favorable prognosis (p = 0.004; hazard ratio 0.225). Cluster B tumors (p = 0.009; hazard ratio 3.4) and tumors with copy number gain of all analyzed genes on chromosome 17 (p = 0.005; hazard ratio 4.8) were also independent predictors of poor 123 Breast Cancer Res Treat (2012) 135:49–58 survival. The multivariate models are supplied in supple- mentary format (Supplementary Table 3). Discussion Gene ampliﬁcation is an important mechanism of oncogene activation and is crucial for the development and progres- sion of cancer. The identiﬁcation of frequent copy number change in certain chromosomal regions can lead to iden- tiﬁcation of functional important genes in carcinogenesis, reveal distinctive groups of breast cancer and can be used as prognostic markers. Knowledge of gene proﬁling in male breast cancer is sparse, because male breast cancer is a rare disease and most studies are based on small single institutional series. In the present study we used the high throughput technique MLPA to study gene copy number alterations of 21 breast cancer related genes in a large multi-institutional cohort of 106 male breast cancer patients. The average amount of genes that showed copy number gain was four (range 0–12), of which one (range 0–8) was ampliﬁed. 18 cases (17 %) did not show any copy number change in the studied genes. These 18 cases tended to be low grade cancers with few mitoses and seem to have favorable prognosis compared to male breast cancers with gene copy number gain. The number of genes with copy number gain was correlated with high grade and a high mitotic count. This is in line with female breast cancers, as the genome in high grade female breast cancers is also more rearranged and these patients have a poor outcome [23, 24]. Simultaneous copy number gain of all analyzed genes on chromosome 8, 11, and 17 was seen in 12, 10, and 5 % of the cases, respectively. This points to polysomy or gain of whole chromosome arms, a ﬁnding often seen in male breast cancer [14]. This is interesting, as polysomy of e.g., chromosome 17 has been refuted in female breast cancer [25–30]. In our group of male breast cancer copy number gain of all genes located on chromosome 17 was an independent predictor of adverse prognosis. Using unsupervised hierarchical cluster analysis a small sub-cluster was formed by FGFR1 and ADAM9. In female breast cancer, co-ampliﬁcation of these chromosomal regions is also a common ﬁnding [24, 31]. In addition, two stable clusters of male breast cancer patients were identi- ﬁed with additional prognostic value to classical clinico- pathological prognosticators. HER2 ampliﬁcation deﬁned by MLPA in the present study strongly correlated with HER2 ampliﬁcation status deﬁned by CISH on TMA slides. Small differences found could be due to heterogeneity of tumors which could be missed or overrepresented in TMA slides. We have also previously validated MPLA against CISH and FISH [16].