Multiple breast cancer gene expression profiles have been developed that appear to provide similar abilities to predict outcome and may outperform clinical-pathologic criteria; however, the extent to which seemingly disparate profiles provide additive prognostic information is not known, nor do we know whether prognostic profiles perform equally across clinically defined breast cancer subtypes. We evaluated whether combining the prognostic powers of standard breast cancer clinical variables with a large set of gene expression signatures could improve on our ability to predict patient outcomes.Using clinical-pathological variables and a collection of 323 gene expression "modules", including 115 previously published signatures, we build multivariate Cox proportional hazards models using a dataset of 550 node-negative systemically untreated breast cancer patients. Models predictive of pathological complete response (pCR) to neoadjuvant chemotherapy were also built using this approach.We identified statistically significant prognostic models for relapse-free survival (RFS) at 7 years for the entire population, and for the subgroups of patients with ER-positive, or Luminal tumors. Furthermore, we found that combined models that included both clinical and genomic parameters improved prognostication compared with models with either clinical or genomic variables alone. Finally, we were able to build statistically significant combined models for pathological complete response (pCR) predictions for the entire population.Integration of gene expression signatures and clinical-pathological factors is an improved method over either variable type alone. Highly prognostic models could be created when using all patients, and for the subset of patients with lymph node-negative and ER-positive breast cancers. Other variables beyond gene expression and clinical-pathological variables, like gene mutation status or DNA copy number changes, will be needed to build robust prognostic models for ER-negative breast cancer patients. This combined clinical and genomics model approach can also be used to build predictors of therapy responsiveness, and could ultimately be applied to other tumor types. Genomic profiles have significantly improved our ability to prognosticate in breast cancer patients ,2. SeverMany studies have examined the prognostic significance of genomic biomarkers along with clinical-pathological variables and often shown that both provide independent information -44; howeIn this study, we developed a prognostic model in systematically untreated node-negative breast cancer patients derived from multiple commonly used clinical variables and a large database of gene expression modules, and we confirmed that models that incorporate both clinical and genomic variables are the most accurate for outcome predictions for newly diagnosed patients with node-negative breast cancer. Importantly, this approach to model development can also be used to predict responsiveness to therapy and could be generalized to other tumor types.A single large dataset of homogenously treated patients was created by combining 5 different publicly available clinically-annotated microarray datasets of node-negative breast cancer patients treated with local therapy only, and no adjuvant systemic therapy: van de Vijver et al., Wang etOf the 666 patients included in this combined dataset, 550 (83%) had complete data on relapse-free survival  and clinical variables , and were included for further analyses using a 7-year cut-point for RFS. For the HER2 "clinical status", which was not available for most studies, we used a gene expression surrogate based on the mRNA levels of the HER2 gene, using the top 20% rank order highest expressers as the cutoff value for calling a tumor "HER2-positive"; the chosen cutoff value for HER2 positivity was based on a population-based study of breast cancer.We examined the ability of this approach to predict pathological complete response (pCR) rate to neoadjuvant chemotherapy in a dataset from Popovici et al. (MDACC),53, whic1) 221 modules were built using the median expression of all genes within the module that homogeneously expresses these genes . The sources of the selected homogenous gene lists were the following: 50 were identified by bicluster analyses[2) 77 modules were represented as the first Principal Component of previously published gene lists [3) 22 modules were correlations to previously published training dataset centroids [4) 3 modules were built from previously published gene expression prognostic models [We defined modules as sets of co-expressed genes that were considered as a functional unit. Using multiple approaches, we built a collection of 323 gene expression modules, including 115 gene lists obtained from 53 publications:  were evaluated by the Least Absolute Shrinkage and Selection Operator (LASSO) method to build prognostic models using a Cox proportional hazards approach. For allThe prognostic ability of the reproducible models and known prognostic predictors was further characterized by calculating the concordance index (C-index) . The C-ihttp://www.r-project.org.To predict pCR, we randomly split the MDACC225 ,53 databIn order to examine the potential of new prognostic models, a large and homogenously treated data set of breast cancer patients was created by combining together systematically untreated (i.e. no systemic adjuvant therapy) patients; the clinical and pathologic characteristics of the 550 patient dataset of node-negative, local therapy-only, breast cancer patients from the public domain are described in Table Along with clinical-pathological variables we applied 323 different modules to the combined dataset  all patients Figure  and (b) In order to robustly identify prognostic variables, 200 rounds of training and testing were performed for each patient subset discussed above. Examination of the most frequently selected modules and clinical variables for the "all patients' group was revealing of the underlying biology Figure . StartinIncreased expression of other unpublished modules was associated with good outcome. Several of these were highly enriched with genes involved in immune response , extracellular space (HS_Green19 and Fibrinogen_Cluster), transcription (MHistone) and cation transport (MM_Red21). Of note, the IGG_Cluster (Immunoglobulin) 14-gene module, which was identified by unsupervised clustering of human breast tumors, was selected in 196 of 200 combined models (98%). Consistent with our findings, data from a recent report suggests that, besides proliferation, the combination of immunity and RNA splicing processes may have a high prognostic impact in breast cancer .Among the rest of the modules that frequently comprised the combined model for all patients, it is interesting to note that two of them were derived from a single study that evaluated the transcription factor E2F1-dependent gene expression program , two othThe majority of combined models built for ER-positive patients included grade, tumor size, and various modules whose expression was associated with either poor or good prognosis Figure . Among tWe also repeated the analysis without the four best-known prognostic profiles   in the tThe performance of the clinical, genomic and combined models in all patients and ER-positive patients was also compared to the other known 4 prognostic signatures with and without the addition of clinical variables Figure . It shouTo show the potential application of this combined approach in predicting treatment response, we applied the different genomic modules, along with clinical variables , to 225 patients with breast cancer treated with a taxane and anthracycline containing neoadjuvant chemotherapy regimen ,53 i.e.. Among tBoth genomic biomarkers and clinical parameters provide prognostic powers ,7,41-43,While proliferation-related genes are an important part of many prognostic gene sets, these signatures show no prognostic value when the analysis is limited to ER-negative patients, as has been shown before . The majAlthough the gene lists of the majority of "selected" modules/signatures were largely non-overlapping, significant agreement in outcome predictions for individual patients was observed, which confirms and extends our previous observations  that difWhile we have recapitulated known biology and made novel observations that are relevant to breast cancer patient management, there are limitations to this study. First, the sample size of the entire data set was sufficient to build strong predictors when considering all patients and for ER-positive patients. However, when conducting stratified analyses, sample size significantly decreased in some cases , thus limiting power for those subsets. This represents an alternative explanation for the failure to identify prognostic signatures within those groups, although it is quite possible that almost all Basal-like patients do have an inherently poor prognosis in the untreated setting and that no identifiable "good prognosis signature" exists for Basal-like tumors. Second, a challenge of this study was the implementation of many published predictors onto a common dataset, many of which used unique statistical methods; we strove to implement each predictor as published, however, almost all predictors "lost" genes due to combining data across microarray platforms, and thus, almost all predictors differed somewhat from their original specification. Lastly, this dataset is comprised primarily of tissue bank samples and therefore factors such as patient selection may differ from what is observed in a true population-based sampling of incident cases.Despite these caveats, our analyses did identify robust predictors for all patients and for ER-positive patients, and confirmed the prognostic abilities of many previously published signatures. In addition, we demonstrated that models built using clinical variables can be improved with genomic information, and showed that the best models are a combination of genomic and clinical variables. Another implication of these data is that prognostication for breast cancer patients is possibly only relevant for ER-positive/Luminal patients; it does not adequately prognosticate in ER-negative or HER2-positive breast cancers. Similarly, a recent study suggests a markedly poorer prognosis in even the smallest node-negative HER2-positive tumors . AdditioClearly models focused on predicting chemotherapy responsiveness would be highly relevant to ER-negative tumors and breast cancer patients in general. To address this predictive need, we successfully applied our model building approach on a set of 225 T/FAC treated patients and achieved AUC values of ~0.8. However, we were unable to test if pCR predictors are of great value for HER2-positive and Basal-like patients survival outcomes due to the lack of a dataset that included outcomes, neoadjuvant response and microarray data.Lastly, some individual models, like the NKI 70-gene signature ,4, the OIn this study, we have identified significant prognostic models using clinical-pathological variables and 323 gene expression "modules" in patients with lymph node-negative ER-positive breast cancer. Specifically, we show that 1) the clinical variables alone build the least accurate models, 2) genomic models alone are better than clinical variables alone, and 3) a combined genomic and clinical model is best, and thus, would be the most helpful in the process of adjuvant decision-making for node-negative ER-positive breast cancers. These findings reinforce that clinical information still plays a key role for prognostication in node-negative breast cancer , but also that genomic variables provide important information not provided by the classical clinical variables. Interestingly, we also found that single module/signatures built for prognosis  can perform nearly as well as a combination of hundreds of signatures. Finally, although huge advancements in prognostic models for breast cancer are unlikely given the amount of work that has already gone into biomarkers for breast cancer, we feel that our advances are significant, even if the magnitude of the effect is not large.CMP is the co-founder of University Genomics and is a major stock holder of University Genomics and Bioclassifier LLC. JSP and CMP are also listed as inventors on patents filed pertaining to the PAM50 and ROR score assays.CF analyzed the data and performed the statistical analysis. AP analyzed the data and wrote the manuscript. JSP and YL provided technical support. CMP, LAC and MAT participated in the design of the study and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1755-8794/4/3/prepubSupplemental materials and methods. This file contains all the gene lists and methods used for the 323 modules evaluated in this article.Click here for fileSupplemental data. This file contains additional analyses and results.Click here for file


Here, we discuss how IRGM might contribute to human autophagy upon viral infection, and why its targeting might be beneficial to virus replication.Autophagy is a cell autonomous process allowing each individual cell to fight intracellular pathogens. Autophagy can destroy pathogens within the cytosol, and can elicit innate and adaptive immune responses against microorganisms. Nevertheless, numerous pathogens have developed molecular strategies enabling them to avoid or even exploit autophagy for their own benefit. IRGM (immunity-related GTPase family M) is a human protein recently highlighted for its contribution to autophagy upon infections. The physical association of IRGM with mitochondria and different autophagy-regulating proteins, ATG5, ATG10, SH3GLB1, and LC3, contribute to explain how IRGM could regulate autophagy. Whereas IRGM is involved in autophagy-mediated immunity against bacteria, certain viruses seem to have developed strategies to manipulate autophagy through the selective targeting of this protein. Furthermore,  Figure 1). Autophagy can destroy infectious virions or virus components that are essential for replication -1 or the cytomegalovirus (CMV) can inhibit autophagy induction by targeting BECLIN1 (BECN1), an essential autophagy-associated protein, through interactions with viral proteins . Other virgm was recently found to be involved.The understanding of the molecular pathways linked to the proviral or antiviral functions of autophagy is still at its beginning, especially regarding the molecular interplay between viruses and autophagy proteins. The human immunity-related GTPase family M (IRGM) protein was shown to be widely targeted by RNA viruses, several among which can exploit autophagy in human cells to improve their replication ,2012. Hoirgm gene expression is not under the control of IFN-γ (irg) genes exist in mice and play immune-related functions, the IRGM-mediated cell autonomous resistance mechanisms have been first lost in primates due to deleterious mutations. However, irgm gene function was restored in some primates including Homo sapiens, possibly due to the insertion of a retroviral element that promotes its expression . All these proteins contribute to autophagy, supporting the idea that IRGM plays an essential role in this process -kinase VPS34 to promote autophagosome biogenesis . Through the dampening of antiviral IFN-I synthesis, this targeting might be of further benefit to viruses as discussed below.To date, only four cellular proteins were identified to interact with IRGM: ATG5, ATG10, MAP1LC3C, and SH3GLB1 . Indeed, MFN2 contributes to the supply of mitochondria membranes for the biogenesis of autophagosome  plays an important role in cross-presentation of phagocytosed protein antigens to conventional CD8+ T cells without impacting antigen presentation to CD4+ T cells (It recently became clear that autophagy which is constitutively active in antigen-presenting cells (APCs), can regulate adaptive immune responses by promoting the access of antigens from intracellular pathogens to compartments that assemble peptide:MHC class II complexes for presentation to CD4 T cells . Among vimpaired . The neg T cells .irgm variants as susceptibility genes for Crohn’s disease (CD), tuberculosis (TB), gastric cancer and autoimmune systemic lupus erythematosus (SLE).Recent studies identified atg16L1 and irgm, that are linked to CD. CD-associated irgm polymorphisms, that influence or not the primary protein sequence, were both reported (E. coli (AIEC) and IRGM-dependent autophagy contributes to fight pathogenic AIEC  of IRGM in autophagy-mediated immunity could be of crucial importance to fight infectious viruses and human pathologies.In an infected cell a virus has to counteract cell autonomous defense mechanisms while exploiting elementary cellular processes to replicate efficiently. By selectively targeting autophagy, viruses might accomplish both. As discussed here, IRGM could be a key protein for autophagy manipulation upon viral infection. The molecular organization involving IRGM in autophagy during viral infections requires further investigations. While interactions of IRGM with its protein partners were only observed in transfected cells for the time being, it would be important to visualize these interactions between endogenous proteins and during productive infections. It would also be crucial to understand why and how IRGM plays an antibacterial function, whereas it seems to act as a proviral factor. Furthermore, the role of IRGM could be cell type-specific and, as described for several autophagy-related proteins, IRGM might have non-autophagy-related functions upon infections. In regards of the link of numerous The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The detection and enumeration of circulating tumor cells (CTCs) has shown significant clinical utility with respect to prognosis in breast, colorectal and prostate cancers. Emerging studies show that CTCs can provide pharmacodynamic information to aid therapy decision making. CTCs as a ‘virtual and real-time biopsy’ have clear potential to facilitate exploration of tumor biology, and in particular, the process of metastasis. The challenge of profiling CTC molecular characteristics and generating CTC signatures using current technologies is that they enrich rather than purify CTCs from whole blood; we face the problem of looking for the proverbial ‘needle in the haystack’. This review summarizes the current methods for CTC detection and enumeration, focuses on molecular characterization of CTCs, unveils some aspects of CTC heterogeneity, describes attempts to purify CTCs and scans the horizon for approaches leading to comprehensive dissection of CTC biology.  Metastatic disease is responsible for over 90% of cancer related deaths . The pre6 and current technologies enrich rather than purify CTCs from whole blood. Thus molecular profiling of CTCs, which might reveal important predictive information and report changes in CTC biology, for example during tumor relapse, is frustrated by wbc signatures that overwhelm those emanating from the CTC minority. This review will outline the current cytometric methods for CTC detection and enumeration, describe with some examples from our laboratory, how additional information with potential use for drug development can be obtained from CTCs, focus on approaches used to purify CTCs to allow their molecular characterization and conclude with our horizon scanning with respect to achieving a comprehensive dissection of CTC biology.However, even in cancer patients with high CTC numbers, such as those with extensive stage Small Cell Lung Cancer (SCLC) (where thousands of CTCs are detected in 7.5 mL blood ), the maMethods of CTC detection are broadly divided into cytometric and nucleic-acid based approaches . CytometTM technology  employs a ferrofluid consisting of nanoparticles with a magnetic core surrounded by a polymeric layer coated with antibodies targeting epithelial cell adhesion molecule (EpCAM) [TM AutoPrep System into a cartridge that is inserted into a MagNestTM cell presentation device, wherein the magnetic field pulls ferrofluid-tagged EpCAM antibody-labeled epithelial cells to the surface of the cartridge. Sample analysis is performed by the CellTracksTM Analyzer II, a four-color automated fluorescence microscope that scans the entire surface of the cartridge, acquires images and displays any event to the user where CK-PE and DAPI fluorescence are co-located. Images are presented in a gallery format for final classification by a trained analyst. Sensitivity is in the order of 1 CTC per 7.5 mL of whole blood and CellSearchTM technology has been demonstrated to be reproducible, sensitive and validated in numerous and increasing numbers of studies [The CellSearch (EpCAM) . After i studies ,13. HoweAnother innovative and exciting micro-fluidic technology that has recently been developed and applied for CTC detection is a micro-fluidic device, the ‘CTC-chip’, capable of efficient and selective separation of viable CTCs from whole blood samples ,15,16. T9 to 8.04 × 103 cells per mL, and using this approach CTCs were detected from 20 out of 26 head and neck cancer patients. The average number of CTCs detected was 22 per mL of blood with the number ranging from 282 to 1 [In contrast to the positive selection strategy for CTC detection, either based on EpCAM and cytokeratin expression profile, or cell size based discrimination (discussed below), Chalmers and colleagues have optimized comprehensively a purely negative enrichment methodology in which only normal blood cells are targeted and removed, thereby allowing rare, non-hematopoietic cells to be enriched. The enrichment process uses a red cell lysis step followed by immunomagnetic labeling, and subsequent depletion, of CD45 positive cells. They reported that the system was able to reduce the number of blood cells in a cancer patient's sample from 4.05 × 10282 to 1 . Another282 to 1 . CompareIsolation by Size of Epithelial Tumor cells (ISET by ScreenCell) allows direct enrichment of epithelial cells using filtration and size exclusion thereby releasing the dependence for detection on the expression of a selected epithelial marker(s) and thus the potential to uncover CTC heterogeneity. Peripheral blood is diluted with ISET buffer, loaded onto polycarbonate membranes with 8µm calibrated pores and processed by gentle filtration under vacuum . This teMost solid cancers are of epithelial origin ,26. Usinet al. conducted several case studies on cytomorphologic analysis of CTCs detected from patients with metastatic colorectal cancer, metastatic breast cancer, or relapsed lung adenocarcinoma using fiber-optic array scanning technology. The detected CTCs were subsequently stained with a Wright-Giemsa stain or Papanicolau stain and compared to the corresponding primary and metastatic tumors. Both the striking similarity and remarkable pleomorphism were found in the CTCs in comparison with the primary tumor tissues or metastatic biopsies [Hematoxylin and Eosin stain (H&E) of ISET isolated CTCs showed that cell morphology is retained and CTCs are characterized by large size, high nucleus/cytoplasmic ratio and irregular nuclear shape A. Marrinbiopsies ,35,36. 4-6-Diamidino-2-phenylindole (DAPI), the DNA specific dye used in the Veridex CellSearch platform to confirm presence of nucleated cells, also allows discrimination of apoptotic cells with condensed chromatin and nuclear fragmentation . Our SCLThe 4th channel of the CellSearch platform enables further molecular characterization of CTCs and has been used for phenotyping isolated CTCs in several studies. CD56, one of the diagnostic markers for SCLC, was analyzed in CTCs using Alexa Fluor 488-conjugated mouse anti-human CD56 antibody. The antibody concentration was 12 μg/mL, which was optimized by titration, and the integration time for the 4th channel of the CellSearch Analyzer II was configured to 0.4 seconds. The positivity of CD56 in SCLC CTCs was obtained using the research mode of the CellSearch Analyzer II and the results showed that in all blood samples that contained CTCs there were CD56 positive cells, consistent with the CD56 staining profiles from matched tumor biopsies and confirming the dual epithelial and neuroendocrine phenotype and neoplastic origin of CTCs . SimilarCirculating tumor micro-emboli (CTM) are observed as multi-cellular aggregates or clusters of epithelial-originated tumor cells present in circulation. The presence of CTM is suggested to be a marker of highly metastatic potential . Our recet al. investigated the expression profile of EGFR, HER2, PI3K and Akt in CTCs in breast cancer patients. EGFR and HER2 expression was detected in a subpopulation of CTCs. More importantly, activated EGFR and downstream pathway activation of PI3K/Akt was also observed in CTCs which suggested that EGFR- or HER2-driven PI3K/Akt activation may be involved in the regulation of the malignant and metastatic potential of CTCs. These molecules could serve as therapeutic targets or pharmacodynamic biomarkers for mechanism based therapeutics [As a potential ‘virtual’ biopsy with availability pre- and post-drug treatment, the molecular characteristics of CTCs have been utilized in several studies to serve as predictive biomarkers and/or to provide pharmacodynamic data to inform on targeted therapy. For example, being a predictive biomarker for HER2 targeted therapy, HER2 expression was assessed in advanced breast cancer patients and the results showed that a bi-directional shift in HER2 status between the primary tumor and corresponding CTCs occurred which may help to identify additional patients who could benefit from HER2 targeted therapy . This stapeutics . Similarapeutics . EGFR gene mutation in a tumor as a strong predictor of a better outcome for EGFR inhibitor treatment. Equally important findings showed that patients with wild type EGFR who received first-line EGFR inhibitor treatment had the Hazard Ratio for progression free survival of 2.85  compared with patients received carboplatin-paclitaxel chemotherapy [in situ hybridization (FISH). More recently CTC-chip technology showed that both the classical EGFR activating mutation, and the emergence of the T790M resistance mutation were detected in CTCs [Disease staging and personalized cancer therapy depends on molecular information derived from tumors. A clear example of this was the large randomized trial that demonstrated the presence of the otherapy . This pa in CTCs . K-ras thus hijack the pathway with loss of EGFR dependence, thus rendering EGFR inhibitors ineffective [et al. established a platform-weighted chemi-luminescent membrane array and demonstrated detection of CTCs harboring K-ras mutations in the blood of colorectal cancer patients which could potentially aid to identify K-ras wild type patients who are more likely to benefit from treatment with EGFR inhibitors [K-Ras, the small G-protein acting downstream of EGFR signaling is an essential component of the EGFR signaling cascade. Activating mutations of ffective . Yang ethibitors .et al. developed a method for assessment of chromosome 1, 7, 8, and 17 copy numbers on CTCs from metastatic castration resistant prostate cancer patients and showed that the enriched cells were indeed cancerous and heterogeneity was observed with respect to the aberrancy of the copy number of these chromosomes between patients, as well as between CTCs in the individual patient [MYC, AR, PTEN, as well as TMPRSS2/ER gene rearrangements on prostate cancer CTCs. The results confirmed that CTCs isolated from castration-resistant prostate cancer (CRPC) patients are malignant in origin and indicated that hormone-regulated expression of ERG persists in CRPC [AR and relative gain of MYC were respectively detected in 38% and 56% of CTC samples collected from castration-resistant metastatic prostate cancer patients. These data indicated that androgen signaling continues to play an important role in late-stage prostate cancer [Several studies of CTC cytogenetics have been conducted regarding prostate cancer. Swennenhuis  patient . Attard  in CRPC . Similare cancer . Bcl-2 genetic abnormalities such as amplification, Bcl-2/IGH translocation, and triploidy can be detected in CTCs from SCLC patients  approach. Cultured H146 SCLC cells were spiked into whole blood, which was previously collected into EDTA tube but not CellSave tube, and then the spiked samples were processed by the CellSearch system  so as to enrich spiked unstained tumor cells. To overcome the contamination of wbc, recovered H146 cells were subsequently stained with AlexaFluor 488 conjugated anti-CD56 antibody (a neuroendocrine marker) and with 7-AAD to assess cell viability. Sorting of viable (7-AAD negative) and CD56 positive cells by FACS was optimized and the RNA extracted from the sorted cells was of good enough quality for gene expression profile analysis , which gex vivo culture. However, considerable method development is required for ex vivo expansion of CTCs immediately after their isolation. If viable CTCs/CTM can be isolated , and can be labeled, their behavior after injection into nude mice might be revealing. Kojima and colleagues developed a biological imaging system for detecting viable human CTCs using a telomerase-specific replication-selective adenovirus expressing GFP (OBP-401). The detection method involved a three-step procedure, including the lysis of red blood cells, the subsequent addition of OBP-401 to the cell pellets, and an automated scan using fluorescence microscopy. This GFP-expressing virus-based method enables relatively precise enumeration of CTCs and more importantly the simple and potentially efficient way of isolating viable CTCs. However, the impact of the manipulation of CTCs with the adenovirus infection on the molecular profile of CTCs needs to be comprehensively addressed [Another avenue for CTC research is ddressed . (i)will baseline CTC signatures reveal predictive biomarkers for targeted therapy? (ii)versus those from chemo-resistant patients inform on mechanisms of drug resistance? will signatures from CTCs from chemo-sensitive patients (iii)versus disease relapse after initial responses to therapy inform on acquired drug resistance and/or identify a ‘stem like’ tumor cell?will longitundinal CTC signatures at diagnosis (iv)will comparative signatures from matched primary and secondary tumors and CTCs shed additional light on molecular events important for metastasis? (v)will CTC signatures aid discovery and development of anti-metastatic drugs? With continuous improvement to CTCs technologies, CTC research is no longer limited to CTC enumeration and an era of molecular characterization of CTCs has begun. It is intuitive that viable CTCs, as an intermediate both temporally and spatially between primary tumor and metastasis, possess the unique advantages for researchers to study the tumor biology, especially the process of metastasis. However the real significance of CTCs in tumor biology can only be revealed by well designed, properly matched, studies adopting cutting edge techniques such as comparative genomic hybridization (CGH)/array CGH, deep-sequencing, microarray-based expression profile analysis, proteomic expression profile analysis. Some outstanding questions for CTCs can then be specifically addressed: examples include:Ultimately, it seems highly likely given the current momentum of CTC research underpinned by technology breakthroughs that accurate and sensitive detection together with comprehensive investigation of CTC biology is on the horizon. CTC research is set to expand our knowledge of metastasis and assist in the development of more effective therapeutics for cancer patients.


Thus our results do not support the prediction of generally high mfr and suggest that intersexual constraints on the evolution of colouration traits may be weaker than expected. We hypothesise that observed patterns of genetic correlations result from sex-specific selective pressures acting on nestling plumage colouration.In some bird species, both adult and juvenile individuals are often brightly coloured. It has been commonly assumed that identical plumage colouration present in both sexes results from strong intersexual genetic correlations in colour-related traits. Here, we aimed at testing this hypothesis in juvenile individuals and looked at genetic parameters describing carotenoid-based colouration of blue tit nestlings in a wild population. To separate genetic and environmental sources of phenotypic variation we performed a cross-fostering experiment. Our analyses confirmed the existence of sexual dichromatism in blue tit nestlings and revealed a significant, although low, genetic component of carotenoid-based colouration. However, genetic effects are expressed differently across sexes as indicated by low cross-sex genetic correlations ( In this species, both adults and juveniles express yellow colouration of breast feathers Our aim was to explore genetic patterns in sexually dimorphic traits in blue tit   were recorded. Nestlings were uniquely marked by nail clipping on day 2 followed by ringing on day 11. Two days post-hatching nestlings were blood-sampled for molecular sex identification (see th day post-hatching) and placed between two glass slides together with colour standards helping to calibrate subsequent colour sampling. The arrangement of feathers resembled that on the living bird in such a way that they formed two superimposed layers. Samples were photographed  in a black-box using an unidirectional halogen light source. Photographs were analysed blindly with respect to the origin of the sample using Colour Analysis Tool software (http://wwwfxc.btinternet.co.uk). We averaged RGB colour values from ten 2×2 mm squares per individual. Measurements were highly repeatable . We made all efforts to minimize the time required to handle the nestlings and thus to decrease the stress associated with all procedures.Colour data were analysed with using linear mixed models implemented using the Markov Chain Monte Carlo method in R 2.9.2 The models fitted experimental treatment (brood-size manipulation) and year as fixed effects. Additionally, sex was introduced in the univariate models. All models contained random effects of nest of origin, nest of rearing and dyad. Inclusion of each of these effects was decided upon the deviance information criterion (DIC) (Table S2 in 2H). Since we defined sex-specific variances for nest-of-origin effect we were able to estimate also related covariances. Dividing genetic covariance by geometrical mean of the respective variances yielded genetic correlations Using such kind of full-sib analyses assumes that there is no relatedness between the analysed families. In such a case, nest-of-origin approximates half of additive genetic variance plus quarter of dominance variance and maternal effects (ME) if present V = 1, covariance COV = 0, ν = 1.002). However, all models were robust to prior modification – changing priors by doubling variances had no effect on qualitative conclusions drawn from our analyses.All analyses were checked for possible problems resulting from autocorrelations in MCMC time series by visual inspection of the time-series plots and by the calculation of autocorrelations between successive samples. No autocorrelation-related issues were detected in any of the models. All models were fitted with relatively uninformative, inverse-Wishart distributed priors  of about 8% . Thus, our estimates of heritability and genetic correlations might be downwardly biased. It is hard to quantify the strength of this bias as, to our knowledge, the influence of misassigned paternities on the estimates of heritability and genetic correlations has rarely been rigorously assessed 2 for colour related traits reaches 8% ), and does not differ significantly between sexes (see respective CIs in mfr) in all three colour traits, with mfr for brightness being even weakly negative  and yellower  selection acting on adults would require demonstrating that juvenile and adult plumage traits are strongly correlated genetically. Compelling evidence for such correlations in blue tits does not exist. Only one study estimated phenotypic juvenile-adult correlation  in carotenoid-based plumage traits in a closely related species – the great tit - Firstly, juvenile plumage may be ontogenetically correlated with that of adult birds. In such a case juvenile plumage would be a mere dysfunctional side-product of selection acting on adult individuals – and all genetic patterns observed in offspring plumage would in fact reflect underlying genetics of adult traits. Usually, strong genetic correlations between analogous traits expressed throughout the ontogeny are expected mfr – remains to be studied.Secondly, carotenoid-based plumage colouration in juvenile blue tit might be adaptive at this stage of life independently of its function in the adulthood. It is unlikely that plumage colouration plays any role in the competition inside a nest or in parental favouritism as tits breed in cavities with limited light availability To conclude, our results provide first evidence that genetic constraints on the independent evolution of male and female ornamentation observed among nestlings may be weak. Lack of strong intersexual genetic correlation is contrary to a common assumption of shared genetic background of the sexes. However, the potential sex-specific selection forces responsible for diminishing the expected genetic correlation between sex remains to be identified. Further studies, focusing on sex-specific function of carotenoid-based plumage colouration in juveniles, should explain evolutionary mechanisms that produce low cross-sex genetic correlations among nestlings, and will certainly open new and exciting research perspectives.File S1Tables S1–3 providing repeatabilites of analysed traits, fixed-effects estimates and DIC-based model selection.(DOC)Click here for additional data file.


Autoimmune cholangitis remains an elusive manifestation of immunoglobulin G4-associated systemic disease most commonly encountered in patients with autoimmune pancreatitis. No strict diagnostic criteria have been described to date and diagnosis mainly relies on a combination of clinical and histopathologic findings. It is hence even more challenging to diagnose autoimmune cholangitis in patients with late or atypical presentations, such as without concomitant pancreatic involvement. Early diagnosis of this rare disorder can significantly improve outcomes considering high rates of surgical intervention, as well as high relapse rates in the absence of steroid treatment. To the best of our knowledge the literature is quite sparse on cases with atypical presentations of autoimmune cholangitis.We report a case of a previously healthy 65-year-old man of Middle-Eastern origin, with a history of pancreatic insufficiency of unknown etiology, evaluated for elevated liver function tests found incidentally on a routine physical examination. Imaging studies revealed an atrophic pancreas and biliary duct dilatation consistent with obstruction. Subsequent endoscopic retrograde cholangiopancreatography showed a bile duct narrowing pattern suggestive of cholangiocarcinoma, but brushings failed to reveal malignant cells. Our patient proceeded to undergo surgical resection. Histological examination of the resected mass revealed lymphoplasmacytic infiltrate with no malignant features. Our patient returned three months later with persistently high liver function tests and no evidence of biliary obstruction on imaging. A presumptive diagnosis of autoimmune cholangitis was made and our patient's symptoms resolved after a short course of an oral steroid regimen. Post factum staining of the resection specimen revealed an immunoglobulin G4 antibody positive immune cell infiltrate, consistent with the proposed diagnosis.Our case thus highlights the importance of clinician awareness of the autoimmune spectrum of biliary pathologies when confronted with atypical clinical presentations, the paucity of diagnostic measures and the benefit from long-term steroid and/or immunosuppressive treatment. Autoimmune pancreatitis (AIP) was initially described by Yoshida and colleagues in 1995 to represent a type of chronic pancreatitis with characteristic histopathologic and imaging findings . Type IIAlthough several diagnostic criteria have been proposed for type I AIP such as HISORt  and the The importance of recognizing the possibility of AIP-associated disease is particularly important in patients with atypical as well as non-pancreatic presentation, or those caught in later stages of the disease. Prompt initiation of steroid treatment in these individuals could greatly improve outcomes, at least in part, by the avoidance of surgery. We would thus like to share our experience in encountering a case of serum IgG4-negative, steroid-responsive sclerosing cholangitis in a patient with pancreatic insufficiency.Helicobacter pylori-associated gastritis, which was treated. On a follow-up appointment our patient described a history of diarrhea with passage of greasy stool of several months duration associated with 13.6 kg weight loss but no associated abdominal pain. On further questioning, our patient described a history of binge alcohol consumption over five years prior to presentation. His physical exam was unremarkable and laboratory investigations revealed mild anemia, with hemoglobin (Hg) 127 g/L, normal liver function tests (LFTs), negative stool cultures, increased fecal fat of 72 g/day, fecal elastase below 15 ug/g of stool, and elevated blood sugar. Ultrasound investigation of his abdomen was unremarkable and computed tomography (CT) of his abdomen revealed an atrophic pancreas with no focal lesions. Our patient was presumed to have chronic alcoholic pancreatitis, although the absence of abdominal pain was puzzling, and was prescribed pancreatic enzyme replacement therapy with significant clinical improvement.A previously healthy 65-year-old man of Middle-Eastern origin, with a past medical history of hypertension, was initially referred to our gastroenterology service for evaluation of a suspected gastrointestinal bleed. Upper and lower endoscopies failed to reveal an active source of bleeding at that time, however he did have One year later our patient was referred again for an evaluation of malaise and elevated LFTs; again there was no complaint of abdominal pain. His alkaline phosphatase (ALP) level was found to be 265 U/L, gamma- glutamyltransferase (γ-GT) 236 U/L, alanine transaminase (ALT) 138 U/L and aspartate transaminase (AST) 94 U/L in the setting of normal lipase and bilirubin levels. Our patient was not jaundiced on exam; carbohydrate antigen (CA) 19-9 was elevated at 210 U/ml with a normal carcinoembryonic antigen (CEA) level. Abdominal ultrasound revealed right-sided intrahepatic bile duct dilatation suggestive of an obstruction. A subsequent abdominal CT scan showed an irregular soft tissue mass in his right distal hepatic duct proximal to the confluence Figure , and a sSubsequent results of the pathology revealed a fibroinflammatory rather than malignant nature of the resected mass, with predominance of a lymphoplasmacytic infiltrate Figure . The livThe patient returned three months after surgery with malaise, fatigue and a reduced appetite. His laboratory studies revealed abnormal liver enzymes: total/direct bilirubin 21/9 umol/L, ALP 821 U/L, γ-GT 624 U/L, ALT 127 U/L and AST 143 U/L. Abdominal ultrasound revealed the normal appearance of the residual liver with no biliary tract dilatation. Given the histology and positive immunochemical staining for IgG4 positive cells, our patient was then prescribed a trial of a steroid regimen, which led to complete normalization of liver enzymes within three months. After tapering of the steroid therapy three months later our patient developed significant submandibular gland swelling, which again responded to re-introduction of corticosteroids. He now remains well on maintenance azathioprine treatment, 15 months after presenting with unilateral bile duct obstruction.Autoimmune cholangitis (AIC) is a term that has recently been coined to describe a sclerosing cholangitis associated with IgG4-associated systemic disease. Thus far no diagnostic criteria have been proposed and it remains largely a clinical diagnosis based on histopathological and laboratory parameters. The occasional absence of pancreatic involvement in these patients has been previously reported ,8.Despite the rarity of AIC, it remains crucial to be alert to its possibility, especially in patients with cholangitis and underlying unexplained pancreatic disease. We have described a case of a serum IgG4-negative patient who underwent laparotomy for a suspected Klatskin tumor that revealed a lymphocytoplasmic infiltrate staining heavily for IgG4. A previous diagnosis of both endocrine and exocrine pancreatic insufficiency in this patient, with a remote binge drinking history and no history of abdominal pain, is worth re-examining and possibly attributing to the late stage of autoimmune pancreatitis. The remarkable response to steroids further supports an autoimmune origin.The natural course of AIP-associated cholangitis involves relapses. An examination of a series of 53 AIC patients with a mean follow-up of 38 months demonstrated relapse of 44% and 53% postoperative and post-steroid-withdrawal respectively . SimilarWe have then reviewed the literature in an attempt to identify what could have aided in the early diagnosis of this patient and could potentially reduce morbidity associated with unnecessary surgical treatment.et al., where radiographic findings in 15 patients with a postoperative diagnosis of AIP were reviewed and failed to demonstrate specific nuances that would differentiate AIC from other biliary disease [The difficulty in preoperative diagnosis was explored by Itoh  disease . This suMeasurement of serum IgG4 levels is not currently recommended as routine without a high index of suspicion for autoimmune origin in patients with gastrointestinal complaints. In addition, as mentioned previously, only 44% of patients with IgG4-associated disease confirmed by biopsy had elevated levels of serum IgG4, rendering this test of equivocal value.We then hypothesized that perhaps steroid treatment should have been initiated right after the histology of the resected mass described a lymphocytoplasmic infiltrate. We searched the literature for similar cases, and found several cases of patients who have undergone laparotomies for suspected malignant bile duct strictures with postoperative histopathological findings suggestive of AIC ,13. In aOur case report highlights the importance of clinician awareness of the possibility of an autoimmune origin of pancreatitis and cholangitis, especially in patients presenting with atypical or late presentations. The challenge still lies in the lack of early diagnostic tests to evaluate biliary strictures that could prevent significant surgical interventions in these patients. Routine IgG4 staining on benign lesions obtained at biopsy in patients with obstructive cholangitis could potentially aid in early initiation of steroid treatment.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.BS provided patient information and was a major contributor in writing and editing the manuscript. AS collected data and composed the preliminary manuscript. DO performed the histological examination of the specimen. CZ provided the CT scan and ERCP images. All authors read and approved the final manuscript.


Antibody-mediated rejection (AMR) is gaining increasing recognition as a major complication after heart transplantation, posing a significant risk for allograft failure, cardiac allograft vasculopathy, and poor survival. AMR results from activation of the humoral immune arm and the production of donor-specific antibodies (DSA) that bind to the cardiac allograft causing myocardial injury predominantly through complement activation. The diagnosis of AMR has evolved from a clinical diagnosis involving allograft dysfunction and the presence of DSA to a primarily pathologic diagnosis based on histopathology and immunopathology. Treatment for AMR is multifaceted, targeting inhibition of the humoral immune system at different levels with emerging agents including proteasome and complement inhibitors showing particular promise. While there have been significant advances in our current understanding of the pathogenesis, diagnosis, and treatment of AMR, further research is required to determine optimal diagnostic tools, therapeutic agents, and timing of treatment. Antibody-mediated rejection (AMR) is a diagnostic and therapeutic challenge in human heart transplantation. Although the true incidence of AMR is unknown, it has been reported in 10–20% of patients after heart transplant, typically occurring within a few months after transplant .AMR was first described as a clinical entity in 1987 by Herskowitz et al. who identified a subset of heart transplant patients with arteriolar vasculitis and poor outcomes . HammondAMR occurs due to a humoral immune response with antibodies binding to endothelium on the transplanted heart . The antDSA binding to the allograft causes myocardial injury and allograft dysfunction predominantly through immune complex activation of the classical pathway of the complement cascade . AntigenAnti-HLA antibody binding may also lead to endothelial cell activation by complement independent mechanisms. Direct cross-linking of HLA molecules on the cell surface can activate endothelial cells and lead to the production of growth factors such as fibroblast growth factor, platelet-derived growth factor, monocyte chemotactic protein as well as cytokines and adhesion molecules , 14. ImmThe role of non-HLA antibodies in AMR remains an area of contention. Recently, Nath et al. showed that non-HLA antibodies directed against cardiac myosin and vimentin were elevated in heart transplant recipients who subsequently developed AMR and CAV . The appThe diagnostic criteria for AMR have undergone significant revision. A key difference between the 2005 and 2011 ISHLT guidelines is the proposed shift from a clinical to pathologic diagnosis. The 2005 guidelines recommended diagnosis of AMR based on interpretation of pathologic changes in conjunction with cardiac allograft dysfunction and/or hemodynamic compromise and the presence of DSA . AMR hasScreening for DSA is an important component of immune surveillance following heart transplantation. Screening is typically performed with solid phase assays using recombinant HLA-protein-coated beads . DetectiHistologic features of AMR as defined by the 2005 ISHLT guidelines include “myocardial injury with endothelial cell swelling and intravascular macrophage accumulation.” Additional features include “interstitial edema and hemorrhage” as well as “intravascular thrombi and myocyte necrosis” . ImportaDetection of AMR may be improved by immunopathologic examination of endomyocardial biopsy specimens. The 2005 ISHLT guidelines recommended immunofluorescence or immunohistochemical staining on frozen and paraffin sections, respectively. Immunofluorescence stains detect immunoglobulin and complement  deposition within capillaries, while immunohistochemistry stains detect capillary CD68 positive macrophages and C4d deposition .C4d deposition has been evaluated in multiple studies as a potential marker for AMR. Gupta et al. evaluated the routine use of C4d immunofluorescence on endomyocardial biopsies after transplant and found that positive C4d staining correlated with a positive retrospective crossmatch and the presence of DSA . RodriguA potentially useful marker of AMR is CD68, a cell surface antigen present on macrophages. Rodriguez et al. showed that patients with graft dysfunction likely related to AMR had positive staining for intravascular macrophages while patients with preserved graft function did not display CD68 staining . SeveralThe ISHLT recently proposed a new AMR grading scheme that is focussed on a shift towards a pathologic diagnosis . The proA proposed timeline for AMR screening is also proposed in the new ISHLT guidelines . The guiThere has been growing interest in the use of microarray technology to monitor gene expression and diagnose AMR. Microarrays were used to study endothelial-associated gene transcripts (ENDATs) in kidney allograft biopsies , 33. In The development of evidence-based guidelines for the management of AMR in heart transplantation is greatly needed. Persisting controversy over the diagnostic criteria for AMR, continued development of diagnostic techniques and a paucity of robust data to substantiate traditional therapies represent important challenges. At present, the management of AMR is nonstandardized, and optimal surveillance, treatment, and timing of intervention are undefined. AMR is difficult to treat, and therapy remains empiric with supporting evidence limited to small, nonrandomized studies with short followup. Furthermore, although it is widely accepted that AMR associated with allograft dysfunction should be aggressively treated, the management of asymptomatic AMR is less clear. While close surveillance is advocated for this group of patients, the ideal frequency and mode of assessment including evaluation of allograft function, performance of endomyocardial biopsies, and HLA antibody screening are unknown. Additionally, although studies suggest that asymptomatic AMR is associated with reduced patient survival, and an increased risk for development of CAV, implementation of surveillance and treatment has not been proven to improve outcomes , 21.A proposed management algorithm for AMR is summarized in A potential DSA monitoring algorithm is shown in Altered humoral immunity with plasma cell production of antibodies directed against donor antigens on the allograft is primarily responsible for myocardial injury in AMR. Plasma cells are generated by T-lymphocyte-dependent activation and generation of memory B-lymphocytes. Treatment strategies for AMR are directed at inhibiting the humoral response at various levels by targeting (1) removal and blockade of circulating antibodies, (2) depletion of B-lymphocytes, (3) depletion of plasma cells, (4) suppression of T-lymphocyte dependent antibody responses, and (5) inhibition of the complement cascade. While several agents have been used successfully and others show promise, AMR remains difficult to treat and is often refractory to therapy.B-lymphocyte responses, including B-lymphocyte activation, the generation of memory B-lymphocytes, and high-affinity antibodies are dependent on T-lymphocytes. Suppression of T-lymphocytes, therefore, indirectly inhibits B-lymphocytes. Cytolytic antibodies , corticosteroids, calcineurin inhibitors , mycophenolate mofetil, and proliferation signal inhibitors  inhibit T-cell proliferation and/or activation through a variety of mechanisms and may be used to treat AMR. Many of these drugs also exhibit direct effects on B-lymphocytes. For example, thymoglobulin is a polyclonal antibody containing multiple anti-T-lymphocyte antibodies that target classes I and II, HLA molecules, associated ligands , costimulatory molecules, cell adhesion molecules as well as surface molecules expressed on B-lymphocytes (CD20) and plasma cells (CD38 and CD138) . CalcinePlasmapheresis removes circulating plasma antibodies by extracorporeal separation of plasma from cellular blood components using either centrifugation or membrane filtration. As antibody production is unaffected, antibody levels may increase after discontinuation of plasmapheresis. Potential adverse effects include complications related to vascular access, hypotension due intravascular volume contraction, bleeding from depletion of clotting factors as well as allergic reactions and blood-borne infection from plasma repletion with replacement fluids such as albumin and fresh frozen plasma.n < 15) followed. Although these small studies have consistently demonstrated that plasmapheresis is an effective treatment for AMR, variation in protocols with differing duration and frequency of treatment and adjunctive treatment confounds interpretation. Similarly, an ideal plasmapheresis treatment regimen has not been established with the number of treatments in studies varying from days to weeks.In 2001, Grauhan et al. published the first clinical case series of plasmapheresis for the treatment of AMR after heart transplantation . BetweenIntravenous immunoglobulin (IVIG) is comprised of 90% IgG, extracted from pooled plasma from between 50,000 and 100,000 blood donors . A numbeExperience with IVIG in transplantation is largely derived from renal transplantation. In this patient population, IVIG has mostly been evaluated for desensitization and shown to reduce PRA and increase conversion to a negative complement dependent cytotoxic crossmatch . RecentlRituximab is a chimeric monoclonal IgG antibody, comprised of human constant regions and mouse variable regions directed against the CD20 pan-B lymphocyte surface molecule. Rituximab depletes B-lymphocytes by antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and induction of apoptosis through CD20 cellular crosslinking and caspase activation . Rituximn = 8) [2/week for up to 4 weeks. Pharmacodynamic studies in patients with lymphoma have shown maximal B-lymphocyte depleting effects at three to four months with low or undetectable numbers in the peripheral blood for up to six months and full recovery within 12 months [The efficacy of Rituximab for AMR treatment after heart transplant has only been examined in case reports and one small case series (n = 8) . The maj2 months . Interes2 months . Further2 months , 51.The safety and tolerability of rituximab is well established in the context of hematologic malignancies. Mild-to-moderate first dose infusion-related reaction with fever and hives account for the majority of adverse effects. Concerns have, however, been raised over an increased risk of serious infections with rituximab therapy after transplantation. In a study of 22 renal transplant recipients, rituximab was shown to be effective in treating AMR but associated with a high incidence of infection . Eighty-Proteasomes are responsible for the processing and degradation of ubiquitin-labeled proteins. Bortezomib is a selective 26S proteasome inhibitor depleting plasma cells and antibody production by inducing apoptosis via cell cycle arrest as well as inducing the unfolded protein response due to increased endoplasmic reticulum stress from accumulation of misfolded proteins , 54. Addn = 81), but also included five heart transplant recipients. Bortezomib was administered in four doses, each preceded by plasmapheresis, and all patients also received a single dose of rituximab. Over an average 9 month followup, 58% of patients showed histologic improvement, with 81% and 96% graft and patient survival, respectively. Additionally, there was a >50% reduction in DSA in 40% of patients at 14 days after treatment. An 11.2% incidence of opportunistic infection was reported. Studies in transplantation and multiple myeloma series have reported high tolerability of Bortezomib with common adverse effects including fatigue, gastrointestinal toxicity, thrombocytopenia, neutropenia, and peripheral neuropathy.Bortezomib is currently approved for the treatment of multiple myeloma. Small studies in renal transplant patients have shown that bortezomib effectively decreases HLA antibodies in patients with stable renal function and is effective in treating refractory AMR –59. WoodEculizumab is a humanized monoclonal antibody directed against C5, thereby preventing complement activation by inhibiting cleavage of C5 to C5a and C5b, and formation of the membrane attack complex. Eculizumab is currently approved in the United States for the treatment of paroxysmal nocturnal hemoglobinuria. Experimental studies in sensitized mice have shown that eculizumab as part of a multi-immunosuppressive regimen prevents allograft rejection and improves allograft survival . At presThe diagnosis and management of AMR in heart transplantation has evolved significantly over the last decade. Current international multidisciplinary consensus recommend a pathologic-based diagnosis, combining specific histologic and immunopathologic features on endomyocardial biopsy and utilizing accompanying allograft dysfunction and/or circulating DSA as additional supporting evidence. AMR as a disease entity will continue to evolve and become better defined with advances in diagnostic techniques and improved understanding of key pathogenic molecular and immunologic mechanisms. Current available therapeutic options modify the humoral immune response by targeting the removal and blockade of antibodies, and the depletion of T- and B-lymphocytes. Emerging therapies showing promise include agents that directly deplete plasma cells and antibody production, such as bortezomib, as well as complement inhibiting agents such as eculizumab. The scarcity of heart transplantation as a clinical resource and current poor patient and allograft outcomes associated with AMR provide significant impetus for the development of effective treatment strategies. It is anticipated that the 2011 ISHLT consensus guidelines on AMR diagnosis and management will further facilitate the goal of developing a standardized evidence based management approach to AMR in heart transplantation.


Damage to the intestinal lining is thought to be mediated by increased shedding of epithelial cells and enhanced gut permeability. These processes remain poorly understood at the molecular level, in part because of a lack of suitable animal models. Now, Mark Pritchard and colleagues have established a robust model for examining intestinal epithelial cell shedding, using lipopolysaccharide (LPS) treatment to induce intestinal injury in mice. They show that LPS-treated mice develop diarrhoea, which correlates with intestinal epithelial cell shedding and caspase-3-driven apoptosis. By examining the effects of LPS in knockout mice, they demonstrate a role for TNFR1 and NFκB2 signalling in these molecular events. The model provides a powerful tool for investigating the mechanisms underlying intestinal epithelial damage, which could guide the development of new therapies for gastrointestinal disorders.


The growth of contemporary cell biology is due in large part to technologicaladvances. In the 1950s, electron micrographs of thin sections first providedunrivaled in situ views of the delicate intracellular architecture and finestructure of organelles, whereas new subcellular fractionation methods gave accessto various biochemical components—especially proteins—enriched indifferent cellular fractions. A central tenet that emerged from these pioneeringstudies is that the intracellular biosynthetic and endocytic membrane systems ofeukaryotic cells are functionally interconnected, and exchange of material betweenthem often occurs in small (50–100 nm diameter), roughly spherical membranoustransport vesicles. In electron micrographs, these vesicles are typically seen inclose proximity to a membrane compartment and are frequently covered on theircytosolic face with a fuzzy proteinaceous coating. Subsequent technical advancesfacilitated further discovery and progress: genetic screens in model organisms andrefinement of subcellular fractionation to facilitate cell-free reconstitution oftransport reactions allowed the identification and purification of key regulatoryand structural components. Persuasively, many of the genes discovered in geneticscreens encoded the proteins purified biochemically. More recently, genomesequencing and proteomics efforts have bolstered the identification of sortingcomponents, leading to long lists of evolutionarily conserved proteins that areinvolved in specific sorting operations at different membranes.Clathrin-mediated endocytosis is the archetype of a vesicular transport reaction thatsorts specific cargo for transportation to another intracellular compartment  At least 40 different proteins participate in the construction of a clathrin-coatedendocytic vesicle Saccharomyces cerevisiae,assembled clathrin caps the ends of regularly sized, bullet-shaped invaginations ofplasma membrane, termed cortical actin patches, which are involved in endocyticuptake S. cerevisaeSchizosaccharomyces pombeS. pombeactin outnumbers clathrin by >100-fold in cortical patches Past insights into clathrin-mediated endocytosis have come mainly from studies ofchemically fixed or ground-up cells PLoS Biology, Merrifield and colleagues present a tour de forceanalysis of the latter type To deal with these confounding issues, investigators in the field have pursued twodirections. One is to define carefully the temporal parameters of clathrin coatassembly in an atypical mammalian cell line (BS-C-1), chosen for the inherentuniformity of the size and behavior of the clathrin structures on the surface ofthese cells To begin with, the pHlourin–transferrin receptor accumulates at bothdiffraction-limited spots and large patches By determining precisely, to within two seconds, when a cargo-laden vesicle detaches,Taylor et al. have produced a functionally defined, unmistakable temporal landmarkfor vesicle scission. This differs importantly from simply following the abruptdisappearance of the clathrin signal from the TIR-FM illumination field, which couldalso be due to (later) vesicle uncoating or physical movement out of the evanescentfield. Moreover, upon budding not all clathrin-positive structures lose completelytheir surface-apposed clathrin: there are also budding events in which a smallresidue of the coat remains at the bud site to nucleate a second round of vesicleassembly within ∼40 seconds Dynamin is a large GTPase that plays an essential role in vesicle release from theplasma membrane. By expressing a red fluorescently tagged dynamin protein instead ofthe clathrin, and again using two-color TIR-FM, Taylor et al. show that dynaminappears immediately preceding the occurrence of a pH 5-stablepHlourin–transferrin receptor signal, nicely validating the assay. Accuratelypinpointing the moment of scission also provides the opportunity to catalog otherparticipants in the process; so, with the two-color TIR-FM assay in hand, theMerrifield group turned their attention to 33 other proteins. What they learned isinstructive and far-reaching. Cluster analysis of kinetic profiles of the manyproteins examined defines seven operational modules on the basis of their similardynamics Messenger RNA analysis confirms that the bulk of the proteins followed by Taylor etal. are endogenously expressed in NIH-3T3 cells and the transfected, fluorescentlytagged versions are detectable in the majority of clathrin-coated structures If there is indeed only one basic mode of clathrin vesicle budding, it still leavesopen why sometimes these arise de novo and sometimes from preexisting largerclathrin assemblies .Perhaps


Diet is a major source of cadmium intake among the non-smoking general population. Recent studies have determined that cadmium exposure may produce adverse health effects at lower exposure levels than previously predicted. We conducted a meta-analysis to combine and analyze the results of previous studies that have investigated the association of dietary cadmium intake and cancer risk.We searched PubMed, EMBASE, and MEDLINE database for case-control and cohort studies that assessed the association of dietary cadmium intake and cancer risk. We performed a meta-analysis using eight eligible studies to summarize the data and summary relative risks (RRs) and 95% confidence intervals (CIs) were calculated using a random effects model.Overall, dietary cadmium intake showed no statistically significant association with cancer risk . However, there was strong evidence of heterogeneity, and subgroup analyses were conducted using the study design, geographical location, and cancer type. In subgroup analyses, the positive associations between dietary cadmium intake and cancer risk were observed among studies with Western populations  and studies investigating some hormone-related cancers .Our analysis found a positive association between dietary cadmium intake and cancer risk among studies conducted in Western countries, particularly with hormone-related cancers. Additional experimental and epidemiological studies are required to verify our findings. Cadmium has been recognized as a carcinogen for many decades based on studies of occupationally exposed individuals . FindingDiet is the main source of environmental cadmium among non-occupationally exposed non-smokers . Additioin vitro cell culture and in vivo animal studies demonstrated that cadmium exposure results in cell transformation and induces cancer in various organs ; 2) exposure ; 3) outcome ; and 4) covariates used in multivariate analysisP-values less than 0.05 were considered statistically significant. We estimated the summary association between dietary cadmium intake and cancer incidence using a random effects model. The Q-statistic and I2 statistics were calculated to estimate heterogeneity. Potential sources of heterogeneity were determined via subgroup or sensitivity analyses. The data were stratified by study design (case-control/cohort), geographic location (Western/Asian), and cancer type . We also performed sensitivity analyses to determine the effects of individual studies on the summary estimate by repeating the meta-analysis. For each subgroup analyses, we used a fixed or random effects model based on the results of the Q-statistics to calculate the summary RRs and 95% CIs [P < 0.05 for heterogeneity. We also conducted a cumulative meta-analysis to evaluate whether the trend of summary RRs (95% CIs) changed over time as more data were collected. Studies were added one at a time according to the publication year. The results were summarized as each new study was added. Publication bias was examined using analyses described by Egger [All statistical analyses were performed using STATA software , and two-sided  95% CIs ; we usedby Egger  and Beggby Egger  and a viOverall, the meta-analysis conducted in this manuscript contained eight studies, including two case-control studies (746 cases and 1069 controls) ,16 and sP = 0.020; I2 = 58%). Therefore, we calculated the summary estimate using a random effects model. Overall, dietary cadmium intake showed no statistically significant association with cancer risk . To identify the source of heterogeneity, we conducted subgroup analyses based on study design, geographic location, and cancer type . The funnel plot of studies was shown in We also conducted a cumulative meta-analysis based on publication year. The association between dietary cadmium intake and cancer risk did not change significantly as more data were collected 3. AdditDiet is the major source of cadmium for general populations that are not exposed to cadmium occupationally and do not live in cadmium polluted areas. We identified eight studies that investigated the role of dietary cadmium intake, but the findings of these studies are inconsistent. Several studies have investigated the role of cadmium in cancer development in female reproductive organs -12. In aMany studies have investigated the role of cadmium in hormone-related cancers  in non-occupationally exposed populations using biomarkers -30 TablS2. Dietin vitro cell culture or in vivo animal models provide evidence of the estrogenic or androgenic effects of cadmium on cell growth and gene expression [+) breast tumor but not an estrogen receptor negative (ER-) breast tumor in a cohort of postmenopausal women [It has been suggested that cadmium may induce cancer through multiple pathways, such as via aberrant gene expression, inhibition of oxidative stress, inhibition of DNA damage repair, or inhibition of apoptosis . Recent pression -35. Furtpression . Initialal women . Severalal women ,12,13 oral women . Howeveral women . Additioal women .Several factors may influence the role of dietary cadmium in cancer development. Therefore, it is important to identify the high-risk population for cadmium-induced carcinogenesis―a population that is defined by increased exposure to cadmium or a higher absorbed dose of cadmium. First, dietary habits affect the level of cadmium exposure. Individuals who regularly eat crustaceans, mollusks, and cephalopods or consume large amounts of whole grains and vegetables have the highest exposure to dietary cadmium . AlthougTo the best of our knowledge, this is the first meta-analysis combining the results of existing studies that have investigated the effect of dietary cadmium on cancer risk. Most are population-based cohort studies, which may eliminate recall and selection biases. However, we recognize several limitations in interpreting our results. The most important limitation is the validity of the estimated cadmium intake values. Estimates of dietary cadmium intake could vary widely among the populations studied based on the method of diet assessment and the cadmium database used . FFQ mayOur meta-analysis supports the findings of existing studies regarding the role of dietary cadmium intake in hormone-related cancer risk in Western countries. We cautiously suggest that chronic exposure to cadmium and other metalloestrogens may partly explain the risk of developing hormone-related cancers, particularly in Western populations. To reduce cadmium-induced cancer risk, it is important to identify the high-risk population  and to provide an appropriate medical intervention. Although this meta-analysis of epidemiological studies suggests a link between cadmium and hormone-related cancers, more experimental and epidemiological studies using diverse populations are needed to establish a causal association, as well as to verify the underlying mechanisms.Table S1The association between environmental cadmium exposure and breast cancer risk in studies using biomarkers.(DOCX)Click here for additional data file.Table S2The association between environmental cadmium exposure and prostate cancer risk in studies using biomarkers.(DOCX)Click here for additional data file.


Ischaemic left ventricular (LV) dysfunction can arise from myocardial stunning, hibernation, or necrosis. Imaging modalities have become front-line methods in the assessment of viable myocardial tissue, with the aim to stratify patients into optimal treatment pathways. Initial studies, although favorable, lacked sufficient power and sample size to provide conclusive outcomes of viability assessment. Recent trials, including the STICH and HEART studies, have failed to confer prognostic benefits of revascularisation therapy over standard medical management in ischaemic cardiomyopathy. In lieu of these recent findings, assessment of myocardial viability therefore should not be the sole factor for therapy choice. Optimization of medical therapy is paramount, and physicians should feel comfortable in deferring coronary revascularisation in patients with coronary artery disease with reduced LV systolic function. Newer trials are currently underway and will hopefully provide a more complete understanding of the pathos and management of ischaemic cardiomyopathy. Ischaemic heart disease (IHD) is the leading cause of morbidity and mortality in Western society, with an overrepresentation in the primary healthcare burden –3. In paThe myocardium is exquisitely sensitive to ischemia, with contractile dysfunction occurring shortly after an ischaemic stimulus. The degree of contractile impairment remains strongly under the influence of the severity and duration of the ischaemic event, with irreversible myocardial necrosis representing the end pathway of prolonged and significant coronary ischemia . Hence, Despite early intervention, patients with IHD have a predisposition to develop structural heart disease, with impairment of myocardial function leading to cardiac failure, a condition termed as “ischaemic cardiomyopathy” . Given tThe ability to distinguish whether dysfunctional myocardium is “viable” and thus able to recover following revascularisation, however, presents a clinical challenge in current practice.This review examines the concept of myocardial viability, with focus on imaging modalities and principal outcome trials.Viability of myocardial tissue is the central principal which underpins reperfusion therapies, whether in the acute phase following myocardial infarction or in chronic ischemia-mediated LV dysfunction. Should “viable” myocardial tissue be present, restoration of adequate coronary blood flow should in theory improve myocardial performance and LV ejection fraction (EF), with the hope of translating into improved long-term outcomes.Early work into CAD and myocardial flow limitation supported the hypothesis that myocardial ischemia results in significant myocyte injury . myocardial stunning, was defined as a prolonged and completely reversible dysfunction of the ischaemic myocardium that continued after restoration of coronary arterial flow [Heyndrickx and coinvestigators first demonstrated the impact of reversible ischemia on myocardial contractile reserve. Utilising animal models, they demonstrated that short (5- or 15-minute) induced episodes of ischemia to the myocardium, with a subsequent reperfusion period , resulted in regional deficits in contractile function that persisted despite reperfusion . This phial flow . Stunnedial flow .Myocardial stunning has also been found in clinical practice, particularly in the setting of increased myocardial demand or reduced coronary supply such as following coronary artery spasm, postmyocardial infarction, or postcardiopulmonary bypass secondary to “cardiac off-time.” Myocardial stunning is also prominent in patients following successful revascularisation postinfarct, wherein there is prolonged systolic dysfunction which takes several days to normalise after the incident event –15.Myocardial hibernation represents a condition of sustained depression of myocardial function in the setting of CAD, which is amenable to improvement in function postrevascularisation. This term was first coined by Diamond and colleagues in 1978 , and was repetitive stunning hypothesis. In this theory, multiple bouts of demand ischemia in context of flow limitation result in repetitive episodes of ischaemic myocardial dysfunction (or stunning), which eventually creates an environment of sustained depression of contractile function [An alternate hypothesis offered for the mechanism of sustained contractile depression is thefunction .Resting myocardial perfusion is normal or near normal in stunning but is reduced in hibernation. Stunning of the myocardium is frequently represented as transient regional LV wall motion abnormality persisting for hours to days following reperfusion after short-term but significant impairment of coronary blood flow. Hibernating myocardium, on the other hand, is a state of persistently impaired myocardial performance at rest due to a chronic reduction in coronary blood flow that can be restored by favorably altering the supply/demand relationship of the myocardium . AlthougIdentifying myocardial hibernation is of clinical relevance, as it represents potentially salvageable myocardial tissue. Coronary revascularisation in this context is likely to improve contractile performance, LV systolic function, and, in turn, overall morbidity and mortality. However, hibernating myocardium, if left untreated, has the potential to transform into clinically overt heart failure. Revascularisation, via either percutaneous angioplasty or coronary bypass surgery, is the primary avenue of restoring coronary blood flow, unless natural collaterals are formed from the primary diseased vessel.Pathologic Q waves, deep initial negative deflections of the QRS complex, were traditionally thought to be secondary to chronic transmural ischemia and representative of “dead myocardium.” On subsequent analysis, it has been demonstrated that presence of pathologic Q waves has a poor correlation with the lack of residual viable myocardial tissue, with a relatively low sensitivity (41–65%) and specificity (69–79%) relative to other imaging modalities , 20.Utility of exercise electrocardiography improves viability detection, with elevation of the ST segment during exercise in infarct-related leads being representative of viable myocardium (sensitivity 82% and specificity 100%) . A similUse of normalisation of abnormal T waves during exercise electrocardiography for viability assessment, on the other hand, has conflicting reports in the literature , 24, witAssessment of echocardiographic parameters at rest is important in assessment of viability. Severe dilatation of the LV is a marker of nonviable myocardium, with higher end-systolic volume indices associated with poor ventricular functional recovery . These f (i)μg/kg/min) can increase contractility in dysfunctional segments which are still viable. At higher doses (10–40 μg/kg/min), wall motion in these segments may further improve or paradoxically diminish, reflecting tachycardia-induced ischemia. This phenomenon is referred to as a biphasic response and has been shown to be highly predictable of functional recovery postrevascularisation. This finding is suggestive of limited, but present, myocardial reserve in the hibernating myocardium.Biphasic response: low-dose dobutamine (defined as 5–10  (ii)Worsening contractile function with lack of initial improvement with dobutamine: this response is suggestive of a hibernating myocardium which is supplied by a critically limited arterial supply, with no contractile reserve. (iii)Sustained improvement with increasing dobutamine dose: this response is traditionally seen in the setting of myocardial stunning. (iv)No response to dobutamine: this response is indicative of lack of functional reserve and, thus, lack of viable myocardial tissue.Dysfunctional areas with resting end-diastolic wall thickness of less than 6 mm are thought to reflect significant scar. They are not known to show functional improvement with DSE and do not improve postrevascularisation.Dobutamine stress echocardiography (DSE) is a valuable tool in the assessment of viability of the myocardium. Classically, four responses are noted in a dysfunctional myocardial response to dobutamine. These are as follows –33: (i)BDSE has been shown to have a sensitivity and specificity range for prediction of contractile recovery that is modestly high , with the biphasic response having the greatest predictive capability of the four responses .Myocardial contrast echocardiography (MCE) utilises acoustically reflective high molecular weight inert gases which form microbubbles and act as a contrast agent. These bubbles remain within the intravascular space and help attenuate the borders of the left ventricle. Tissue capillary blood flow, a determinant of myocardial perfusion, is the byproduct of capillary blood volume and myocardial blood velocity.Once the microbubbles reach a steady-state concentration, high-burst ultrasonography is used to displace the microbubbles, with subsequent replenishment within myocardial segments over the following cardiac cycles reflective of myocardial blood velocity. Segments are deemed viable if there is homogeneity of contrast intensity, which is in keeping with intact myocardial microvasculature. Nonviable segments, however, lack contrast enhancement and represent necrotic myocardial cells causing obstruction and collapse of the microcirculation –35.MCE has been shown to have a high sensitivity (78–90%), however, low specificity (51–72%), of myocardial contractile recovery postrevascularisation relative to DSE (which on average has a relatively high specificity but lower sensitivity) –39. A coMyocardial deformation indices, including tissue Doppler imaging (TDI) and strain assessment, are new echocardiographic modalities in the assessment of myocardial function, which allow for a more complete appraisal of myocardial motion and overcome traditional challenges of two-dimensional echocardiography with regard to regional myocardial assessment , 41. StrMyocardial deformation (strain) and deformation rate (strain rate) provide multidimensional evaluation of myocardial mechanics  and have the added advantage of being able to detect subtle wall motion abnormalities of regional function that do not decrease global LVEF , 43. ThiBoth TDI and speckle-tracking echocardiography have been shown to be facilitative in prediction of myocardial viability. This is of relevance given the limitations of subjective assessment of wall thickness as well as operator dependence with traditional two-dimensional stress echocardiographic methods. Bansal and colleagues (2010) revealed that longitudinal and circumferential strain and strain rate measurements at rest and low-dose dobutamine concentrations were predictive of functional recovery postrevascularisation using strain-based imaging. Furthermore, only tissue velocity imaging was found to have incremental value over wall motion analysis .18FDG [Based on a study by Hoffmann et al. (2002), an increase of peak systolic strain rate greater than or equal to 0.23/s had a sensitivity of 83% and specificity of 84% in discerning viable myocardium as determined by 18FDG . Additio18FDG . FurtherAdvantages of echocardiography include ease of procedure and widespread availability as well as its noninvasive qualities. Furthermore, with DSE, there is an ability to monitor functional response to accurate uptitration of inotropic therapy. Limitations of echocardiography include its high operator dependency with resultant inter- and intraobserver variability. Patients with comorbidities such as obesity, chronic obstructive airflow limitation, and thoracic chest wall abnormalities limit the acoustic window and thus impair LV views. Furthermore, with respect to DSE, assessment relies heavily on subjective visual interpretation of wall motion abnormalities.Single-photon emission CT (SPECT) is a modality which utilises radionuclide-labeled tracer compounds to measure myocardial uptake. Initial acquisition signifies delivery of the tracer throughout the circulation. The images acquired following this  reflect myocardial sarcolemmal integrity .99mTc-sestamibi, 99mTc-tetrofosmin, and 201Thallium. These molecules are lipophilic and permeate through myocardial cellular membranes via passive diffusion or active uptake from Na+/K+ ATPase systems. Intracellular retention, however, requires intact function of the mitochondrion with preservation of the action potential, and as such serves as a marker of viability. These tracer agents emit high-energy photons, which are captured via gated SPECT, and provide information of global LV function and viability of the myocardium [Primary tracers include ocardium , 48. late distribution imaging) [Viability assessment with SPECT can be performed at rest, following physical exercise or chemical coronary stress. With stress testing, physical exertion or chemical agents  are used. Imaging is performed immediately following the test, with delayed imaging repeated 3 to 4 hours later, allowing for adequate redistribution of the tracer agent. If warranted, imaging may be repeated at 24 hours after stress (termed asimaging) .Viability is seen with myocardial segments which reveal defective uptake immediately following stress, with subsequent replenishment of uptake at 3 to 4 hours. Critically hypoperfused myocardial segments may still be viable if defective uptake is seen at this delayed time-point, warranting repeat imaging at 24 hours after stress to allow for redistribution of the tracer to significantly hypoperfused myocardial regions. Nonviable myocardium reveals fixed defective uptake throughout a 24-hour imaging cycle .SPECT has been shown to provide a higher sensitivity (64–72%) however lower specificity (45–88%) than modalities based on evaluation of residual contractile recovery , 50. Pri13N-labeled ammonia (13NH3) and 18F-fluorodeoxyglucose (18FDG), are utilised in standard practice.Positron emission tomography (PET) imaging is based on the shift of myocardial perfusion energetics, whereby chronically underperfused myocardial tissue shifts from utilization of free fatty acids (that require high oxygenation for use) to that of glucose metabolism, which uses a more anaerobic process at the expense of poor energetic efficiency. This translates into uptake of perfusion tracers in myocardial segments which are hypoperfused. Perfusion tracers, including 18FDG uptake. Regions of myocardium where there is a concordance between reduction of myocardial blood flow and 18FDG uptake (flow-metabolism match) reflect irreversible myocardial injury. In contrast, areas where FDG uptake (reflective of metabolism) is preserved or increased despite perfusion deficits reflect viable myocardium [Regions are classified according to the degree of “flow-metabolism” matching, which is reflected by concordance between myocardial blood flow and ocardium   . ReducedCine cardiovascular magnetic resonance (CMR) sequencing provides information on global left ventricular function and regional wall motion. It can be used in conjunction with dobutamine stress and gadolinium-chelated contrast. Gadolinium-chelated contrast agents have been utilised to detect perfusion deficits, microvascular obstruction, and myocardial scarring. Accumulation of contrast agents have a paramagnetic effect, which form bright signal intensities in areas of accumulation. These agents are unable to penetrate cardiac myocytes with intact membranes; however, they easily diffuse and accumulate into extracellular membranes with increased volume of distribution  or ruptured cellular membranes  during the “late” steady-state phase .The transmural extent of scarring is inversely correlated with functional recovery of the dysfunctional myocardium postrevascularisation, whereas the absence of late gadolinium enhancement in a hypokinetic myocardium is associated with functional recovery postrevascularisation , 53 Fig.Benefits of CMR over alternate imaging modalities include excellent spatial imaging, ability to discern transmural variations in viability, and provision of accurate quantification of nonviable or necrotic tissue. The ability of CMR for detection of scar (nonviable tissue) is robust, with a sensitivity of 83% and specificity of 88% , 54. PriA summary of trials evaluating the utility of different imaging modalities in viability assessment is shown in Numerous nonrandomized retrospective studies in the early 1990s evaluated the value of viability testing. A meta-analysis of these trials revealed a significant association between revascularisation and improvement in mortality utilising viability testing in patients with known ischaemic cardiomyopathy. This finding was shared irrespective of imaging modality chosen . PrimaryThere was significant clinical uncertainty with regard to the impact of viability on survival given the lack of large, heavily powered randomized trials. These questions were largely addressed in the Surgical Treatment for Ischaemic Heart Failure (STICH) trial (2011). The STICH trial was designed to evaluate the impact of coronary artery bypass grafting (CABG) in management of patients with CAD with reduced LVEF.n = 130), or SPECT (n = 321), or both (n = 150). Of the viability subgroup, 298 participants were randomly assigned to receive medical therapy plus surgical revascularisation (cardiac bypass) and 303 received solitary medical management. Participants were followed up at intervals  with a median length of follow-up of 56 months  [In this multicenter , nonblinded, randomized trial, 1212 participants were enrolled, with 601 undergoing myocardial viability assessments. Participants were enrolled on the basis of echocardiographic evidence of LV systolic dysfunction (defined as LVEF ≤ 35%) and coronary angiography revealing CAD amenable to surgical intervention. Myocardial viability assessment was provided via DSE (p = 0.21). Furthermore, assessment of myocardial viability did not provide a differential benefit for surgical intervention (p = 0.53). That is to say that viability assessment did not recognize participants who would benefit from CABG relative to medical therapy [Despite an association of viable myocardium to likelihood of survival in this cohort, multivariate analysis did not find a statistically significant mortality benefit with surgical intervention ( therapy .p = 0.05), composite death from any cause and hospitalization from cardiovascular causes . Long-term follow-up (>4 years) of both cohorts revealed a reduction in all-cause mortality in the surgical revascularisation cohort compared to solitary medical therapy; however, this finding was not statistically significant (p = 0.12). These positive secondary findings should be interpreted with caution given a negative primary outcome measure [Secondary endpoints were more forgiving towards revascularisation, with bypass surgery having a significant reduction in cardiovascular mortality  in the viability subgroup having single-vessel disease. Given the scope of the paper , this differential profile may have selected out participants for whom viability assessment may not have been required. Thirdly, analysis in this study was limited to DSE and SPECT modalities, with no analysis of PET or CMR on viability assessment. This creates difficulty with extrapolation of these results to other imaging modalities of viability assessment.Despite these limitations, this study represents the largest analysis of the influence of myocardial viability on clinical endpoints in persons with ischaemic cardiomyopathy to date,and was the first to assess the differential effect of viability on revascularisation versus medical management.The Heart Failure Revascularisation Trial (HEART) (2011) was a multicenter study comparing the efficacy of surgical revascularisation with optimal medical treatment in the management of persons with clinically diagnosed cardiac failure with reduced EF (LVEF < 35%) and evidence of CAD. Participants were screened for viable myocardium via DSE. An inclusionary prerequisite was the presence of at least 5 viable LV segments with reduced contractility using a 17-segment model .n = 65) and medical arms (n = 69) and followed up over a five-year period. The primary outcome revealed noninferiority of medical therapy. This study was, however, underpowered secondary to a relatively small sample size. Furthermore, the primary modality of viability assessment was DSE, which has a lower sensitivity for viability detection relative to other imaging modalities. Additionally, randomization had not occurred prior to viability assessment, therefore clouding the impact of viability assessment on treatment outcomes [138 participants were randomized to interventional (outcomes .The PET and Recovery Following Revascularisation-2 (PARR-2) trial (2007) evaluated the efficacy of perfusion FDG-mediated PET imaging in risk stratification and identification of patients who would most benefit from revascularisation .p = 0.15), with post hoc analysis showing a statistically significant reduction in adverse events in the FDG PET-assisted group  [The study enrolled 430 participants, with an inclusionary criterion of a LVEF <35% and suspected or confirmed CAD. Participants were randomly placed to receive FDG and perfusion PET imaging versus standard care . Effect of PET scanning on appropriate decision showed a nonsignificant trend towards a reduction in the predefined composite endpoint  at one year . The results of this substudy illustrated prognostic benefit with the utilization of FDG PET viability imaging in ischaemic cardiomyopathy when used in centers with experience in PET imaging [The Ottawa-FIVE substudy  (2010) evaluated 111 participants with LV systolic dysfunction  and suspected or confirmed CAD in a single center with experience with FDG PET imaging . A stati imaging .Despite the relatively disappointing results of the aforementioned trials, the 2013 American Heart Association/American College of Cardiology guidelines for management of heart failure remain unaltered in their Class IIa (Level of Evidence B) recommendation for viability testing in the work-up for revascularisation in patients with ischaemic cardiomyopathy. This is in keeping with the belief that there may still be diagnostic and prognostic benefit in the utility of viability studies which have not become apparent given the limitations of the aforementioned primary trials.Ischaemic LV dysfunction can arise from myocardial stunning, hibernation, or necrosis. In line with technological advances, noninvasive imaging modalities have become front-line methods in the assessment of viable myocardial tissue, with each modality conferring a variable advantage in terms of sensitivity and specificity, culminating in the overriding goal of accurate stratification of patients into optimal treatment pathways.Despite determined research efforts, however, many questions remain unanswered with regard to myocardial viability. Initial studies, although favorable, lacked sufficient power and sample size to provide conclusive outcomes of viability assessment. More recent trials, including the STICH and HEART studies, have failed to confer prognostic benefits of revascularisation therapy over standard medical management in ischaemic cardiomyopathy but have their own limitations. In lieu of these recent findings, however, assessment of myocardial viability therefore should not be the arbitrating factor for therapy choice. Optimization of medical therapy for all patients is paramount, and physicians should feel comfortable in deferring coronary revascularisation in patients with CAD with reduced LVEF at present.It is clear that further trials are needed to better our understanding of the mechanistic underpinnings of the viable myocardium as well as the underlying pathos of ischaemic cardiomyopathy. Newer trials such as the AIMI-HF  study, the largest randomized trial to date evaluating the role of imaging in the treatment of ischaemic cardiomyopathy, are currently underway and will hopefully decipher some of these uncertainties .