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Metastasis is associated with poor prognosis in breast cancer. Although some studies suggest beta-blockers increase survival by delaying metastasis, others have been discordant. This study provides both insights into the anomalous findings and identifies potential biomarkers that may be treatment targets. Cell line models of basal-type and oestrogen receptor-positive breast cancer were profiled for basal levels of adrenoceptor gene/protein expression, and β2-adrenoceptor mediated cell behaviour including migration, invasion, adhesion, and survival in response to adrenoceptor agonist/antagonist treatment. Protein profiling and histology identified biomarkers and drug targets. Baseline levels of adrenoceptor gene expression are higher in basal-type rather than oestrogen receptor-positive cancer cells. Norepinephrine (NE) treatment increased invasive capacity in all cell lines but did not increase proliferation/survival. Protein profiling revealed the upregulation of the pro-metastatic gene Ly6/PLAUR Domain-Containing Protein 3 (LYPD3) in norepinephrine-treated MDA-MB-468 cells. Histology confirmed selective LYPD3 expression in primary and metastatic breast tumour samples. These findings demonstrate that basal-type cancer cells show a more aggressive adrenoceptor-β2-activated phenotype in the resting and stimulated state, which is attenuated by adrenoceptor-β2 inhibition. This study also highlights the first association between ADRβ2 signalling and LYPD3; its knockdown significantly reduced the basal and norepinephrine-induced activity of MCF-7 cells in vitro. The regulation of ADRβ2 signalling by LYPD3 and its metastasis promoting activities, reveal LYPD3 as a promising therapeutic target in the treatment of breast and other cancers.

Cell-cell adhesions constitute the structural "glue" that retains cells together and contributes to tissue organisation and physiological function. The integrity of these structures is regulated by extracellular and intracellular signals and pathways that act on the functional units of cell adhesion such as the cell adhesion molecules/adhesion receptors, the extracellular matrix (ECM) proteins and the cytoplasmic plaque/peripheral membrane proteins. In advanced cancer, these regulatory pathways are dysregulated and lead to cell-cell adhesion disassembly, increased invasion and metastasis. The Metastasis suppressor protein 1 (MTSS1) plays a key role in the maintenance of cell-cell adhesions and its loss correlates with tumour progression in a variety of cancers. However, the mechanisms that regulate its function are not well-known. Using a system biology approach, we unravelled potential interacting partners of MTSS1. We found that the secretory carrier-associated membrane protein 1 (SCAMP1), a molecule involved in post-Golgi recycling pathways and in endosome cell membrane recycling, enhances Mtss1 anti-invasive function in HER2+/ER-/PR- breast cancer, by promoting its protein trafficking leading to elevated levels of RAC1-GTP and increased cell-cell adhesions. This was clinically tested in HER2 breast cancer tissue and shown that loss of MTSS1 and SCAMP1 correlates with reduced disease-specific survival. In summary, we provide evidence of the cooperative roles of MTSS1 and SCAMP1 in preventing HER2+/ER-/PR- breast cancer invasion and we show that the loss of Mtss1 and Scamp1 results in a more aggressive cancer cell phenotype.

Background: Preclinical studies have demonstrated that propranolol inhibits several pathways involved in breast cancer progression and metastasis. We investigated whether breast cancer patients who used propranolol, or other non-selective beta-blockers, had reduced breast cancer-specific or all-cause mortality in eight European cohorts., Methods: Incident breast cancer patients were identified from eight cancer registries and compiled through the European Cancer Pharmacoepidemiology Network. Propranolol and non-selective beta-blocker use was ascertained for each patient. Breast cancer-specific and all-cause mortality were available for five and eight cohorts, respectively. Cox regression models were used to calculate hazard ratios (HR) and 95% confidence intervals (CIs) for cancer-specific and all-cause mortality by propranolol and non-selective beta-blocker use. HRs were pooled across cohorts using meta-analysis techniques. Dose-response analyses by number of prescriptions were also performed. Analyses were repeated investigating propranolol use before cancer diagnosis., Results: The combined study population included 55,252 and 133,251 breast cancer patients in the analysis of breast cancer-specific and all-cause mortality respectively. Overall, there was no association between propranolol use after diagnosis of breast cancer and breast cancer-specific or all-cause mortality (fully adjusted HR = 0.94, 95% CI, 0.77, 1.16 and HR = 1.09, 95% CI, 0.93, 1.28, respectively). There was little evidence of a dose-response relationship. There was also no association between propranolol use before breast cancer diagnosis and breast cancer-specific or all-cause mortality (fully adjusted HR = 1.03, 95% CI, 0.86, 1.22 and HR = 1.02, 95% CI, 0.94, 1.10, respectively). Similar null associations were observed for non-selective beta-blockers., Conclusions: In this large pooled analysis of breast cancer patients, use of propranolol or non-selective beta-blockers was not associated with improved survival.

Background: There remains a need to identify and validate biomarkers for predicting prostate cancer (CaP) outcomes using robust and routinely available pathology techniques to identify men at most risk of premature death due to prostate cancer. Previous immunohistochemical studies suggest the proliferation marker Ki67 might be a predictor of survival, independently of PSA and Gleason score. We performed a validation study of Ki67 as a marker of survival and disease progression and compared its performance against another candidate biomarker, DLX2, selected using artificial neural network analysis., Methods: A tissue microarray (TMA) was constructed from transurethral resected prostatectomy histology samples (n=192). Artificial neural network analysis was used to identify candidate markers conferring increased risk of death and metastasis in a public cDNA array. Immunohistochemical analysis of the TMA was carried out and univariate and multivariate tests performed to explore the association of tumour protein levels of Ki67 and DLX2 with time to death and metastasis., Results: Univariate analysis demonstrated Ki67 as predictive of CaP-specific survival (DSS; P=0.022), and both Ki67 (P=0.025) and DLX2 (P=0.001) as predictive of future metastases. Multivariate analysis demonstrated Ki67 as independent of PSA, Gleason score and D'Amico risk category for DSS (HR=2.436, P=0.029) and both Ki67 (HR=3.296, P=0.023) and DLX2 (HR=3.051, P=0.003) as independent for future metastases., Conclusions: High Ki67 expression is only present in 6.8% of CaP patients and is predictive of reduced survival and increased risk of metastasis, independent of PSA, Gleason score and D'Amico risk category. DLX2 is a novel marker of increased metastasis risk found in 73% patients and 8.2% showed co-expression with a high Ki67 score. Two cancer cell proliferation markers, Ki67 and DLX2, may be able to inform clinical decision-making when identifying patients for active surveillance.

Epithelial-mesenchymal transition (EMT) is a key event that is involved in the invasion and dissemination of cancer cells. Although typically considered as having tumour-suppressive properties, transforming growth factor (TGF)-β signalling is altered during cancer and has been associated with the invasion of cancer cells and metastasis. In this study, we report a previously unknown role for the cytoplasmic promyelocytic leukaemia (cPML) tumour suppressor in TGF-β signalling-induced regulation of prostate cancer-associated EMT and invasion. We demonstrate that cPML promotes a mesenchymal phenotype and increases the invasiveness of prostate cancer cells. This event is associated with activation of TGF-β canonical signalling pathway through the induction of Sma and Mad related family 2 and 3 (SMAD2 and SMAD3) phosphorylation. Furthermore, the cytoplasmic localization of promyelocytic leukaemia (PML) is mediated by its nuclear export in a chromosomal maintenance 1 (CRM1)-dependent manner. This was clinically tested in prostate cancer tissue and shown that cytoplasmic PML and CRM1 co-expression correlates with reduced disease-specific survival. In summary, we provide evidence of dysfunctional TGF-β signalling occurring at an early stage in prostate cancer. We show that this disease pathway is mediated by cPML and CRM1 and results in a more aggressive cancer cell phenotype. We propose that the targeting of this pathway could be therapeutically exploited for clinical benefit.

The Nottingham prognostic index plus (NPI+) is based on the assessment of biological class combined with established clinicopathologic prognostic variables providing improved patient outcome stratification for breast cancer superior to the traditional NPI. This study aimed to determine prognostic capability of the NPI+ in predicting risk of development of distant disease. A well-characterised series of 1073 primary early-stage BC cases treated in Nottingham and 251 cases from Budapest were immunohistochemically assessed for cytokeratin (Ck)5/6, Ck18, EGFR, oestrogen receptor (ER), progesterone receptor, HER2, HER3, HER4, Mucin 1 and p53 expression. NPI+ biological class and prognostic scores were assigned using individual algorithms for each biological class incorporating clinicopathologic parameters and investigated in terms of prediction of distant metastases-free survival (MFS). The NPI+ identified distinct prognostic groups (PG) within each molecular class which were predictive of MFS providing improved patient outcome stratification superior to the traditional NPI. NPI+ PGs, between series, were comparable in predicting patient outcome between series in luminal A, basal p53 altered and HER2+/ER+ (p > 0.01) tumours. The low-risk groups were similarly validated in luminal B, luminal N, basal p53 normal tumours (p > 0.01). Due to small patient numbers the remaining PGs could not be validated. NPI+ was additionally able to predict a higher risk of metastases at certain distant sites. This study may indicate the NPI+ as a useful tool in predicting the risk of metastases. The NPI+ provides accurate risk stratification allowing improved individualised clinical decision making for breast cancer.

The Nottingham Prognostic Index Plus (NPI+) is a clinical decision making tool in breast cancer (BC) that aims to provide improved patient outcome stratification superior to the traditional NPI. This study aimed to validate the NPI+ in an independent series of BC. Eight hundred and eighty five primary early stage BC cases from Edinburgh were semi-quantitatively assessed for 10 biomarkers [Estrogen Receptor (ER), Progesterone Receptor (PgR), cytokeratin (CK) 5/6, CK7/8, epidermal growth factor receptor (EGFR), HER2, HER3, HER4, p53, and Mucin 1] using immunohistochemistry and classified into biological classes by fuzzy logic-derived algorithms previously developed in the Nottingham series. Subsequently, NPI+ Prognostic Groups (PGs) were assigned for each class using bespoke NPI-like formulae, previously developed in each NPI+ biological class of the Nottingham series, utilising clinicopathological parameters: number of positive nodes, pathological tumour size, stage, tubule formation, nuclear pleomorphism and mitotic counts. Biological classes and PGs were compared between the Edinburgh and Nottingham series using Cramer's V and their role in patient outcome prediction using Kaplan-Meier curves and tested using Log Rank. The NPI+ biomarker panel classified the Edinburgh series into seven biological classes similar to the Nottingham series (p > 0.01). The biological classes were significantly associated with patient outcome (p < 0.001). PGs were comparable in predicting patient outcome between series in Luminal A, Basal p53 altered, HER2+/ER+ tumours (p > 0.01). The good PGs were similarly validated in Luminal B, Basal p53 normal, HER2+/ER- tumours and the poor PG in the Luminal N class (p > 0.01). Due to small patient numbers assigned to the remaining PGs, Luminal N, Luminal B, Basal p53 normal and HER2+/ER- classes could not be validated. This study demonstrates the reproducibility of NPI+ and confirmed its prognostic value in an independent cohort of primary BC. Further validation in large randomised controlled trial material is warranted.

Background: Recent laboratory and epidemiological evidence suggests that beta-blockers could inhibit prostate cancer progression., Methods: We investigated the effect of beta-blockers on prostate cancer-specific mortality in a cohort of prostate cancer patients. Prostate cancer patients diagnosed between 1998 and 2006 were identified from the UK Clinical Practice Research Database and confirmed by cancer registries. Patients were followed up to 2011 with deaths identified by the Office of National Statistics. A nested case-control analysis compared patients dying from prostate cancer (cases) with up to three controls alive at the time of their death, matched by age and year of diagnosis. Odds ratios (OR) and 95% confidence intervals (CI) were calculated using conditional logistic regression., Results: Post-diagnostic beta-blocker use was identified in 25% of 1184 prostate cancer-specific deaths and 26% of 3531 matched controls. There was little evidence (P=0.40) of a reduction in the risk of cancer-specific death in beta-blocker users compared with non-users (OR=0.94 95% CI 0.81, 1.09). Similar results were observed after adjustments for confounders, in analyses by beta-blocker frequency, duration, type and for all-cause mortality., Conclusions: Beta-blocker usage after diagnosis was not associated with cancer-specific or all-cause mortality in prostate cancer patients in this large UK study., (Copyright © 2014. Published by Elsevier Ltd.)

Inflammation is an important component of various cancers and its inflammatory cells and mediators have been shown to have prognostic potential. Tumor-infiltrating mast cells can promote tumor growth and angiogenesis, but the mechanism of mast cell activation is unclear. In earlier studies, we demonstrated that immunoglobulin free light chains (FLC) can trigger mast cells in an antigen-specific manner. Increased expression of FLC was observed within stroma of various human cancers including those of breast, colon, lung, pancreas, kidney and skin, and FLC expression co-localized with areas of mast cell infiltration. In a large cohort of breast cancer patients, FLC expression was shown associated with basal-like cancers with an aggressive phenotype. Moreover, lambda FLC was found expressed in areas of inflammatory infiltration and its expression was significantly associated with poor clinical outcome. Functional importance of FLCs was shown in a murine B16F10 melanoma model, where inhibition of FLC-mediated mast cell activation strongly reduced tumor growth. Collectively, this study identifies FLCs as a ligand in the pro-tumorigenic activation of mast cells. Blocking this pathway may open new avenues for the inhibition of tumor growth, while immunohistochemical staining of FLC may be helpful in the diagnosis and prognosis of cancer.

Introduction: Recent observational studies indicate that post-diagnostic use of aspirin in breast cancer patients may protect against cancer progression perhaps by inhibiting cyclooxygenase-2 dependent mechanisms. Evidence also supports a crucial role for interactions between tumour cells and circulating platelets in cancer growth and dissemination, therefore, use of low-dose aspirin may reduce the risk of death from cancer in breast cancer patients., Methods: A cohort of newly diagnosed breast cancer patients (1998 to 2006) were identified in the UK Clinical Practice Research Datalink (and confirmed by cancer registry linkage). Cancer-specific deaths were identified up to 2011 from Office for National Statistics mortality data. A nested case-control analysis was conducted using conditional logistic regression to compare post-diagnostic aspirin exposure using General Practice prescription data in 1,435 cases (breast cancer deaths) with 5,697 controls (matched by age and year of diagnosis)., Results: After breast cancer diagnosis, 18.3% of cancer-specific deaths and 18.5% of matched controls received at least one prescription for low-dose aspirin, corresponding to an odds ratio (OR) of 0.98 (95% CI 0.83, 1.15). Adjustment for potential confounders (including stage and grade) had little impact on this estimate. No dose response relationship was observed when the number of tablets was investigated and no associations were seen when analyses were stratified by receipt of prescriptions for aspirin in the pre-diagnostic period, by stage at diagnosis or by receipt of prescriptions for hormone therapy., Conclusions: Overall, in this large population-based cohort of breast cancer patients, there was little evidence of an association between receipt of post-diagnostic prescriptions for low-dose aspirin and breast cancer-specific death. However, information was not available on medication compliance or over-the-counter use of aspirin, which may have contributed to the null findings.

Background: Current management of breast cancer (BC) relies on risk stratification based on well-defined clinicopathologic factors. Global gene expression profiling studies have demonstrated that BC comprises distinct molecular classes with clinical relevance. In this study, we hypothesised that molecular features of BC are a key driver of tumour behaviour and when coupled with a novel and bespoke application of established clinicopathologic prognostic variables can predict both clinical outcome and relevant therapeutic options more accurately than existing methods., Methods: In the current study, a comprehensive panel of biomarkers with relevance to BC was applied to a large and well-characterised series of BC, using immunohistochemistry and different multivariate clustering techniques, to identify the key molecular classes. Subsequently, each class was further stratified using a set of well-defined prognostic clinicopathologic variables. These variables were combined in formulae to prognostically stratify different molecular classes, collectively known as the Nottingham Prognostic Index Plus (NPI+). The NPI+ was then used to predict outcome in the different molecular classes., Results: Seven core molecular classes were identified using a selective panel of 10 biomarkers. Incorporation of clinicopathologic variables in a second-stage analysis resulted in identification of distinct prognostic groups within each molecular class (NPI+). Outcome analysis showed that using the bespoke NPI formulae for each biological BC class provides improved patient outcome stratification superior to the traditional NPI., Conclusion: This study provides proof-of-principle evidence for the use of NPI+ in supporting improved individualised clinical decision making.

Background: Beta-blockers have potential antiangiogenic and antimigratory activity. Studies have demonstrated a survival benefit in patients with malignant melanoma treated with beta-blockers., Objectives: To investigate the association between postdiagnostic beta-blocker usage and risk of melanoma-specific mortality in a population-based cohort of patients with malignant melanoma., Methods: Patients with incident malignant melanoma diagnosed between 1998 and 2010 were identified within the U.K. Clinical Practice Research Datalink and confirmed using cancer registry data. Patients with malignant melanoma with a melanoma-specific death (cases) recorded by the Office of National Statistics were matched on year of diagnosis, age and sex to four malignant melanoma controls (who lived at least as long after diagnosis as their matched case). A nested case-control approach was used to investigate the association between postdiagnostic beta-blocker usage and melanoma-specific death and all-cause mortality. Conditional logistic regression was applied to generate odds ratios (ORs) and 95% confidence intervals (CIs) for beta-blocker use determined from general practitioner prescribing., Results: Beta-blocker medications were prescribed after malignant melanoma diagnosis to 20·2% of 242 patients who died from malignant melanoma (cases) and 20·3% of 886 matched controls. Consequently, there was no association between beta-blocker use postdiagnosis and cancer-specific death (OR 0·99, 95% CI 0·68-1·42), which did not markedly alter after adjustment for confounders including stage (OR 0·87, 95% CI 0·56-1·34). No significant associations were detected for individual beta-blocker types, by defined daily doses of use or for all-cause mortality., Conclusions: Contrary to some previous studies, beta-blocker use after malignant melanoma diagnosis was not associated with reduced risk of death from melanoma in this U.K. population-based study., (© 2014 British Association of Dermatologists.)

Background & Aims: Individuals who began taking low-dose aspirin before they were diagnosed with colorectal cancer were reported to have longer survival times than patients who did not take this drug. We investigated survival times of patients who begin taking low-dose aspirin after a diagnosis of colorectal cancer in a large population-based cohort study., Methods: We performed a nested case-control analysis using a cohort of 4794 patients diagnosed with colorectal cancer from 1998 through 2007, identified from the UK Clinical Practice Research Datalink and confirmed by cancer registries. There were 1559 colorectal cancer-specific deaths, recorded by the Office of National Statistics; these were each matched with up to 5 risk-set controls. Conditional logistic regression was used to calculate odds ratios (OR) and 95% confidence intervals (CI), based on practitioner-recorded aspirin usage., Results: Overall, low-dose aspirin use after a diagnosis of colorectal cancer was not associated with colorectal cancer-specific mortality (adjusted OR = 1.06; 95% CI: 0.92-1.24) or all-cause mortality (adjusted OR = 1.06; 95% CI: 0.94-1.19). A dose-response association was not apparent; for example, low-dose aspirin use for more than 1 year after diagnosis was not associated with colorectal cancer-specific mortality (adjusted OR = 0.98; 95% CI: 0.82-1.19). There was also no association between low-dose aspirin usage and colon cancer-specific mortality (adjusted OR = 1.02; 95% CI: 0.83-1.25) or rectal cancer-specific mortality (adjusted OR = 1.10; 95% CI: 0.88-1.38)., Conclusions: In a large population-based cohort, low-dose aspirin usage after diagnosis of colorectal cancer did not increase survival time., (Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: Oestrogen receptor (ER) positive (luminal) tumours account for the largest proportion of females with breast cancer. Theirs is a heterogeneous disease presenting clinical challenges in managing their treatment. Three main biological luminal groups have been identified but clinically these can be distilled into two prognostic groups in which Luminal A are accorded good prognosis and Luminal B correlate with poor prognosis. Further biomarkers are needed to attain classification consensus. Machine learning approaches like Artificial Neural Networks (ANNs) have been used for classification and identification of biomarkers in breast cancer using high throughput data. In this study, we have used an artificial neural network (ANN) approach to identify DACH1 as a candidate luminal marker and its role in predicting clinical outcome in breast cancer is assessed., Materials and Methods: A reiterative ANN approach incorporating a network inferencing algorithm was used to identify ER-associated biomarkers in a publically available cDNA microarray dataset. DACH1 was identified in having a strong influence on ER associated markers and a positive association with ER. Its clinical relevance in predicting breast cancer specific survival was investigated by statistically assessing protein expression levels after immunohistochemistry in a series of unselected breast cancers, formatted as a tissue microarray., Results: Strong nuclear DACH1 staining is more prevalent in tubular and lobular breast cancer. Its expression correlated with ER-alpha positive tumours expressing PgR, epithelial cytokeratins (CK)18/19 and 'luminal-like' markers of good prognosis including FOXA1 and RERG (p<0.05). DACH1 is increased in patients showing longer cancer specific survival and disease free interval and reduced metastasis formation (p<0.001). Nuclear DACH1 showed a negative association with markers of aggressive growth and poor prognosis., Conclusion: Nuclear DACH1 expression appears to be a Luminal A biomarker predictive of good prognosis, but is not independent of clinical stage, tumour size, NPI status or systemic therapy.

Purpose: Aspirin use is associated with reduced risk of, and death from, prostate cancer. Our aim was to determine whether low-dose aspirin use after a prostate cancer diagnosis was associated with reduced prostate cancer-specific mortality., Methods: A cohort of newly diagnosed prostate cancer patients (1998-2006) was identified in the UK Clinical Practice Research Datalink (confirmed by cancer registry linkage). A nested case-control analysis was conducted using conditional logistic regression to compare aspirin usage in cases (prostate cancer deaths) with up to three controls (matched by age and year of diagnosis)., Results: Post-diagnostic low-dose aspirin use was identified in 52 % of 1,184 prostate cancer-specific deaths and 39 % of 3,531 matched controls (unadjusted OR 1.51, 95 % CI 1.19, 1.90; p < 0.001). After adjustment for confounders including treatment and comorbidities, this association was attenuated (adjusted OR  1.02 95 % CI 0.78, 1.34; p = 0.86). Adjustment for estrogen therapy accounted for the majority of this attenuation. There was also no evidence of dose-response association after adjustments. Compared with no use, patients with 1-11 prescriptions and 12 or more prescriptions had adjusted ORs of 1.07 (95 % CI 0.78, 1.47; p = 0.66) and 0.97 (95 % CI 0.69, 1.37; p = 0.88), respectively. There was no evidence of a protective association between low-dose aspirin use in the year prior to diagnosis and prostate cancer-specific mortality (adjusted OR 1.04 95 % CI 0.89, 1.22; p = 0.60)., Conclusion: We found no evidence of an association between low-dose aspirin use before or after diagnosis and risk of prostate cancer-specific mortality, after potential confounders were accounted for, in UK prostate cancer patients.

Background: To investigate the association between post-diagnostic beta-blocker usage and risk of cancer-specific mortality in a large population-based cohort of female breast cancer patients., Methods: A nested case-control study was conducted within a cohort of breast cancer patients identified from cancer registries in England(using the National Cancer Data repository) and diagnosed between 1998 and 2007. Patients who had a breast cancer-specific death(ascertained from Office of National Statistics death registration data) were each matched to four alive controls by year and age at diagnosis. Prescription data for these patients were available through the Clinical Practice Research Datalink. Conditional logistic regression models were used to investigate the association between breast cancer-specific death and beta-blocker usage., Results: Post-diagnostic use of beta-blockers was identified in 18.9% of 1435 breast cancer-specific deaths and 19.4% of their 5697 matched controls,indicating little evidence of association between beta-blocker use and breast cancer-specific mortality [odds ratio (OR) = 0.97,95% confidence interval (CI) 0.83, 1.13]. There was also little evidence of an association when analyses were restricted to cardio non-selective beta-blockers (OR = 0.90, 95% CI 0.69, 1.17). Similar results were observed in analyses of drug dosage frequency and duration, and beta-blocker type., Conclusions: In this large UK population-based cohort of breast cancer patients,there was little evidence of an association between post-diagnostic beta-blocker usage and breast cancer progression. Further studies which include information on tumour receptor status are warranted to determine whether response to beta-blockers varies by tumour subtypes.

Background: Epidemiological and laboratory studies suggest that β-blockers may reduce cancer progression in various cancer sites. The aim of this study was to conduct the first epidemiological investigation of the effect of post-diagnostic β-blocker usage on colorectal cancer-specific mortality in a large population-based colorectal cancer patient cohort., Patients and Methods: A nested case-control analysis was conducted within a cohort of 4794 colorectal cancer patients diagnosed between 1998 and 2007. Patients were identified from the UK Clinical Practice Research Datalink and confirmed using cancer registry data. Patients with a colorectal cancer- specific death (data from the Office of National Statistics death registration system) were matched to five controls. Conditional logistic regression was applied to calculate odds ratios (OR) and 95% confidence intervals (95% CIs) according to β-blocker usage (data from GP-prescribing records)., Results: Post-diagnostic β-blocker use was identified in 21.4% of 1559 colorectal cancer-specific deaths and 23.7% of their 7531 matched controls, with little evidence of an association (OR = 0.89 95% CI 0.78-1.02). Similar associations were found when analysing drug frequency, β-blocker type or specific drugs such as propranolol. There was some evidence of a weak reduction in all-cause mortality in β-blocker users (adjusted OR = 0.88; 95% CI 0.77-1.00; P = 0.04) which was in part due to the marked effect of atenolol on cardiovascular mortality (adjusted OR = 0.62; 95% CI 0.40-0.97; P = 0.04)., Conclusions: In this novel, large UK population-based cohort of colorectal cancer patients, there was no evidence of an association between post-diagnostic β-blocker use and colorectal cancer-specific mortality., Clinical Trials Number: NCT00888797.

Background: The most important challenge of performing insitu transcriptional profiling of the human ocular surface epithelial regions is obtaining samples in sufficient amounts, without contamination from adjacent tissue, as the region of interest is microscopic and closely apposed to other tissues regions. We have effectively collected ocular surface (OS) epithelial tissue samples from the Limbal Epithelial Crypt (LEC), limbus, cornea and conjunctiva of post-mortem cadaver eyes with laser microdissection (LMD) technique for gene expression studies with spotted oligonucleotide microarrays and Gene 1.0 ST arrays., Methods: Human donor eyes (4 pairs for spotted oligonucleotide microarrays, 3 pairs for Gene 1.0 ST arrays) consented for research were included in this study with due ethical approval of the Nottingham Research Ethics Committee. Eye retrieval was performed within 36 hours of post-mortem period. The dissected corneoscleral buttons were immersed in OCT media and frozen in liquid nitrogen and stored at -80°C till further use. Microscopic tissue sections of interest were taken on PALM slides and stained with Toluidine Blue for laser microdissection with PALM microbeam systems. Optimisation of the laser microdissection technique was crucial for efficient and cost effective sample collection., Results: The starting concentration of RNA as stipulated by the protocol of microarray platforms was taken as the cut-off concentration of RNA samples in our studies. The area of LMD tissue processed for spotted oligonucleotide microarray study ranged from 86,253 μm2 in LEC to 392,887 μm2 in LEC stroma. The RNA concentration of the LMD samples ranged from 22 to 92 pg/μl. The recommended starting concentration of the RNA samples used for Gene 1.0 ST arrays was 6 ng/5 μl. To achieve the desired RNA concentration the area of ocular surface epithelial tissue sample processed for the Gene 1.0 ST array experiments was approximately 100,0000 μm2 to 130,0000 μm2. RNA concentration of these samples ranged from 10.88 ng/12 μl to 25.8 ng/12 μl, with the RNA integrity numbers (RIN) for these samples from 3.3 to 7.9. RNA samples with RIN values below 2, that had failed to amplify satisfactorily were discarded., Conclusions: The optimised protocol for sample collection and laser microdissection improved the RNA yield of the in situ ocular surface epithelial regions for effective microarray studies on spotted oligonucleotide and affymetrix platforms.

Background: Breast cancer is a heterogeneous disease characterised by complex molecular alterations underlying the varied behaviour and response to therapy. However, translation of cancer genetic profiling for use in routine clinical practice remains elusive or prohibitively expensive. As an alternative, immunohistochemical analysis applied to routinely processed tissue samples could be used to identify distinct biological classes of breast cancer., Methods: In this study, 1073 archival breast tumours previously assessed for 25 key breast cancer biomarkers using immunohistochemistry and classified using clustering algorithms were further refined using naïve Bayes classification performance. Criteria for class membership were defined using the expression of a reduced panel of 10 proteins able to identify key molecular classes. We examined the association between these breast cancer classes with clinicopathological factors and patient outcome., Results: We confirm patient classification similar to established genotypic biological classes of breast cancer in addition to novel sub-divisions of luminal and basal tumours. Correlations between classes and clinicopathological parameters were in line with expectations and showed highly significant association with patient outcome. Furthermore, our novel biological class stratification provides additional prognostic information to the Nottingham Prognostic Index., Conclusion: This study confirms that distinct molecular phenotypes of breast cancer can be identified using robust and routinely available techniques and both the luminal and basal breast cancer phenotypes are heterogeneous and contain distinct subgroups.

We have shown previously that norepinephrine induces migratory activity of tumour cells from breast, colon and prostate tissue via activation of beta-2 adrenergic receptors. Consequently, this effect can be inhibited pharmacologically by clinically established beta-blockers. Tumour cell migration is a prerequisite for metastasis formation, and accordingly we and others have shown that breast cancer patients, which take beta-blockers due to hypertension, have reduced metastasis formation and increased survival probability as compared to patients without hypertension or using other anti-hypertensive medication. Unlike the aforementioned tumour cells, pancreatic cancer cells show a reduced migratory activity upon norepinephrine treatment. By means of our three-dimensional, collagen-based cell migration assay, we have investigated the signal transduction pathways involved in this phenomenon. We have found that this conflicting effect of norepinephrine on pancreatic cancer cells is due to an imbalanced activation of the two pathways that usually mediate a pro-migratory effect of norepinephrine in other tumour cell types. Firstly, the inhibitory effect results from activation of a pathway which causes a strong increase of the secondary cell signalling molecule, cAMP. In addition, activation of phospholipase C gamma and the downstream protein kinase C alpha were shown to be already activated in pancreatic cancer cells and cannot be further activated by norepinephrine. We hypothesize that this constitutive activation of the phospholipase C gamma pathway is due to a cross-talk with receptor tyrosine kinase signalling, and this might also deliver an explanation for the unusual high spontaneous migratory activity of pancreatic cancer cells., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objectives: Recent studies of breast cancer data have identified seven distinct clinical phenotypes (groups) using immunohistochemical analysis and a range of different clustering techniques. Consensus between unsupervised classification algorithms has been successfully used to categorise patients into these specific groups, but often at the expenses of not classifying the whole set. It is known that fuzzy methodologies can provide linguistic based classification rules. The objective of this study was to investigate the use of fuzzy methodologies to create an easy to interpret set of classification rules, capable of placing the large majority of patients into one of the specified groups., Materials and Methods: In this paper, we extend a data-driven fuzzy rule-based system for classification purposes (called 'fuzzy quantification subsethood-based algorithm') and combine it with a novel class assignment procedure. The whole approach is then applied to a well characterised breast cancer dataset consisting of ten protein markers for over 1000 patients to refine previously identified groups and to present clinicians with a linguistic ruleset. A range of statistical approaches was used to compare the obtained classes to previously obtained groupings and to assess the proportion of unclassified patients., Results: A rule set was obtained from the algorithm which features one classification rule per class, using labels of High, Low or Omit for each biomarker, to determine the most appropriate class for each patient. When applied to the whole set of patients, the distribution of the obtained classes had an agreement of 0.9 when assessed using Kendall's Tau with the original reference class distribution. In doing so, only 38 patients out of 1073 remain unclassified, representing a more clinically usable class assignment algorithm., Conclusion: The fuzzy algorithm provides a simple to interpret, linguistic rule set which classifies over 95% of breast cancer patients into one of seven clinical groups., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The coactivator-associated arginine methyltransferase-1 (CARM1) is implicated in regulation of oestrogen receptor (ER) α-mediated gene pathways in response to ER activation. It plays an important role in breast cancer growth by regulating the E2F1 expression suggesting that CARM1 could be a target in the subclassification of oestrogen-dependent breast cancer. This study aims to investigate the clinical and biological importance of CARM1 protein expression in a large (1,130 patients), well-characterised and annotated series of invasive breast cancers using tissue microarrays and immunohistochemistry. In the whole series, increased CARM1 expression is correlated with features associated with aggressive behaviour such as young age, premenopausal status, large tumour size and high tumour grade. There is a positive correlation between CARM1 expression and biomarkers associated with non-luminal phenotype and poor prognosis such as HER2, basal cytokeratins, EGFR, p53 and the proliferation markers Ki67, TK1, CD71 and Cyclin E. Negative associations with the luminal-associated markers including steroid receptors and luminal cytokeratins are found. Similar associations are identified in the ER-positive/luminal subgroup (n = 767). Outcome analyses indicate that CARM1 expression is an independent predictor of shorter breast cancer-specific survival and disease-free interval in the whole series and in the ER-positive subgroup. CARM1 shows an oncogenic effect in breast cancer and its expression is associated with poor prognosis. CARM1 could be a potential marker of luminal class subclassification and for target therapy, particularly in the ER-positive luminal-like subgroup.

Recent preclinical studies have associated beta-adrenergic receptor (β-AR) signaling with breast cancer pathways such as progression and metastasis. These findings have been supported by clinical and epidemiological studies which examined the effect of beta-blocker therapy on breast cancer metastasis, recurrence and mortality. Results from these studies have provided initial evidence for the inhibition of cell migration in breast cancer by beta-blockers and have introduced the beta-adrenergic receptor pathways as a target for therapy. This paper analyzes gene expression profiles in breast cancer patients, utilising Artificial Neural Networks (ANNs) to identify molecular signatures corresponding to possible disease management pathways and biomarker treatment strategies associated with beta-2-adrenergic receptor (ADRB2) cell signaling. The adrenergic receptor relationship to cancer is investigated in order to validate the results of recent studies that suggest the use of beta-blockers for breast cancer therapy. A panel of genes is identified which has previously been reported to play an important role in cancer and also to be involved in the beta-adrenergic receptor signaling.

Breast cancer is a heterogeneous and complex disease. Although the use of tumor biomarkers has improved individualized breast cancer care, i.e., assessment of risk, diagnosis, prognosis, and prediction of treatment outcome, new markers are required to further improve patient clinical management. In the present study, a search for novel breast cancer-associated genes was performed by mining the UniGene database for expressed sequence tags (ESTs) originating from human normal breast, breast cancer tissue, or breast cancer cell lines. Two hundred and twenty-eight distinct breast-associated UniGene Clusters (BUC1-228) matched the search criteria. Four BUC ESTs (BUC6, BUC9, BUC10, and BUC11) were subsequently selected for extensive in silico database searches, and in vitro analyses through sequencing and RT-PCR based assays on well-characterized cell lines and tissues of normal and cancerous origin. BUC6, BUC9, BUC10, and BUC11 are clustered on 10p11.21-12.1 and showed no homology to any known RNAs. Overall, expression of the four BUC transcripts was high in normal breast and testis tissue, and in some breast cancers; in contrast, BUC was low in other normal tissues, peripheral blood mononuclear cells (PBMCs), and other cancer cell lines. Results to-date suggest that BUC11 and BUC9 translate to protein and BUC11 cytoplasmic and nuclear protein expression was detected in a large cohort of breast cancer samples using immunohistochemistry. This study demonstrates the discovery and expression analysis of a tissue-restricted novel transcript set which is strongly expressed in breast tissue and their application as clinical cancer biomarkers clearly warrants further investigation., (Copyright © 2012 Wiley Periodicals, Inc.)

Advances in the understanding of the molecular basis of breast cancer have necessitated a definition of more sensitive and specific indicators of prognosis that are central to the underlying cancer biology and that reflect the complicated and heterogeneous nature of the disease. This study investigates the expression of epithelial cell adhesion molecule (EpCAM) in breast cancer particularly basal-like phenotype group which remains unclear. EpCAM expression was assessed using immunohistochemistry in a large well-characterised series of breast carcinomas prepared as tissue microarrays. Relationships between EpCAM expression with molecular subtypes, clinicopathological variables and patients' outcome were examined. EpCAM expression was associated with higher tumour grade (P < 0.001), larger tumour size (P < 0.001) and basal phenotype (P = 0.03). Importantly, within the basal-like tumours those positive for EpCAM showed a significantly shorter DFI (LR = 7.97, P = 0.005) and MFS (LR 4.01, P = 0.045). EpCAM may play a role in breast cancer progression and its expression is associated with poor patient outcome in basal-like breast cancer, independent of other prognostic factors.

Local allergic rhinitis (LAR) is a localized nasal allergic response in the absence of systemic atopy characterized by local production of specific IgE (sIgE) antibodies, a T(H)2 pattern of mucosal cell infiltration during natural exposure to aeroallergens, and a positive nasal allergen provocation test response with release of inflammatory mediators (tryptase and eosinophil cationic protein). Although the prevalence remains to be established, a number of patients previously given a diagnosis of nonallergic rhinitis or idiopathic rhinitis are now being classified as having LAR. Culprit allergens responsible include house dust mite, grass and olive pollens, and many others. For the diagnosis of LAR, neither skin prick testing nor determination of the presence of serum sIgE antibodies is useful, and a nasal allergen provocation test is needed to identify the culprit allergen or allergens. In a certain proportion of cases, local sIgE can be detected, and conjunctivitis, asthma, or both can be associated. Whether patients with LAR will have systemic atopy in the future is a matter of debate. Further studies are needed for examine the prevalence of this phenomenon in different areas, to improve the diagnostic methods to better identify these patients, and to develop therapeutic approaches, including the use of immunotherapy., (Published by Mosby, Inc.)

Global gene expression profiling (GEP) studies of breast cancer have identified distinct biological classes with different clinical and therapeutic implications. Oestrogen receptor (ER) has been found to be a central marker of the molecular signature. GEP studies have consistently recognized a molecularly distinct class of tumours that is characterized by high-level expression of ER and other biomarkers recognized to be characteristic of normal luminal cells of the breast. This class is the largest of the GEP-defined molecular subclasses, comprising 60-70% of breast cancer cases. Moreover, it has been proposed that this group of tumours is composed of at least two subclasses distinguished by differing GEP profiles. At present, there is no consensus on the definition of the luminal subclasses and, in clinical practice, luminal-like tumours and ER-positive tumours are frequently considered to be the same. A better understanding of the biological features of luminal tumours could lead to their improved characterization and consistent identification. In this review, we explore the concept and definitions of the luminal-like class of breast carcinoma and their contribution to our understanding of their molecular features, clinical significance and therapeutic implications., (© 2011 Blackwell Publishing Limited.)

The migratory activity of tumor cells and their ability to extravasate from the blood stream through the vascular endothelium are important steps within the metastasis cascade. We have shown previously that norepinephrine is a potent inducer of the migration of MDA-MB-468 human breast carcinoma cells and therefore investigated herein, whether the interaction of these cells as well as MDA-MB-231 and MDA-MB-435S human breast carcinoma cells with the vascular endothelium is affected by this neurotransmitter as well. By means of a flow-through assay under physiologic flow conditions, we show that norepinephrine induces an increase of the adhesion of the MDA-MB-231 cells, but not of MDA-MB-468 and MDA-MB-435S cells to human pulmonary microvascular endothelial cells (HMVEC). The adhesion of MDA-MB-231 cells was based on a norepinephrine-mediated release of GROα from HMVECs. GROα caused a β1-integrin-mediated increase of the adhesion of MDA-MB-231 cells. Most interestingly, this effect of norepinephrine, similar to the aforementioned induction of migration in MDA-MB-468 cells, was mediated by β-adrenergic receptors and therefore abrogated by β-blockers. In conclusion, norepinephrine has cell line-specific effects with regard to certain steps of the metastasis cascade, which are conjointly inhibited by clinically established β-blockers. Therefore, these results may deliver a molecular explanation for our recently published retrospective data analysis of patients with breast cancer which shows that β-blockers significantly reduce the development of metastases., (©2011 AACR.)

Purpose of Review: Due to the overlap of symptoms and signs in the various types of rhinitis, arriving at a precise diagnosis can be difficult. A diagnosis of idiopathic rhinitis can only be used when all other causes have been excluded and this review tries to clarify whom this term should be applied to, and the reasons why., Recent Findings: The current literature on idiopathic rhinitis is sparse. Haematological and immunological tests can help to distinguish which individuals have a predisposition to allergic mediated disease, but these results should be interpreted with caution because of the prevalence of false-positives. It has recently been shown that some patients previously labelled as 'idiopathic' suffer from a highly localized form of IgE-mediated allergy known as 'entopy'. Patients with idiopathic rhinitis probably represent a disparate group who may be suffering from a range of immunological or neuroregulatory disease processes in which an inability to down-regulate inflammatory processes may be as important as the process that initiates it., Summary: To arrive at a diagnosis of idiopathic rhinitis all other forms of rhinitis must be excluded. With further research and a better understanding of the pathological processes involved in rhinitis, we may need to use the term 'idiopathic' less frequently but be more assured of its correct usage.

Breast cancer mortality is frequently associated with metastatic disease. Metastasis models have shown adrenoceptor (AR) stimulation induces cell migration which is inhibited by adrenoceptor antagonist drugs. We investigated adrenoceptor protein expression in clinical breast tumours and its association with disease progression and prognosis. Immunohistochemistry on tissue microarrays was used to characterise α1b, α2c and β(2)2 adrenoceptor protein expression in operable breast tumours. Associations with tumour-relevant biological markers and clinical outcome were statistically assessed. Strong α1b expression occurred in large high grade (P < 0.0001), HER2+ (P < 0.0001) or basal-like (CK5/6, P = 0.0005; CK14, P = 0.0001; EGFR, P = 0.003) cancers, showing increased proliferation (Mib1, P = 0.002), decreased apoptosis (Bcl2, P < 0.0001) and poor NPI membership (P = 0.001). α1b expression correlated with poor cancer-specific survival (LR = 7.628, P = 0.022) and tumour recurrence (LR = 6.128, P = 0.047). Strong α2c was over-expressed in high grade (P = 0.007), HER3+ (P = 0.002) and HER4+ (P < 0.0001) cancers with borderline increase in EGFR, p53 and MIB1 proteins, and inverse association with hormonal (PgR, P = 0.002) phenotype. In contrast, strong β(2) expression occurred in small-size, luminal-like (ER+, P < 0.001) tumours of low grade (P < 0.001) and lymph node stage (P = 0.027) that showed poor prognosis when hormonal treatment was withheld. Adrenoceptors were not found to be independent predictors of clinical outcome. Alpha1b and α2c AR is over-expressed in basal-like breast tumours of poor prognosis. Strong β(2) adrenoceptor expression is seen in patients with a luminal (ER+) tumour phenotype and good prognosis, due to benefits derived from hormonal therapy. These findings suggest a possible role for targeted therapy using adrenoceptor antagonists.

Three recent population studies have translated laboratory investigations into a clinical setting and concur in presenting evidence that suggest a dramatic new role for β-blockers in reducing metastases, tumor recurrence and specific mortality in breast cancer. Should we be skeptical about these controversial findings?

Oestrogen receptor (ER)-positive breast cancer (BC) constitutes a heterogeneous group of tumours with regard to outcome and response to therapy. Accurate stratification of ER-positive BC according to risk of relapse and response to therapy will be achieved through an improved understanding of ER and ER-related biological pathways. Recent studies have identified Forkhead box O3a (FOXO3a) transcription factor as an intracellular mediator of ERα expression and as an important downstream target of the Akt/PI3K pathway indicating a biological and potential clinical role for FOXO3a in ER-positive BC. In this study, we investigated the clinical relevance and biological associations of FOXO3a protein expression, using tissue microarrays and immunohistochemistry, in a large series of patients with invasive breast cancer. FOXO3a protein expression showed both nuclear and/or cytoplasmic staining patterns. FOXO3a predominant nuclear expression was positively associated with biomarkers of good prognosis including PgR, FOXA1 and p27 expression. There was an inverse association with mitotic counts, MIB1 growth fraction, C-MYC and PIK3CA expression. With respect to patient outcome, FOXO3a nuclear localisation was associated with longer BC specific survival (P < 0.001) and longer distant metastasis free interval (P = 0.001), independently of the well-established breast cancer prognostic factors. In conclusion, our results demonstrate the biological and prognostic role of FOXO3a protein expression and its subcellular localisation in ER-positive/luminal-like BC possibly through its involvement in controlling cell proliferation.

Global gene expression profiling studies have classified breast cancer into a number of distinct biological and molecular classes with clinical relevance. The heterogeneous luminal group, which is largely characterised by oestrogen receptor (ER) expression, appears to contain distinct subgroups with differing behaviour. In this study, we analysed 47,293 gene transcripts in 128 invasive breast carcinomas (BC) using Artificial Neural Networks and a cross-validation analysis in combination with an ensemble sample classification to identify genes that can be used to subclassify ER+ luminal tumours. The results were validated using immunohistochemistry on TMAs containing 1,140 invasive breast cancers. Our results showed that the RERG gene is one of the highest ranked genes to differentiate between ER+ luminal-like and ER- non-luminal cancers based on a 10-fold external cross-validation analysis with an average classification accuracy of 89%. This was confirmed in our protein expression studies that showed RERG positive associations with markers of luminal differentiation including ER, luminal cytokeratins (CK19, CK18 and CK7/8) and FOXA1 (P = 0.004) and other markers of good prognosis in BC including small size, lower histologic grade and positive expression of androgen receptor, nuclear BRCA1, FHIT and cell cycle inhibitors p27 and p21. RERG expression was inversely associated with the proliferation marker MIB1 (P = 0.005) and p53. Strong RERG expression showed an association with longer breast cancer specific survival and distant metastasis free interval in the whole series as well as in the ER+ luminal group and these associations were independent of other prognostic variables. In conclusion, we used novel bioinformatics methods to identify candidate genes to characterise ER+ luminal-like breast cancer. RERG gene is a key marker of the luminal BC class and can be used to separate distinct prognostic subgroups.

Akt/PKB serine/threonine kinase is a leading signalling modulator for several cellular processes including metabolism, growth, proliferation and survival. However, complexity and diversity in the upstream/downstream arms of Akt pathway, as recent genetic studies reported, challenge considerably the evolvement of effective targeted therapies. The aim was to study the expression of phospho-Akt1 (pAkt) in breast cancer (BC), with respect to different component proteins upstream/downstream of Akt pathway activation, clinicopathologic parameters and patients' outcome. pAkt (Ser473) was evaluated by immunohistochemistry, on tissue microarrays containing 1,202 early invasive BC with long-term clinical follow-up. Seventy-six percent of the studied tumours overexpressed pAkt, where it was associated with expression of oestrogen and androgen receptors, PIK3CA, cytokeratin (CK)18, CK19 and PTEN. Loss of pAkt was correlated with high grade, CK5/6, p53 and high Ki67 labelling index. Higher proportions of luminal tumours were pAkt positive relative to triple negative/basal subtypes. However, pAkt overexpression was not associated with breast cancer specific (BCSS) or metastasis-free survival (MFS). Four tumour phenotypes were identified based on PIK3CA and pAkt expression, with substantial proportions being PIK3CA⁻/pAkt⁺ or PIK3CA⁺/pAkt⁻. These four combinatorial phenotypes were significantly associated with BCSS (p = 0.001) and MFS (p = 0.002). Although pAKT is an oncogene correlated with poor prognostic variables, it was not a prognostic marker. Combinatorial phenotypic groups of PIK3CA/pAkt denoted functional complexity, at translational level, within the upstream and downstream arms of Akt activation with significant impact on patients' outcome. These findings may help development more adequate therapeutic regimens for specific subgroups of this key cancer pathway.

Histological grade is recognized as one of the strongest prognostic factors in operable breast cancer (BC). Although grade 1 and grade 3 tumours are biologically and clinically distinct, grade 2 tumours bear considerable difficulty in outcome prediction and planning therapies. Several attempts such as genomic grade index have been performed to subclassify grade 2 into two subgroups with clinical relevance. Here, we present evidence that the routinely evaluable immunohistochemical MIB1/Ki67 labelling index (MIB-LI) can classify grade 2 tumours into two clinically distinct subgroups. In this study, growth fractions of 1,550 primary operable invasive breast carcinomas were immunohistochemically assayed on full-face tissue sections using the MIB1 clone of Ki-67. Growth fractions were assessed as number of MIB1 positive nuclei in 1,000 tumour nuclei at high-power magnification and expressed as MIB1-LI. Using a 10% cut-point of MIB1-LI, grade 2 BCs were classified into low (49.8%) and high (50.2%) proliferative subgroups. Univariate and multivariate survival analysis revealed statistically significant differences between these subgroups regarding patients' BC specific survival (P < 0.001), and metastasis free survival (P < 0.001) which was independent of the well-established prognostic factors (HR = 2.944, 95% CI = 1.634-5.303, P < 0.001). In conclusion, our results further demonstrate that grade 2 BCs may represent at least two biological or behaviourally different entities. Assay of growth fraction in BC using MIB1/Ki67 immunohistochemistry is a robust cost-effective diagnostic tool that subdivides grade 2 tumours into low and high risk populations providing additional prognostic information in planning therapies and outcome prediction.

Background: Some breast cancer patients receiving anti-angiogenic treatment show increased metastases, possibly as a result of induced hypoxia. The effect of hypoxia on tumor cell migration was assessed in selected luminal, post-EMT and basal-like breast carcinoma cell lines., Methods: Migration was assessed in luminal (MCF-7), post-EMT (MDA-MB-231, MDA-MB-435S), and basal-like (MDA-MB-468) human breast carcinoma cell lines under normal and oxygen-deprived conditions, using a collagen-based assay. Cell proliferation was determined, secreted cytokine and chemokine levels were measured using flow-cytometry and a bead-based immunoassay, and the hypoxic genes HIF-1α and CA IX were assessed using PCR. The functional effect of tumor-cell conditioned medium on the migration of neutrophil granulocytes (NG) was tested., Results: Hypoxia caused increased migratory activity but not proliferation in all tumor cell lines, involving the release and autocrine action of soluble mediators. Conditioned medium (CM) from hypoxic cells induced migration in normoxic cells. Hypoxia changed the profile of released inflammatory mediators according to cell type. Interleukin-8 was produced only by post-EMT and basal-like cell lines, regardless of hypoxia. MCP-1 was produced by MDA-MB-435 and -468 cells, whereas IL-6 was present only in MDA-MB-231. IL-2, TNF-α, and NGF production was stimulated by hypoxia in MCF-7 cells. CM from normoxic and hypoxic MDA-MB-231 and MDA-MB-435S cells and hypoxic MCF-7 cells, but not MDA-MB-468, induced NG migration., Conclusions: Hypoxia increases migration by the autocrine action of released signal substances in selected luminal and basal-like breast carcinoma cell lines which might explain why anti-angiogenic treatment can worsen clinical outcome in some patients.