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Abstract Background Male breast cancer (MBC) is an uncommon and relatively uncharacterised disease accounting for BRCA2 mutations. Here we describe clinicopathological features and genomic BRCA1 and BRCA2 mutation status in a large cohort of familial MBCs. Methods Cases (n=60) included 3 BRCA1 and 25 BRCA2 mutation carries, and 32 non-BRCA1/2 (BRCAX) carriers with strong family histories of breast cancer. The cohort was examined with respect to mutation status, clinicopathological parameters including TNM staging, grade, histological subtype and intrinsic phenotype. Results Compared to the general population, MBC incidence was higher in all subgroups. In contrast to female breast cancer (FBC) there was greater representation of BRCA2 tumours (41.7% vs 8.3%, p=0.0008) and underrepresentation of BRCA1 tumours (5.0% vs 14.4%, p=0.0001). There was no correlation between mutation status and age of onset, disease specific survival (DSS) or other clincopathological factors. Comparison with sporadic MBC studies showed similar clinicopathological features. Prognostic variables affecting DSS included primary tumour size (p=0.003, HR:4.26 95%CI 1.63-11.11), age (p=0.002, HR:4.09 95%CI 1.65-10.12), lymphovascular (p=0.019, HR:3.25 95%CI 1.21-8.74) and perineural invasion (p=0.027, HR:2.82 95%CI 1.13-7.06). Unlike familial FBC, the histological subtypes seen in familial MBC were more similar to those seen in sporadic MBC with 46 (76.7%) pure invasive ductal carcinoma of no special type (IDC-NST), 2 (3.3%) invasive lobular carcinomas and 4 (6.7%) invasive papillary carcinoma. A further 8 (13.3%) IDC-NST had foci of micropapillary differentiation, with a strong trend for co-occurrence in BRCA2 carriers (p=0.058). Most tumours were of the luminal phenotype (89.7%), with infrequent HER2 (8.6%) and basal (1.7%) phenotype tumours seen. Conclusion MBC in BRCA1/2 carriers and BRCAX families is different to females. Unlike FBC, a clear BRCA1 phenotype is not seen but a possible BRCA2 phenotype of micropapillary histological subtype is suggested. Comparison with sporadic MBCs shows this to be a high-risk population making further recruitment and investigation of this cohort of value in further understanding these uncommon tumours.

Tumor neovascularization acquires their vessels through a number of processes including angiogenesis, vasculogenesis, vascular remodeling, intussusception, and possibly vascular mimicry in certain tumors. The end result of the tumor vasculature has been quantified by counting the number of immunohistochemically identified microvessels in areas of maximal vascularity, so-called hot spot. Other techniques have been developed such as Chalkley counting and the use of image analysis systems that are robust and reproducible as well as being more objective. Many of the molecular pathways that govern tumor neovascularization have been identified and many reagents are now available to study these tissue sections. These include angiogenic growth factors and their receptors and cell adhesion molecules, proteases, and markers of activated, proliferating, cytokine-stimulated, or angiogenic vessels, such as CD105. It is also possible to differentiate quiescent from active vessels. Other reagents that can identify proteins involved in microenvironmental influences such as hypoxia have also been generated. Although the histological assessment of tumor vascularity is used mostly in the research context, it may also have clinical applications if appropriate methodology and trained observers perform the studies.

Aim: To investigate the cellular and immunophenotypic basis of mammographic density in women at high risk of breast cancer., Methods: Mammograms and targeted breast biopsies were accrued from 24 women at high risk of breast cancer. Mammographic density was classified into Wolfe categories and ranked by increasing density. The histological composition and immunophenotypic profile were quantified from digitized haematoxylin and eosin-stained and immunohistochemically-stained (ERα, ERβ, PgR, HER2, Ki-67, and CD31) slides and correlated to mammographic density., Results: Increasing mammographic density was significantly correlated with increased fibrous stroma proportion (rs (22) = 0.5226, p = 0.0088) and significantly inversely associated with adipose tissue proportion (rs (22) = -0.5409, p = 0.0064). Contrary to previous reports, stromal expression of ERα was common (19/20 cases, 95%). There was significantly higher stromal PgR expression in mammographically-dense breasts (p=0.026)., Conclusions: The proportion of stroma and fat underlies mammographic density in women at high risk of breast cancer. Increased expression of PgR in the stroma of mammographically dense breasts and frequent and unexpected presence of stromal ERα expression raises the possibility that hormone receptor expression in breast stroma may have a role in mediating the effects of exogenous hormonal therapy on mammographic density.

Introduction: DNA methylation is a well-studied biomarker in invasive breast cancer, but its role in ductal carcinoma in situ (DCIS) is less well characterized. The aims of this study are to assess the methylation profile in DCIS for a panel of well-characterized genes that are frequently methylated in breast cancer, to investigate the relationship of methylation with pathological features, and to perform a proof-of-principle study to evaluate the practicality of methylation as a biomarker in diagnostic DCIS material., Methods: Promoter CpG island methylation for a panel of 11 breast cancer-related genes was performed by methylation-sensitive high resolution melting (MS-HRM). Formalin-fixed, paraffin-embedded (FFPE) biopsies from 72 samples of pure DCIS (DCIS occurring in the absence of synchronous invasive carcinoma), 10 samples of mixed DCIS (DCIS adjacent to invasive carcinoma), and 18 samples of normal breast epithelium adjacent to a DCIS lesion were micro-dissected prior to DNA extraction., Results: Methylation was seen for all the tested genes except BRCA1. RASSF1A was the most frequently methylated gene (90% of DCIS samples) and its methylation was associated with comedo necrosis (p = 0.018). Cluster analysis based on the methylation profile revealed four groups, the highly methylated cluster being significantly associated with high nuclear grade, HER2 amplification, negative estrogen receptor (ER) α status, and negative progesterone receptor (PgR) status, (p = 0.038, p = 0.018, p <0.001, p = 0.001, respectively). Methylation of APC (p = 0.017), CDH13 (p = 0.017), and RARβ (p <0.001) was associated with negative ERα status. Methylation of CDH13 (p <0.001), and RARβ (p = 0.001) was associated with negative PgR status. Methylation of APC (p = 0.013) and CDH13 (p = 0.026) was associated with high nuclear grade. Methylation of CDH13 (p = 0.009), and RARβ (p = 0.042) was associated with HER2-amplification., Conclusions: DNA methylation can be assessed in FFPE-derived samples using suitable methodologies. Methylation of a panel of genes that are known to be methylated in invasive breast cancer was able to classify DCIS into distinct groups and was differentially associated with phenotypic features in DCIS.

We have investigated the potential for the p16-cyclin D-CDK4/6-retinoblastoma protein pathway to be exploited as a therapeutic target in melanoma. In a cohort of 143 patients with primary invasive melanoma, we used fluorescence in situ hybridization to detect gene copy number variations (CNVs) in CDK4, CCND1, and CDKN2A and immunohistochemistry to determine protein expression. CNVs were common in melanoma, with gain of CDK4 or CCND1 in 37 and 18% of cases, respectively, and hemizygous or homozygous loss of CDKN2A in 56%. Three-quarters of all patients demonstrated a CNV in at least one of the three genes. The combination of CCND1 gain with either a gain of CDK4 and/or loss of CDKN2A was associated with poorer melanoma-specific survival. In 47 melanoma cell lines homozygous loss, methylation or mutation of CDKN2A gene or loss of protein (p16(INK) (4A) ) predicted sensitivity to the CDK4/6 inhibitor PD0332991, while RB1 loss predicted resistance., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The aim of this study was to compare HER2 amplification, as determined by the HER2 copy number (CN) and the HER2/CEP17 ratio, with protein expression in gastric and gastro-oesophageal junction (G/GOJ) adenocarcinoma.HER2 immunohistochemistry (IHC) and silver in situ hybridisation (SISH) were performed in 185 cases. Modified gastric criteria were used for IHC scoring. HER2 and CEP17 CNs were counted in at least 20 cancer cells and the ratio calculated as per previously defined protocols. These two SISH methods were statistically compared against the different IHC scores.Thirty-four cases showed amplification, by both methods in 29, and either method in five. IHC score was 3+ in 29 cases; 26 showed amplification by both methods, one by ratio only and two were not amplified. IHC score was 2+ in 24 cases; three showed amplification by both methods and two by either. One each of IHC 1+ and 0 showed an increased ratio but not CN. The HER2 CN and ratio for IHC score 3+ compared to scores 2+, 1+ and 0 were significantly different (all p < 0.01). The CN for IHC 2+ vs IHC 1+ and IHC 0 was significantly different (both p < 0.01) but the ratio was not (p = 0.5711 and p = 0.2857, respectively). The CN and the ratio for scores 1+ and 0 were not significantly different (p = 0.9823 and p = 0.9910, respectively).The HER2 CN differentiates between the different IHC scores better than the HER2:CEP17 ratio. Cases that show IHC3+ and high CN may not require calculation of the ratio. Furthermore, consideration should be given to the CN when IHC negative cases appear amplified by the ratio only.

Introduction: Although a substantial proportion of male breast cancers (MBCs) are hereditary, the molecular pathways that are activated are unknown. We therefore examined the frequency and clinicopathological associations of the PIK3CA/mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase (MAPK) pathways and their regulatory genes in familial MBC., Methods: High resolution melting analysis and confirmatory sequencing was used to determine the presence of somatic mutations in PIK3CA (exon 9 and 20), AKT1 (exon 4), KRAS (exon 2) and BRAF (exon 15) genes in 57 familial MBCs. Further analysis of the PIK3CA/mTOR pathway was performed using immunohistochemistry for the pAKT1, pS6 and p4EBP1 biomarkers., Results: PIK3CA somatic mutations were identified in 10.5% (6 of 57) of cases; there were no AKT1, KRAS or BRAF somatic mutations. PIK3CA mutations were significantly more frequent in cancers from BRCAX patients (17.2%, 5/29) than BRCA2 (0%, 0/25) carriers (P = 0.030). Two BRCAX patients had an E547K mutation which has only been reported in one female breast cancer previously. PIK3CA mutation was significantly correlated with positive pS6 (83.3% vs. 32.0%, P = 0.024) and negative p4EBP1 (100% vs. 38.0%, P = 0.006) expression, but not pAKT expression. Expression of nuclear p4EBP1 correlated with BRCA2 mutation carrier status (68.0% vs. 38.7%, P = 0.035)., Conclusions: Somatic PIK3CA mutation is present in familial male breast cancer but absent in BRCA2 carriers. The presence of two of the extremely rare E547K PIK3CA mutations in our cohort may have specific relevance in MBCs. Further study of PIK3CA in MBCs, and in particular BRCAX patients, may contribute to further establishing the relevance of specific PIK3CA mutations in MBC aetiology and in the identification of particular patient groups most likely to benefit from therapeutic targeting with the novel PIK3CA inhibitors that are currently in development.

Aims: A number of clinicopathological features have been attributed to the CD20 positive subset of plasma cell myeloma (PCM). CD20 is an appealing therapeutic target given the success with monoclonal antibody regimens in a spectrum of B cell lymphomas. To date, a small number of reports have described CD20 PCM as a unique subset, and these are not conclusive, especially taking into consideration reporting bias. This study aims to further identify the clinicopathological features of CD20 PCM., Methods: A retrospective analysis of all newly diagnosed PCM between 2003 and 2010 was undertaken. Trephine material was retrieved and reviewed for CD20, and for positive cases an extended immunohistochemical (IHC) panel including cyclin D1 was subsequently performed., Results: The review of our 40 cases and those described in the literature demonstrated that these are heterogeneous with regard to clinical features, morphology, biochemical features, immunophenotype, and cytogenetics., Conclusion: Based on our study and review of the literature, CD20 PCM cases represent a heterogeneous disease and not a unique clinicopathological entity.

Introduction: Basal-like breast cancers behave more aggressively despite the presence of a dense lymphoid infiltrate. We hypothesised that immune suppression in this subtype may be due to T regulatory cells (Treg) recruitment driven by hypoxia-induced up-regulation of CXCR4 in Treg., Methods: Immunoperoxidase staining for FOXP3 and CXCL12 was performed on tissue microarrays from 491 breast cancers. The hypoxia-associated marker carbonic anhydrase IX (CA9) and double FOXP3/CXCR4 staining were performed on sections from a subset of these cancers including 10 basal-like and 11 luminal cancers matched for tumour grade., Results: High Treg infiltration correlated with tumour CXCL12 positivity (OR 1.89, 95% CI 1.22 to 2.94, P = 0.004) and basal phenotype (OR 3.14, 95% CI 1.08 to 9.17, P = 0.004) in univariate and multivariate analyses. CXCL12 positivity correlated with improved survival (P = 0.005), whereas high Treg correlated with shorter survival for all breast cancers (P = 0.001), luminal cancers (P < 0.001) and basal-like cancers (P = 0.040) that were confirmed in a multivariate analysis (OR 1.61, 95% CI 1.02 to 2.53, P = 0.042). In patients treated with hormone therapy, high Treg were associated with a shorter survival in a multivariate analysis (OR 1.78, 95% CI 1.01 to 3.15, P = 0.040). There was a tendency for luminal cancers to show CXCL12 expression (102/138, 74%) compared to basal-like cancers (16/27, 59%), which verged on statistical significance (P = 0.050). Up-regulation of CXCR4 in Treg correlated with the basal-like phenotype (P = 0.029) and tumour hypoxia, as indicated by CA9 expression (P = 0.049)., Conclusions: Our data show that in the setting of hypoxia and CXCR4 up-regulation in Treg, CXCL12 expression may have the negative consequence of enhancing Treg recruitment and suppressing the anti-tumour immune response.