Estrogen receptor 1 chromatin profiling in human breast tumors reveals high inter-patient heterogeneity with enrichment of risk SNPs and enhancer activity at most-conserved regions.

Estrogen Receptor 1 (ESR1; also known as ERα, encoded by ESR1 gene) is the main driver and prime drug target in luminal breast cancer. ESR1 chromatin binding is extensively studied in cell lines and a limited number of human tumors, using consensi of peaks shared among samples. However, little is known about inter-tumor heterogeneity of ESR1 chromatin action, along with its biological implications. Here, we use a large set of ESR1 ChIP-seq data from 70 ESR1+ breast cancers to explore inter-patient heterogeneity in ESR1 DNA binding to reveal a striking inter-tumor heterogeneity of ESR1 action. Of note, commonly shared ESR1 sites show the highest estrogen-driven enhancer activity and are most engaged in long-range chromatin interactions. In addition, the most commonly shared ESR1-occupied enhancers are enriched for breast cancer risk SNP loci. We experimentally confirm SNVs to impact chromatin binding potential for ESR1 and its pioneer factor FOXA1. Finally, in the TCGA breast cancer cohort, we can confirm these variations to associate with differences in expression for the target gene. Cumulatively, we reveal a natural hierarchy of ESR1-chromatin interactions in breast cancers within a highly heterogeneous inter-tumor ESR1 landscape, with the most common shared regions being most active and affected by germline functional risk SNPs for breast cancer development.

The Diagnostic Value of microRNA Expression Analysis in Detecting Intraductal Papillomas in Patients with Pathological Nipple Discharge.

Patients with pathological nipple discharge (PND) often undergo local surgical procedures because standard radiologic imaging fails to identify the underlying cause. MicroRNA (MiRNA) expression analysis of nipple fluid holds potential for distinguishing between breast diseases. This study aimed to compare miRNA expression levels between nipple fluids from patients with PND to identify possible relevant miRNAs that could differentiate between intraductal papillomas and no abnormalities in the breast tissue. Nipple fluid samples from patients with PND without radiological and pathological suspicion for malignancy who underwent a ductoscopy procedure were analyzed. We used univariate and multivariate regression analyses to identify nipple fluid miRNAs differing between pathologically confirmed papillomas and breast tissue without abnormalities. A total of 27 nipple fluid samples from patients with PND were included for miRNA expression analysis. Out of the 22 miRNAs examined, only miR-145-5p was significantly differentially expressed (upregulated) in nipple fluid from patients with an intraductal papilloma compared to patients showing no breast abnormalities (OR 4.76, p = 0.046), with a diagnostic accuracy of 92%. miR-145-5p expression in nipple fluid differs for intraductal papillomas and breast tissue without abnormalities and, therefore, has potential as a diagnostic marker to signal presence of papillomas in PND patients. However, further refinement and validation in clinical trials are necessary to establish its clinical applicability.

Analytical Validation of a Novel 6-Gene Signature for Prediction of Distant Recurrence in Estrogen Receptor-Positive, HER2-Negative, Early-Stage Breast Cancer.

BACKGROUND: OncoMasTR is a recently developed multigene prognostic test for early-stage breast cancer. The test has been developed in a kit-based format for decentralized deployment in molecular pathology laboratories. The analytical performance characteristics of the OncoMasTR test are described in this study. METHODS: Expression levels of 6 genes were measured by 1-step reverse transcription-quantitative PCR on RNA samples prepared from formalin-fixed, paraffin-embedded (FFPE) breast tumor specimens. Assay precision, reproducibility, input range, and interference were determined using FFPE-derived RNA samples representative of low and high prognostic risk scores. A pooled RNA sample derived from 6 FFPE breast tumor specimens was used to establish the linear range, limit of detection, and amplification efficiency of the individual gene expression assays. RESULTS: The overall precision of the OncoMasTR test was high with an SD of 0.16, which represents less than 2% of the 10-unit risk score range. Test results were reproducible across 4 testing sites, with correlation coefficients of 0.94 to 0.96 for the continuous risk score and concordance of 86% to 96% in low-/high-risk sample classification. Consistent risk scores were obtained across a > 100-fold RNA input range. Individual gene expression assays were linear up to quantification cycle values of 36.0 to 36.9, with amplification efficiencies of 80% to 102%. Test results were not influenced by agents used during RNA isolation, by low levels of copurified genomic DNA, or by moderate levels of copurified adjacent nontumor tissue. CONCLUSION: The OncoMasTR prognostic test displays robust analytical performance that is suitable for deployment by local pathology laboratories for decentralized use.

Patient-centered research: how do women tolerate nipple fluid aspiration as a potential screening tool for breast cancer?

BACKGROUND: Nipple fluid aspiration (NFA) is a technique to acquire nipple aspirate fluid (NAF), which is considered a rich source of breast-specific biomarkers. Originating directly from the mammary ducts, this liquid biopsy can offer insight into the process of carcinogenesis at its earliest stage and therefore could be of added value to the current imaging-based breast cancer screening tools. With that in mind, it is necessary to know how well NFA is tolerated. AIM: To evaluate the participants' tolerability of NFA compared to breast imaging screening methods and blood draws. MATERIALS AND METHODS: Three cohorts of women underwent NFA: healthy women (n = 190), women diagnosed with breast cancer (n = 137) and women at high risk of developing breast cancer (n = 48). A 0-10 discomfort score of NFA, mammography, breast MRI and blood draws, was filled in at the study visits, which took place once or annually. RESULTS: The median discomfort rate of NFA was 1, which was significantly lower than the median discomfort of mammography and breast MRI (5 and 3, respectively, p < 0.001), but significantly higher than median discomfort for blood draws (0, p < 0.001). The great majority of women would undergo the procedure again (98%) and recommend it to others (97%). CONCLUSION: This study shows that NFA was well tolerated by healthy women, women diagnosed with breast cancer and high-risk women. This makes NFA a feasible method to pursue as a potential future breast cancer early detection tool, based on resident biomarkers. TRIAL REGISTRATION: NL41845.041.12 , NL57343.041.16 and NL11690.041.06 in trialregister.nl.

The Physiological MicroRNA Landscape in Nipple Aspirate Fluid: Differences and Similarities with Breast Tissue, Breast Milk, Plasma and Serum.

BACKGROUND: MicroRNAs (miRNAs) target 60% of human messenger RNAs and can be detected in tissues and biofluids without loss of stability during sample processing, making them highly appraised upcoming biomarkers for evaluation of disease. However, reporting of the abundantly expressed miRNAs in healthy samples is often surpassed. Here, we characterized for the first time the physiological miRNA landscape in a biofluid of the healthy breast: nipple aspirate fluid (NAF), and compared NAF miRNA expression patterns with publically available miRNA expression profiles of healthy breast tissue, breast milk, plasma and serum. METHODS: MiRNA RT-qPCR profiling of NAF (n = 41) and serum (n = 23) samples from two healthy female cohorts was performed using the TaqMan OpenArray Human Advanced MicroRNA 754-Panel. MiRNA quantification data based on non-targeted or multi-targeted profiling techniques for breast tissue, breast milk, plasma and serum were retrieved from the literature by means of a systematic search. MiRNAs from each individual study were orderly ranked between 1 and 50, combined into an overall ranking per sample type and compared. RESULTS: NAF expressed 11 unique miRNAs and shared 21/50 miRNAs with breast tissue. Seven miRNAs were shared between the five sample types. Overlap between sample types varied between 42% and 62%. Highly ranked NAF miRNAs have established roles in breast carcinogenesis. CONCLUSION: This is the first study to characterize and compare the unique physiological NAF-derived miRNA landscape with the physiological expression pattern in breast tissue, breast milk, plasma and serum. Breast-specific sources did not mutually overlap more than with systemic sources. Given their established role in carcinogenesis, NAF miRNA assessment could be a valuable tool in breast tumor diagnostics.

Application of Nipple Aspirate Fluid miRNA Profiles for Early Breast Cancer Detection and Management.

The authors of the recently published review, "Why the Gold Standard Approach by Mammography Demands Extension by Multiomics [...].

Role of columnar cell lesions in breast carcinogenesis: analysis of chromosome 16 copy number changes by multiplex ligation-dependent probe amplification.

Columnar cell lesions have been proposed as precursor lesions of low-grade breast cancer. The molecular characteristic of low-grade breast neoplasia is whole-arm loss of chromosome 16q. Copy number changes of 6 genes on 16p and 20 genes on 16q were analysed by multiplex ligation-dependent probe amplification in 165 lesions of 103 patients. Twenty-three columnar cell lesions and 19 atypical ducal hyperplasia lesions arising in columnar cell lesions were included, as well as cases of usual ductal hyperplasia, blunt duct adenosis, ductal carcinoma in situ, lobular neoplasia and invasive carcinoma. Usual ductal hyperplasia and blunt duct adenosis lacked whole-arm losses of 16q. In contrast, columnar cell lesions without atypia, columnar cell lesions with atypia, atypical ductal hyperplasia, low-grade ductal carcinoma in situ and low-grade invasive carcinomas increasingly harboured whole-arm losses of 16q (17%, 27%, 47% and 57%, respectively). However, no recurrent losses in specific genes could be identified. In several patients, columnar cell lesions and atypical ductal hyperplasia harboured similar losses as related ductal carcinoma in situ or invasive carcinomas within the same breast. There were indications for 16q breakpoints near the centromere. Whole-arm gains on 16p were relatively scarce and there was no relation between whole-arm gains of 16p and progression of lesions of the low-grade breast neoplasia family. In conclusion, columnar cell lesions (with and without atypia) often harbour whole-arm losses of 16q, which underlines their role as precursors in low-grade breast carcinogenesis, in contrast with usual ductal hyperplasia and blunt duct adenosis. However, no recurrent losses in specific genes could be identified, pointing to minor events in multiple tumour suppressor genes rather than major events in a single 16q gene contributing to low-grade breast carcinogenesis.

BRCA promoter methylation in sporadic versus BRCA germline mutation-related breast cancers.

BACKGROUND: In breast cancer, BRCA promoter hypermethylation and BRCA germline mutations are said to occur together rarely, but this property has not yet been translated into a clinical test. Our aim in this study was to investigate the diagnostic value of BRCA1/2 methylation in distinguishing breast carcinomas of BRCA1 and BRCA2 germline mutation carriers from sporadic breast carcinomas using a recently developed BRCA methylation assay based on methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). METHODS: MS-MLPAs were performed to assess BRCA1 and BRCA2 methylation in breast carcinoma tissues from 39 BRCA1 and 33 BRCA2 germline mutation carriers, 80 patients with sporadic breast cancer, and normal breast tissues from 5 BRCA1 and 4 BRCA2 mutation carriers and 5 nonmutation carriers. RESULTS: Methylation frequencies varied considerably between CpG sites across the BRCA1 and BRCA2 promoters. Some CpG sites were methylated more frequently in BRCA1/2-related than in sporadic carcinomas, whereas other CpG sites were methylated more frequently in sporadic carcinomas, with large variances in sensitivity and specificity as a consequence. CONCLUSIONS: The diagnostic value of BRCA promoter methylation analysis in distinguishing BRCA1/2-related from sporadic breast carcinomas seems to be considerably dependent on the targeted CpG sites. These findings are important for adequate use of BRCA methylation analysis as a prescreening tool for BRCA germline genetic testing or to identify BRCAness patients who may benefit from targeted therapies such as poly(adenosine diphosphate-ribose) polymerase inhibitors.

Influence of decalcification procedures on immunohistochemistry and molecular pathology in breast cancer.

Distant breast cancer metastases are nowadays routinely biopsied to reassess receptor status and to isolate DNA for sequencing of druggable targets. Bone metastases are the most frequent subgroup. Decalcification procedures may negatively affect antigenicity and DNA quality. We therefore evaluated the effect of several decalcification procedures on receptor status and DNA/RNA quality. In 23 prospectively collected breast tumors, we compared ERα, PR and HER2 status by immunohistochemistry in (non-decalcified) tissue routinely processed for diagnostic purposes and in parallel tissue decalcified in Christensen's buffer with and without microwave, EDTA and Formical-4. Furthermore, HER2 fluorescence in situ hybridization and DNA/RNA quantity and quality were assessed. We found that the percentage of ERα-positive cells were on average lower in EDTA (P=0.049) and Formical-4 (P=0.047) treated cases, compared with controls, and PR expression showed decreased antigenicity after Christensen's buffer treatment (P=0.041). Overall, a good concordance (weighted kappa) was seen for ERα, PR and HER2 immunohistochemistry when comparing the non-decalcified control tissues with the decalcified tissues. For two patients (9%), there was a potential influence on therapeutic decision making with regard to hormonal therapy or HER2-targeted therapy. HER2 fluorescence in situ hybridization interpretation was seriously hampered by Christensen's buffer and Formical-4, and DNA/RNA quantity and quality were decreased after all four decalcification procedures. Validation on paired primary breast tumor specimens and EDTA-treated bone metastases showed that immunohistochemistry and fluorescence in situ hybridization were well assessable and DNA and RNA yield and quality were sufficient. With this, we conclude that common decalcification procedures have only a modest negative influence on hormone and HER2 receptor immunohistochemistry in breast cancer. However, they may seriously affect DNA/RNA-based diagnostic procedures. Overall, EDTA-based decalcification is therefore to be preferred as it best allows fluorescence in situ hybridization and DNA/RNA isolation.

Promoter hypermethylation profiling of distant breast cancer metastases.

Promoter hypermethylation of tumor suppressor genes seems to be an early event in breast carcinogenesis and is potentially reversible. This makes methylation a possible therapeutic target, a marker for treatment response and/or a prognostic factor. Methylation status of 40 tumor suppressor genes was compared between 53 primary breast tumors and their corresponding metastases to brain, lung, liver, or skin. In paired analyses, a significant decrease in methylation values was seen in distant metastases compared to their primaries in 21/40 individual tumor suppressor genes. Furthermore, primary tumors that metastasized to the liver clustered together, in line with the finding that primary breast carcinomas that metastasized to the brain, skin, or lung, showed higher methylation values in up to 27.5 % of tumor suppressor genes than primary carcinomas that metastasized to the liver. Conversion in methylation status of several genes from the primary tumor to the metastasis had prognostic value, and methylation status of some genes in the metastases predicted survival after onset of metastases. Methylation levels for most of the analyzed tumor suppressor genes were lower in distant metastases compared to their primaries, pointing to the dynamic aspect of methylation of these tumor suppressor genes during cancer progression. Also, specific distant metastatic sites seem to show differences in methylation patterns, implying that hypermethylation profiles of the primaries may steer site-specific metastatic spread. Lastly, methylation status of the metastases seems to have prognostic value. These promising findings warrant further validation in larger patient cohorts and more tumor suppressor genes.

Clonal intratumor heterogeneity of promoter hypermethylation in breast cancer by MS-MLPA.

Intratumor heterogeneity may lead to sampling bias and may present major challenges to personalized medicine and biomarker development. Despite many studies investigating genetic heterogeneity, epigenetic intratumor heterogeneity of promoter hypermethylation has only rarely been examined in breast cancer. To examine clonal intratumor heterogeneity of promotor hypermethylation, we performed methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) for 24 established tumor-suppressor genes on multiple spatially separated samples obtained from 21 primary breast carcinomas. Multiregion analysis was performed, representing at least two and a maximum of five tumor blocks per patient and four areas per tumor block. Methylation results were heterogeneous at one or more genetic loci between different tumor regions in 95% of breast carcinomas. The most heterogeneous loci in decreasing frequency were RASSF1A (62%), CDKN2B (43%), APC (38%), GSTP1 (33%), CDH13 (24%), DAPK1 (19%), and CDKN1B (5%). Heterogeneity lead to a methylation status change in at least one locus in 65% of the tumors. For most genes, the relative contribution of between-patients and between-block variability to the total variation in methylation results was similar. Regardless of the gene, contribution of within-block variability was of little importance. In conclusion, although most variation in methylation status is present between individual breast cancers, clonal epigenetic heterogeneity is seen within most primary breast carcinomas, indicating that methylation results from a single random sample may not be representative of the whole tumor.

Upregulation of Claudin-4, CAIX and GLUT-1 in distant breast cancer metastases.

BACKGROUND: Several studies have shown that the immunophenotype of distant breast cancer metastases may differ significantly from that of the primary tumor, especially with regard to differences in the level of hormone receptor protein expression, a process known as receptor conversion. This study aimed to compare expression levels of several membrane proteins between primary breast tumors and their corresponding distant metastases in view of their potential applicability for molecular imaging and drug targeting. METHODS: Expression of Claudin-4, EGFR, CAIX, GLUT-1 and IGF1R was assessed by immunohistochemistry on tissue microarrays composed of 97 paired primary breast tumors and their distant (non-bone) metastases. RESULTS: In both the primary cancers and the metastases, Claudin-4 was most frequently expressed, followed by GLUT-1, CAIX and EGFR.From primary breast cancers to their distant metastases there was positive to negative conversion, e.g. protein expression in the primary tumor with no expression in its paired metastasis, in 6%, 19%, 12%, 38%, and 0% for Claudin-4 (n.s), GLUT-1 (n.s), CAIX (n.s), EGFR (n.s) and IGF1R (n.s) respectively. Negative to positive conversion was seen in 65%, 47%, 43%, 9% and 0% of cases for Claudin-4 (p = 0.049), GLUT-1 (p = 0.024), CAIX (p = 0.002), EGFR (n.s.) and IGF1R (n.s.) respectively. Negative to positive conversion of Claudin-4 in the metastasis was significantly associated with tumor size (p = 0.015), negative to positive conversion of EGFR with negative PR status (p = 0.046) and high MAI (p = 0.047) and GLUT-1 negative to positive conversion with (neo)adjuvant chemotherapy (p = 0.039) and time to metastasis formation (p = 0.034). CAIX and GLUT-1 expression in the primary tumor were significantly associated with high MAI (p = 0.008 and p = 0.038 respectively). CONCLUSION: Claudin-4 is frequently expressed in primary breast cancers but especially in their metastases and is thereby an attractive membrane bound molecular imaging and drug target. Conversion in expression of the studied proteins from the primary tumor to metastases was fairly frequent, except for IGF1R, implying that the expression status of metastases cannot always be reliably predicted from the primary tumor, thereby necessitating biopsy for reliable assessment.

Proteomics on malignant pleural effusions reveals ERα loss in metastatic breast cancer associates with SGK1-NDRG1 deregulation.

Breast cancer (BCa) is a highly heterogeneous disease, with hormone receptor status being a key factor in patient prognostication and treatment decision-making. The majority of primary tumours are positive for oestrogen receptor alpha (ERα), which plays a key role in tumorigenesis and disease progression, and represents the major target for treatment of BCa. However, around one-third of patients with ERα-positive BCa relapse and progress into the metastatic stage, with 20% of metastatic cases characterised by loss of ERα expression after endocrine treatment, known as ERα-conversion. It remains unclear whether ERα-converted cancers are biologically similar to bona fide ERα-negative disease and which signalling cascades compensate for ERα loss and drive tumour progression. To better understand the biological changes that occur in metastatic BCa upon ERα loss, we performed (phospho)proteomics analysis of 47 malignant pleural effusions derived from 37 BCa patients, comparing ERα-positive, ERα-converted and ERα-negative cases. Our data revealed that the loss of ERα-dependency in this metastatic site leads to only a partial switch to an ERα-negative molecular phenotype, with preservation of a luminal-like proteomic landscape. Furthermore, we found evidence for decreased activity of several key kinases, including serum/glucocorticoid regulated kinase 1 (SGK1), in ERα-converted metastases. Loss of SGK1 substrate phosphorylation may compensate for the loss of ERα-dependency in advanced disease and exposes a potential therapeutic vulnerability that may be exploited in treating these patients.

A YAP-centered mechanotransduction loop drives collective breast cancer cell invasion.

Dense and aligned Collagen I fibers are associated with collective cancer invasion led by protrusive tumor cells, leader cells. In some breast tumors, a population of cancer cells (basal-like cells) maintain several epithelial characteristics and express the myoepithelial/basal cell marker Keratin 14 (K14). Emergence of leader cells and K14 expression are regarded as interconnected events triggered by Collagen I, however the underlying mechanisms remain unknown. Using breast carcinoma organoids, we show that Collagen I drives a force-dependent loop, specifically in basal-like cancer cells. The feed-forward loop is centered around the mechanotransducer Yap and independent of K14 expression. Yap promotes a transcriptional program that enhances Collagen I alignment and tension, which further activates Yap. Active Yap is detected in invading breast cancer cells in patients and required for collective invasion in 3D Collagen I and in the mammary fat pad of mice. Our work uncovers an essential function for Yap in leader cell selection during collective cancer invasion.

Analysis of copy number changes on chromosome 16q in male breast cancer by multiplex ligation-dependent probe amplification.

Gene copy number changes have an important role in carcinogenesis and could serve as potential biomarkers for prognosis and targets for therapy. Copy number changes mapping to chromosome 16 have been reported to be the most frequent alteration observed in female breast cancer and a loss on 16q has been shown to be associated with low grade and better prognosis. In the present study, we aimed to characterize copy number changes on 16q in a group of 135 male breast cancers using a novel multiplex ligation-dependent probe amplification kit. One hundred and twelve out of 135 (83%) male breast cancer showed copy number changes of at least one gene on chromosome 16, with frequent loss of 16q (71/135; 53%), either partial (66/135; 49%) or whole arm loss (5/135; 4%). Losses on 16q were thereby less often seen in male breast cancer than previously described in female breast cancer. Loss on 16q was significantly correlated with favorable clinicopathological features such as negative lymph node status, small tumor size, and low grade. Copy number gain of almost all genes on the short arm was also significantly correlated with lymph node negative status. A combination of 16q loss and 16p gain correlated even stronger with negative lymph node status (n=112; P=0.012), which was also underlined by unsupervised clustering. In conclusion, copy number loss on 16q is less frequent in male breast cancer than in female breast cancer, providing further evidence that male breast cancer and female breast cancer are genetically different. Gain on 16p and loss of 16q identify a group of male breast cancer with low propensity to develop lymph node metastases.

Papillomatous breast lesions with atypical columnar cell features.

AIMS: Columnar cell lesions (CCLs) are recognised breast cancer precursor lesions. Intraductal papillomas are usually lined by benign (polyclonal) cells. Although papillomas with monoclonal lesions (atypical ductal hyperplasia (ADH)/ductal carcinoma in situ (DCIS)) have been described, CCLs have not been described in papillomas. METHODS: We present two papillary breast lesions lined by a single layer of luminal cells resembling atypical CCL/flat epithelial atypia (FEA). We compared these two lesions with 13 benign intraductal papillomas, and 2 papillomas with ADH/DCIS grade 1 features as controls were immunohistochemically stained for the oestrogen receptor alpha (oestrogen receptor) and progesterone receptors (PR), cytokeratin 5 (CK5) and cyclin D1. RESULTS: Oestrogen receptor/PR expression was variable, with areas with ≥85% hormone receptor positivity in both morphologically normal papillomas and papillomas with ADH. In ADH areas, CK5 expression was seen in ≤5% of cells while cyclin D1 expression was high (>60%). The two papillary lesions with FEA were 100% oestrogen receptor and 90% cyclin D1 positive, and low on PR/CK5. There was only one morphologically normal papilloma with similar areas of low CK5 (5%) and high cyclin D1 expression; in all other morphologically benign papillomas CK5 expression varied between 10% and 50% and cyclin D1 expression was ≤50%. The papillary lesion with FEA that could be tested showed 16q losses, the hallmark genetic change in low nuclear grade breast neoplasias, in contrast to nine morphologically benign papillomas that could be tested. CONCLUSION: We present two papillomatous breast lesions with atypical CCL morphology and 16q loss, for which we propose the term papillary FEA.

[Breast cancer during pregnancy and the postpartum period: challenging diagnosis of an aggressive disease]. / Borstkanker tijdens en na de zwangerschap.

Breast cancer is the most common malignancy diagnosed during pregnancy. Breast cancer during pregnancy or within the first postpartum year is commonly described as Pregnancy-Associated Breast Cancer (PABC). PABC often exhibits poorer histopathologic features and has a worse prognosis when compared to young breast cancer patients without a current or recent pregnancy. Here, we describe two cases of PABC in which the presenting symptoms of the patients were interpreted as pregnancy-related changes, causing a diagnostic delay. Therefore, we argue that every pregnant or postpartum woman with changes in the breast must be thoroughly evaluated to exclude the possibility of a malignancy. In case of any suspicion, patients must be referred to a breast cancer center for further evaluation.

A tumor cell specific Zona Pellucida glycoprotein 3 RNA transcript encodes an intracellular cancer antigen.

Background: Expression of Zona Pellucida glycoprotein 3 (ZP3) in healthy tissue is restricted to the extracellular Zona Pellucida layer surrounding oocytes of ovarian follicles and to specific cells of the spermatogenic lineage. Ectopic expression of ZP3 has been observed in various types of cancer, rendering it a possible therapeutic target. Methods: To support its validity as therapeutic target, we extended the cancer related data by investigating ZP3 expression using immunohistochemistry (IHC) of tumor biopsies. We performed a ZP3 transcript specific analysis of publicly available RNA-sequencing (RNA-seq) data of cancer cell lines (CCLs) and tumor and normal tissues, and validated expression data by independent computational analysis and real-time quantitative PCR (qPCR). A correlation between the ZP3 expression level and pathological and clinical parameters was also investigated. Results: IHC data for several cancer types showed abundant ZP3 protein staining, which was confined to the cytoplasm, contradicting the extracellular protein localization in oocytes. We noticed that an alternative ZP3 RNA transcript, which we term 'ZP3-Cancer', was annotated in gene databases that lacks the genetic information encoding the N-terminal signal peptide that governs entry into the secretory pathway. This explains the intracellular localization of ZP3 in tumor cells. Analysis of publicly available RNA-seq data of 1339 cancer cell lines (CCLs), 10386 tumor tissues (The Cancer Genome Atlas) and 7481 healthy tissues (Genotype-Tissue Expression) indicated that ZP3-Cancer is the dominant ZP3 RNA transcript in tumor cells and is highly enriched in many cancer types, particularly in rectal, ovarian, colorectal, prostate, lung and breast cancer. Expression of ZP3-Cancer in tumor cells was confirmed by qPCR. Higher levels of the ZP3-Cancer transcript were associated with more aggressive tumors and worse survival of patients with various types of cancer. Conclusion: The cancer-restricted expression of ZP3-Cancer renders it an attractive tumor antigen for the development of a therapeutic cancer vaccine, particularly using mRNA expression technologies.

Promoter hypermethylation in male breast cancer: analysis by multiplex ligation-dependent probe amplification.

INTRODUCTION: Epigenetic events are, along with genetic alteration, important in the development and progression of cancer. Promoter hypermethylation causes gene silencing and is thought to be an early event in carcinogenesis. The role of promoter hypermethylation in male breast cancer has not yet been studied. METHODS: In a group of 108 male breast cancers, the methylation status of 25 genes was studied using methylation-specific multiplex ligation-dependent probe amplification. Methylation of more than 15% was regarded indicative for promoter hypermethylation. Methylation status was correlated with clinicopathological features, with patients' outcome and with 28 female breast cancer cases. RESULTS: Promoter hypermethylation of the genes MSH6, WT1, PAX5, CDH13, GATA5 and PAX6 was seen in more than 50% of the cases, but was uncommon or absent in normal male breast tissue. High overall methylation status was correlated with high grade (P = 0.003) and was an independent predictor of poor survival (P = 0.048; hazard ratio 2.5). ESR1 and GSTP1 hypermethylation were associated with high mitotic count (P = 0.037 and P = 0.002, respectively) and high grade (both P = 0.001). No correlation with survival was seen for individual genes. Compared with female breast cancers (logistic regression), promoter hypermethylation was less common in a variety of genes, particularly ESR1 (P = 0.005), BRCA1 (P = 0.010) and BRCA2 (P < 0.001). The most frequently hypermethylated genes (MSH6, CDH13, PAX5, PAX6 and WT1) were similar for male and female breast cancer. CONCLUSION: Promoter hypermethylation is common in male breast cancer and high methylation status correlates with aggressive phenotype and poor survival. ESR1 and GSTP1 promoter hypermethylation seem to be involved in development and/or progression of high-grade male breast cancer. Although female and male breast cancer share a set of commonly methylated genes, many of the studied genes are less frequently methylated in male breast cancer, pointing towards possible differences between male and female breast carcinogenesis.

Prognostic value of estrogen receptor α and progesterone receptor conversion in distant breast cancer metastases.

BACKGROUND: Changes in the receptor profile of primary breast cancers to their metastases (receptor conversion) have been described for the estrogen receptor α (ERα) and progesterone receptor (PR). The purpose of this study was to evaluate the impact of receptor conversion for ERα and PR on survival in a large group of distant non-bone breast cancer metastases. METHODS: Receptor conversion was studied by immunohistochemistry in a group of 233 metastatic breast cancer patients. Kaplan-Meier overall survival curves were plotted, and differences between the curves were analyzed by log-rank analysis. The additional prognostic value of conversion to established prognosticators was studied by Cox regression. RESULTS: Overall survival of patients showing conversion from positive to negative ERα or PR, or from negative to positive ERα or PR, or remaining receptor negative was comparable, and significantly worse than patients remaining receptor positive. ERα or PR receptor conversion from positive in the primary breast tumor to negative in distant metastases has independent negative prognostic value. CONCLUSIONS: ERα or PR receptor conversion from positive in the primary breast cancer to negative in distant metastases has negative prognostic value.