Indwelling pleural catheter infection and colonisation: a clinical practice review.

Indwelling pleural catheters (IPCs) are used in the management of malignant pleural effusions, but they can become infected in 5.7% of cases. This review aims to provide a summary of the development of IPC infections and their microbiology, diagnosis and management. IPC infections can be deep, involving the pleural space, or superficial. The former are of greater clinical concern. Deep infection is associated with biofilm formation on the IPC surface and require longer courses of antibiotic treatment. Mortality from infections is low and it is common for patients to undergo pleurodesis following a deep infection. The diagnosis of pleural infections is based upon positive IPC pleural fluid cultures, changes in pleural fluid appearance and biochemistry, and signs or symptoms suggestive of infection. IPCs can also become colonised, where bacteria are grown from pleural fluid drained via an IPC but without evidence of infection. It is important to distinguish between infection and colonisation clinically, and though infections require antibiotic treatment, colonisation does not. It is unclear what proportion of IPCs become colonised. The most common causes of IPC infection and colonisation are Staphylococcus aureus and Coagulase-negative Staphylococci respectively. The management of deep IPC infections requires prolonged antibiotic therapy and the drainage of infected fluid, usually via the IPC. Intrapleural enzyme therapy (DNase and fibrinolytics) can be used to aid drainage. IPCs rarely need to be removed and patients can generally be managed as outpatients. Work is ongoing to study the incidence and significance of IPC colonisation. Other topics of interest include topical mupirocin to prevent IPC infections, and whether IPCs can be designed to limit infection risk.

Digital image analysis and machine learning-assisted prediction of neoadjuvant chemotherapy response in triple-negative breast cancer.

BACKGROUND: Pathological complete response (pCR) is associated with favorable prognosis in patients with triple-negative breast cancer (TNBC). However, only 30-40% of TNBC patients treated with neoadjuvant chemotherapy (NAC) show pCR, while the remaining 60-70% show residual disease (RD). The role of the tumor microenvironment in NAC response in patients with TNBC remains unclear. In this study, we developed a machine learning-based two-step pipeline to distinguish between various histological components in hematoxylin and eosin (H&E)-stained whole slide images (WSIs) of TNBC tissue biopsies and to identify histological features that can predict NAC response. METHODS: H&E-stained WSIs of treatment-naïve biopsies from 85 patients (51 with pCR and 34 with RD) of the model development cohort and 79 patients (41 with pCR and 38 with RD) of the validation cohort were separated through a stratified eightfold cross-validation strategy for the first step and leave-one-out cross-validation strategy for the second step. A tile-level histology label prediction pipeline and four machine-learning classifiers were used to analyze 468,043 tiles of WSIs. The best-trained classifier used 55 texture features from each tile to produce a probability profile during testing. The predicted histology classes were used to generate a histology classification map of the spatial distributions of different tissue regions. A patient-level NAC response prediction pipeline was trained with features derived from paired histology classification maps. The top graph-based features capturing the relevant spatial information across the different histological classes were provided to the radial basis function kernel support vector machine (rbfSVM) classifier for NAC treatment response prediction. RESULTS: The tile-level prediction pipeline achieved 86.72% accuracy for histology class classification, while the patient-level pipeline achieved 83.53% NAC response (pCR vs. RD) prediction accuracy of the model development cohort. The model was validated with an independent cohort with tile histology validation accuracy of 83.59% and NAC prediction accuracy of 81.01%. The histological class pairs with the strongest NAC response predictive ability were tumor and tumor tumor-infiltrating lymphocytes for pCR and microvessel density and polyploid giant cancer cells for RD. CONCLUSION: Our machine learning pipeline can robustly identify clinically relevant histological classes that predict NAC response in TNBC patients and may help guide patient selection for NAC treatment.

Centrosome amplification promotes cell invasion via cell-cell contact disruption and Rap-1 activation.

Centrosome amplification (CA) is a prominent feature of human cancers linked to tumorigenesis in vivo. Here, we report mechanistic contributions of CA induction alone to tumour architecture and extracellular matrix (ECM) remodelling. CA induction in non-tumorigenic breast cells MCF10A causes cell migration and invasion, with underlying disruption of epithelial cell-cell junction integrity and dysregulation of expression and subcellular localisation of cell junction proteins. CA also elevates expression of integrin ß-3, its binding partner fibronectin-1 and matrix metalloproteinase enzymes, promoting cell-ECM attachment, ECM degradation, and a migratory and invasive cell phenotype. Using a chicken embryo xenograft model for in vivo validation, we show that CA-induced (+CA) MCF10A cells invade into the chick mesodermal layer, with inflammatory cell infiltration and marked focal reactions between chorioallantoic membrane and cell graft. We also demonstrate a key role of small GTPase Rap-1 signalling through inhibition using GGTI-298, which blocked various CA-induced effects. These insights reveal that in normal cells, CA induction alone (without additional oncogenic alterations) is sufficient to confer early pro-tumorigenic changes within days, acting through Rap-1-dependent signalling to alter cell-cell contacts and ECM disruption.

UPR-Induced miR-616 Inhibits Human Breast Cancer Cell Growth and Migration by Targeting c-MYC.

C/EBP homologous protein (CHOP), also known as growth arrest and DNA damage-inducible protein 153 (GADD153), belongs to the CCAAT/enhancer-binding protein (C/EBP) family. CHOP expression is induced by unfolded protein response (UPR), and sustained CHOP activation acts as a pivotal trigger for ER stress-induced apoptosis. MicroRNA-616 is located within an intron of the CHOP gene. However, the regulation of miR-616 expression during UPR and its function in breast cancer is not clearly understood. Here we show that the expression of miR-616 and CHOP (host gene of miR-616) is downregulated in human breast cancer. Both miR-5p/-3p arms of miR-616 are expressed with levels of the 5p arm higher than the 3p arm. During conditions of ER stress, the expression of miR-616-5p and miR-616-3p arms was concordantly increased primarily through the PERK pathway. Our results show that ectopic expression of miR-616 significantly suppressed cell proliferation and colony formation, whereas knockout of miR-616 increased it. We found that miR-616 represses c-MYC expression via binding sites located in its protein coding region. Furthermore, we show that miR-616 exerted growth inhibitory effects on cells by suppressing c-MYC expression. Our results establish a new role for the CHOP locus by providing evidence that miR-616 can inhibit cell proliferation by targeting c-MYC. In summary, our results suggest a dual function for the CHOP locus, where CHOP protein and miR-616 can cooperate to inhibit cancer progression.

Digital image analysis and machine learning-assisted prediction of neoadjuvant chemotherapy response in triple-negative breast cancer.

Background: Pathological complete response (pCR) is associated with favorable prognosis in patients with triple-negative breast cancer (TNBC). However, only 30-40% of TNBC patients treated with neoadjuvant chemotherapy (NAC) show pCR, while the remaining 60-70% show residual disease (RD). The role of the tumor microenvironment (TME) in NAC response in patients with TNBC remains unclear. In this study, we developed a machine learning-based two-step pipeline to distinguish between various histological components in hematoxylin and eosin (H&E)-stained whole slide images (WSIs) of TNBC tissue biopsies and to identify histological features that can predict NAC response. Methods: H&E-stained WSIs of treatment-naïve biopsies from 85 patients (51 with pCR and 34 with RD) were separated through a stratified 8-fold cross validation strategy for the first step and leave one out cross validation strategy for the second step. A tile-level histology label prediction pipeline and four machine learning classifiers were used to analyze 468,043 tiles of WSIs. The best-trained classifier used 55 texture features from each tile to produce a probability profile during testing. The predicted histology classes were used to generate a histology classification map of the spatial distributions of different tissue regions. A patient-level NAC response prediction pipeline was trained with features derived from paired histology classification maps. The top graph-based features capturing the relevant spatial information across the different histological classes were provided to the radial basis function kernel support vector machine (rbfSVM) classifier for NAC treatment response prediction. Results: The tile-level prediction pipeline achieved 86.72% accuracy for histology class classification, while the patient-level pipeline achieved 83.53% NAC response (pCR vs. RD) prediction accuracy. The histological class pairs with the strongest NAC response predictive ability were tumor and tumor tumor-infiltrating lymphocytes for pCR and microvessel density and polyploid giant cancer cells for RD. Conclusion: Our machine learning pipeline can robustly identify clinically relevant histological classes that predict NAC response in TNBC patients and may help guide patient selection for NAC treatment.

ONEST (Observers Needed to Evaluate Subjective Tests) Analysis of Stromal Tumour-Infiltrating Lymphocytes (sTILs) in Breast Cancer and Its Limitations.

Tumour-infiltrating lymphocytes (TILs) reflect antitumour immunity. Their evaluation of histopathology specimens is influenced by several factors and is subject to issues of reproducibility. ONEST (Observers Needed to Evaluate Subjective Tests) helps in determining the number of observers that would be sufficient for the reliable estimation of inter-observer agreement of TIL categorisation. This has not been explored previously in relation to TILs. ONEST analyses, using an open-source software developed by the first author, were performed on TIL quantification in breast cancers taken from two previous studies. These were one reproducibility study involving 49 breast cancers, 23 in the first circulation and 14 pathologists in the second circulation, and one study involving 100 cases and 9 pathologists. In addition to the estimates of the number of observers required, other factors influencing the results of ONEST were examined. The analyses reveal that between six and nine observers (range 2-11) are most commonly needed to give a robust estimate of reproducibility. In addition, the number and experience of observers, the distribution of values around or away from the extremes, and outliers in the classification also influence the results. Due to the simplicity and the potentially relevant information it may give, we propose ONEST to be a part of new reproducibility analyses.

miRNA-378 Is Downregulated by XBP1 and Inhibits Growth and Migration of Luminal Breast Cancer Cells.

X-box binding protein 1 (XBP1) is a transcription factor that plays a crucial role in the unfolded protein response (UPR), a cellular stress response pathway involved in maintaining protein homeostasis in the endoplasmic reticulum (EnR). While the role of XBP1 in UPR is well-characterised, emerging evidence suggests its involvement in endocrine resistance in breast cancer. The transcriptional activity of spliced XBP1 (XBP1s) is a major component of its biological effects, but the targets of XBP1s in estrogen receptor (ER)-positive breast cancer are not well understood. Here, we show that the expression of miR-378 and PPARGC1B (host gene of miR-378) is downregulated during UPR. Using chemical and genetic methods, we show that XBP1s is necessary and sufficient for the downregulation of miR-378 and PPARGC1B. Our results show that overexpression of miR-378 significantly suppressed cell growth, colony formation, and migration of ER-positive breast cancer cells. Further, we found that expression of miR-378 sensitised the cells to UPR-induced cell death and anti-estrogens. The expression of miR-378 and PPARGC1B was downregulated in breast cancer, and higher expression of miR-378 is associated with better outcomes in ER-positive breast cancer. We found that miR-378 upregulates the expression of several genes that regulate type I interferon signalling. Analysis of separate cohorts of breast cancer patients showed that a gene signature derived from miR-378 upregulated genes showed a strong association with improved overall and recurrence-free survival in breast cancer. Our results suggest a growth-suppressive role for miR-378 in ER-positive breast cancer where downregulation of miR-378 by XBP1 contributes to endocrine resistance in ER-positive breast cancer.

Activity of antibacterial phytochemicals and their potential use as natural food preservatives.

The risk to human health from bacterial foodborne infection is presently controlled by the addition of antimicrobial preservatives to food. However, the use of chemical preservatives such as sodium nitrite poses a health risk in themselves with concerns around carcinogenic effects. This makes the development of improved preservatives a priority for the food industry.One promising source of novel antimicrobial compounds can be found in nature; phytochemicals, in particular polyphenols are secondary metabolites produced by plants for numerous purposes including antimicrobial defence. There has been significant study of phytochemicals; including quantifying their antimicrobial activity, potential to synergise with current antibiotics and the feasibility of their application as natural food preservatives. However, there remains significant uncertainty about the relative antimicrobial efficacy of different phytochemicals, their mechanisms of action (MOA) and the potential for emergence of bacterial resistance to their effects. This review summarizes recent work relevant to the potential development of phytochemicals as antimicrobial agents.

Single-cell genomics reveals population structures from in vitro evolutionary studies of Salmonella.

Single-cell DNA sequencing has the potential to reveal detailed hierarchical structures in evolving populations of cells. Single cell approaches are increasingly used to study clonal evolution in human ageing and cancer but have not yet been deployed to study evolving clonal microbial populations. Here, we present an approach for single bacterial genomic analysis for in vitro evolution experiments using FACS isolation of individual bacteria followed by whole-genome amplification and sequencing. We apply this to the experimental evolution of a hypermutator strain of Salmonella in response to antibiotic stress (ciprofloxacin). By analysing sequence polymorphisms in individual cells from populations we identified the presence and prevalence of sub-populations which have acquired polymorphisms in genes previously demonstrated to be associated with ciprofloxacin susceptibility. We were also able to identify that the population exposed to antibiotic stress was able to develop resistance whilst maintaining diversity. This population structure could not be resolved from bulk sequence data, and our results show how high-throughput single-cell sequencing can enhance experimental studies of bacterial evolution.

Intra-Tumour Heterogeneity Is One of the Main Sources of Inter-Observer Variation in Scoring Stromal Tumour Infiltrating Lymphocytes in Triple Negative Breast Cancer.

Stromal tumour infiltrating lymphocytes (sTILs) are a strong prognostic marker in triple negative breast cancer (TNBC). Consistency scoring sTILs is good and was excellent when an internet-based scoring aid developed by the TIL-WG was used to score cases in a reproducibility study. This study aimed to evaluate the reproducibility of sTILs assessment using this scoring aid in cases from routine practice and to explore the potential of the tool to overcome variability in scoring. Twenty-three breast pathologists scored sTILs in digitized slides of 49 TNBC biopsies using the scoring aid. Subsequently, fields of view (FOV) from each case were selected by one pathologist and scored by the group using the tool. Inter-observer agreement was good for absolute sTILs (ICC 0.634, 95% CI 0.539-0.735, p < 0.001) but was poor to fair using binary cutpoints. sTILs heterogeneity was the main contributor to disagreement. When pathologists scored the same FOV from each case, inter-observer agreement was excellent for absolute sTILs (ICC 0.798, 95% CI 0.727-0.864, p < 0.001) and good for the 20% (ICC 0.657, 95% CI 0.561-0.756, p < 0.001) and 40% (ICC 0.644, 95% CI 0.546-0.745, p < 0.001) cutpoints. However, there was a wide range of scores for many cases. Reproducibility scoring sTILs is good when the scoring aid is used. Heterogeneity is the main contributor to variance and will need to be overcome for analytic validity to be achieved.

Emergence of ciprofloxacin heteroresistance in foodborne Salmonella enterica serovar Agona.

BACKGROUND: Bacterial heteroresistance has been increasingly identified as an important phenomenon for many antibiotic/bacterium combinations. OBJECTIVES: To investigate ciprofloxacin heteroresistance in Salmonella and characterize mechanisms contributing to ciprofloxacin heteroresistance. METHODS: Ciprofloxacin-heteroresistant Salmonella were identified by population analysis profiling (PAP). Target mutations and the presence of PMQR genes were detected using PCR and sequencing. Expression of acrB, acrF and qnrS was conducted by quantitative RT-PCR. Competition ability and virulence were also compared using pyrosequencing, blue/white screening, adhesion and invasion assays and a Galleria model. Two subpopulations were whole-genome sequenced using Oxford Nanopore and Illumina platforms. RESULTS: PAP identified one Salmonella from food that yielded a subpopulation demonstrating heteroresistance to ciprofloxacin at a low frequency (10-9 to 10-7). WGS and PFGE analyses confirmed that the two subpopulations were isogenic, with six SNPs and two small deletions distinguishing the resistant from the susceptible. Both subpopulations possessed a T57S substitution in ParC and carried qnrS. The resistant subpopulation was distinguished by overexpression of acrB and acrF, a deletion within rsxC and altered expression of soxS. The resistant population had a competitive advantage against the parental population when grown in the presence of bile salts but was attenuated in the adhesion and invasion of human intestinal cells. CONCLUSIONS: We determined that heteroresistance resulted from a combination of mutations in fluoroquinolone target genes and overexpression of efflux pumps associated with a deletion in rsxC. This study warns that ciprofloxacin heteroresistance exists in Salmonella in the food chain and highlights the necessity for careful interpretation of antibiotic susceptibility.

A 16-year Longitudinal Cohort Study of Incidence and Bacteriology of Necrotising Fasciitis in England.

BACKGROUND: Necrotising fasciitis (NF) is a rapidly progressive, destructive soft tissue infection with high mortality. The primary aim of this study was to evaluate the incidence and mortality of NF amongst patients admitted to English National Health Service (NHS) hospitals. The secondary aims included the identification of risk factors for mortality and causative pathogens. METHODS: The Hospital Episodes Statistics database identified patients with NF admitted to English NHS Trusts from 1/1/2002 to 31/12/2017. Information on patient demographics, co-morbid conditions, microbiology specimens, surgical intervention and in-hospital mortality was collected. Uni- and multivariable analyses were performed to investigate factors related to in-hospital mortality. RESULTS: A total of 11,042 patients were diagnosed with NF. Age-standardised incidence rose from 9 per million in 2002 to 21 per million in 2017 (annual percentage change = 6.9%). Incidence increased with age and was higher in men. Age-standardised mortality rate remained at 16% over the study period, while in-hospital mortality declined. On multivariable analysis, the following factors were associated with increased risk of in-hospital mortality: emergency admission, female sex, history of congestive heart failure, peripheral vascular disease, chronic kidney disease and cancer. Admission year and diabetes, which was significantly prevalent at 27%, were not associated with increased risk of mortality. Gram-positive pathogens, particularly Staphylococci, decreased over the study period with a corresponding increase in Gram-negative pathogens, predominantly E. coli. CONCLUSION: The incidence of NF increased markedly from 2002 to 2017 although in-hospital mortality did not change. There was a gradual shift in the causative organisms from Gram-positive to Gram-negative.

Outcome for triple negative breast cancer in a retrospective cohort with an emphasis on response to platinum-based neoadjuvant therapy.

PURPOSE: The rate of pathological complete response (pCR) for patients with triple negative breast cancer (TNBC) is increased when carboplatin is added to neo-adjuvant chemotherapy (NACT). However, while phase III trial data showing a survival benefit are awaited, carboplatin is not yet standard-of-care for TNBC. The aim of this study was to examine the rate of pCR and the outcome for those treated with carboplatin and to examine the predictors of response to therapy. METHODS: The retrospective series comprised 333 consecutive patients with TNBC (median follow-up time, 43 months). Adjuvant chemotherapy was given to 51% (n = 168) of patients and 29% (n = 97) received anthracycline-taxane NACT with carboplatin given to 9% (n = 31) of patients. RESULTS: Overall, 25% (n = 78) of patients experienced a breast cancer recurrence and 22% (n = 68) died from disease. A pCR breast and pCR breast/axilla was more common in those who received carboplatin (n = 18, 58% and n = 17, 55%, respectively) compared those who did not (n = 23, 36% and n = 18, 28%, respectively) (p = 0.041 and p = 0.011, respectively). By multivariable analysis, carboplatin and high tumor grade were independent predictors of pCR breast/axilla (ORnon-pCR = 0.17; 95% CI 0.06-0.54; p = 0.002; and ORnon-pCR = 0.05, 95% CI 0.01-0.27; p < 0.001, respectively). pCR breast/axilla was an independent predictor of DFS (HRnon-pCR=6.23; 95% CI 1.36-28.50; p = 0.018), metastasis-free survival (HRnon-pCR = 5.08; 95% CI 1.09-23.65; p = 0.038) and BCSS (HRnon-pCR = 8.52; 95% CI 1.09-66.64; p = 0.041). CONCLUSION: Carboplatin therapy and high tumor grade are associated with a significant increase in the rate of pCR, which is an independent predictor of outcome. These data support the use of carboplatin in NACT for TNBC, while results from phase III studies are awaited.

Impact of inducible nitric oxide synthase (iNOS) expression on triple negative breast cancer outcome and activation of EGFR and ERK signaling pathways.

Inflammation is implicated in triple negative breast cancer (TNBC) progression. TNBC carries a worse prognosis than other breast cancer subtypes, and with the clinical and molecular heterogeneity of TNBC, there is a lack of effective therapeutic targets available. Identification of molecular targets for TNBC subtypes is crucial towards personalized patient stratification. Inducible nitric oxide synthase (iNOS) has been shown to induce p53 mutation accumulation, basal-like gene signature enrichment and transactivation of the epidermal growth factor receptor (EGFR) via s-nitrosylation. Herein we report that iNOS is associated with disease recurrence, distant metastasis and decreased breast cancer specific survival in 209 cases of TNBC. Employing TNBC cell lines representing normal basal breast, and basal-like 1 and basal-like 2 tumors, we demonstrate that nitric oxide (NO) induces EGFR-dependent ERK phosphorylation in basal-like TNBC cell lines. Moreover NO mediated cell migration and cell invasion was found to be dependent on EGFR and ERK activation particularly in basal-like 2 TBNC cells. This occurred in conjunction with NF-κB activation and increased secretion of pro-inflammatory cytokines IL-8, IL-1ß and TNF-α. This provides substantial evidence for EGFR as a therapeutic target to be taken into consideration in the treatment of a specific subset of basal-like TNBC overexpressing iNOS.

Antibacterial Activity of Blue Light against Nosocomial Wound Pathogens Growing Planktonically and as Mature Biofilms.

UNLABELLED: The blue wavelengths within the visible light spectrum are intrinisically antimicrobial and can photodynamically inactivate the cells of a wide spectrum of bacteria (Gram positive and negative) and fungi. Furthermore, blue light is equally effective against both drug-sensitive and -resistant members of target species and is less detrimental to mammalian cells than is UV radiation. Blue light is currently used for treating acnes vulgaris and Helicobacter pylori infections; the utility for decontamination and treatment of wound infections is in its infancy. Furthermore, limited studies have been performed on bacterial biofilms, the key growth mode of bacteria involved in clinical infections. Here we report the findings of a multicenter in vitro study performed to assess the antimicrobial activity of 400-nm blue light against bacteria in both planktonic and biofilm growth modes. Blue light was tested against a panel of 34 bacterial isolates (clinical and type strains) comprising Acinetobacter baumannii, Enterobacter cloacae, Stenotrophomonas maltophilia, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Enterococcus faecium, Klebsiella pneumoniae, and Elizabethkingia meningoseptica All planktonic-phase bacteria were susceptible to blue light treatment, with the majority (71%) demonstrating a ≥5-log10 decrease in viability after 15 to 30 min of exposure (54 J/cm(2) to 108 J/cm(2)). Bacterial biofilms were also highly susceptible to blue light, with significant reduction in seeding observed for all isolates at all levels of exposure. These results warrant further investigation of blue light as a novel decontamination strategy for the nosocomial environment, as well as additional wider decontamination applications. IMPORTANCE: Blue light shows great promise as a novel decontamination strategy for the nosocomial environment, as well as additional wider decontamination applications (e.g., wound closure during surgery). This warrants further investigation.

Prediction of Oncotype DX and TAILORx risk categories using histopathological and immunohistochemical markers by classification and regression tree (CART) analysis.

Oncotype DX is an RT-PCR assay used to predict which patients with ER-positive node-negative (NN) disease will benefit from chemotherapy. Each patient is stratified into a risk category based on a recurrence score (RS) and the TAILORx trial is determining the benefit of chemotherapy for patients with mid-range RSs. We tested if Oncotype DX and TAILORx risk categories could be predicted by standard pathological features and protein markers corresponding to 10 genes in the assay (ER, PR, Ki67, HER2, BCL2, CD68, Aurora A kinase, survivin, cyclin B1 and BAG1) on 52 patients who enrolled on TAILORx. Immunohistochemistry for the protein markers was performed on whole tissue sections. Classification and regression tree (CART) analysis correctly classified 69% of cases into Oncotype DX risk categories based on the expression of PR, survivin and nuclear pleomorphism. All tumours with PR staining (Allred score ≥ 2) and marked nuclear pleomorphism were in the high-risk category. No case with PR <2, low survivin (≤ 15.5%) and nuclear pleomorphism <3 was high-risk. Similarly, 77% of cases were correctly classified into TAILORx categories based on nuclear pleomorphism, survivin, BAG1 and cyclin B1. Ki67 was the only variable that predicted the absolute RS with a cut-off for positivity of 15% (p = 0.003). In conclusion, CART revealed key predictors including proliferation markers, PR and nuclear pleomorphism that correctly classified over two thirds of ER-positive NN cancers into Oncotype DX and TAILORx risk categories. These variables could be used as an alternative to the RT-PCR assay to reduce the number of patients requiring Oncotype DX testing.

Prognostic significance of deregulated dicer expression in breast cancer.

BACKGROUND: Dicer, an RNase III-type endonuclease, is the key enzyme involved in RNA interference and microRNA pathways. Aberrant expression of Dicer is reported in several human cancers. Our aim was to assess the prognostic role of Dicer in breast cancer. METHODS: The entire series comprised 666 invasive breast cancers (IBCs), 480 DCIS cases (397 associated with IBC and 83 pure DCIS) and 305 lymph node metastases. Cytoplasmic Dicer expression by immunohistochemistry was scored as negative (no staining) and positive (weak, moderate or strong staining). RESULTS: Dicer staining was assessable in 446 IBC, 128 DCIS and 101 lymph node metastases. Expression of Dicer was observed in 33% (145/446) of IBCs, 34% (44/128) of DCIS and 57% (58/101) of lymph node metastases. Dicer expression was increased in nodal metastases compared to primary tumours (p<0.001); and was associated with ER negativity (p<0.001), HER2 positivity (p<0.001), high Ki67 labeling index (p<0.001) and expression of basal-like biomarkers (p = 0.002). Dicer positivity was more frequent in the HER2 overexpressing (p<0.001) and basal-like (p = 0.002) subtypes compared to luminal A subtype. Dicer expression was associated with reduced overall survival (OS) on univariate analysis (p = 0.058) and remained an independent predictor of OS on multivariate analysis (HR 2.84, 95% CI 1.43-5.62, p = 0.003), with nodal status (HR 2.61, 95% CI 1.18-5.80, p = 0.018) and PR (HR 0.28, 95% CI 0.13-0.59, p = 0.001). Further, moderate or strong expression of Dicer was associated with improved disease-free survival in the HER2-overexpressing subtype compared to negative or weak expression (p = 0.038). CONCLUSION: Deregulated Dicer expression is associated with aggressive tumour characteristics and is an independent prognostic factor for OS. Our findings suggest that Dicer is an important prognostic marker in breast cancer and that its prognostic role may be subtype specific.

Meta-analysis confirms BCL2 is an independent prognostic marker in breast cancer.

BACKGROUND: A number of protein markers have been investigated as prognostic adjuncts in breast cancer but their translation into clinical practice has been impeded by a lack of appropriate validation. Recently, we showed that BCL2 protein expression had prognostic power independent of current used standards. Here, we present the results of a meta-analysis of the association between BCL2 expression and both disease free survival (DFS) and overall survival (OS) in female breast cancer. METHODS: Reports published in 1994-2006 were selected for the meta-analysis using a search of PubMed. Studies that investigated the role of BCL2 expression by immunohistochemistry with a sample size greater than 100 were included. Seventeen papers reported the results of 18 different series including 5,892 cases with an average median follow-up of 92.1 months. RESULTS: Eight studies investigated DFS unadjusted for other variables in 2,285 cases. The relative hazard estimates ranged from 0.85 - 3.03 with a combined random effects estimate of 1.66 (95%CI 1.25 - 2.22). The effect of BCL2 on DFS adjusted for other prognostic factors was reported in 11 studies and the pooled random effects hazard ratio estimate was 1.58 (95%CI 1.29-1.94). OS was investigated unadjusted for other variables in eight studies incorporating 3,910 cases. The hazard estimates ranged from 0.99-4.31 with a pooled estimate of risk of 1.64 (95%CI 1.36-2.0). OS adjusted for other parameters was evaluated in nine series comprising 3,624 cases and the estimates for these studies ranged from 1.10 to 2.49 with a pooled estimate of 1.37 (95%CI 1.19-1.58). CONCLUSION: The meta-analysis strongly supports the prognostic role of BCL2 as assessed by immunohistochemistry in breast cancer and shows that this effect is independent of lymph node status, tumour size and tumour grade as well as a range of other biological variables on multi-variate analysis. Large prospective studies are now needed to establish the clinical utility of BCL2 as an independent prognostic marker.

The AcrAB-TolC efflux system of Salmonella enterica serovar Typhimurium plays a role in pathogenesis.

The ability of an isogenic set of mutants of Salmonella enterica serovar Typhimurium L354 (SL1344) with defined deletions in genes encoding components of tripartite efflux pumps, including acrB, acrD, acrF and tolC, to colonize chickens was determined in competition with L354. In addition, the ability of L354 and each mutant to adhere to, and invade, human embryonic intestine cells and mouse monocyte macrophages was determined in vitro. The tolC and acrB knockout mutants were hyper-susceptible to a range of antibiotics, dyes and detergents; the tolC mutant was also more susceptible to acid pH and bile and grew more slowly than L354. Complementation of either gene ablated the phenotype. The tolC mutant poorly adhered to both cell types in vitro and was unable to invade macrophages. The acrB mutant adhered, but did not invade macrophages. In vivo, both the acrB mutant and the tolC mutant colonized poorly and did not persist in the avian gut, whereas the acrD and acrF mutant colonized and persisted as well as L354. These data indicate that the AcrAB-TolC system is important for the colonization of chickens by S. Typhimurium and that this system has a role in mediating adherence and uptake into target host cells.

Spinal-supraspinal serotonergic circuits regulating neuropathic pain and its treatment with gabapentin.

Not all neuropathic pain patients gain relief from current therapies that include the anticonvulsant, gabapentin, thought to modulate calcium channel function. We report a neural circuit that is permissive for the effectiveness of gabapentin. Substance P-saporin (SP-SAP) was used to selectively ablate superficial dorsal horn neurons expressing the neurokinin-1 receptor for substance P. These neurons project to the brain as shown by retrograde labelling and engage descending brainstem serotonergic influences that enhance spinal excitability via a facilitatory action on 5HT(3) receptors. We show the integrity of this pathway following nerve injury contributes to the behavioural allodynia, neuronal plasticity of deep dorsal horn neurons and the injury-specific actions of gabapentin. Thus SP-SAP attenuated the tactile and cold hypersensitivity and abnormal neuronal coding (including spontaneous activity, expansion of receptive field size) seen after spinal nerve ligation. Furthermore the powerful actions of gabapentin after neuropathy were blocked by either ablation of NK-1 expressing neurones or 5HT(3) receptor antagonism using ondansetron. Remarkably, 5HT(3) receptor activation provided a state-dependency (independent of that produced by neuropathy) allowing GBP to powerfully inhibit in normal uninjured animals. This circuit is therefore a crucial determinant of the abnormal neuronal and behavioural manifestations of neuropathy and importantly, the efficacy of gabapentin. As this spino-bulbo-spinal circuit contacts areas of the brain implicated in the affective components of pain, this loop may represent a route by which emotions can influence the degree of pain in a patient, as well as the effectiveness of the drug treatment. These hypotheses are testable in patients.