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Purpose: Tamoxifen remains therapy of choice for premenopausal estrogen receptor α–positive breast cancer. However, resistance and recurrence are serious problems. Our previous work indicated that carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) was significantly up-regulated in tamoxifen-resistant (TAMr) MCF-7 derivatives. The aim of this study was to determine the functional role of CEACAM6 in endocrine-resistant breast cancer and to retrospectively test whether it was predictive of resistance in a large cohort of breast cancers with long-term follow-up.Experimental Design: siRNA silencing of CEACAM6 was done in TAMr cells and effects on clonogenicity and endocrine sensitivity were determined. CEACAM6 immunohistochemistry was done on a tissue microarray comprising 108 relapsed primary human breast cancers and 243 tamoxifen-sensitive controls.Results: siRNA-mediated silencing of CEACAM6 reduced both clonogenicity and anchorage-dependent and anchorage-independent growth of TAMr cells. Importantly, CEACAM6 silencing restored sensitivity of TAMr cells to 4-hydroxytamoxifen and proliferative response to 17β-estradiol. Immunohistochemistry showed significantly more CEACAM expression in the relapsed group compared with nonrelapsed controls [35 of 108 (33.3%) and 32 of 243 (13.2%), respectively; odds ratio, 3.16 (95% confidence interval, 1.83-5.47); P < 0.0001]. Additionally, we derived an outcome predictor model based on CEACAM expression that restratified patients in the Nottingham prognostic index intermediate-risk group into either higher-risk or lower-risk group.Conclusions: Our data support an important role for CEACAM6 in endocrine resistance, which can serve as a powerful predictor of future recurrence.

Supplementary Table S1 from Carcinoembryonic Antigen Cell Adhesion Molecule 6 Predicts Breast Cancer Recurrence following Adjuvant Tamoxifen

Supplementary Table 1 from Estrogen Receptor β1 Expression Is Regulated by miR-92 in Breast Cancer

Supplementary Figure 3 from Estrogen Receptor β1 Expression Is Regulated by miR-92 in Breast Cancer

Supplementary Figure 1 from Estrogen Receptor β1 Expression Is Regulated by miR-92 in Breast Cancer

Supplementary Figure 2 from Estrogen Receptor β1 Expression Is Regulated by miR-92 in Breast Cancer

Supplementary Table 2 from Estrogen Receptor β1 Expression Is Regulated by miR-92 in Breast Cancer

Supplementary Figure 4 from Estrogen Receptor β1 Expression Is Regulated by miR-92 in Breast Cancer

Ovarian cancer typically presents at an advanced stage, and although the majority of cases initially respond well to platinum-based therapies, chemoresistance almost always occurs leading to a poor long-term prognosis. While various cellular autonomous mechanisms contribute to intrinsic or acquired platinum resistance, the tumour microenvironment (TME) plays a central role in resistance to therapy and disease progression by providing cancer stem cell niches, promoting tumour cell metabolic reprogramming, reducing chemotherapy drug perfusion and promoting an immunosuppressive environment. As such, the TME is an attractive therapeutic target which has been the focus of intense research in recent years. This review provides an overview of the unique ovarian cancer TME and its role in disease progression and therapy resistance, highlighting some of the latest preclinical and clinical data on TME-targeted therapies. In particular, it focuses on strategies targeting cancer-associated fibroblasts, tumour-associated macrophages, cancer stem cells and cancer cell metabolic vulnerabilities.

Aim: Tamoxifen (TAM) resistance remains a clinical issue in breast cancer. The authors previously reported that 15-hydroxyprostaglandin dehydrogenase (HPGD) was significantly downregulated in tamoxifen-resistant (TAMr) breast cancer cell lines. Here, the authors investigated the relationship between HPGD expression, TAM resistance and prediction of outcome in breast cancer. Methods: HPGD overexpression and silencing studies were performed in isogenic TAMr and parental human breast cancer cell lines to establish the impact of HPGD expression on TAM resistance. HPGD expression and clinical outcome relationships were explored using immunohistochemistry and in silico analysis. Results: Restoration of HPGD expression and activity sensitised TAMr MCF-7 cells to TAM and 17β-oestradiol, whilst HPGD silencing in parental MCF-7 cells reduced TAM sensitivity. TAMr cells released more prostaglandin E2 (PGE2) than controls, which was reduced in TAMr cells stably transfected with HPGD. Exogenous PGE2 signalled through the EP4 receptor to reduce breast cancer cell sensitivity to TAM. Decreased HPGD expression was associated with decreased overall survival in ERα-positive breast cancer patients. Conclusions: HPGD downregulation in breast cancer is associated with reduced response to TAM therapy via PGE2-EP4 signalling and decreases patient survival. The data offer a potential target to develop combination therapies that may overcome acquired tamoxifen resistance.

e12518 Background: Despite its purported prognostic significance in breast cancer, Ki67 index assessment remains poorly standardized and features high discordance rates amongst pathologists. Moreover, its clinical utility is presently confined to low stage, ER+/HER2- tumors in determining the advisability of adjuvant chemotherapy. Unfortunately, established cut-offs limit its utility to tumors with either high or low Ki67 index extremes. While Ki67 is a marker of actively dividing cells, it fails to capture the detail of cell cycle phase and dynamics which could be informative in terms of prognosis and tumor sensitivity to specific therapies (e.g. CDK4/6 inhibitors). This study therefore developed two novel indices as surrogates of (i) Ki67 index and (ii) quiescent cell population load (QPL). Methods: Breast cancer (comprising evenly distributed hormone receptor/HER2 status cases) H&E slides ( n = 79 cases/108 slides) were digitized on an Aperio DT3 scanner, and used to extract surrogate Ki67 and QPL indices. Sections were also stained for Ki67 by immunohistochemistry (IHC) using a clinically validated assay and digitized. Whole slide image (WSI) tumor Ki67 counts were performed on QuPath and used to validate the surrogate Ki67 index (Cohen’s kappa score). Indices (i) and (ii) were related to progression free survival (PFS). Survival analyses were performed using Kaplan-Meier (KM; with median cut-off) and Cox Proportional Hazards (as a continuous variable) models. Results: The surrogate Ki67 index showed good concordance with IHC scores (kappa = 0.76; 95%CI 0.61-0.91; P< 0.00001). However, this surrogate index performed better as a prognostic indicator of PFS compared to conventional Ki67 IHC (KM P = 0.048; HR = 1.35, P = 0.015 vs. KM P = 0.70; HR = 1.23, P = 0.08). Prognostically, the QPL index outperformed both Ki67 indices (KM P = 0.03; HR = 3.22, P = 0.001). Conclusions: We have developed two novel surrogate indices of Ki67 and QPL that can be readily automated to analyze H&E breast cancer WSIs. Our results show that both outperformed conventional Ki67 IHC evaluation in terms of prognostication, applied across molecular subtypes, improved informativeness of mid-range Ki67 index calls, and could potentially have predictive merit in selecting patients for cell cycle targeted therapies such as CDK4/6 inhibitors.

e12517 Background: HER2 amplification or overexpression is negative prognostic factor that occurs in circa 15% of primary invasive breast cancers, with positive results opening up eligibility for HER2 targeted (e.g. trastuzumab) combination therapy. However, HER2 intratumoral heterogeneity has been proposed as an explanation for the development of resistance to anti-HER2 targeted therapy. The aim of this retrospective study was to test the prognostic value of a HER2 heterogeneity index on ER+ breast cancer H&E whole slide images (WSIs). Methods: ER+ breast cancers slides ( n=60 cases) of known HER2 status (immunohistochemically assessed over the 0 to 3+ range, with HER2 2+ borderline cases validated by fluorescence in situ hybridization) were digitized on an Aperio AT3 scanner. A subset of these patients ( n=29) was accounted for by HER2 amplified/borderline cases. A HER2 heterogeneity index was tested and compared to standard clinical HER2 status in these cases and related to progression free survival (PFS). Survival analyses were performed using Kaplan-Meier (KM; with median cut-off) and Cox Proportional Hazards (as a continuous variable) models. Results: The HER2 heterogeneity index performed better in terms of prognostication than clinical HER2 status alone in relation to PFS (KM P=0.019; HR 8.06, P=0.01 vs. KM P=0.02; HR 6.75, P=0.048). These differences were more pronounced in the HER2 amplified/borderline tumor subset, where the HER2 heterogeneity index outperformed clinical HER2 status (KM P=0.04; HR 6.38, P=0.001 vs. KM P=0.10; HR 0.64, P=0.20). Conclusions: These findings highlight the merit of identifying HER2 heterogeneity from H&E slides alone and establish this index as a robust prognostic factor in determining PFS in ER+ breast tumors. Future work will extend this investigation into ER- tumors and evaluate patient-specific tumor responses to anti-HER2 targeted therapy.

Genomic technologies are increasingly used clinically for both diagnosis and guiding cancer therapy. However, formalin fixation can compromise DNA quality. This study aimed to optimise tissue fixation using normal colon, liver and uterus (n=8 each) by varying neutral buffered formalin (NBF) concentration (1%–5% w/v) and fixation time (24–48 hours). Fixation using 4% NBF improved DNA quality (assessed by qPCR) compared with routine (4% unbuffered formal saline-fixed) specimens (p

Simple Summary Immunotherapy hinges on stimulating patients’ immune system to fight cancer. This treatment has led to improved survival in patients with malignancies such as melanoma and lung cancer but, disappointingly, its benefits have not been as forthcoming in ovarian cancer. This review summarises how the ovarian cancer tumour microenvironment hinders the efficacy of immunotherapy by modulating immunoregulatory pathways, reorchestrating metabolism and featuring specific cancer cell genomic aberrations. The impact of novel targeted drugs and combination therapies aiming to overcome these obstacles and improve the clinical success of immunotherapy in ovarian cancer are considered. Abstract A lack of explicit early clinical signs and effective screening measures mean that ovarian cancer (OC) often presents as advanced, incurable disease. While conventional treatment combines maximal cytoreductive surgery and platinum-based chemotherapy, patients frequently develop chemoresistance and disease recurrence. The clinical application of immune checkpoint blockade (ICB) aims to restore anti-cancer T-cell function in the tumour microenvironment (TME). Disappointingly, even though tumour infiltrating lymphocytes are associated with superior survival in OC, ICB has offered limited therapeutic benefits. Herein, we discuss specific TME features that prevent ICB from reaching its full potential, focussing in particular on the challenges created by immune, genomic and metabolic alterations. We explore both recent and current therapeutic strategies aiming to overcome these hurdles, including the synergistic effect of combination treatments with immune-based strategies and review the status quo of current clinical trials aiming to maximise the success of immunotherapy in OC.

e12575 Background: The determination of breast cancer grade remains a problematic diagnostic issue in biopsies due to limited tissue sampling and the inherent inter-observer variability associated with the Nottingham (histologic) score. Significantly, biopsy grading corresponds only moderately with that based on excision specimens, with discordance rates of 21-30.0%, which is a concern for cases managed neoadjuvantly or with minimally ablative therapy. Although the advent of digital pathology has encouraged endeavors to automate breast cancer detection and grading, no method has yet received regulatory approval for clinical use or proven to be a platform agnostic solution. Methods: This study was the blinded clinical validation a novel, FDA breakthrough designation approved automated device-based predictive solution which uses a surrogate scale to determine breast carcinoma grade in core biopsies using H&E slide whole slide images (WSIs) only. Non-preselected malignant breast core biopsy clinical cases (n=173 WSIs; 107 cases) covering a broad spectrum of breast cancer morphological subtypes were scanned at x20 magnification on both on Aperio high-throughput T2/T3 systems (.SVS files) and Roche-Ventana DP200 scanners (.BIF files). The diagnostic gold standard reference was the reports of tertiary referral center breast subspecialty consultant histopathologists. Diagnostic outputs were also compared to case-matched resection specimen grades. Comparison of diagnostic outcomes with pathologists as gold standard was done using inter observer agreement (cohen kappa) with 95% CI and statistically significant chi-squared test p-value for two different scanner platforms. Results: Although biopsy-based diagnostic concordance with pathologists was very good, the device actually delivered a much better concordance between case-matched biopsy and resection specimen (accuracy: 95%, cohen kappa: 0.91) relative to pathologists’ assessments (accuracy: 80%). Conclusions: This indicates that the use of a single continuous surrogate grading scale is much less affected by limited tissue sampling than conventional ordinal morphological scales. This level of performance was independent of file format analyzed, demonstrating the device’s platform agnosia.

e12535 Background: Categorical combinations of ER, PR, HER2, and Ki67 levels are traditionally used to classify patients into luminal A and B-like subtypes in order to inform treatment choice. Accounting for nearly 70% of all breast malignancies, luminal cancer is heterogeneous, harboring subtypes with distinct molecular profiles and clinical outcomes. Although most patients with luminal-type disease respond well to endocrine therapy alone, some develop recurrences benefiting from additional cytotoxic therapy. Identifying such cases a priori remains a challenge but would enable patients to be spared the debilitating side-effects of ineffective chemotherapy. In this regard, the efficacy of chemotherapy and disease recurrence relate to (i) ER driven G1/S perturbations and/or (ii) quiescent cell populations arrested in the G0/G1 phase of cell cycle. This study aimed to develop a histopathology whole slide image (WSI)-based, low cost, rapid and automated approach to: (i) predict ER/PR/Ki67 status, (ii) quantify quiescence burden, (iii) develop a G1/S-based patient stratification system for luminal A/B patients, and (iv) achieve a quiescence burden-based stratification of TNBC patients. Methods: This investigation centered on the initial clinical validation of a novel, immunostaining-free technology which uses information extracted from pre-treatment hematoxylin and eosin (H&E) stained slide WSIs alone to achieve these aims. Unlike conventional artificial intelligence-based approaches, the underlying proprietary algorithm and its prediction criteria are based on deterministic, hard-coded observational relationships of continuous scales drawn from WSI morphological features. In this instance, these represent tumor-related biological pathway disruptions and mitotic checkpoint perturbations, where G1/S perturbations enable luminal subtype stratification, and G0/G1 perturbations reflect quiescence burden. Back projecting the algorithm’s quiescence burden output on to the original WSIs enables morphological patterns to be mapped to quiescence burden.

593 Background: Digital pathology has fostered the development of automated diagnostic solutions. However, current technologies in breast cancer remain unable to determine HER2 amplification status, which is established by immunohistochemistry (IHC) and/or fluorescent in situ hybridization (FISH). These ancillary tests carry a significant cost, prolong diagnostic time and fail to capture HER2 tumor heterogeneity and tumor infiltrating lymphocyte (TIL) burden, both of which determine the effectiveness anti-HER2 targeted therapy. Methods: This study describes the real-life clinical context development and validation of a patented novel, universal, automated, white-box, scanning platform agnostic solution that determines HER2 amplification status, prognostically significant TIL levels and tumor heterogeneity index (HI) from hematoxylin and eosin (H&E) stained malignant breast biopsy whole slide images (WSIs) alone. Unlike conventional artificial intelligence-based approaches, the underlying proprietary algorithm’s prediction criteria are explainable and are based on deterministic, hard-coded observational relationships of scale constructed from image morphological features mapped to observables representing underlying tumor-related perturbations in biological pathways/mitotic checkpoints.This includes G1/S deregulation signatures reflecting oncogenic HER2-neu. Results: Blinded validation of HER2 status prediction (n = 197 WSIs; 118 independent cases/patients) showed excellent diagnostic performance (κ = 0.85) relative to existing standard-of-care methodologies. This was independent of WSI file format, background histology, tumor subtype/grade or hormone receptor status. The device also displayed good accuracy (92%) in determining TIL profiles, and its combined HER2-TIL and HER2-HI prediction scales both exhibited a significant association with progression-free survival (CoxPH hazard ratio: 1.856; 95% CI: 1.002-3.438; p = 0.049 and CoxPH hazard ratio: 3.45; 95% CI: 0.95-12.55; p = 0.060). Conclusions: This technology opens up the future possibility of bypassing existing ancillary HER2 profiling investigations, thus potentially reducing laboratory workloads/healthcare costs while accelerating diagnostic turnaround times for patient benefit. In the interim, if used as an adjunct tool, this device could provide an objective HER2 testing reference scale while the robustness of its prediction of patient response to anti-HER2 targeted therapy is fully explored.

Eicosanoids comprise a diverse group of bioactive lipids which orchestrate inflammation, immunity and tissue homeostasis, and whose dysregulation has been implicated in carcinogenesis. Among the various eicosanoid metabolic pathways, studies of their role in endometrial cancer (EC) have very much been confined to the COX‐2 pathway. This study aimed to determine changes in epithelial eicosanoid metabolic gene expression in endometrial carcinogenesis, to integrate these with eicosanoid profiles in matched clinical specimens and, finally, to investigate the prognostic value of candidate eicosanoid metabolic enzymes. Eicosanoids and related mediators were profiled using liquid chromatography‐tandem mass spectrometry in fresh frozen normal, hyperplastic and cancerous (Types I and II) endometrial specimens (n=192). Sample‐matched epithelia were isolated by laser capture microdissection and whole genome expression analysis was performed using microarrays. Integration of eicosanoid and gene expression data showed that the accepted paradigm of increased COX‐2 mediated prostaglandin production does not apply in EC carcinogenesis. Instead, there was evidence for decreased PGE2/PGF2α inactivation via 15‐hydroxyprostaglandin dehydrogenase (HPGD) in Type II ECs. Increased expression of 5‐lipoxygenase (ALOX5) mRNA was also identified in Type II ECs, together with proportional increases in its product, 5‐hydroxyeicosatetraenioic acid (5‐HETE). Decreased HPGD and elevated ALOX5 mRNA expression were associated with adverse outcome, which was confirmed by immunohistochemical tissue microarray analysis of an independent series of EC specimens (n=419). While neither COX‐1 nor COX‐2 protein expression had prognostic value, low HPGD combined with high ALOX5 expression associated with the worst overall and progression‐free survival. These findings highlight HPGD and ALOX5 as potential therapeutic targets in aggressive EC subtypes.

Eicosanoids comprise a diverse group of bioactive lipids which orchestrate inflammation, immunity, and tissue homeostasis, and whose dysregulation has been implicated in carcinogenesis. Among the various eicosanoid metabolic pathways, studies of their role in endometrial cancer (EC) have very much been confined to the COX-2 pathway. This study aimed to determine changes in epithelial eicosanoid metabolic gene expression in endometrial carcinogenesis; to integrate these with eicosanoid profiles in matched clinical specimens; and, finally, to investigate the prognostic value of candidate eicosanoid metabolic enzymes. Eicosanoids and related mediators were profiled using liquid chromatography-tandem mass spectrometry in fresh frozen normal, hyperplastic, and cancerous (types I and II) endometrial specimens (n = 192). Sample-matched epithelia were isolated by laser capture microdissection and whole genome expression analysis was performed using microarrays. Integration of eicosanoid and gene expression data showed that the accepted paradigm of increased COX-2-mediated prostaglandin production does not apply in EC carcinogenesis. Instead, there was evidence for decreased PGE

Laser capture microdissection (LCM) allows expression profiling of specific cell populations within tissues. However, isolation of high-quality RNA from laser capture microdissected frozen tissue is beset by problems arising from intrinsic tissue RNase activity. Herein, we describe an optimized staining/LCM/RNA extraction protocol developed for the isolation of epithelial RNA from frozen tissue sections using human endometrial cancer as a model tissue. This method combines excellent, reproducible visualization of tissue morphology with the isolation of high-integrity RNA suitable for downstream applications such as expression microarray analysis. We present quantitative and qualitative RNA data obtained from200 endometrial epithelial samples (normal, hyperplastic, and cancerous), where 92% of samples had RIN values of 7 and above and highlight common pitfalls faced by investigators. This method should also be broadly applicable to a range of other tissue types.

Laser capture microdissection (LCM) allows expression profiling of specific cell populations within tissues. However, isolation of high-quality RNA from laser capture microdissected frozen tissue is beset by problems arising from intrinsic tissue RNase activity. Herein, we describe an optimized staining/LCM/RNA extraction protocol developed for the isolation of epithelial RNA from frozen tissue sections using human endometrial cancer as a model tissue. This method combines excellent, reproducible visualization of tissue morphology with the isolation of high-integrity RNA suitable for downstream applications such as expression microarray analysis. We present quantitative and qualitative RNA data obtained from >200 endometrial epithelial samples (normal, hyperplastic, and cancerous), where 92% of samples had RIN values of 7 and above and highlight common pitfalls faced by investigators. This method should also be broadly applicable to a range of other tissue types.

Background: Variations in systemic inflammatory response biomarker levels have been associated with adverse clinical outcome in various malignancies. This study determined the prognostic significance of preoperative neutrophil:lymphocyte (NLR), platelet:lymphocyte (PLR) and monocyte:lymphocyte (MLR) ratios in endometrial cancer. Methods: Clinicopathological and 5-year follow-up data were obtained for a retrospective series of surgically treated endometrial cancer patients (n=605). Prognostic significance was determined for overall (OS) and cancer-specific survival (CSS) using Cox proportional hazards models and Kaplan–Meier analysis. Receiver–operator characteristic and log-rank functions were used to optimise cut-offs. NLR, PLR and MLR associations with clinicopathological variables were determined using non-parametric tests. Results: Applying cut-offs of ⩾2.4 (NLR), ⩾240 (PLR) and ⩾0.19 (MLR), NLR and PLR (but not MLR) had independent prognostic significance. Combining NLR and PLR scores stratified patients into low (NLR-low and PLR-low), intermediate (NLR-high or PLR-high) and high risk (NLR-high and PLR-high) groups: multivariable hazard ratio (HR) 2.51; P

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has long been used as a default reference gene in quantitative mRNA profiling experiments. However, its expression reportedly varies in response to a range of pathophysiological variables (inflammation, oxidative stress, hyperinsulinaemia, hypoxia) which feature prominently in sepsis. We therefore assessed the applicability of using GAPDH as a reference gene for expression studies in sepsis compared to other housekeeping genes (succinate dehydrogenase complex subunit A (SDHA), hypoxanthine phosphoribosyltransferase (HPRT)-1). Severe sepsis resulted in a 42.4-fold increase in median GAPDH expression (P

Cytokines are key regulators of ovarian physiology, particularly in relation to folliculogenesis and ovulation, where they contribute to creating an environment supporting follicle selection and growth. Their manifold functions include regulating cellular proliferation/differentiation, follicular survival/atresia, and oocyte maturation. Several cytokines, such as TGF-β-superfamily members, are involved at all stages of folliculogenesis while the production of others is stage-dependent. This review draws upon evidence from both human and animal models to highlight the species-specific roles at each milestone of follicular development. Given these pivotal roles and their ease of detection in follicular fluid, cytokines have been considered as attractive biomarkers of oocyte maturational status and of successful assisted reproductive outcome. Despite this, our understanding of cytokines and their interactions remains incomplete, and is still frequently limited to overly simplistic descriptions of their interrelationships. Given our increased appreciation of cytokine activity in complex and highly regulated networks, we put forward the case for using Bayesian modelling approaches to describe their hierarchical relationships in order to predict causal physiological interactions in vivo.

This is the accepted manuscript. The final version is available at http://onlinelibrary.wiley.com/doi/10.1002/gcc.22131/abstract.

Estrogen receptor β1 (ERβ1) downregulation occurs in many breast cancers, but the responsible molecular mechanisms remain unclear. Here, we report that levels of ERβ1 expression are negatively regulated by the microRNA miR-92. Expression analysis in a cohort of primary breast tumors confirmed a significant negative correlation between miR-92 and both ERβ1 mRNA and protein. Inhibition of miR-92 in MCF-7 cells increased ERβ1 expression in a dose-dependent manner, whereas miR-92 overexpression led to ERβ1 downregulation. Reporter constructs containing candidate miR-92 binding sites in the 3′-untranslated region (UTR) of ERβ1 suggested by bioinformatics analysis confirmed that miR-92 downregulated ERβ1 via direct targeting of its 3′-UTR. Our results define a potentially important mechanism for downregulation of ERβ1 expression in breast cancer. Cancer Res; 70(11); 4778–84. ©2010 AACR.

Purpose: Tamoxifen remains therapy of choice for premenopausal estrogen receptor α–positive breast cancer. However, resistance and recurrence are serious problems. Our previous work indicated that carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) was significantly up-regulated in tamoxifen-resistant (TAMr) MCF-7 derivatives. The aim of this study was to determine the functional role of CEACAM6 in endocrine-resistant breast cancer and to retrospectively test whether it was predictive of resistance in a large cohort of breast cancers with long-term follow-up. Experimental Design: siRNA silencing of CEACAM6 was done in TAMr cells and effects on clonogenicity and endocrine sensitivity were determined. CEACAM6 immunohistochemistry was done on a tissue microarray comprising 108 relapsed primary human breast cancers and 243 tamoxifen-sensitive controls. Results: siRNA-mediated silencing of CEACAM6 reduced both clonogenicity and anchorage-dependent and anchorage-independent growth of TAMr cells. Importantly, CEACAM6 silencing restored sensitivity of TAMr cells to 4-hydroxytamoxifen and proliferative response to 17β-estradiol. Immunohistochemistry showed significantly more CEACAM expression in the relapsed group compared with nonrelapsed controls [35 of 108 (33.3%) and 32 of 243 (13.2%), respectively; odds ratio, 3.16 (95% confidence interval, 1.83-5.47); P < 0.0001]. Additionally, we derived an outcome predictor model based on CEACAM expression that restratified patients in the Nottingham prognostic index intermediate-risk group into either higher-risk or lower-risk group. Conclusions: Our data support an important role for CEACAM6 in endocrine resistance, which can serve as a powerful predictor of future recurrence.

Background Cytokine-hormone network deregulations underpin pathologies ranging from autoimmune disorders to cancer, but our understanding of these networks in physiological/pathophysiological states remains patchy. We employed Bayesian networks to analyze cytokine-hormone interactions in vivo using murine lactation as a dynamic, physiological model system. Results Circulatory levels of estrogen, progesterone, prolactin and twenty-three cytokines were profiled in post partum mice with/without pups. The resultant networks were very robust and assembled about structural hubs, with evidence that interleukin (IL)-12 (p40), IL-13 and monocyte chemoattractant protein (MCP)-1 were the primary drivers of network behavior. Network structural conservation across physiological scenarios coupled with the successful empirical validation of our approach suggested that in silico network perturbations can predict in vivo qualitative responses. In silico perturbation of network components also captured biological features of cytokine interactions (antagonism, synergy, redundancy). Conclusion These findings highlight the potential of network-based approaches in identifying novel cytokine pharmacological targets and in predicting the effects of their exogenous manipulation in inflammatory/immune disorders. Electronic supplementary material The online version of this article (doi:10.1186/s12918-015-0226-3) contains supplementary material, which is available to authorized users.

The endometrium is a dynamic tissue, demonstrating cyclical growth/remodelling in preparation for implantation. In mice, seminal constituents trigger mechanisms to prepare the endometrium, a process dubbed ‘seminal priming’ that modifies immune system components and mediates endometrial remodelling in preparation for pregnancy. An array of cytokines has been reported to mediate this interaction, although much of the literature relates toin vitrostudies on isolated endometrial epithelial cells. This study measured changes in immune-related gene expression in endometrial epithelial and stromal cellsin vivofollowing natural mating. CD1 mice were naturally mated and sacrificed over the first 4 dayspost-coitum(n=3 each day). Endometrial epithelial and stromal compartments were isolated by laser capture microdissection. Labelled cRNA was generated and hybridised to genome-wide expression microarrays. Pathway analysis identified several immune-related pathways active within epithelial and stromal compartments, in particular relating to cytokine networks, matrix metalloproteinases and prostaglandin synthesis. Cluster analysis demonstrated that the expression of factors involved in immunomodulation/endometrial remodelling differed between the epithelial and stromal compartments in a temporal fashion. This study is the first to examine the disparate responses of the endometrial epithelial and stromal compartments to seminal plasmain vivoin mice, and demonstrates the complexity of the interactions between these two compartments needed to create a permissive environment for implantation.

The K562 leukaemic cell line expresses an inherent survival signal due to the antiapoptotic properties of Bcr-abl, which is, in part, mediated by prolonging the G2M checkpoint and allowing DNA repair mechanisms to operate post genotoxic insult. Arrest of the cell cycle is mediated by retaining an inactivating state of phosphorylation of cyclin-dependent kinase 1 (Cdk1) on tyrosine 15. Our data confirmed that cell survival in K562 was promoted by cell cycle arrest at G2M in response to the genotoxin etoposide. There was no predicted cell cycle arrest in Bcr-abl-positive derivative cell lines of K562 that did not survive the same genotoxic insult but, paradoxically, Cdk1 tyrosine phosphorylation was enhanced to a higher extent compared with the parental cell line where arrest of the cell cycle was observed. To ascertain that this was not an anomaly of the derivative lines, HL60 cells were treated with concentrations of etoposide that induced arrest of the cell cycle or apoptosis. Only HL60 cells that subsequently underwent apoptosis elicited the same effect of increased Cdk1 tyrosine phosphorylation. It is proposed that the augmented tyrosine phosphorylation status of Cdk1 is associated with the abolition of cell survival, in addition to the previously reported induction of cell cycle arrest in myeloid cell lines.

BCR-ABL confers apoptotic resistance to a range of genotoxic agents, and this protection is mediated in part by prolonging the G2 checkpoint. The p53 tumour suppressor protein regulates the transcription of regulatory genes involved in cell cycle arrest and apoptosis. To investigate the effect of p53 on the BCR-ABL-mediated G2M checkpoint response, we transiently transfected the BCR-ABL-positive, p53-negative cell line K562 with wild-type human p53. The p53-transfected cells showed a decreased ability to arrest in G2 and an increase in apoptosis in response to etoposide treatment, relative to the control mock-transfected cells. p53-transfected and control cells were treated with etoposide and trapped at mitosis with nocodazole. The mitotic index of p53-transfected cells was higher than that of the control cells, which suggests that p53 abrogates the G2 checkpoint response to etoposide treatment in K562 cells. We found that the expression of the cell cycle checkpoint protein Chk1 was reduced in the etoposide-treated p53-transfected cells by 24 h, and this correlated with a reduction in the extent of etoposide-induced phosphorylation of CDK1 at tyrosine 15 (Y15). We conclude, therefore, that p53 overrides the strong G2 checkpoint response to etoposide in K562 cells, by directly or indirectly downregulating Chk1 expression, which, in turn, contributes to the proapoptotic effect of p53.

Background Conception assisted by intracytoplasmic sperm injection (ICSI) requires oocyte stripping for morphological evaluation of maturity status. However, this approach prevents further maturation and poorly predicts fertilisability, so more robust assessment strategies are needed. Given that cytokines orchestrate oocyte development, we aimed to assess the association of follicular fluid cytokine profiles with maturation stage and develop predictive machine learning-based methods to identify those with the greatest fertilisation potential. Methods In this retrospective study, follicular fluid was collected at oocyte retrieval from 64 women and linked to oocyte maturity status or fate—namely, germinal vesicle (n=26), metaphase I (51), metaphase II not fertilised (51), and metaphase II fertilised (84). 51 follicular fluid cytokines were profiled by multiplex immunoassay. Machine learning-based classifiers to predict oocyte fertilisability were subjected to iterative feature reduction to a threshold suitable for developing a clinically viable assessment of oocyte maturity. Women gave written, informed consent. Findings Cytokine profiles varied dynamically throughout maturation. When applied to naive samples with known outcome, classifiers developed using tumour necrosis factor-related apoptosis-inducing ligand and interleukin 18 profiles alone correctly discriminated 89% of metaphase II not fertilised oocytes (ie, those with the highest fertilisability) with high confidence from all other maturation stages. Interpretation These classifiers offer the prospect of cost-effective, point-of-care testing, and streamlined ICSI-based workflows. This assessment circumvents stripping such that immature or low fertilisability oocytes could benefit from in-vitro maturation and increase the pool of usable oocytes. Further studies will confirm the robustness of these classifiers in women with a broader morbidity spectrum and their translational value across a range of clinical settings. Funding Infertility Research Trust.

Laser capture microdissection of frozen tissue sections allows homogeneous cell populations to be isolated for expression profiling. However, this requires striking a balance between retaining adequate morphology for accurate microdissection and maintaining RNA integrity. Various staining protocols were applied to frozen endometrial carcinoma tissue sections. Although alcohol-based methods were superior to aqueous stains for maintaining RNA integrity, they suffered from irreproducible staining intensity. We developed a modified alcohol-based, buffered cresyl violet staining protocol that provides reproducible staining with minimal RNA degradation suitable for tissues with moderate to high levels of intrinsic RNase activity.

The ovary is an unique organ which fulfils a plethora of physiological functions ranging from its well-recognized roles in steroid hormone biosynthesis, oocyte production and the support of early pregnancy through to its rather less well-known involvement in the regulation of growth, behaviour and immune function in response to an array of endogenous and environmental cues. While multiple cytokine-mediated interactions between the granulosa/theca cell compartments and the oocyte of developing follicles are central to the coordination of ovarian cyclicity, its systemic effects are instead attributable to exquisitely well-honed interactions between ovarian follicles, the hypothalamus and pituitary implicating an array of peptide and steroid hormones. During the follicular phase, primordial follicles are called upon to initiate a process of follicular development which culminates in ovulation. However, the release of an oocyte belies the fact that the postovulatory follicle has yet another role to play in the luteal phase. Therein, it adopts a new keystone position in supporting the establishment and maintenance of pregnancy through its production of progesterone. Once the ovary has exhausted its reserve of follicles throughout reproductive life, many of its functions are lost as women enter the menopause. This Chapter aims to explore the ovary’s developmental origins and functional roles to highlight the complexity of this remarkable product of reproductive evolution.

Genomic representational difference analysis (RDA) was carried out on a total of nine Wilms tumors and one cystic partially differentiated nephroblastoma (CPDN; a sub-type of Wilms) to look for novel genetic deletions involving tumor suppressor genes. Genomic DNA from either short-term cultured Wilms tumor cells or a WT xenograft was used to create driver representations, and genomic DNA from matched normal kidney or normal kidney cells grown in short-term culture was used to create the tester. Genuine difference products were obtained from only one of the tumors. However, none of these fragments were found to be deleted in the original tumor biopsy, microdissected tumor or in the lung metastasis from this patient. It is, therefore, likely that the deletions were due to random losses associated with the genetic instability of the cultured cells from this particular tumor. We did not isolate difference products from any of the other tumors, showing that they did not have chromosomal losses, homozygous deletions or regions of LOH that were detectable by RDA.

Germ cell tumors (GCTs) are rare but clinically and pathologically diverse tumors that occur in an extensive range of age groups, from children to older adults and which include both seminomatous and nonseminomatous tumors. Current clinical management for both male and female teenagers and young adults (TYAs) with GCTs remains inconsistent, alternating between pediatric and adult multidisciplinary oncology teams, based on locally defined age cutoffs. Therefore, we reviewed available literature to determine the biological similarities and differences between GCTs in young children (0-12 years), TYAs (13-24 years), and older adults (24 years). GCTs arising in pediatric and adult populations in general showed marked molecular biological differences within identical histological subtypes, whereas there was a distinct paucity of available data for GCTs in the TYA population. These findings highlight that clinical management based simply on chronological age may be inappropriate for TYA and suggests that the optimal future management of GCTs should consider specific molecular biological factors in addition to clinical parameters in the context of patient-specific age group rather than medical specialty.

Cytokines are key regulators of ovarian physiology, particularly in relation to folliculogenesis and ovulation, where they contribute to creating an environment supporting follicle selection and growth. Their manifold functions include regulating cellular proliferation/differentiation, follicular survival/atresia, and oocyte maturation. Several cytokines, such as TGF-β-superfamily members, are involved at all stages of folliculogenesis while the production of others is stage-dependent. This review draws upon evidence from both human and animal models to highlight the species-specific roles at each milestone of follicular development. Given these pivotal roles and their ease of detection in follicular fluid, cytokines have been considered as attractive biomarkers of oocyte maturational status and of successful assisted reproductive outcome. Despite this, our understanding of cytokines and their interactions remains incomplete, and is still frequently limited to overly simplistic descriptions of their interrelationships. Given our increased appreciation of cytokine activity in complex and highly regulated networks, we put forward the case for using Bayesian modelling approaches to describe their hierarchical relationships in order to predict causal physiological interactions in vivo.

ERβ1 is often down-regulated in breast cancer compared to normal breast but mechanisms surrounding this are unclear. We examined whether loss of heterozygosity (LOH) or methylation at ERβ promoters (0N, 0K) and/or untranslated exon 0N were involved in ERβ down-regulation in breast cancer tissues and cell lines and if treatment with the de-methylating agent 5-aza-deoxycytidine and/or the histone deacetylase inhibitor Trichostatin A could influence expression in vitro. We found no evidence of correlation between LOH at 14q22-24 (genomic locus containing ERβ/ESR2), and ERβ1 expression in primary breast cancers. A negative correlation between ERβ1 mRNA expression and methylation status was observed for promoter 0N in BT-20, MDA-MB-453 and T47D cells. Promoter 0K was consistently unmethylated. In primary breast tumours, methylation of the untranslated exon 0N, downstream of promoter 0N, but not of promoter 0N itself, correlated with down-regulation of ERβ. In MDA-MB-453 cells, treatment with 5-aza-deoxycytidine was sufficient to induce ERβ1 expression from the 0N promoter while in BT-20 both agents were required. Examination of various sites on ESR2 highlighted epigenetic but not genetic regulation of ERβ1. In particular methylation adjacent to promoter 0N was a key regulatory event for ERβ1 silencing. A combination of de-methylating agents and histone deacetylase inhibitors fully restored ERβ1 expression which may offer a novel therapeutic angle for breast cancer management.

Estrogen receptor (ER) action is modulated by posttranslational modifications. Although ERalpha phosphorylation correlates with patient outcome, ERbeta is similarly phosphorylated but its significance in breast cancer has not been addressed. We investigated whether ERbeta that is phosphorylated at serine 105 (S105-ERbeta) is expressed in breast cancer and assessed potential clinical implications of this phosphorylation. Following antibody validation, S105-ERbeta expression was studied in tissue microarrays comprising 108 tamoxifen-resistant and 351 tamoxifen-sensitive cases and analyzed against clinical data. S105-ERbeta regulation in vitro was assessed by Western blot, flow cytometry, and immunofluorescence. Nuclear S105-ERbeta was observed in breast carcinoma and was associated with better survival (Allred scoreor =3), even in tamoxifen-resistant cases, and additionally correlated with ERbeta1 and ERbeta2 expression. Distinct S105-ERbeta nuclear speckles were seen in some higher grade tumors. S105-ERbeta levels increased in MCF-7 cells in response to 17beta-estradiol, the ERbeta-specific agonist diarylpropionitrile, and the partial ERbeta-agonist genistein. S105-ERbeta nuclear speckles were also seen in MCF-7 cells and markedly increased in size and number at 24 hours following 17beta-estradiol and, in particular diarylpropionitrile, treatment. These speckles were coexpressed with ERbeta1 and ERbeta2. Presence of S105-ERbeta in breast cancer and association with improved survival, even in endocrine resistant breast tumors suggest S105-ERbeta might be a useful additional prognostic marker in this disease.

Although ERs (oestrogen receptors) mediate breast tumour behaviour, the precise role of ERbeta remains unclear. This is mainly because analyses have been complicated by the presence in breast tissue of three ERbeta protein variants (ERbeta1, ERbeta2 and ERbeta5) that derive from differential 3' splicing. We have recently identified the first known mechanisms responsible for the differential control of isoform expression, involving regulation of translation via 5'-UTRs (untranslated regions). In the present study, we have uncovered further complexity involving the influence of multiple promoters and cross-talk between 5'- and 3'-UTRs. We demonstrate that full-length ERbeta mRNAs are transcribed from three separate promoters; two promoters are well-established within the literature, whereas the third represents a novel finding. Each promoter produces transcripts with distinct 5'-UTRs. The differential 3' splicing that produces transcripts coding for the ERbeta isoforms also defines isoform-specific 3'-UTRs. We identified exact 3'-UTR sequences for each isoform, and have shown that alternative polyadenylation sites are used in a cell-type specific manner to produce transcripts with 3'-UTRs of different lengths. Critically, we show that 5'- and 3'-UTRs combine to specify the efficiencies with which individual transcripts are translated, with 3'-UTR length having a key influence. In addition, we demonstrate how 17beta-oestradiol, a key driver of breast cancer development, affects the regulation of ERbeta expression at both transcriptional and translational levels.

ERβ1 is often downregulated in cancer compared with normal cells, suggesting that it may function as a tumour suppressor and could play an important role in carcinogenesis. The expression of ERβ1 is regulated by multiple mechanisms such as methylation. Five splice variants of ERβ mRNA have been identified, ERβ1 to ERβ5. However, it is unclear whether and how the full-length version, ERβ1, is regulated post-transcriptionally. MicroRNAs are a class of nonprotein coding small RNAs that regulate expression of genes at post-transcriptional levels. Using rapid amplification of 3' complementary DNA ends (3'RACE), we have confirmed 3' untranslated region (3'UTR) expression and sequences of ERβ1 mRNA in MCF-7 cells. Based on miRNA expression profiling of human breast cancer studies, we found that miR-92 is upregulated in malignant breast. In silico analysis using the miRGen database and RNA hybrid predicted that there are two putative miR-92 target sequences within the 3'UTR of human ERβ1 mRNA. Firstly, we profiled the expression of ERβ1 mRNA and miR-92 in breast cancer tissue and cell lines. miR-92 levels were higher in ERβ1-negative MDA-MB-453 cells than ERβ1-positive MCF-7 cells. We observed miR-92 upregulation in breast tumours while ERβ1 mRNA expression was decreased compared with matched adjacent normal tissues. We found a significant negative correlation between miR-92 and ERβ1 mRNA and protein in breast tumour (r = -0.5, P = 0.001 and r = -0.39, P = 0.037), respectively. Transfection of MCF-7 cells with anti-miR-92 increased endogenous ERβ1 mRNA and reduced cell proliferation. EGFP report experiment also confirms that the 3'UTR of ERβ1 carries the directly binding sites of miR-92. Finally, we showed that miR-92 expression is modulated by the ER ligands 17β-estradiol and tamoxifen in MCF-7 cells. These findings prove that ERβ1 expression is negatively regulated at a post-transcriptional level by miR-92. This miRNA could be considered a potential therapeutic target in breast cancer.

Tamoxifen resistance (TAMr) in breast cancer is a serious clinical dilemma, with no satisfactory explanation. We hypothesised that changes in the expression of steroid hormone receptors (ERalpha, ERbeta), their downstream target genes (PR, pS2) and their associated co-regulators (AIB-1, SRC-1, SRA, NCoR-1, SMRT and REA) could be related to the acquisition of TAMr. To test this hypothesis, we developed in vitro TAMr cell line models by continuous exposure of MCF-7 cells to 4-hydroxytamoxifen (4-HT) over 12 (MCF-7MMU1) and 21 (MCF-7MMU2) months, respectively and examined the expression of the above by Western blotting and immunohistochemistry. In addition, we further examined the changes in global gene expression in TAMr cells in comparison with TAM-sensitive cells by microarray analysis. We report here that acquisition of TAMr is associated with changes in the expression of PR, pS2 and several co-activators, but not ERs. In addition, genes associated with cell cycle, cell adhesion and extracellular matrix, were up-regulated while those associated with apoptosis or growth factors/hormones were down-regulated. Based on our results, it appears that increased co-activator expression, in concert with alterations in genes associated with controlling cell proliferation and survival contribute to TAMr in breast cancer.

Nucleotide excision repair is the principal mechanism for the removal of bulky DNA adducts caused by a range of chemotherapeutic drugs, and contributes to cisplatin resistance. In this study, we used synthetic siRNAs targeted to XPA and ERCC1 and compared their effectiveness in sensitising mismatch repair deficient prostate cancer cell lines to cisplatin and mitomycin C. Downregulation of ERCC1 sensitised DU145 and PC3 cells to cisplatin and mitomycin C. In contrast, XPA downregulation did not sensitise either cell line to mitomycin C, and only sensitised DU145 cells to cisplatin. The effects of ERCC1 downregulation may be due to its role in homologous recombination repair. Excision repair of cisplatin adducts in PC3 cells was attenuated to a similar extent by XPA and ERCC1 downregulation. Downregulation of XPA but not ERCC1 caused an increase in the number of cisplatin-induced RAD51 foci in PC3 cells, suggesting that HRR is able to substitute for NER in these cells. We observed co-localisation of ERCC1 and RAD51 in cisplatin treated PC3 cells by immunofluorescence and co-immunoprecipitation, which may represent recruitment of ERCC1/XPF to sites of recombination repair. These results indicate that ERCC1 is a broader therapeutic target than XPA with which to sensitise cancer cells to chemotherapy because of its additional role in recombination repair.

Although most advanced cancers are incurable, the majority of testicular germ cell tumors can be cured using cisplatin-based combination chemotherapy. The nucleotide excision repair (NER) pathway removes most DNA adducts produced by cisplatin, and the low levels of NER in testis tumor cells may explain why these cancers are curable. Three NER proteins: ERCC1, XPF, and XPA, are present at low levels in testis tumor cell lines, and addition of these proteins to protein extracts of testis tumor cells increases their in vitro DNA repair capacity to normal levels. The aim of this study was to identify the mechanism responsible for the low levels of these DNA repair proteins. The levels of the mRNA transcripts for ERCC1, XPF, and XPA were measured in a panel of 14 different human cancer cell lines, using real-time PCR. Three ERCC1 splice variants were identified and quantitated. Three alternative transcription start points (TSPs) were identified for ERCC1 but none were testis-specific. The significantly lower levels of ERCC1, XPF, and XPA protein in testis tumor cell lines cannot be explained solely by differences in transcriptional efficiency or mRNA stability. For ERCC1, post-transcriptional control by alternative splicing does not account for the testis-specific low levels of protein expression. Pulse-chase experiments showed that the half-life of ERCC1 protein in a testis tumor cell line was not significantly different to that in a prostate cancer cell line. Taken together, these results suggest that constitutive levels of these DNA repair proteins are controlled at the level of translation.