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Purpose: Breast cancer affects both genders, but is understudied in men. Although still rare, male breast cancer (MBC) is being diagnosed more frequently. Treatments are wholly informed by clinical studies conducted in women, based on assumptions that underlying biology is similar. Experimental Design: A transcriptomic investigation of male and female breast cancer was performed, confirming transcriptomic data in silico Biomarkers were immunohistochemically assessed in 697 MBCs ( n = 477, training; n = 220, validation set) and quantified in pre- and posttreatment samples from an MBC patient receiving everolimus and PI3K/mTOR inhibitor. Results: Gender-specific gene expression patterns were identified. eIF transcripts were upregulated in MBC. eIF4E and eIF5 were negatively prognostic for overall survival alone (log-rank P = 0.013; HR = 1.77, 1.12-2.8 and P = 0.035; HR = 1.68, 1.03-2.74, respectively), or when coexpressed ( P = 0.01; HR = 2.66, 1.26-5.63), confirmed in the validation set. This remained upon multivariate Cox regression analysis [eIF4E P = 0.016; HR = 2.38 (1.18-4.8), eIF5 P = 0.022; HR = 2.55 (1.14-5.7); coexpression P = 0.001; HR = 7.04 (2.22-22.26)]. Marked reduction in eIF4E and eIF5 expression was seen post BEZ235/everolimus, with extended survival. Conclusions: Translational initiation pathway inhibition could be of clinical utility in MBC patients overexpressing eIF4E and eIF5. With mTOR inhibitors that target this pathway now in the clinic, these biomarkers may represent new targets for therapeutic intervention, although further independent validation is required. Clin Cancer Res; 23(10); 2575-83. ©2016 AACR ., (©2016 American Association for Cancer Research.)

Background: A large proportion of women with lymph node negative breast cancer treated with systemic adjuvant treatment do not benefit from such therapy since the patient is already cured by local treatment. Several studies have suggested that proliferation markers are strong prognostic factors in early breast cancer. Cyclins are probably the most specific markers of cell proliferation. Previously high expression of cyclin E has been associated with breast cancer recurrence., Materials and Methods: In this study we investigate the prognostic value of cyclin E1 in node negative breast cancer patients. In a population-based cohort 186 women who died from breast cancer were defined as cases and 186 women alive at the corresponding time as controls. Inclusion criteria were tumour size ≤ 50 mm, no lymph node metastases and no adjuvant chemotherapy. The study was designed to detect an odds ratio of 2.5 with a power of 90% and significance level of 0.05. Cyclin E1 was determined with immunohistochemistry (IHC) on tissue microarray (TMA)., Results: High expression of cyclin E1 was significantly associated with breast cancer death, in both uni- and multivariate analyses with odds ratios (OR) 2.3 [univariate, 95% confidence interval (CI) 1.5-3.6] and 2.1 (multivariate, 95% CI 1.2-3.5)., Discussion: Cyclin E1 is a strong prognostic factor for breast cancer death in a population-based and node negative patient cohort and can identify high-risk patients in this group.

Background: Although Osteopontin has been known as a marker for cancer progression, the elevated production of this cytokine is not specific for cancer. We have identified the splice variant Osteopontin-c as being absent from healthy tissue but associated with about 75% of breast cancer cases. However, in previous studies of Osteopontin-c, follow-up information was not available., Methods: Here we have analysed 671 patients, comprising a cohort of 291 paraffin blocks plus a population-based case-control study of 380 arrayed breast tumor tissues., Results: We find that high staining intensity of nuclear Osteopontin-c is strongly associated with mortality in patients with early breast cancer. Cytosolic staining for exon 4, reflective of Osteopontin-a and -b also predicts poor outcome. By contrast, total Osteopontin does not correlate with prognosis. These diverse assessments of Osteopontin also do not correlate with each other, suggesting distinct expression patterns for the variant forms. Consistent with its role in tumor progression, not tumor initiation, Osteopontin-c is not correlated with proliferation markers (Ki-67, cyclin A, cyclin B, cyclin E and cyclin D), neither is it correlated with ER, PR or HER2., Conclusions: The addition of Osteopontin-c immunohistochemistry to standard pathology work-ups may have prognostic benefit in early breast cancer diagnosis.

Male breast cancer is poorly understood with a large proportion arising in the familial context particularly with the BRCA2 germline mutation. As phenotypic and genotypic differences between sporadic and familial male breast cancers have been noted, we investigated the importance of a hypoxic drive in these cancers as this pathway has been shown to be of importance in familial female breast cancer. Expression of two major hypoxia-induced proteins, the hypoxia-inducible factor-1α (HIF1A) and the carbonic anhydrase IX (CA9), examined within a large cohort including 61 familial (3 BRCA1, 28 BRCA2, 30 BRCAX) and 225 sporadic male breast cancers showed that 31% of all male breast cancers expressed either HIF1A (25%) and/or CA9 (8%) in the combined cohort. Expression of HIF1A correlated with an increased incidence of a second-major malignancy (P=0.04), histological tumor type (P=0.005) and basal phenotype (P=0.02). Expression of CA9 correlated with age (P=0.004) in sporadic cases and an increased tumor size (P=0.003). Expression of HIF1A was prognostic for disease-specific survival in sporadic male breast cancers (HR: 3.8, 95% CI: 1.5-9.8, P=0.006) but not within familial male breast cancer, whereas CA9 was only prognostic in familial male breast cancers (HR: 358.0, 95% CI: 9.3-13781.7, P=0.002) and not in sporadic male breast cancer. This study found that hypoxic drive is less prevalent in male breast cancer compared with female breast cancer, possibly due to a different breast microenvironment. The prognostic impact of HIF1A is greatest in sporadic male breast cancers with an alternate dominant mechanism for the oncogenic drivers suggested in high risk familial male breast cancers.

Assessment of proliferation is important in female breast cancer and individual treatment decisions are based upon its results, especially in the luminal subgroups. Gene expression analyses fail to group male breast cancer into the intrinsic subgroups previously established in female breast cancer. Even though proliferation has been shown to divide male breast cancer into molecular subgroups with different prognoses, the clinical importance of proliferation markers has not yet been elucidated. Previous studies in male breast cancer have demonstrated contradictory results regarding the prognostic impact of histological grade and Ki-67, parameters strongly associated with proliferation. The aim of the present project was to study proliferation in male breast cancer by assessing other proliferation-related markers viz. cyclins A, B, D1 and mitotic count. A total of 197 male breast cancer cases with accessible paraffin-embedded material and outcome data were investigated. Immunohistochemical stainings were performed on tissue microarrays. Kaplan-Meier estimates and the Cox proportional regression models were used for survival analyses with breast cancer death as the event. The subset of patients with high expression of cyclin A (hazard ratio (HR) 3.7; P=0.001) and B (HR 2.7; P=0.02) demonstrated a poorer survival. Furthermore, high mitotic count was associated with an increased risk of breast cancer death (HR 2.5; P=0.01). In contrast, cyclin D1 overexpression was predictive of better breast cancer survival (HR 0.3; P=0.001). In conclusion, high levels of cyclin A and B expression and an elevated mitotic count result in a two to threefold higher risk for breast cancer death, whereas cyclin D1 overexpression halves the risk. The clinical utility of these proliferation markers needs further elucidation.

Background: Expression of the peptide hormones ghrelin and obestatin has previously been demonstrated in human mammary glands. However, the clinical implications of the expression of these peptides in breast cancer are unclear. The aim of this study was to investigate the potential clinical value of ghrelin and obestatin as breast cancer biomarkers., Methods: A tissue microarray containing breast cancer specimens from 144 patients was immunostained with antibodies directed towards ghrelin and obestatin. Using varying cut-offs, the expression of the two peptides was evaluated and correlated to previously known prognostic factors in breast cancer and to the outcome. Cox regression analysis was used to assess whether these markers may predict survival of breast cancer patients., Results: Moderate to strong immunoreactivity for ghrelin and obestatin was observed in 71.5% and 77.1% of the cases, respectively. Ghrelin and obestatin expression was significantly but weakly correlated to low histological grade, estrogen receptor positivity, small tumor size and low proliferation. Only ghrelin expression was significantly correlated to better recurrence-free and breast cancer-specific survival (HR = 0.3-0.4, p = 0.02-0.05) in both uni- and multivariate analyses. The optimal cut-off was any ghrelin expression versus none. Reproducibility between the two readers was very good for both stainings with kappa values of 0.94-1.00., Conclusions: Patients with tumors expressing ghrelin had 2.5-3 times lower risk for recurrence or breast cancer death than those lacking ghrelin expression. Ghrelin expression is easily assessable with high reproducibility using immunohistochemistry. Further investigations are needed to establish the clinical significance of ghrelin as a biomarker in breast cancer.

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.

A large proportion of women with lymph node negative breast cancer do not benefit from chemotherapy. Proliferation markers have been shown to recognize patients at high risk for recurrence. The Ki67 protein has recently been included in the St Gallen guidelines. The authors investigated the prognostic importance of cyclin B1 in node negative breast cancer and included a study of reproducibility. In a population-based case-control study, 190 women who died from breast cancer were defined as cases and 190 women alive at the time for the corresponding case's death were defined as controls. Inclusion criteria were tumor size

Unlabelled: Patients with low-risk node negative breast cancer have an excellent prognosis with 5% breast cancer mortality at 10 years. However, prognostic factors are needed to identify poor prognostic patients who might benefit from adjuvant systemic therapy. Proliferation has been identified as the most important component of gene expression profiles. Cyclin B is a proliferative marker easily assessed by immunohistochemistry. We wanted to examine cyclin B as a prognostic factor in low-risk breast cancer patients., Patients and Methods: Using an experimental study design, we compared women dying early from their breast cancer (n=17) with women free from relapse more than eight years after initial diagnosis (n=24). All women had stage I, node negative and hormone receptor positive disease. None had received adjuvant chemotherapy. Tumor samples were immunostained for cyclin B using commercial antibodies., Results: The mean percentage of cyclin B (12%) was significantly higher (p=0.001) in women dying from their breast cancer compared with women free from relapse (5%). High cyclin B (> or =9%) identified 11/17 patients dying from breast cancer and low cyclin B identified 22/24 patients free from relapse. The sensitivity and specificity of cyclin B was 65% and 92%, respectively., Discussion: We found that low-risk node negative patients with high expression of cylin B had a significantly worse outcome than patients with low expression of cyclin B. Cyclin B could separate patients with poor survival from those with good survival with 80% accuracy. We suggest that cyclin B might be a potent prognostic factor in this low-risk patient group.

Background: Proliferative markers are not recommended as prognostic factors for clinical use in breast cancer due to lack of standardization in methodology. However, proliferation is driving several gene expression signatures emphasizing the need for a reliable proliferative marker for clinical use. Studies suggest that cyclin A is a prognostic marker with satisfying reproducibility. We investigated cyclin A as a prognostic marker in node-negative breast cancer using previously defined cutoff values., Patients and Methods: In a case-control study, we defined 190 women who died from breast cancer as cases and 190 women alive at the time for the corresponding case's death as controls. Inclusion criteria were tumor size