Gene expression profiling of primary male breast cancers reveals two unique subgroups and identifies N-acetyltransferase-1 (NAT1) as a novel prognostic biomarker

Abstract

Introduction: Male breast cancer (MBC) is a rare and inadequately characterized disease. The aim of the present study was to characterize MBC tumors transcriptionally, to classify them into comprehensive subgroups, and to compare them with female breast cancer (FBC).

Methods: A total of 66 clinicopathologically well-annotated fresh frozen MBC tumors were analyzed using Illumina Human HT-12 bead arrays, and a tissue microarray with 220 MBC tumors was constructed for validation using immunohistochemistry. Two external gene expression datasets were used for comparison purposes: 37 MBCs and 359 FBCs.

Results: Using an unsupervised approach, we classified the MBC tumors into two subgroups, luminal M1 and luminal M2, respectively, with differences in tumor biological features and outcome, and which differed from the intrinsic subgroups described in FBC. The two subgroups were recapitulated in the external MBC dataset. Luminal M2 tumors were characterized by high expression of immune response genes and genes associated with estrogen receptor (ER) signaling. Luminal M1 tumors, on the other hand, despite being ER positive by immunohistochemistry showed a lower correlation to genes associated with ER signaling and displayed a more aggressive phenotype and worse prognosis. Validation of two of the most differentially expressed genes, class 1 human leukocyte antigen (HLA) and the metabolizing gene N-acetyltransferase-1 (NAT1), respectively, revealed significantly better survival associated with high expression of both markers (HLA, hazard ratio (HR) 3.6, P = 0.002; NAT1, HR 2.5, P = 0.033). Importantly, NAT1 remained significant in a multivariate analysis (HR 2.8, P = 0.040) and may thus be a novel prognostic marker in MBC.

Conclusions: We have detected two unique and stable subgroups of MBC with differences in tumor biological features and outcome. They differ from the widely acknowledged intrinsic subgroups of FBC. As such, they may constitute two novel subgroups of breast cancer, occurring exclusively in men, and which may consequently require novel treatment approaches. Finally, we identified NAT1 as a possible prognostic biomarker for MBC, as suggested by NAT1 positivity corresponding to better outcome.

Figure 1

Unsupervised hierarchical clustering (HCL) of male breast cancers based on 1,652 differential expressed genes. (A) HCL revealed two stable subgroups, luminal M1 (right) and luminal M2 (left). Annotations with the prefix Hu indicate the result of the centroid classification based on the Hu genes [21]. Annotations with the prefix cent were derived from the centroid classification with the genes for ER+ luminal female breast cancer (FBC). NHG, Nottingham histological grade. (B) Unsupervised HCL based on co-clustering frequencies revealed two stable subgroups. Co-clustering frequencies close to 1 are red, close to 0 are green and equal to 0.5 are black. (C) Difference in age at diagnosis between the subgroups. (D) Kaplan-Meier survival analysis suggesting better distant metastasis free survival (DMFS) in the luminal M2 subgroup. The numbers below the plot indicate the number of patients at risk in each group at the given time points.

Figure 2

Gene expression (GEX) modules associated with key biological processes. The module scores of GEX modules representing key biological processes involved in FBC tumorigenesis [32] in the two subgroups of MBC (A), in the intrinsic subgroups of FBC (B), and in the MBC validation dataset (C), respectively. Proliferation (Wilcoxon test, P = 0.064), HER2 (Wilcoxon test, P = 0.0057), tumor invasion and metastasis (Wilcoxon test, P = 1.0 × 10^-5), ER (Wilcoxon test, P = 1.3 × 10^-8) and immune response (Wilcoxon test, P = 0.16) displayed a significant or borderline significant difference between the two subgroups of MBC (A). The ANOVA test was used to calculate P-values (B).

Figure 3

Male breast cancer (MBC) subgroup specific genes. Validation of two stable MBC subgroups in an external dataset. The heatmaps of the MBC subgroup-derived centroid genes revealed identical distribution frequencies and similar transcriptional profiles in our dataset (A) and the external validation dataset (B). Red corresponds to up-regulation and green to down-regulation. The MBC sample order was derived from the unsupervised hierarchical clustering and the annotations are from the centroid classification with the MBC subgroup-derived centroid genes.

Figure 4

Kaplan-Meier survival analyses. Distant metastasis free survival of the 220 male breast cancer patients included in the TMA stratified by HLA expression (A) and NAT1 expression (B), respectively. The numbers below the plots indicate the number of patients at risk in each group at the given time points.