52 number of gene ampliﬁcation and clinicopathological fea- tures were calculated with Spearman’s rho. Unsupervised hierarchical clustering using the statistical program R (www.r-project.org) was performed to identify relevant clusters and co-ampliﬁcation. We used the maximum dis- tance and Ward’s clustering method and calculated the stability of the clusters with pvclust. Logistic regression analysis was performed to compare gene ampliﬁcation in male and female breast cancer, taking signiﬁcant differ- ences in clinicopathological features between the two groups into account. Information regarding prognosis and therapy was requested from the Integral Cancer registration The Netherlands (IKNL). Survival data were available for 101 cases with a mean follow up of 5.7 years. Therefore, survival analysis was based on 5 years survival rates. For univariate survival analysis Kaplan–Meier curves were plotted and analyzed with the log rank test. Multivariate survival analysis was done with Cox regression including the variables that were signiﬁcant in univariate analysis. Results Copy number analysis by MLPA In 4 cases the amount of DNA was insufﬁcient, leaving 106 cases of male breast cancer for further analysis. Gene copy number status of the 21 analyzed genes is presented in Table 1 and Fig. 1. All genes analyzed showed copy number alterations with varying frequencies. The average number of genes with copy number gain was four (range 0–12), of which one (range 0–8) showed ampliﬁcation. Copy number gain was most frequently seen in the genes MTDH (52/106; 49 %) and CCND1 (49/106; 46 %), and these genes were also frequently ampliﬁed (13/106; Fig. 1 Copy number change of 21 genes with corresponding chromosome in 106 male breast cancer patients. Copy number gain (Gain, [1.3); Ampliﬁcation ([2.0); Loss (\0.7) 123 Breast Cancer Res Treat (2012) 135:49–58 12 % and 19/106; 18 % respectively). The genes analyzed on chromosome 8 (FGFR1, ADAM9, IKBKB, PRDM14, MTDH and MYC) were also frequently affected with high rates of copy number gain and ampliﬁcation. Thirteen cases (12 %) showed copy number gain of all genes analyzed on chromosome 8. Also the genes located on chromosome 17 were often affected, particularly TRAF4, CDC6, and BIRC5 with copy number gain in 37, 37 and 26 % of cases, respectively. However, ampliﬁcation of these genes was rare (\4 %). Ampliﬁcation of HER2 was also rare (4/106; 4 %). In ﬁve cases (5 %), all genes analyzed on chromo- some 17 showed copy number gain. In 17 % of cases (18/ 106) no gene copy number changes were found. Losses were rare and seen in only seven genes of which CDH1 (10/106; 9 %) and AURKA (13/106; 12 %) were most frequently affected. Correlation with clinicopathological features Tumors with a copy number gain in one or more genes tended to have a more aggressive phenotype with more mitoses (p = 0.004) and a higher histological grade (p = 0.007) compared to tumors without gene copy num- ber alterations. The number of genes with copy number gain was signiﬁcantly correlated with a high mitotic count (p = 0.001) and a high histological grade (p \ 0.001). Copy number gain in the genes MED1 (p \ 0.001), BIRC5 (p \ 0.001), PRDM14 (p = 0.003), and MTDH (p = 0.003) were signiﬁcantly correlated with high grade male breast cancer. MED1 and HER2 copy number gain were signiﬁcantly correlated with high mitotic count (p \ 0.001 and p = 0.003, respectively). We found trends for other genes, which did not remain signiﬁcant after correction for multiple comparisons (Table 2). Three out of the four tumors with HER2 ampliﬁcation (deﬁned by CISH) also showed HER2 ampliﬁcation using MLPA (p \ 0.001). Loss of CDH1 was not correlated with any clinicopathological feature and loss of the CDH1 gene did not correlate with E-cadherin expression. Comparison with female breast cancer Because breast cancer is a heterogeneous disease only luminal type male and female breast cancers (deﬁned by ER and/or PR expression) were compared. In this approach mitotic count (11 vs 13 mitoses) and grade (37 vs 33 % grade 3 tumors) were quite similar in male and female breast cancers. Only age was signiﬁcantly different, as male breast cancer patients were signiﬁcantly older (p \ 0.001). Figure 2 illustrates gene copy number gain and gene ampliﬁcation in 101 male and 73 female breast cancer cases. EGFR (p = 0.005) and CCND1 (p = 0.041) copy number gain were independent predictors of gender in