ß2-Adrenergic Signalling Promotes Cell Migration by Upregulating Expression of the Metastasis-Associated Molecule LYPD3.

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 ß2-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-ß2-activated phenotype in the resting and stimulated state, which is attenuated by adrenoceptor-ß2 inhibition. This study also highlights the first association between ADRß2 signalling and LYPD3; its knockdown significantly reduced the basal and norepinephrine-induced activity of MCF-7 cells in vitro. The regulation of ADRß2 signalling by LYPD3 and its metastasis promoting activities, reveal LYPD3 as a promising therapeutic target in the treatment of breast and other cancers.

Nottingham Prognostic Index Plus: Validation of a clinical decision making tool in breast cancer in an independent series.

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.

KI67 and DLX2 predict increased risk of metastasis formation in prostate cancer-a targeted molecular approach.

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.

Immunoglobulin free light chains are biomarkers of poor prognosis in basal-like breast cancer and are potential targets in tumor-associated inflammation.

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.

DACH1: its role as a classifier of long term good prognosis in luminal breast cancer.

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.

Optimised laser microdissection of the human ocular surface epithelial regions for microarray studies.

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.

The oestrogen receptor coactivator CARM1 has an oncogenic effect and is associated with poor prognosis in breast cancer.

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.

A quantifier-based fuzzy classification system for breast cancer patients.

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.

Norepinephrine inhibits the migratory activity of pancreatic cancer cells.

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.

Biomarker identification in breast cancer: Beta-adrenergic receptor signaling and pathways to therapeutic response.

Recent preclinical studies have associated beta-adrenergic receptor (ß-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.

Local allergic rhinitis: concept, pathophysiology, and management.

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.

EpCAM expression is an indicator of recurrence in basal-like breast cancer.

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.

A review of the biological and clinical characteristics of luminal-like oestrogen receptor-positive breast cancer.

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.

Norepinephrine promotes the ß1-integrin-mediated adhesion of MDA-MB-231 cells to vascular endothelium by the induction of a GROα release.

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 ß1-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 ß-adrenergic receptors and therefore abrogated by ß-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 ß-blockers. Therefore, these results may deliver a molecular explanation for our recently published retrospective data analysis of patients with breast cancer which shows that ß-blockers significantly reduce the development of metastases.

Idiopathic rhinitis.

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.

Targeted therapies: Using ß-blockers to inhibit breast cancer progression.

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

Luminal and basal-like breast cancer cells show increased migration induced by hypoxia, mediated by an autocrine mechanism.

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.

Tumor-infiltrating CD8+ lymphocytes predict clinical outcome in breast cancer.

Breast carcinomas are often infiltrated by inflammatory cells, particularly macrophages and T lymphocytes, but the significance of these cells remains unclear. One possible role of these inflammatory cells is that they represent a cell-mediated immune response against the carcinoma. CD8(+) lymphocytes are a known crucial component of cell-mediated immunity. The purpose of this study was to explore the prognostic value of tumor-infiltrating CD8(+) cytotoxic lymphocytes in breast cancer. Tumor-infiltrating CD8(+) lymphocytes were assessed by immunohistochemical staining of tissue microarray cores from 1,334 unselected breast tumors from patients with long-term follow-up. The number of CD8(+) T cells was counted in tumor nests (intratumoral), in stroma adjacent to tumor cells, and in stroma distant to tumor cells, and their relationship with clinical outcome was determined. The total number of CD8(+) cells was positively correlated with tumor grade (r(s) = 0.20; P < .001) and inversely correlated with patient's age at diagnosis, estrogen receptor-alpha (ER-α), and progesterone receptor (PgR) expression (Mann-Whitney U test, P < .001). The total patient cohort was randomly divided into two separate training and validation sets before performing univariate survival analysis. Total number and distant stromal CD8(+) lymphocytes were associated with better patient survival (P = .041 and P < .001, respectively) in the training set. In multivariate analysis, total CD8(+) T-cell count was an independent prognostic factor in both training and validation sets. These results suggest that tumor-infiltrating CD8(+) T lymphocytes have antitumor activity as judged by their favorable effect on patients' survival and could potentially be exploited in the treatment of breast cancer.

Heterotrimeric G protein signaling in cancer cells with regard to metastasis formation.

The signal transduction mediated by heterotrimeric G proteins is involved in the regulation of a plethora of cell functions ranging from the sensation of light, taste and odor to chemotaxis, inflammation and the coordination of immune responses. These reactions have in common that they occur fast and are short-lived. Apart from this, it becomes increasingly evident, that the signaling of heterotrimeric G proteins has an imminent function in gene regulation, too, and therefore mediates even long-term effects. Herein, we illustrate the pathways of the four classes of α subunits and of the ßγ subunits of these heterotrimeric G proteins especially with regard to their function in cancer. G protein signaling is crucial for the development and localization of metastases and furthermore has been shown to be involved in tumor growth and angiogenesis. We summarize the current knowledge, how these processes are regulated by the short-term cellular response and the long-term gene regulation in cancer cells, and we discuss possible strategies for a therapeutic intervention.

FOXO3a nuclear localisation is associated with good prognosis in luminal-like breast cancer.

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.