Sex steroid hormone signaling is critical in the development of breast cancers, although the role of the androgen receptor remains unclear. This study evaluated androgen receptor (AR) expression in normal breast tissue as a potential marker of breast cancer risk. We conducted a nested case-control study of women with benign breast disease (BBD) within the Nurses' Health Studies. Epithelial AR expression was assessed by immunohistochemistry in normal tissue from the BBD biopsy and the percent of positive nuclei was estimated in ordinal categories of 10% for 78 breast cancer cases and 276 controls. Logistic regression models adjusting for the matching factors and BBD lesion type were used to calculate odds ratios (ORs) for the association between AR expression (tertiles: ≤10%, 11-30%, and >30%) and breast cancer risk. AR expression in normal breast tissue was not associated with subsequent breast cancer risk (OR = 0.9, 95% CI = 0.4-1.8, trend = 0.68). In comparison with low AR/low ER women, ORs of 0.4 (95% CI = 0.1-1.2) for high AR/high ER women, 1.8 (95% CI = 0.4-7.8) for low AR/high ER women, and 0.7 (95% CI = 0.3-1.6) for high AR/low ER women were observed ( interaction = 0.21). Ki67 did not modify the association between AR expression and breast cancer risk ( interaction = 0.75). There was little evidence for an overall association between AR expression in normal breast tissue and breast cancer risk. These findings did not show that the AR association varied by Ki67 expression in normal breast tissue, though there was suggestive heterogeneity by ER expression.

The contribution of basal and luminal cells to cancer progression and metastasis is poorly understood. We report generation of reporter systems driven by either keratin-14 (K14) or keratin-8 (K8) promoter that not only express a fluorescent protein but also an inducible suicide gene. Transgenic mice express the reporter genes in the right cell compartments of mammary gland epithelia and respond to treatment with toxins. In addition, we engineered the reporters into 4T1 metastatic mouse tumor cell line and demonstrate that K14+ cells, but not K14- or K8+, are both highly invasive in three-dimensional (3D) culture and metastatic in vivo. Treatment of cells in culture, or tumors in mice, with reporter-targeting toxin inhibited both invasive behavior and metastasis in vivo. RNA sequencing (RNA-seq), secretome, and epigenome analysis of K14+ and K14- cells led to the identification of amphoterin-induced protein 2 (Amigo2) as a new cell invasion driver whose expression correlated with decreased relapse-free survival in patients with TP53 wild-type (WT) breast cancer.

BACKGROUND: Alcohol consumption is an established risk factor for breast cancer and the association generally appears stronger among estrogen receptor (ER)-positive tumors. However, the biological mechanisms underlying this association are not completely understood. METHODS: We analyzed messenger RNA (mRNA) microarray data from both invasive breast tumors (N = 602) and tumor-adjacent normal tissues (N = 508) from participants diagnosed with breast cancer in the Nurses' Health Study (NHS) and NHSII. Multivariable linear regression, controlling for other known breast cancer risk factors, was used to identify differentially expressed genes by pre-diagnostic alcohol intake. For pathway analysis, we performed gene set enrichment analysis (GSEA). Differentially expressed genes or enriched pathway-defined gene sets with false discovery rate (FDR) 0.1 identified in tumors were validated in RNA sequencing data of invasive breast tumors (N = 166) from The Cancer Genome Atlas. RESULTS: No individual genes were significantly differentially expressed by alcohol consumption in the NHS/NHSII. However, GSEA identified 33 and 68 pathway-defined gene sets at FDR 0.1 among 471 ER+ and 127 ER- tumors, respectively, all of which were validated. Among ER+ tumors, consuming 10+ grams of alcohol per day (vs. 0) was associated with upregulation in RNA metabolism and transport, cell cycle regulation, and DNA repair, and downregulation in lipid metabolism. Among ER- tumors, in addition to upregulation in RNA processing and cell cycle, alcohol intake was linked to overexpression of genes involved in cytokine signaling, including interferon and transforming growth factor (TGF)-β signaling pathways, and translation and post-translational modifications. Lower lipid metabolism was observed in both ER+ tumors and ER+ tumor-adjacent normal samples. Most of the significantly enriched gene sets identified in ER- tumors showed a similar enrichment pattern among ER- tumor-adjacent normal tissues. CONCLUSIONS: Our data suggest that moderate alcohol consumption (i.e. 10+ grams/day, equivalent to one or more drinks/day) is associated with several specific and reproducible biological processes and pathways, which adds potential new insight into alcohol-related breast carcinogenesis.

The histopathological evaluation of morphological features in breast tumours provides prognostic information to guide therapy. Adjunct molecular analyses provide further diagnostic, prognostic and predictive information. However, there is limited knowledge of the molecular basis of morphological phenotypes in invasive breast cancer. This study integrated genomic, transcriptomic and protein data to provide a comprehensive molecular profiling of morphological features in breast cancer. Fifteen pathologists assessed 850 invasive breast cancer cases from The Cancer Genome Atlas (TCGA). Morphological features were significantly associated with genomic alteration, DNA methylation subtype, PAM50 and microRNA subtypes, proliferation scores, gene expression and/or reverse-phase protein assay subtype. Marked nuclear pleomorphism, necrosis, inflammation and a high mitotic count were associated with the basal-like subtype, and had a similar molecular basis. Omics-based signatures were constructed to predict morphological features. The association of morphology transcriptome signatures with overall survival in oestrogen receptor (ER)-positive and ER-negative breast cancer was first assessed by use of the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) dataset; signatures that remained prognostic in the METABRIC multivariate analysis were further evaluated in five additional datasets. The transcriptomic signature of poorly differentiated epithelial tubules was prognostic in ER-positive breast cancer. No signature was prognostic in ER-negative breast cancer. This study provided new insights into the molecular basis of breast cancer morphological phenotypes. The integration of morphological with molecular data has the potential to refine breast cancer classification, predict response to therapy, enhance our understanding of breast cancer biology, and improve clinical management. This work is publicly accessible at www.dx.ai/tcga_breast. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Molecular pathological epidemiology (MPE) is a transdisciplinary and relatively new scientific discipline that integrates theory, methods, and resources from epidemiology, pathology, biostatistics, bioinformatics, and computational biology. The underlying objective of MPE research is to better understand the etiology and progression of complex and heterogeneous human diseases with the goal of informing prevention and treatment efforts in population health and clinical medicine. Although MPE research has been commonly applied to investigating breast, lung, and colorectal cancers, its methodology can be used to study most diseases. Recent successes in MPE studies include: (1) the development of new statistical methods to address etiologic heterogeneity; (2) the enhancement of causal inference; (3) the identification of previously unknown exposure-subtype disease associations; and (4) better understanding of the role of lifestyle/behavioral factors on modifying prognosis according to disease subtype. Central challenges to MPE include the relative lack of transdisciplinary experts, educational programs, and forums to discuss issues related to the advancement of the field. To address these challenges, highlight recent successes in the field, and identify new opportunities, a series of MPE meetings have been held at the Dana-Farber Cancer Institute in Boston, MA. Herein, we share the proceedings of the Third International MPE Meeting, held in May 2016 and attended by 150 scientists from 17 countries. Special topics included integration of MPE with immunology and health disparity research. This meeting series will continue to provide an impetus to foster further transdisciplinary integration of divergent scientific fields.

The heterogeneity of spontaneous preterm birth (SPTB) requires an interdisciplinary approach to determine potential predictive risk factors of early delivery. The aim of this study was to investigate maternal whole blood gene expression profiles associated with spontaneous preterm birth (SPTB, 37 weeks) in asymptomatic pregnant women. The study population was a matched subgroup of women (51 SPTBs, 114 term delivery controls) who participated in the All Our Babies community based cohort in Calgary (n = 1878). Maternal blood at 17-23 (sampling time point 1, T1) and 27-33 weeks of gestation (T2) were collected. Total RNA was extracted and microarray was performed on 326 samples (165 women). Univariate analyses determined significant clinical factors and differential gene expression associated with SPTB. Thirteen genes were validated using qRT-PCR. Three multivariate logistic models were constructed to identify gene expression at T1 (Model A), T2 (Model B), and gene expression fold change from T1 to T2 (Model C) associated with SPTB. All models were adjusted for clinical factors. Model C can predict SPTB with 65% sensitivity and 88% specificity in asymptomatic women after adjusting for history of abortion and anaemia (occurring before T2). Clinical data enhanced the sensitivity of the Models to predict SPTB. In conclusion, clinical factors and whole blood gene expression are associated with SPTB in asymptomatic women. An effective screening tool for SPTB during pregnancy would enable targeted preventive approaches and personalised antenatal care.

Preterm birth (PTB; birth before 37 completed weeks of gestation) remains the major cause of neonatal morbidity and mortality. The current generation of biomarkers predictive of PTB have limited utility. In pregnancy, the human cervicovaginal fluid (CVF) proteome is a reflection of the local biochemical milieu and is influenced by the physical changes occurring in the vagina, cervix and adjacent overlying fetal membranes. Term and preterm labor (PTL) share common pathways of cervical ripening, myometrial activation and fetal membranes rupture leading to birth. We therefore hypothesize that CVF biomarkers predictive of labor may be similar in both the term and preterm labor setting. In this review, we summarize some of the existing published literature as well as our team's breadth of work utilizing the CVF for the discovery and validation of putative CVF biomarkers predictive of human labor. Our team established an efficient method for collecting serial CVF samples for optimal 2-dimensional gel electrophoresis resolution and analysis. We first embarked on CVF biomarker discovery for the prediction of spontaneous onset of term labor using 2D-electrophoresis and solution array multiple analyte profiling. 2D-electrophoretic analyses were subsequently performed on CVF samples associated with PTB. Several proteins have been successfully validated and demonstrate that these biomarkers are associated with term and PTL and may be predictive of both term and PTL. In addition, the measurement of these putative biomarkers was found to be robust to the influences of vaginal microflora and/or semen. The future development of a multiple biomarker bed-side test would help improve the prediction of PTB and the clinical management of patients.

Threatened preterm labor (TPTL) accounts for ∼30% of pregnancy-related hospital admissions. Maternal peripheral leukocytes can be used to monitor a variety of physiological processes occurring in the body. Two high-throughput mass spectrometry methodologies, SWATH and iTRAQ, were used to study differentially expressed peripheral blood leukocyte lysate proteins in symptomatic women admitted for TPTL who had a preterm birth within 48 h (n = 16) and those who did not (n = 24). The SWATH spectral library consisted of 783 proteins. SWATH methodology quantified 258 proteins (using ≥2 peptides) and 5 proteins (ALBU, ANXA6, HNRPK, HSP90A, and PDIA1) were differentially expressed (p 0.05, Mann-Whitney U). iTRAQ workflow identified 765 proteins; 354 proteins were quantified and 14 proteins (MIF, UBIQ, HXK3, ALBU, HNRPD, ST1A2, RS15A, RAP1B, CAN1, IQGA2, ST1A1, COX5A, ADDA, and UBQL1) were significantly different between the two groups of women (p 0.05, Mann-Whitney U). Albumin was the only common differentially expressed protein in both SWATH (28% decrease) and iTRAQ studies (45% decrease). This decrease in albumin was validated using ELISA (11% decrease, p 0.05, Mann-Whitney U) in another 23 TPTL women. This work suggests that albumin is a broad indicator of leukocyte activation with impending preterm birth and provides new future work directions to understand the pathophysiology of TPTL.

Threatened preterm labor (TPTL) is defined as persistent premature uterine contractions between 20 and 37 weeks of gestation and is the most common condition that requires hospitalization during pregnancy. Most of these TPTL women continue their pregnancies to term while only an estimated 5% will deliver a premature baby within ten days. The aim of this work was to study differential whole blood gene expression associated with spontaneous preterm birth (sPTB) within 48 hours of hospital admission. Peripheral blood was collected at point of hospital admission from 154 women with TPTL before any medical treatment. Microarrays were utilized to investigate differential whole blood gene expression between TPTL women who did (n = 48) or did not have a sPTB (n = 106) within 48 hours of admission. Total leukocyte and neutrophil counts were significantly higher (35% and 41% respectively) in women who had sPTB than women who did not deliver within 48 hours (p0.001). Fetal fibronectin (fFN) test was performed on 62 women. There was no difference in the urine, vaginal and placental microbiology and histopathology reports between the two groups of women. There were 469 significant differentially expressed genes (FDR0.05); 28 differentially expressed genes were chosen for microarray validation using qRT-PCR and 20 out of 28 genes were successfully validated (p0.05). An optimal random forest classifier model to predict sPTB was achieved using the top nine differentially expressed genes coupled with peripheral clinical blood data (sensitivity 70.8%, specificity 75.5%). These differentially expressed genes may further elucidate the underlying mechanisms of sPTB and pave the way for future systems biology studies to predict sPTB.