Therapy-related acute myeloid leukemia (t-AML) is a secondary, generally incurable, malignancy attributable to chemotherapy exposure. Although there is a genetic component to t-AML susceptibility in mice, the relevant loci and the mechanism(s) by which they contribute to t-AML are largely unknown. An improved understanding of susceptibility factors and the biological processes in which they act may lead to the development of t-AML prevention strategies.Parp2, Casp9, Polr1b) are the most likely to have a non-redundant role in the pathogenesis of t-AML. While identifying these networks, we found that current CNVR and SNP-based haplotype maps in mice represented distinct sources of genetic variation contributing to expression variation, implying that mapping studies utilizing either source alone will have reduced sensitivity.In this work we applied an integrated genomics strategy in inbred strains of mice to find novel factors that might contribute to susceptibility. We found that the pre-exposure transcriptional state of hematopoietic stem/progenitor cells predicts susceptibility status. More than 900 genes were differentially expressed between susceptible and resistant strains and were highly enriched in the apoptotic program, but it remained unclear which genes, if any, contribute directly to t-AML susceptibility. To address this issue, we integrated gene expression data with genetic information, including single nucleotide polymorphisms (SNPs) and DNA copy number variants (CNVs), to identify genetic networks underlying t-AML susceptibility. The 30 t-AML susceptibility networks we found are robust: they were validated in independent, previously published expression data, and different analytical methods converge on them. Further, the networks are enriched in genes involved in cell cycle and DNA repair , suggesting that these processes contribute to t-AML susceptibility. Within these networks, the putative regulators (e.g., The identification and prioritization of genes and networks not previously implicated in t-AML generates novel hypotheses on the biology and treatment of this disease that will be the focus of future research. NF1 are associated with increased risk of t-AML in humans and mice [de novo AML.Therapy-related acute myeloid leukemia (t-AML) is a secondary malignancy attributable to chemotherapy and/or radiation exposure. t-AML comprises 5-20% of adult AML cases and its prevalence is increasing along with the size of the population undergoing chemotherapy ,2. Whileand mice . GainingN-nitroso-N-ethylurea (ENU), a potent mutagen with a propensity to cause AT:TA transversions and AT:GC transitions [2 = 0.10, P-value < 0.001), supporting the hypothesis that there is a strong genetic component in t-AML susceptibility.One class of chemotherapeutics associated with t-AML is the alkylators . The therapeutic effect of alkylator agents is believed to result from the formation of DNA adducts and single and double-strand breaks, which trigger apoptosis or growth arrest . Based onsitions . Mice weWe hypothesized that the pre-exposure transcriptional state of hematopoietic stem and progenitor cells, the putative target of leukemogenesis , underliIn this study, we apply an integrated genomics approach  to identhttp://www.broadinstitute.org/mouse/hapmap/. The SNP resource includes 132,285 SNPs per genome, of which 115,009 we considered informative (as defined in Methods). We used these data to derive haplotype blocks, which are ancestral regions of shared genetic background among strains. Haplotype blocks facilitate trait mapping because they robustly and efficiently represent un-typed genetic variation at a locus. The practical benefit of utilizing haplotype blocks instead of individual SNPs is a reduced number of statistical tests. We used a simple merging algorithm to iteratively join adjacent SNPs into haplotype blocks. This algorithm results in haplotype blocks in which the genotypes of a complete set of SNPs are predictable to a given level of accuracy. We selected a threshold such that for a given block, we can accurately predict the genotype of every SNP in all 48 strains with at most one error  by a SNP-based haplotype  in mice using inherited DNA copy number variant regions (CNVRs) as genetic markers . This cir Figure . The 23,r Figure . Only 21e Figure . We specWe performed gene expression profiling (GEP) in c-kit+/lineage- (KL) bone marrow cells (a population enriched in hematopoietic stem/progenitors) from 20 inbred strains listed as Tier 1-4 from the Mouse Phenome Database . Two-to-Next, we asked which genes were differentially expressed between susceptible and resistant strains in KL cells. We identified 917 differentially expressed genes (976 probes) at an FDR threshold of 5%  in KL cells that were associated with 214 CNVRs . We repeThere are 45 genes (45 probes) that are both differentially expressed and linked to at least one eQTL. We refer to these genes as anchors throughout the text. 37 are linked to CNVR-eQTLs; the remaining 8 are linked to haplotype-eQTLs. To validate the cis-eQTL associations, we mined publicly available expression data representing hematopoietic stem, progenitor, erythroid and myeloid populations from the BXD recombinant inbred panel . Becausetrans) on multiple downstream genes (targets). For each anchor, we identified correlated expression profiles (FDR < 1%), resulting in 30 sets of co-expressed genes or modules. The number of targets per module ranged from 3 to 607 . We reasoned that true response genes will exhibit association with anchor expression even when the remaining genome is randomly shuffled, as it is in the BXD recombinant inbred cross. For each module, we tested the association between expression of the anchor and each response transcript in each of the BXD hematopoietic populations. We removed target genes from modules that were not associated with anchor gene expression in at least one population (FDR < 25%) . These genes are enriched in several, independent biological processes, most notably apoptosis. Among the differentially expressed intrinsic apoptosis genes are Caspase 9 (Casp9), B-cell leukemia/lymphoma 2 (Bcl-2), BCL2-associated agonist of cell death (Bad), BCL2-associated X protein (Bax), and mutS homolog 6 (Msh6). Msh6 is a member of the mutSα DNA mismatch recognition complex that has been shown to mediate apoptosis in certain contexts [O6-methylguanine (O6MeG)-mediated cytotoxicity [Msh6 suggests that upon alkylator exposure, resistant strains may recognize DNA damage and respond appropriately (i.e. undergo apoptosis) whereas the KL cells of susceptible strains may tend to survive, accumulate mutations, and become transformed. In KL cells, almost all susceptible strains have no detectable expression of Casp9, an initiator of programmed cell death, suggesting that these cells (low-to-no Casp9 expression) are less primed for Casp9-dependent apoptosis. Although knockout of Casp9 in mice is embryonic lethal, presumably due to severe brain development defects [Casp9 is not required for apoptosis during normal hematopoiesis [The second step in the integrated approach was to find genes differentially expressed between t-AML susceptible and resistant strains. Because unsupervised clustering using contexts ,36. Notatoxicity . That re defects ,39, Caspopoiesis . Therefotrans are varied. They include altered transcription factor abundance and homeostatic or compensatory forces within and between biological pathways. Regardless of the mechanisms of action, the identification of putative events that influence susceptibility and their linkage to gene networks forms a powerful and practical strategy to both find biological pathways underlying cancer susceptibility and to prioritize candidate mediators. Therefore, as the third step in the integrated genomics approach, we identified networks of genes that are significantly correlated with candidate susceptibility anchors. To validate the networks, we used independent gene expression data from multiple hematopoietic populations, trimming the networks of response genes whose expression was not reproduced.Differential expression and gene enrichment analysis highlighted several biologically plausible pathways that may underlie t-AML susceptibility. However, it remained unclear which pathway members, if any, are causal contributors to the phenotype, as illustrated by the complex expression patterns of the intrinsic apoptosis genes. More broadly, the role and relative importance of each of the 917 differentially expressed genes in susceptibility remained undetermined. We hypothesized that among the 917 differentially expressed genes would be a subset in which expression variation is caused by cis-encoded genetic variation. Further, we posited that these 'anchor' genes cause expression variation of multiple downstream genes, which collectively are associated with t-AML susceptibility (or resistance). The mechanisms by which anchors might act in Casp9 and Bcl2 are differentially expressed, Casp9 is also the candidate anchor of module A_33, the module most strongly associated with susceptibility status. We speculate that perturbation of candidate anchors, such as Casp9, are more likely to be informative in elucidating susceptibility than response genes (i.e. Bcl2).One of the benefits of the integrated genomics approach is that it can implicate biological processes that would not have been detected using differential expression alone. The susceptibility networks that we identified are enriched in genes involved in DNA repair, base excision repair, apoptosis, and cell cycle, among other annotations. A second potential benefit of the integrated approach is that it differentiates between upstream (anchors) and response genes, an advantage over existing approaches that derive gene regulatory networks from expression data alone. While this is a hypothesis that remains to be tested, the identification of candidate upstream factors will be useful in prioritizing among apoptosis-related genes for experimental validation. For example, although Bcl2. Therefore, A630001G21Rik may play previously unknown role in regulation of Bcl2 expression and apoptosis activity. Similarly, Cytoskeleton-associated protein-like 2 (Ckap2l) is the anchor of the largest module, A_16, enriched in both cell cycle and DNA repair genes. Although Ckap2l is highly expressed in hematopoietic progenitors [Ckap2, is highly expressed in mouse stem cell lines and has detectable expression in hematopoietic progenitors, bone marrow, osteoclasts, osteoblasts, and macrophages [Ckap2  is involved in cell cycle progression [Ckap2l contributes to cell cycle regulation in HSCs and progenitors, and that genetic disturbances of its expression impact t-AML susceptibility. Experiments that perturb expression of anchor genes such as Casp9 and Ckap2l to assess their impact on module expression and activity are the next logical steps in determining the role of candidate networks in susceptibility. If such experiments demonstrate a causal link between anchor genes and module expression, then moving forward to formally define their role in leukemia will be warranted.Network analysis allowed us to predict the function of uncharacterized genes. For example, A630001G21Rik is expressed primarily in primitive hematopoietic and B-cells , yet itsgenitors , its funrophages . There igression -44. It iParp2, the anchor) and apurinic/apyrimidinic endonuclease 1 (Apex1), both members of the base excision repair pathway [A drawback to the anchored network approach, as currently implemented, is that it assumes there is only a single anchor per module. In cases where CNVRs disrupt regulatory elements, it is possible that a single genetic event impacts the expression of multiple neighboring genes. For example, in module A_37 Figure  we found pathway ,46. BothA caveat to the current work is that maps of genetic variation in the mouse genome are incomplete, a knowledge gap that promises to be filled by more informative SNP arrays  and nextTo our knowledge, this is the first report of an integrated genomics approach to dissect the role of the pre-exposure transcriptional state in t-AML susceptibility. From a clinical perspective, t-AML is important because the response to treatment is poor and survival is short . But becliftOver. 31 CNVRs were unmapped and dropped from further analysis. To derive haplotype blocks, SNPs for the haplotype map construction were downloaded from the Broad Institute http://www.broadinstitute.org/mouse/hapmap/. Only SNPs from 48 non-wild-derived strains were used. SNPs that were contained within CNVRs, had minor allele frequencies < 5%, or were not genotyped in 25% or more of strains were considered uninformative and were excluded from further analysis. The following steps were performed to simultaneously group SNPs into blocks and to assign haplotype to strains:Genomic coordinates of 1,333 CNVRs were mapped from mm8 to mm9 using (1) Begin with the first informative SNP on a chromosome.(2) If the number of SNPs in the current block is 1 then go to (3). Otherwise, go to (4).(3) Group strains by genotype and add the next consecutive SNP to the current block.Partitioning Around Medioids[(4) Cluster strains by SNP-based distance using  Medioids (number (5) Assign haplotype labels to strains based on the clustering with the maximum average silhouette .(6) Derive consensus haplotypes. For each haplotype cluster, a consensus haplotype is defined as the string comprised of the most frequent genotype at each SNP position.(7) Compare the consensus haplotypes to the actual SNP genotypes.(8) If the number of errors is greater than 1 then go to (9), otherwise go to (10).(9) Remove the most recently added SNP from the current block. Store the haplotyping results from the previous iteration. Start a new block with the current SNP. Go to (3).(10) Add the next consecutive SNP to the current block. Go to (4). If there are no more SNPs on the current chromosome, select a new chromosome and go to (2). The computation is complete when all chromosomes have been analyzed.2 was computed based on haplotype frequencies [SNP-based distances between strains are computed as the sum of SNP differences between strains. The range of number of allowable haplotypes per block was selected based on the estimated number of ancestral haplotypes . Pooled quencies .GEP expression profiling was previously described  and is ahclust function, using 1-Pearson correlation as the distance metric and the complete linkage method for node merging. To assess the non-randomness of the strains clustering according to susceptibility status, we computed the ratio of the mean of the distances among susceptible strains to the mean of the distances between all susceptible and resistant strains. Then, we permuted the strain labels 10,000 times, and recomputed the ratio of distances. The P-value of the observed clustering is the number of random permutations in which the distance test statistic > = observed distance test statistic divided by 10,000. This analysis was performed on the median expression profiles of strain replicates, only in those strains in which the susceptibility status is known. SNP clustering was based on strain-strain pair-wise distances computed by counting the number of SNPs that differ between each of the strains divided by the total number of SNPs that are typed in both strains.Unsupervised hierarchical clustering was performed with R's limma package in R to model the expression of each gene with coefficients representing strain replicates and susceptibility status [Strains with unknown susceptibility status were not included in the differential expression analysis. We used the y status ,54 and ty status . All of y status . Only thCNVR eQTLs previously identified were used in this analysis  and eQTLWGCNA [2 exceeded 0.9. Weighted adjacency matrices were computed, modules were defined using the cut Tree Dynamic function (which selects good dendrogram cutoffs) and similar modules were merged using merge Close Modules (which compensates for the high sensitivity of WGCNA). Eigengenes were computed as the first principal component of a module's expression matrix. Eigengenes were tested for differential expression between susceptible and resistant strains, as described above for individual genes.Normalized gene expression data used for validation of eQTLs and anchored modules was downloaded from GEO (GSE18067). This data set includes profiling on sorted (purified) hematopoietic stem, progenitor, myeloid and erythroid populations from female BXD recombinant inbred mice . Only deWGCNA . BrieflyN-nitroso-N-ethylurea; (CNVRs): copy number variant regions; (GEP): gene expression profiling; (KL): c-kit+/lineage-; (FDR): false discovery rate; (WGCNA): Weighted Gene Co-expression Network Analysis; (Casp9): Caspase 9; (Bcl-2): B-cell leukemia/lymphoma 2; (Bad): BCL2-associated agonist of cell death; (Bax): BCL2-associated X protein; (Msh6): mutS homolog 6; (O6MeG): O6-methylguanine; (Ckap2l): Cytoskeleton-associated protein-like 2; (Parp2): poly (ADP-ribose) polymerase family member 2; (Apex1): apurinic/apyrimidinic endonuclease 1.(t-AML): therapy-related acute myeloid leukemia; (CNV): DNA copy number variation; (eQTL): expression quantitative trait loci; (SNPs): single nucleotide polymorphisms; (ENU): PC and TG designed the study, performed analysis, and wrote the manuscript. Both authors read and approved the final draft.Supplementary Table S1. SNP-derived haplotype blocks in 48 inbred mouse strains.Click here for fileDendrograms showing clustering of strains by gene expression profile or SNP-derived haplotype blocks. (A) Unsupervised clustering of strains using the strain median expression profile in KL cells groups strains by t-AML susceptibility status to an extent greater than expected by chance (see text), and differently than when clustering gene expression profiles of the hypothalamus (B), adipose tissue (C), or when clustering based on SNP-based distance (D).Click here for fileHeatmap of genes differentially expressed in KL cells from t-AML susceptible vs. resistant strains of mice. 917 genes (976 probes) are differentially expressed between t-AML susceptible  and t-AML resistant  mice.Click here for fileSupplementary Table S2. Annotation of genes differentially expressed between t-AML susceptible and resistant strains of mice.Click here for fileUnsupervised clustering of expression profiles of purified hematopoietic compartments. BXD populations are indicated by the enriched population: erythrocytes , myeloid lineage , hematopoietic stem cells , and progenitors . Hematopoietic stem and progenitors from classical inbred strains are indicated by inbred strain name . Each population forms a distinct cluster, with KL cells grouping most closely with stem and progenitor cells.Click here for file


Differentiating between sterile inflammation and bacterial infection in critically ill patients with fever and other signs of the systemic inflammatory response syndrome (SIRS) remains a clinical challenge. The objective of our study was to mine an existing genome-wide expression database for the discovery of candidate diagnostic biomarkers to predict the presence of bacterial infection in critically ill children.n = 21) and patients with sepsis having positive bacterial cultures (n = 60). Differentially expressed genes were subjected to a leave-one-out cross-validation (LOOCV) procedure to predict SIRS or sepsis classes. Serum concentrations of interleukin-27 (IL-27) and procalcitonin (PCT) were compared between 101 patients with SIRS and 130 patients with sepsis. All data represent the first 24 hours of meeting criteria for either SIRS or sepsis.Genome-wide expression data were compared between patients with SIRS having negative bacterial cultures (EBI3) had the highest predictive strength. Computer-assisted image analyses of gene-expression mosaics were able to predict infection with a specificity of 90% and a positive predictive value of 94%. Because EBI3 is a subunit of the heterodimeric cytokine, IL-27, we tested the ability of serum IL-27 protein concentrations to predict infection. At a cut-point value of ≥5 ng/ml, serum IL-27 protein concentrations predicted infection with a specificity and a positive predictive value of >90%, and the overall performance of IL-27 was generally better than that of PCT. A decision tree combining IL-27 and PCT improved overall predictive capacity compared with that of either biomarker alone.Two hundred twenty one gene probes were differentially regulated between patients with SIRS and patients with sepsis. The LOOCV procedure correctly predicted 86% of the SIRS and sepsis classes, and Epstein-Barr virus-induced gene 3 (Genome-wide expression analysis has provided the foundation for the identification of IL-27 as a novel candidate diagnostic biomarker for predicting bacterial infection in critically ill children. Additional studies will be required to test further the diagnostic performance of IL-27.The microarray data reported in this article have been deposited in the Gene Expression Omnibus under accession number GSE4607. Differentiating between sterile inflammation and bacterial infection in critically ill patients with fever and other signs of the systemic inflammatory response syndrome (SIRS) remains a clinical challenge -3. StandWe generated a large genome-wide expression database (transcriptomics) of critically ill children with SIRS, sepsis, and septic shock by way of microarray technology -18. In tn = 17) and was previously described in detail [The study protocol was approved by the Institutional Review Boards of each participating institution  according the manufacturer's specifications. Microarray hybridization was performed by the Affymetrix Gene Chip Core facility at Cincinnati Children's Hospital Research Foundation, as previously described, by using the Human Genome U133 Plus 2.0 GeneChip  .Analyses were performed by using one patient sample per chip. Image files were captured by using an Affymetrix GeneChip Scanner 3000. Raw data files were subsequently preprocessed by using Robust Multiple-array Average (RMA) normalization with GeneSpring GX 7.3 software  . All chiGene-expression mosaics were generated by using the Gene Expression Dynamics Inspector (GEDI) platform. GEDI is a publicly available gene-expression analysis program developed by the Ingber Laboratory at Harvard University ,24. The Individual patient mosaics were compared with SIRS and sepsis reference mosaics by using a publicly available image-analysis platform (ImageJ), as previously described . In brieSerum IL-27  and procalcitonin  protein concentrations were measured by using a magnetic bead multiplex platform and a Luminex 100/200 System , according the manufacturers' specifications.U test, χ2, or Fisher Exact tests, as appropriate. Descriptive statistics and comparisons used SigmaStat Software . Classification and regression tree (CART) analysis was conducted by using the Salford Predictive Modeler v6.6  [Initially, data are described by using medians, interquartile ranges (IQRs), and percentages. Comparisons between study cohorts used the Mann-Whitney CA, USA) . BiomarkCA, USA) .n = 21) or sepsis with positive bacterial cultures (n = 60). All gene-expression data reflect the first 24 hours of meeting clinical criteria for SIRS or sepsis. Fifty-three of the patients with sepsis also met criteria for septic shock. The basic clinical and demographic characteristics of the SIRS and sepsis cohorts are shown in Table Candidate sepsis diagnostic genes were identified by analyzing existing patients in our genome-wide expression database of critically ill children meeting criteria for either SIRS with negative bacterial cultures n = 2 or sepsiThe initial step for identifying candidate sepsis diagnostic genes consisted of an expression filter. Starting with all gene probes on the array , we selected gene probes having ≥2-fold expression between the median values of patients with sepsis and patients with SIRS, respectively. This expression filter yielded 228 gene probes. We next subjected the 228 gene probes to a statistical test  by using the sepsis and SIRS cohorts as the comparison groups. This statistical test yielded 221 gene probes differentially regulated between patients with sepsis and patients with SIRS.EBI3) had the highest predictive strength.We then performed a leave-one-out cross-validation (LOOCV) procedure to determine whether the overall expression patterns of the 221 differentially regulated gene probes could identify SIRS and sepsis classes. The LOOCV procedure correctly predicted 86% of the SIRS or sepsis classes. The top 100 class-predictor genes (based on predictive strength) are provided in Additional file The expression values of the top 100 class-predictor genes were uploaded to the GEDI platform, and reference gene-expression mosaics were generated for patients with SIRS and patients with sepsis, respectively Figure . The refWe next performed computer-assisted image analysis to determine whether the expression mosaics could correctly identify SIRS and sepsis classes. The image-analysis algorithm compared individual patient mosaics with the two reference mosaics and assigned the individual patients to either SIRS or sepsis classes, based on similarity of expression . The tesEBI3 had the highest predictive strength for bacterial infection in this cohort of critically ill children. EBI3 is a subunit of the heterodimeric cytokine, IL-27, which is produced by antigen-presenting cells and plays a role in regulating T-cell function [As previously noted, We measured IL-27 serum protein concentrations in 61 healthy control children and in a cohort of 231 critically ill children. One hundred and one critically ill children met criteria for SIRS and had negative bacterial cultures; 38 met criteria for sepsis and had positive bacterial cultures; and 92 met criteria for septic shock and had positive bacterial cultures. All serum samples represent the first 24 hours of meeting clinical criteria for SIRS, sepsis, or septic shock. The basic clinical and demographic characteristics of this cohort, and the respective median IL-27 concentrations, are shown in Table To determine the ability of serum IL-27 concentrations to predict bacterial infection in critically ill patients, we grouped the patients with sepsis and septic shock as positive cases for infection, and compared them with the SIRS patients as negative cases for infection. The area under the curve (AUC) for the receiver operating characteristic (ROC) curve was 0.811 (0.755 to 0.868). The IL-27 test characteristics for predicting infection in critically ill patients are provided in Table P = 0.049 versus the AUC for IL-27). The PCT test characteristics for predicting infection in critically patients are provided in Table Because procalcitonin (PCT) is currently being used clinically as a biomarker for bacterial infection in critically ill patients, we also measured serum PCT concentrations in the same cohort of patients. As shown in Table We next conducted CART analysis to determine whether a combination of serum IL-27 and PCT concentrations could further improve the ability to predict infection in critically ill patients . The optWe previously reported differential patterns of gene expression across the SIRS, sepsis, and septic-shock clinical spectrum . In thatBased on this analytic approach, we leveraged the discovery potential of microarray-based transcriptomics and generated a list of genes differentially regulated between critically ill patients with SIRS  and critically ill patients with sepsis . This gene list represents a potential working list of candidate diagnostic biomarkers for bacterial infection in critically ill patients. The global expression patterns of the top 100 class-predictor genes were able to predict SIRS and sepsis classes with high specificity and a high positive predictive value.EBI3 had the highest predictive strength for bacterial infection of all genes differentially regulated between patients with SIRS and patients with sepsis. IL-27 is a heterodimeric cytokine belonging the IL-6 and IL-12 family of cytokines and is composed of the IL-27-p28 and EBI3 subunits, which are produced by antigen-presenting cells on exposure to microbial products and inflammatory stimuli [EBI3 or neutralization of IL-27 via a soluble IL-27 receptor fusion protein is protective in a murine model of septic peritonitis [Generating gene-expression data and gene-expression mosaics for 100 genes may not yet be clinically feasible within the time-sensitive constraints of the intensive care unit. Accordingly, we investigated the ability of serum IL-27 protein concentrations to predict bacterial infection in critically ill patients. The rationale for investigating IL-27 is based on the observation that  stimuli , and is  stimuli . Furtheritonitis . Thus, iSerum IL-27 protein levels ≥5 ng/ml, obtained within the first 24 hours of meeting clinical criteria for SIRS/sepsis, had a high specificity and a high positive predictive value for predicting bacterial infection in our cohort of more than 200 critically ill patients with SIRS or sepsis. Thus, serum IL-27 has the potential to serve as an effective "rule-in" test, given that concentrations ≥5 ng/ml had a >90% specificity and positive predictive value for bacterial infection in this cohort of critically ill patients. Conversely, serum IL-27 protein concentrations <5 ng/ml do not necessarily rule out bacterial infection, given that the negative predictive value for a concentration ≥2 ng/ml was 78%. Finally, it does not appear that increased IL-27 protein concentration in critically ill children with bacterial infection reflects increased illness severity, because the median IL-27 concentrations were similar between patients with sepsis and patients with septic shock.et al. [PCT has emerged as a widely used diagnostic biomarker for bacterial infection in clinical practice. However, the performance of PCT varies depending on the patient population in which it is applied, and a meta-analysis by Tang et al.  concludeGiven the biologic complexity and heterogeneity of critical illness, it is unlikely that any one biomarker will consistently predict the presence of bacterial infection. Accordingly, a strategy that combines diagnostic biomarkers may perform better than any single biomarker ,31. WithSeveral strengths of our study design are worthy of discussion. First, we selected IL-27 as a candidate diagnostic marker in an objective manner, by using the discovery potential of transcriptomics. Second, our study cohort was relatively large, and all patients in the sepsis cohort had formal microbiologic confirmation of bacterial infection. Third, the study cohort represents patients from 17 different institutions. Finally, the serum IL-27 data reflect the first 24 hours of meeting criteria for either SIRS or sepsis, which is a clinically relevant time point for the prediction of bacterial infection in critically ill patients.EBI3 mRNA levels had the highest predictive strength, we did not directly measure serum EBI3 protein concentrations, because it was technically more pragmatic to measure serum IL-27 concentrations. Second, EBI3 is also a subunit for IL-35, but we were unable to measure serum IL-35 concentrations for technical reasons. Third, we cannot exclude the possibility that some of the patients in the SIRS cohort had bacterial infections that were undetectable by conventional microbiologic cultures. Fourth, our data are limited to critically ill children and may not be generalizable to other clinical settings. Finally, we have no data regarding the temporal production of IL-27 during the course of bacterial infection. These limitations provide further research opportunities for future studies.Our study also has a number of limitations worthy of discussion. First, although Genome-wide expression analysis has provided the foundation for the identification of IL-27 as a novel candidate diagnostic biomarker for predicting bacterial infection in critically ill patients. We also demonstrated that a combination of IL-27 and PCT improves the overall ability to predict infection, compared with that of either biomarker alone. Additional studies will be required to test further the diagnostic capabilities of IL-27.• By using microarray analysis, we identified 221 gene probes that are differentially regulated between patients with SIRS and patients with sepsis.• The expression patterns of these differentially regulated gene probes can distinguish patients with SIRS and patients with sepsis with a high specificity and positive predictive value.EBI3), a subunit of IL27, has the highest predictive strength among the 221 differentially regulated gene probes.• Epstein-Barr virus-induced gene 3 (• Serum IL27 protein concentrations had a high specificity and positive predictive value for predicting bacterial infection in a cohort of critically ill children.• The performance of IL27 was generally superior to that of procalcitonin in this cohort.EBI3: Epstein-Barr virus-induced gene 3; GEDI: Gene Expression Dynamics Inspector; IL-27: interleukin-27; IQR: interquartile range; LOOCV: leave-one-out cross validation; PCT: procalcitonin; PICU: pediatric intensive care unit; PRISM: pediatric risk of mortality; ROC: receiver-operating characteristic; SIRS: systemic inflammatory response syndrome.AUC: area under the curve; CART: classification and regression tree; HRW and the Cincinnati Children's Hospital Research Foundation have submitted a provisional patent application for the use of IL-27 as a diagnostic biomarker for sepsis.The remaining authors have no competing interests to report.enrolled patients, provided biologic samples and clinical data for the database, and edited the manuscript. SB and EB coordinated patient enrolment among the various study sites, maintained the clinical database, and edited the manuscript. KH coordinated biologic-sample procurement and submission among the various study sites, maintained the biologic-specimen repository, and edited the manuscript. PL conducted all biomarker measurements and edited the manuscript. All authors read and approved the manuscript for publication.HRW conceived and developed the study, obtained funding for the study, conducted the analyses, and wrote the manuscript. NZC, MH, GLA, NJT, RJF, NA, KM, PAC, RL, MTB, AS, JN, MQ, JH, AC, and TPS Top 100 class-predictor genes. A list of the top 100 predictor genes for bacterial infectionClick here for file


Pre-travel health assessments aim to promote risk reduction through preventive measures and safe behavior, including ensuring travelers are up-to-date with their immunizations. However, studies assessing pre-travel health-seeking practices from a variety of medical and non-medical sources and vaccine uptake prior to travel to both developing and developed countries within the Asia-Pacific region are scarce.Cross-sectional surveys were conducted between July and December 2007 to assess pre-travel health seeking practices, including advice from health professionals, health information from other sources and vaccine uptake, in a sample of travelers departing Sydney and Bangkok airports. A two-stage cluster sampling technique was used to ensure representativeness of travelers and travel destinations. Pre-travel health seeking practices were assessed using a self-administered questionnaire distributed at the check-in queues of departing flights. Logistic regression models were used to identify significant factors associated with seeking pre-travel health advice from a health professional, reported separately for Australian residents, residents of other Western countries and residents of countries in Asia.A total of 843 surveys were included in the final sample . Overall, pre-travel health information from any source was sought by 415 (49%) respondents with 298 (35%) seeking pre-travel advice from a health professional, the majority through general practice. Receipt of a pre-travel vaccine was reported by 100 (12%) respondents. Significant factors associated with seeking pre-travel health advice from a health professional differed by region of residence. Asian travelers were less likely to report seeking pre-travel health advice and uptake of pre-travel vaccines than Australian or other Western travelers. Migrant Australians were less likely to report seeking pre-travel health advice than Australian-born travelers.This study highlights differences in health-seeking practices including the uptake of pre-travel health advice by region of residence and country of birth. There is a public health need to identify strategies targeting these travel groups. This includes the promotion of affordable and accessible travel clinics in low resource countries as traveler numbers increase and travel health promotion targeting migrant groups in high resource countries. General practitioners should play a central role. Determining the most appropriate strategies for increasing pre-travel health preparation, particularly for vaccine preventable diseases in travelers is the next stage in advancing travel medicine research. International travel has increased dramatically over the last few decades, in magnitude, speed and geographical reach with 940 million arrivals reported in 2010 [A pre-travel consultation with a health professional can provide the international traveler with the necessary preventive advice on minimizing health risks during travel, including the risk of infectious disease and the opportunity for relevant vaccination and chemoprophylaxis. Travelers who seek pre-travel health advice from a health professional have been found to have better knowledge of infectious disease risk, more accurate risk perceptions and a higher level of intended risk-reducing behaviors -5. Yet sCross-sectional surveys of travelers were conducted prior to their departure from international airports in Sydney, Australia for destinations in Asia between July and September 2007 and Bangkok, Thailand bound for Australia between October and December 2007. Asian destinations were defined according to the United Nations World Macro classification of regions and included countries in Eastern Asia and South-Eastern Asia . Asian dThe second stage of the cluster sampling method involved the distribution of questionnaires to every fifth passenger joining check-in queues of selected flights. Bilingual interviewers attended check-in counters 3 h before scheduled departure until 1 h before departure. A similar method was employed at the Bangkok airport, with selected flights proportionate to the number of traveler arrivals at Australian airports from Thailand and representative of Thai, Australian, and other carriers. Approximately 175 flights were sampled between July and September 2007 at the Sydney site comprising 2.7% of the flights to Asia during this period, and 13 flights between October and December 2007 at the Bangkok site, comprising 2.4% of the flights to Australia from Thailand during this period.Eligible respondents were both visitors and residents aged 18 years or older, departing on the day of interview, excluding passengers in transit. The questionnaire was developed in simplified English and piloted at Sydney Airport. The revised questionnaire was translated into Chinese, Vietnamese and Thai with back-translation employed to ensure consistent interpretation of questions. The self-administered questionnaire included questions on socio-demographic characteristics and travel characteristics of their current trip. Respondents were provided with a list of health professionals and non-medical sources of travel health information. Pre-travel health seeking practices refer to both the seeking of health advice from a health professional as well as health information from other sources. Pre-travel health advice refers only to information obtained from a health professional. A list of common routine and travel vaccines were provided to respondents, who were asked to record vaccines received for the specific purpose of their current trip.The sample obtained at the Sydney study site was weighted by flight destination to reflect the proportion of passenger departures from aviation statistics , due to A total of 843 surveys, including 729 weighted surveys of travelers departing Sydney to destinations in Asia , and 114 surveys of travelers departing Bangkok (response rate 60.0%) were included in the final sample. The number of respondents by flight destination has been reported previously . Of the At least one visit to a health professional in the past year for any reason was reported by 532/843 (63.1%) respondents, including 320 (38.0%) reporting two or more visits. Overall, 415 (49.2%) respondents sought some form of travel health information prior to their present trip. Pre-travel health advice was sought from a health professional by 298 (35.3%) respondents with 237 (79.5%) of these respondents seeking advice from their general practitioner. A travel specialist or travel clinic was attended by 35 (4.2%) respondents prior to travel or 11.7% of those who sought professional pre-travel health advice. Non-medical sources of travel health information were reported by 290 (34.4%) respondents. The Internet was reported as a source of pre-travel health information by 162 (19.2%) respondents, with 71/162 (43.8%) including a government travel advisory website in their web searches. Travel agents were reported as a source of travel health information by 114/843 (13.5%) respondents. Differences in pre-travel health seeking by region of residency are shown in Table Pre-travel vaccination rates were low across both study sites with 100 (11.9%) respondents reporting receipt of one or more vaccines specifically for this trip, including 73/298 (24.5%) of those reporting a pre-travel heath visit. Pre-travel vaccination differed by region of residence  is an established risk factor for acquiring infectious diseases during travel  and for We acknowledge some limitations to our study. A brief self-administered questionnaire design, although appropriate to maximize the response rate in high volume airport surveys, limits the amount of detail obtainable and is also subject to recall bias. Additional factors which have been found to be associated with uptake of pre-travel health advice, such as previous travel experience and economic barriers were not obtained. Due to strict security measures at Sydney Airport, we were not able to gain access beyond customs and conducted our interviews in the departures check-in area. This resulted in a lower response rate than those reported by other airport surveys conducted in the departure lounges ,31,32 inA low proportion of participants in this study reported receipt of pre-travel vaccines 12%) with lower rates of pre-travel vaccination reported by Asian travelers compared to Western travelers. Wilder-Smith et al. also reported low uptake of pre-travel vaccines among Asian travelers, with 5% of Asian resident respondents reporting any pre-travel vaccination and low rates of self-reported prior vaccination against common vaccine-preventable diseases . The fin% with loLack of time has been reported as a reason for not seeking pre-travel advice and vaccinations in other studies -8. OtherThe few travelers seeking advice suggests that public health strategies aimed at travelers may be warranted. These would include increasing uptake of pre-travel medical advice, ensuring vaccines, both routine and those recommended for travel, are up to date as well as the promotion of affordable and accessible travel clinics in low resource countries as traveler numbers increase. Limited data from the Asia-Pacific region are available and few studies assess traveler preparedness in residents of low resource countries, despite the strong growth of the international travel market in Asia. There is variability in pre-travel health advice seeking and vaccine uptake by region of residence and in migrant travelers. The role of general practitioners in the provision of pre-travel health advice and traveler health promotion is central and opportunities for improvement exist. Considering the increasing volume of international travel, a greater integration of travel history and travel plans into general practice consultations would identify those who intend to travel but are not aware of the need for a pre-travel health consultation, particularly migrant travelers. Determining the most appropriate strategies for increasing pre-travel health preparation, particularly for vaccine preventable diseases in travelers is the next stage in advancing travel medicine research.CRM receives funding from GSK and CSL Biotherapies for investigator-driven research. These payments were not associated with this study. The remaining authors have no competing interests.CRM and RW conceived and supervised the study. AH organized airport access in Australia, undertook data collection and analysis and drafted the manuscript. SI and KN organized airport access in Thailand and undertook data collection. CRM, RW, SI and KN reviewed the manuscript. Selected results appearing in this manuscript were presented at the Asia-Pacific International Conference on Travel Medicine, Melbourne, Australia, 24–28 February 2008 and the 13th International Congress on Infectious Diseases, Kuala Lumpur, Malaysia, June 19–22, 2008. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/12/321/prepub


Direct stenting without balloon dilatation may reduce procedural costs and duration, and hypothetically, the restenosis rate. This study was designed to compare the in-hospital and long-term outcomes of direct stenting (DS) versus stenting after pre-dilatation (PS) in our routine clinical practice.The 1 603 patients treated with stenting for single coronary lesions were enrolled into a prospective registry. Patients with acute myocardial infarction (MI) within the preceding 48 hours, and those with highly calcified lesions, total occlusions, or a lesion in a saphenous graft were excluded. The baseline, angiographic and procedural data, inhospital outcomes and follow-up data were recorded in our database and analysed with appropriate statistical methods.p < 0.001). With univariate analysis, dissection and non-Q-wave MI occurred less frequently in this group . However, the cumulative major adverse cardiac events (MACE) did not differ significantly . With multivariate analysis, direct stenting reduced the risk of dissection  nor its constructing components were different between the groups.Eight hundred and fifty-seven patients (53.5%) were treated with DS and 746 (46.5%) underwent PS. In the DS group, lesions were shorter in length, larger in diameter and had lower pre-procedural diameter stenosis. Type C and diffuse lesions and drug-eluting stents were found less often (Direct stenting in the real world has at least similar long-term outcomes in patients treated with stenting after pre-dilatation, and is associated with lower dissection rates. Since the advent of balloon angioplasty, the introduction of coronary stents has been the most important turning point in the percutaneous management of coronary artery lesions. Coronary stents are associated with more effective dilatation and predictable in-hospital outcomes, higher procedural success rates, and a decreased need for target-vessel revascularisation.5The standard stent implantation technique requires routine pre-dilatation with a balloon catheter to allow an easy passage of the stent and to enhance a complete expansion of all stent modules.7The placement of stents without balloon dilatation may reduce the duration of the procedure, the radiation exposure, the amount of contrast media used, and the cost of the disposable supplies.13Animal models have shown that direct implant of a stent reduces the degree of intimal hyperplasia in comparison with prior balloon dilatation.Between March 2003 and 2005, 1 603 patients were enrolled in a prospective registry. The criterion for inclusion into the registry was the implantation of stents for single native coronary lesions with ≥ 50% stenosis in patients with no acute myocardial infarction (MI) within the preceding 48 hours. Patients with a highly calcified lesion, total occlusion, or a lesion in a saphenous graft were excluded from the study. The decision whether or not to pre-dilate was based on the attitudes of the operators. The mean age of participants was 55.96 ± 10.50 years (range: 25–88).In this study, 857 patients (53.5%) were treated with stents without pre-dilatation (direct stenting), whereas 746 (46.5%) underwent stenting after balloon pre-dilatation. Baseline, clinical, angiographic and procedural characteristics, and in-hospital outcomes were obtained by research physicians and entered into a computerised database by computer operators. Finally, 88% of patients agreed to participate in follow-up programmes.Clinical outcomes, most importantly, major adverse cardiac events (MACE) including cardiac death, non-fatal MI and target-vessel revascularisation [bypass surgery or repeated percutaneous coronary intervention (PCI)] were obtained by cardiologists in clinics at one, five and nine months post operation and once a year thereafter, or by formal telephone interviews, and recorded on data sheets, which were later entered into the computerised database.This study was approved by the Tehran Heart Centre Ethics Committee. Informed consent was obtained from all patients before enrolment into this study.All angioplasty procedures were done with a number 6 or 7 French guiding catheter and a femoral approach. Patients received 600 mg of clopidogrel and 325 mg of aspirin before, and 7 500–10 000 IU of heparin at the start of the procedure. The femoral sheath was removed after normalisation (< 40 sec) of the partial thromboplastin time. Patients assigned to stenting after pre-dilatation (PS group) received one or more dilatations, followed by stent implantation to achieve a residual stenosis of less than 20%. The procedure was terminated if the residual stenosis was below 20% and no visible dissection with compromised flow was present.In patients assigned to direct stent implantation (DS group), the target lesion was crossed with an appropriate stent without pre-dilatation. If crossing of the target lesion with the stent delivery system did not succeed, the patient was reallocated to the PS group. After stent placement, ticlopidine (250 mg twice daily) or clopidogrel (75 mg once daily) was given routinely for four weeks with bare metal stents and for six to 12 months with drug-eluting stents. Aspirin was given indefinitely to all patients.Angiographic findings such as vessel dimensions, pre- and post-procedural stenoses, lesion length and thrombolysis in myocardial infarction (TIMI) flow grade were determined by visual estimation using the guiding catheter as a reference object for calibration. The angiographic characteristics were also further analysed by an independent interventional cardiologist not involved in the procedure and checked for inter-observer agreement.Angina symptoms were defined according to the classification of the Canadian Cardiovascular Society.17Angiographic success was defined as residual stenosis of less than 20% plus normal TIMI 3 flow. Procedural success was defined as angiographic success without major complications  during hospitalisation. Major adverse cardiac events (MACE) were defined as the presence of cardiac death, non-fatal MI, or target-vessel revascularisation (TVR) during the follow-up period. Target vessel revascularisation was defined as ischaemia-driven repeat percutaneous intervention or bypass surgery of the target vessel. Target-lesion revascularisation (TLR) was defined as ischaemia-driven repeat percutaneous intervention of the target lesion or bypass surgery of the target vessel.19The primary endpoint was to compare the occurrence of MACE between both groups during follow-up. As a secondary aim, we compared the rates of dissection and rising of cardiac enzymes  after stenting with both strategies, and determined their independent predictors.t-test was used for comparison of continuous variables. Multivariate Cox proportional-hazards models using stepwise selection were developed for comparison of the rates of MACE and its constructing components  in follow-up. Event-free survival curves were drawn using the Kaplan-Meier method. The log-rank test was used to test for differences between survivals. The multivariate logistic regression model was constructed to determine the independent predictors of dissection and non-Q-wave MI in both groups.Statistical testing was performed by the chi-square test or Fisher’s exact test (two-tailed) for categorical variables. The Student’s p < 0.1 in univariate comparisons between the groups. Univariate analyses were performed with SPSS software version 13. Multivariate analyses were conducted with SAS software version 9.1. The variables entered into the multivariate analyses included age, prior MI, stable angina, reference vessel diameter < 3 mm, lesion length, pre-procedural stenosis, type C, proximal and bifurcated lesions, angulated segments, drug-eluting stents, dissection and direct stenting.Results are presented as adjusted odds ratios with 95% confidence intervals (CI). The covariates for both Cox proportional hazard and multivariate logistic models were selected variables that had p = 0.01), while more patients in this group had presented with stable angina .As shown in p < 0.001). Except for the higher prevalence of proximal lesions in the DS group, there were no further significant differences in the location of lesions in these two populations.Lesion characteristics are shown in p = 0.9).In 27 patients, the stents could not pass the lesion directly. These patients were allocated to the PS group without any stent dislodgement or other complications. Stent diameter and length ranges were 2.5–4 mm and 8–39 mm. In the DS group, stents were shorter with a larger diameter, and drug-eluting stents were less frequently used , 3. The p < 0.001). In multivariate analysis, direct stenting was associated with a lower risk of dissection  after adjusting for significant covariates that are marked in p = 0.017), type C lesions , angulated segments  and lesion length .Despite similar angiographic success rates in both groups, the procedural success rate was significantly higher in the DS group p = 0.008). However, stenting technique was not an independent predictor for the occurrence of non-Q-wave MI .In each group, one patient (0.1%) developed unstable angina and one (0.1%) developed Q-wave MI in hospital. More patients in the PS group developed non-Q-wave MI in hospital p = 0.96).The mean follow-up times available for the patients who successfully survived the hospital period were 11.3 ± 4.7 months for the DS and 11.1 ± 4.9 months for the PS groups. There was no difference in MACE between the two groups with univariate analysis. After adjusting for selective variables that had significant differences between the groups, there was still no difference in the occurrence of MACE between the groups. However, reference vessel diameter (RVD) < 3 mm was found to be an independent predictor for MACE in the total population. The long-term adjusted hazard ratios for MACE and its constructing components in the DS versus PS groups are displayed in Table 4. MACE-free survival rates were also similar in both groups at 16 months’ follow-up  were independent predictors for in-hospital MI, and stenting techniques did not have an independent role for the occurrence of this event.Many studies have similarly shown no difference in the rate of non-Q-wave MI between the patients treated with these two techniques.34With the multivariate logistic regression model, we found that direct stenting as an independent risk factor was associated with a lower incidence of dissections compared to stenting after pre-dilatation. This finding is consistent with reports in previous studies. The incidence of dissections after pre-dilatation has ranged from nine to 45% in previous studies.It may be worthwhile noting the results of a meta-analysis of 10 randomised trials, which showed that DS compared with PS in selected coronary lesions was safe, optimised the use of equipment, and might enhance the early results of coronary intervention, while warranting similar late clinical outcomes.39There were several limitations to our study. Firstly, follow-up information was not available in 12% of the patient cohort. However, we have no reason to believe that this would have affected one group more than the other, and would not therefore alter the conclusions in any significant way. Secondly, the study was not randomised and this may have jeopardised the conclusions to some extent. However, we adjusted the outcome data for differences in baseline characteristics, adding further credence to the conclusions.This study demonstrated that patients undergoing direct stenting in the real world had at least similar long-term outcomes to patients treated with stenting after pre-dilatation. Regarding acute procedural success and in-hospital events, our data showed lower rates of dissection and non-Q-wave MI in patients treated with the direct-stenting strategy. However, after statistical adjustments, direct stenting was only accompanied by reduced risk of dissection.


Understanding how stem cells interact with cardiomyocytes is crucial for cell-based therapies to restore the cardiomyocyte loss that occurs during myocardial infarction and other cardiac diseases. It has been thought that functional myocardial repair and regeneration could be regulated by stem cell-cardiomyocyte contact. However, because various contact modes  occur randomly in a conventional coculture system, the particular regulation corresponding to a specific contact mode could not be analyzed. In this study, we used laser-patterned biochips to define cell-cell contact modes for systematic study of contact-mediated cellular interactions at the single-cell level. The results showed that the biochip design allows defined stem cell-cardiomyocyte contact-mode formation, which can be used to determine specific cellular interactions, including electrical coupling, mechanical coupling, and mitochondria transfer. The biochips will help us gain knowledge of contact-mediated interactions between stem cells and cardiomyocytes, which are fundamental for formulating a strategy to achieve stem cell-based cardiac tissue regeneration. Cardiovascular disease, a pervasive clinical problem afflicting more than five million Americans, has an annual mortality rate of more than 20% The structural and functional integrations between cells are closely related to their particular contact-modes, including junction formation, tunneling nanotube connection, and cell fusion. In junction-formation mode, junctional proteins  are distributed at the contact area between stem cells and cardiomyocytes: Connexins play an important role in electrical coupling, and cadherins do so for mechanical coupling To address this issue, we designed laser-patterned biochips to allow only one contact mode to form between stem cells and cardiomyocytes to systematically study their intercellular interactions. In our biochips, two types of microenvironment (contact-promotive and contact-preventive microwells) were created by lithographic microfabrication methods. Individual stem cells and cardiomyocytes were laser-patterned into the microwells using the laser-guided cell micropatterning technique (LGCM) All experiments that involved animal use were performed in compliance with relevant laws and institutional guidelines. These experiments were approved by Clemson University's Institutional Animal Care and Use Committee through protocol AUP2010-032.Neonatal cardiomyocytes were isolated from three-day neonatal rats using a two-day protocol and cultured using high-glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 20% fetal bovine serum (FBS) and 1% penicillin streptomycin The basic design concept was to create a microwell with defined geometrical restrictions that contained one rMSC and one cardiomyocyte. We produced two types of microwell: contact-promotive and contact-preventive. For each biochip, identical 9×9 microwells were created by attaching an elastomeric membrane with through holes onto a glass coverslip. The distance between two neighboring microwells was 200 µm. The microwell that promoted cellular contact was designed in a rectangular shape (50 µm long and 25 µm wide) that allowed the confined cell bodies (a single rMSC and a single cardiomyocyte) to form physical contact. The microwell that prevented cellular contact was designed in a dumbbell shape, with two circles (30 µm in diameter) connected by one channel (30 µm long and 15 µm wide). The curvature between the circular shape and the relatively narrow channel deterred formation of broad contact between a cell on one side of the channel and a cell on the other side. The microwell depth was 40 µm to restrict the cell bodies inside the microwells. Elastomeric membranes were microfabricated with polydimethylsiloxane (PDMS) using the standard procedure of photolithography and soft-lithography. The biochip assembly, treatment, and reuse are described in our previous publication Consecutive placement of single rMSCs and cardiomyocytes into the microwells on the biochip is critical to this research. The high spatiotemporal resolution required in multiple-cell-type positioning was achieved using LGCM, which is thoroughly described in our previous publication 2, and the culture medium was changed every two days.To create heterotypic cell pairs, cardiomyocytes were first laser-patterned into the left side of the microwells on the biochip, one in each microwell, and then rMSCs were patterned into the right side of the microwells, each of which already contained a cardiomyocyte, shown in On Day 5 (four days after laser-patterning), immunostaining was performed on the biochips. The cells were fixed, permeabilized, and blocked in 4% paraformaldehyde (10 min), 0.1% Triton X-100 (15 min), and 2% bovine serum albumin (BSA) with 4% donkey or goat serum. Next, the cells were labeled with the primary antibodies , rabbit anti-connexin 43 , mouse anti-N-cadherin , and mouse anti-CD105 ) at 4°C overnight. Excess primary antibodies were removed by a triple wash in PBS, and cells were stained with secondary antibodies  and Oregon Green 488 goat-anti-rabbit ) at room temperature for 2 hrs. After three washes with PBS, the slides were prepared with ProLong antifade kit mounting medium (Invitrogen Inc.) including cell-nuclei-staining DAPI. The DAPI-labeled nuclei can be used to identify the cell position in the microwells. The nonspecific control groups (one group without primary antibodies and one group without secondary antibodies) were conducted simultaneously with the experimental groups. Immunostained cells were imaged using a confocal fluorescent microscope  equipped with a high-sensitivity quantitative monochrome camera  and NIS-Elements imaging software (Nikon).In cell fusion studies, rMSCs and cardiomyocytes were labeled by DiI and DiO , respectively, before the laser-patterning procedure. The serum culture medium was removed from the rMSC culture flask. The rMSCs were washed with warm PBS solution and then dissociated with 0.25% Trypsin-EDTA. The cells were collected in a 15 mL tube with serum culture medium and centrifuged into a pellet. The cells were resuspended by 2 mL serum-free medium . Then, 10 µL DiI solution was added to the tube, and the cells were incubated in the dark at 37°C for 30 min. The cells were centrifuged into a pellet to remove the DiI solution and washed three times with serum-free medium. Then fresh serum culture medium was added to the tube for laser-patterning. Primary isolated cardiomyocytes were labeled with DiO using, except for the trypsin treatment, the protocol for rMSCs DiI-labeling.To visualize mitochondria transfer from rMSCs to cardiomyocytes, the mitochondria of rMSCs were live-labeled by MitoTracker . The lyophilized MitoTracker was dissolved in high-quality anhydrous dimethylsulfoxide (DMSO) to a final concentration of 1 mM as the stock solution. The labeling protocol was the same as that for cell-membrane labeling except for the use of a final MitoTracker concentration of 400 nM in serum-free medium.The occurrence of each contact mode was statistically analyzed according to our immunostaining results. First, each biochip was observed under bright field, and microwells containing round-shape cells, which were considered to be dead cells, were not used in the confocal examination. The percentage of occurrence of each contact mode was calculated by equation below  for one The biochips with heterotypic cells were created by laser-patterning rMSCs and cardiomyocytes into the contact-promotive and -preventive microwells, shown in Junction formation between contacted cells was determined through imaging data on junctional protein expression: When the fluorescence image of junctional protein expression featured a thin line along the border of two cells in contact rather than a diffusive distribution, junction formation was counted. Two types of junctional proteins, connexin 43 and N-cadherin, were examined because of their importance to electrical and mechanical coupling between cardiac cells. For the heterotypic cell pairs, 84±3.9% were observed with connexin 43 expression, among which 28±6.5% exhibited junction formation, as shown in In general, cell fusion is a natural process present from the time that a spermatozoid fuses with an ovule, and it is closely related to tissue formation, viral infection, and immune response In addition to full cell fusion, we observed partial cell fusion, in which two connected cells exchanged membrane and organelle components at their contact area Long, thin connections have been characterized in cultured PC12 cells The membrane protrusion extending from rMSCs was highlighted by staining rMSCs with specific membrane marker CD105, as shown in By tracking labeled mitochondria from rMSCs, we observed that mitochondria (red) were transferred from rMSCs to distant cardiomyocytes through tunneling nanotubes, as shown in Using our biochips, we isolated different contact modes between MSCs and cardiomyocytes at the single-cell level and investigated MSC-cardiomyocyte interactions through junctional coupling, cell fusion, partial cell fusion, and nanotubular connections. Although directly extrapolating the results to actual tissues is unfeasible, these data demonstrate that the single-cell models, each with a defined contact mode, are significant in providing basic interactional data at the molecular level, such as the direction of mitochondria transfer and the relationship between the molecular transfer rate and the extent of cell fusion. These data cannot be obtained systematically under a complicated cell-cell interactional environment. Our results suggest that 1) cell fusion, observed in microwell studies between stem cells and cardiomyocytes, may be an additional mechanism by which grafted cells can improve the infarcted myocardium; and 2) partial cell fusion and tunneling nanotubes can facilitate transfer of rMSC mitochondria to cardiomyocytes. Mitochondrial transfer from stem cells has been reported to rescue aerobic respiration in mammalian cells and may also promote cardiomyocyte reprogramming back to a progenitor-like state.in-vitro artifact cannot be ruled out Among different culture models, means of cell fusion have varied widely, and therefore extensive cell fusion as an Partial cell fusion is characterized as fusion of the heterocellular plasma membranes and resulted in direct cytoplasmic contact and organelle transfer between the cells. In vivo partial cell fusion has been confirmed in the border zone of a rabbit myocardial infarction, where close contact between cardiac fibroblasts and dedifferentiated cardiomyocytes is accompanied by disruption of the basal lamina Tunneling nanotubes, a novel biological phenomenon in cell-to-cell communication over long distance, allow for transfer of cargo vesicles, mitochondria, and small molecules such as calcium 2+ transport and storage, including Ryanodine Receptor I or II (RyR1 or RyR2), calreticulin, and calsequestrin 2+ concentration. According to the study on Zebrafish heart regeneration, it has been proposed that heart regeneration can be mediated by cardiomyocyte dedifferentiation and proliferation We found that mitochondria from rMSCs can be transferred into cardiomyocyte cytoplasm either through rMSC-origin nanotubes or cardiomyocyte-origin filopodia. The mitochondria from rMSCs accumulated around the cardiomyocyte nucleus after transfer, which may modulate transcription-factor activities in cardiomyocytes through mitochondria-to-nucleus retrograde signaling. Recently, it has been reported that, after being transferred into cardiomyocytes, stem-cell mitochondria may cause somatic reprogramming In our contact-promotive biochips, connexin 43 and N-cadherin expression between two interacting cells was analyzed. The diffusive distribution of these two proteins was frequently observed in our biochips, but junctional distribution remained at one-third of the entire sample. Similar research has been conducted by Dr. Bursac's group In our study, two types of biochips (contact-promotive and -preventive), which were created using laser-guided cell-micropatterning and microfabrication techniques, were used to study contact-mediated cellular interactions between rMSCs and cardiomyocytes. Four in vivo-relevant intercellular interaction modes, including junction formation, cell fusion, partial cell fusion, and tunneling nanotube, were modeled by our biochip systems. Investigation of these contact modes and functional integration of stem cells inside the heart may enhance our understanding of the mechanisms for cardiac-cell therapies.Movie S1One cell was being trapped and guided during laser-patterning procedure.(MP4)Click here for additional data file.Movie S2One cardiomyocyte connected with one rMSC through tunneling nanotube was beating within a contact-preventive microwell.(MP4)Click here for additional data file.Figure S1The cells in the microwells were characterized to demonstrate that the geometric restriction within the microwells did not affect the expression of these cell-type specific markers. The cardiomyocytes were characterized by  myosin heave chain and  cardiac troponin T, and rMSCs were characterized by  CD105 and  CD90.(TIF)Click here for additional data file.Figure S2(A) noncontact cell pair in a contact-promotive microwell , (B) diffusive connexin 43 distribution on noncontact cell pair in a contact-preventive microwell,  cell fusion in a circle-shape microwell with double-nuclei, mixed labels, and membrane reorganization.(TIF)Click here for additional data file.


A peripheral nerve stimulus can enhance or suppress the evoked response to transcranial magnetic stimulation (TMS) depending on the latency of the preceding peripheral nerve stimulation (PNS) pulse. Similarly, somatosensory afference from the passively moving limb can transiently alter corticomotor excitability, in a phase-dependent manner. The repeated association of PNS with TMS is known to modulate corticomotor excitability; however, it is unknown whether repeated passive-movement associative stimulation (MAS) has similar effects.In a proof-of-principle study, using a cross-over design, seven healthy subjects received in separate sessions: (1) TMS  with muscle at rest; (2) TMS paired with cyclic passive movement during extension cyclic passive movement , with the intervention order randomly assigned. Normality was tested using the Kolmogorov-Smirnov test, then compared to pre-intervention baseline using repeated measures ANOVA with a Dunnet multiple comparisons test.2 = 0.6665, P < 0.0001), which was not evident with TMS alone . Post-intervention excitability reduction, only present with MAS intervention, remained for 20min .MAS led to a progressive and significant decrease in the motor evoked potential (MEP) amplitude over the intervention (RThe association of somatosensory afference from the moving limb with TMS over primary motor cortex in healthy subjects can be used to modulate corticomotor excitability, and may have therapeutic implications. A single transcutaneous electrical peripheral nerve stimulus (PNS) generates an afferent volley that can modify the excitability of sensorimotor cortex as assessed by a change in somatosensory evoked potential (SSEP) amplitude following the PNS , or motoSeven right-handed healthy participants  volunteered after giving their written informed consent to the study approved by the Committee for Clinical Investigations at Beth Israel Deaconess Medical Center, Boston, MA and the MIT-COUHES . Participants attended on two occasions separated by 1–7 days, in a within-subjects cross-over study design, with one of two interventions: (1) rTMS at rest and (2) rTMS delivered during passive limb movement. Each intervention (outlined below) was randomly assigned to the first or second visit.Each subject was seated with a robotic device on the right side and secured at the hand, wrist, and above the elbow. Details of the robotic wrist device used have been previously reported . The worThe weight of the wrist was supported by the robotic wrist device. During the movement intervention, the robot moved the wrist in a cyclic pattern of extension and flexion through a range of 90° at a rate of 1Hz. The velocity profile was bell-shaped, peaking as the wrist passed through mid-range flexion and extension  activity was recorded from electrodes positioned over the muscle belly of the right flexor carpi radialis (FCR) muscle. EMG signals were amplified (×1000) and band-pass filtered between 10 and 1000 Hz, before being digitized at 2000 Hz for 100 ms following each stimulation, using a Powerlab 8/30 acquisition and analysis system (ADInstruments).A lycra cap was positioned over the head, with the vertex marked by measuring the mid-point intersection of the nasion-inion and inter-aural lines. Stimulus sites were then marked on the cap using the vertex as a reference point, in 1-cm steps in the coronal and sagittal planes, over the region of the left primary motor cortex. TMS was delivered using a MAGSTIM RAPID (Magstim Corporation) stimulator with a 5 cm diameter figure-of-eight coil, held tangential to the skull and aligned in the para-sagittal plane; the handle was rotated 45° laterally and the coil junction held over the optimal site of stimulation. The optimal site of stimulation for FCR, determined from initial exploration, was defined as the site with the largest MEPs for a given supra-threshold stimulus intensity, and used throughout the experiment. RMT (resting motor threshold) was determined with the wrist supported in the mid-range position, using four stimuli for each 1% increment of stimulator output intensity; it was defined as the minimum TMS intensity that evoked an MEP of at least 50μV amplitude in three of the four stimuli and then again confirmed with decreasing stimulus intensity. Ten single pulses at 120% RMT were delivered over the optimal site for FCR stimulation at baseline and each post-intervention time-point (using the baseline stimulus intensity). For the baseline measurement, a set of 10 stimuli was performed 10 minutes prior, and immediately before the intervention to ensure baseline stability. Post-intervention measurements occurred at 0, 5, 10, 15, 20, 25, and 30 minutes.For both movement and resting interventions, 400 TMS pulses were delivered at 120% RMT and frequency of 1Hz (low frequency ≤1 Hz) ,16. At rThe peak-to-peak amplitude for each MEP was measured. The post-intervention data was grouped into three consecutive 10-minute blocks , and referred to as 0-10 min, 10-20 min and 20-30 min respectively. The mean MEP amplitude for each block was expressed as a percentage of mean pre-intervention amplitude. Group mean data at each time point were tested for normality using the Kolmogorov-Smirnov test, then compared to pre-intervention baseline using repeated measures ANOVA with a Dunnet multiple comparisons test.Change in MEP amplitude over time and trends in mean MEP amplitude with time, during the intervention conditions (stimulation only or combined stimulation and movement), were tested using linear regression analysis. Group mean data for this calculation comprised the average of each successive 10 MEPs, giving 40 data points across the 400 repetitions, each expressed relative to the mean baseline amplitude. Post-intervention changes in MEP amplitude were established by comparison between each time-point post, and the pre-intervention baseline. Group data for pre-intervention and each time-point post-intervention were tested for normality using the Kolmogorov-Smirnov test and subsequent repeated measures ANOVA, with Tukey’s post-hoc comparisons.To test the immediate effect of passive movement on MEP amplitude relative to the same stationary wrist position the mean of each successive 10 stimuli from repetition 1–30 was examined with respect to pre-intervention using repeated measures ANOVA, and the Dunnet multiple comparisons test. Data are presented as mean ± SEM. ‘Normalized’ data refers to expression as a percentage of pre-intervention baseline.A marked reduction in the MEP amplitude was observed during the MAS intervention, but not for the TMS intervention alone. It can be seen in Figure 2 = 0.6665, P < 0.0001). However, when the same stimulation was delivered with the wrist stationary in the mid-range position, no reduction in MEP amplitude was observed with 400 repetitive stimuli . Additionally, movement-related reduction in MEP amplitude was present in some subjects at the onset of cyclic movement, yet the mean amplitude for the group was non significantly reduced until the third block of 10 repetitions . However, relative to pre-intervention baseline taken at rest (0.2Hz stimulation), the amplitude of the MEP response showed a tendency of increasing to the first 10 stimuli delivered during 1Hz resting intervention .During MAS intervention  stimulation alone by delivering 400 stimuli at 120% of the RMT at rest and found no suppression in the MEP size following this intervention. Previous studies of 1Hz stimulation over human primary motor cortex at rest show a short-term reduction in CM excitability ,19. HoweOf note during the intervention, was the different onset and trajectory of MEP amplitude reduction between individuals. The variance in intervention effect time-course has been observed previously with other repetitive non-invasive stimulation protocols . We suIt is possible that the high stimulus intensity results in a net facilitatory effect similar to paired pulse techniques that switch from inhibitory to facilitatory MEP response, with increases in intensity above motor threshold. Furthermore, the number of repetitions at this intensity may be too small to finally result in a decreased excitability. The MEP amplitude fluctuates during low-frequency rTMS protocols ultimately leading to sustained inhibition, including changes in cortical facilitatory and inhibitory network activity, and intervening homeostatic mechanisms. Change in MEP amplitude is not readily observable in most protocols since the stimulus intensity is sub-threshold. Increased MEP amplitude may be a normal phenomenon in the early phase of 1Hz inhibitory protocols, or a result of the relatively high-intensity stimulation used in the current protocol. Increased stimulus intensity from sub-threshold to supra-threshold 80–115% RMT) has previously been shown to augment the post-intervention excitability change (a greater reduction in MEP amplitude), although it is not known if this occurs early in the stimulation period, or if it occurs at intensities above 115% such as in the present study . While b% RMT hasPeripheral limb movement forms a large basis for motor rehabilitation, and repetitive passive movement can lead to a temporary reduction in spasticity and orthopedic benefit -31. In fThe peripheral afference leads to a cumulative and lasting effect that could occur at spinal and/or supraspinal levels. Furthermore, the inhibitory phase of cyclic passive movement need not necessarily be complementary to an inhibitory rTMS protocol, and it may cancel the inhibitory effect, remain unchanged, or lead to an excitatory effect. For example, we know that decreased afferent activity (associated with increased MEP amplitude) also appears to increase the efficacy of low frequency rTMS, as shown during a study of peripheral nerve block . MoreoveOur findings raise the question of whether this type of associative paradigm could be used to increase cortical excitability. In principle, the association of the facilitatory phase of movement with TMS repeatedly, may increase cortical excitability over time consistent with long-term potentiation, as currently is well demonstrated with PAS ,36, yet The association of somatosensory afference from the passively moving limb with TMS over primary motor cortex in healthy human subjects can be used to modulate corticomotor excitability, outlasting the intervention period. Parameters of TMS stimulation  and movement  require further investigation for the development of the optimal effect. It remains to be determined whether our findings could be applied to the treatment of disorders involving increased muscle tone such as spasticity and dystonia.TMS: Transcranial magnetic stimulation; PNS: Peripheral nerve stimulation; MAS: Movement associative stimulation; RMT: Resting motor threshold; MEP: Motor evoked potential; SSEP: Somatosensory evoked potential; ISI: Inter-stimulus interval; EMG: Surface electromyographic; FCR: Flexor carpi radialis; rTMS: Repetitive transcranial magnetic stimulation; CM: Corticomotor.The authors declare that they have no competing interests.DJE: experimental work, concept, study design and manuscript preparation. APL: concept, data interpretation. HIK: concept, experimental work, technical set-up, data analysis. LDP: data analysis, experimental work. GWT: data analysis/interpretation, manuscript preparation. ADT: data collection, data interpretation. AHM: data interpretation, manuscript development and preparation. FLM: concept, data interpretation and manuscript preparation. All authors wrote, read and approved the final manuscript.


Targeted therapies in renal cell carcinoma can have different effects on primary and metastatic tumors. To pave the way for predictive biomarker development, we assessed differences in expression of targets of currently approved drugs in matched primary and metastatic specimens from 34 patients.Four cores from each site were embedded in tissue microarray blocks. Expression of B-Raf, C-Raf, cKIT, FGF-R1, HIF-2α, mTOR, PDGF-Rβ, VEGF-R1, VEGF-R2, VEGF-R3, VEGF, VEGF-B, VEGF-C, VEGF-D, MEK1, and ERK1/2 was studied using a quantitative immunofluorescence method.No significant differences were observed in global expression levels in primary and metastatic renal cell carcinoma tumors, with the exception of MEK, which had higher expression in metastatic than primary specimens. Similarly, more ki67 positive cells were seen in metastatic specimens. Correlations between marker expression in primary and metastatic specimens were variable, with the lowest correlation seen for FGF-R1 and VEGF-D. There were no significant differences in the degree of heterogeneity in primary versus metastatic tumors.Expression of most of the studied markers was similar in primary and metastatic renal cell carcinoma tumors, suggesting that predictive biomarker testing for these markers can be conducted on either the primary or metastatic tumors for most markers. In recent years, the incidence of renal cell carcinoma (RCC) has increased from 38,000 new cases a year in 2006 to over 64,000 estimated for 2012 , suggestSurgery followed by surveillance imaging is the standard of care for RCC patients with localized disease. Fine needle aspiration or core needle biopsies are commonly employed for diagnosis of metastatic disease in the 10-50% of these patients with recurring disease. More than 20% of RCC patients present with metastatic disease without having a previously known localized primary tumor ,7.RCC is very resistant to standard chemotherapy. Despite advances in biological and immune-based therapies, treatment options for patients with unresectable or metastatic RCC (mRCC) are limited; response rates remain at about 15–44%, and five-year survival under 10% for those with distant metastases ,8-10. ImA growing understanding of the pathogenesis of clear cell RCC, the most common histologic subtype, has facilitated development of RCC-targeting therapies. The discovery of Von Hippel-Lindau (VHL) tumor-suppressor gene inactivation and subsequent hypoxia-induced factor (HIF) activation of genes and downstream pathways important to tumor progression have provided the impetus for development of new agents that target angiogenesis and proliferation pathways. A number of drugs that target the vascular endothelial growth factor (VEGF) pathway and downstream signaling molecules have been approved for mRCC. These include the small molecule tyrosine kinase inhibitors (TKIs) sunitinib, sorafenib, pazopanib, and axitinb ,13-15, tResponse of primary RCC (as opposed to metastatic deposits) to targeted therapies has not been well studied, however highly sensitive cases are thought to be relatively uncommon. Several groups have reported that pre-surgical targeted therapy can be effective in mRCC patients who have not had their primary tumors removed -24. HoweAt present no predictive biomarkers have been developed to identify patients whose tumors are more likely to respond to any of the currently available therapies. In addition, biomarkers predictive of discordant response between primary and metastatic tumors are lacking. It is therefore necessary to establish patterns of expression of drug targets in tumors in order to attempt to develop predictive tissue based biomarker assays. Given that these drugs exert their effects on the proteome, protein-level predictive biomarkers are a logical place to start. Patterns of expression of drug targets in primary and metastatic RCC tumors have not been previously well demonstrated. Youssif et al. studied 25 matched primary and metastatic samples for correlations between mTOR pathway targets  and found a strong correlation for phospho-mTOR . Here weThirty-four patients with archived matched primary and metastatic RCC tumors were identified. Resections were done between 1972 and 2011. TMAs were constructed using cores measuring 0.6 mm in diameter, spaced 0.8 mm apart. Tumors from each of the primary and metastatic tumors were represented by four cores from different areas of the specimen  and placed in two TMA blocks. Specimens and clinical information were collected with approval of a Yale University Institutional Review Board.Pairs of slides (containing two cores from different areas of each matching primary and metastatic tumor per patient) were stained individually for the target markers; B-Raf, C-Raf, cKIT, FGF-R1 (fibroblast growth factor receptor-1), HIF-2α (hypoxia induced factor-2α), mTOR  , PDGF-Rβ (platelet-derived growth factor receptor-β), VEGF-R1, VEGF-R2, VEGF-R3, VEGF, VEGF-B, VEGF-C, VEGF-D, MEK1 (mitogen activated protein kinase-1), and ERK1/2 . TMAs were also stained for ki67 as a marker of proliferation. Fluorescent staining for our Automated, Quantitative Analysis (AQUA) method was performed as previously described ,28. BrieImages were acquired and analyzed using previously described algorithms . BrieflyStatview and JMP 5.0 software were used . AQUA scores for replicate tumor cores were averaged. Associations between continuous AQUA scores of the target and clinical and pathological parameters were assessed using ANOVA. Correlations between the AQUA scores of matched primary and metastatic histospots were calculated by the log rank method. Intratumor heterogeneity was assessed by Pearson linear regression.Patient characteristics are summarized in Table Each lot of antibodies was subjected to immunoblotting to verify presence of a single dominant band of the appropriate size (not shown). We note that a number of antibodies to VEGF-R2 are commercially available, and a recent publication demonstrated higher specificity of the 55B11 antibody  than the A-3 antibody  . HoweverFigure Global distribution of scores in primary and metastatic spots was not significantly different for any of the markers with the exception of MEK1. The mean AQUA scores and the differences between primary and metastatic tumors by paired t-tests are shown in Table Given that archival tissue is often available from either the primary or the metastatic site, but not both, we determined the associations between marker expression in paired primary and metastatic samples using the Pearson correlation test, as displayed in Table To determine whether there were differences in intra-tumor heterogeneity in primary and metastatic specimens, we used the four measurements from each tumor. Each core is represented by a vector of measurements of all markers, denoted as the core vector. For each patient we computed the median core vector and measured its L1 distances from the corresponding four core vectors. We defined the composite median absolute deviation (MAD) as the median of these four L1 distances, and used it as a proxy for estimating intra-tumor heterogeneity. For each patient, the composite MAD is computed separately for his/her primary and metastatic tumors. Using the Wilcoxon paired, two-sided signed rank test, we found no significant differences in heterogeneity between primary and metastatic tumors (P = 0.38), as shown in Figure Molecular targeted therapies that inhibit members of the VEGF pathway and mTOR are now widely used for the treatment of metastatic RCC. At present, no predictive biomarkers have been established for this class of drugs. Given that these agents inhibit this pathway at the protein level, target protein expression might be associated with response to therapy. Many metastatic RCC patients have either primary or metastatic tumor tissue (but not both) available for analysis, and our purpose was therefore to determine differences in expression of these drug targets in matched primary and metastatic specimens.Target expression levels were not globally different between primary and metastatic tumors, with the exception of MEK1, which was higher in metastatic specimens. Given that MEK1 is a key component of the major intracellular proliferation signal transduction pathway, we studied ki67 expression in primary and metastatic samples and found that the percentage of ki67 positive cells was also significantly higher in the metastases. This is consistent with the commonly seen clinical picture in which the primary tumor grows over years, yet the course for metastatic disease patients is often much shorter due to rapid metastatic tumor growth.Our data indicate that our ability to predict expression in primary specimens based on measurements from a corresponding metastasis and vice-versa is marker dependent. The intra-patient correlations were variable across markers, with the worst correlation shown for VEGF-D and FGF-R1, while other makers such as C-Raf, VEGF-R2 and cKIT demonstrated excellent correlations between levels in primary and metastatic specimens. These findings are consistent with the only other similar published study of which we are aware in which mTOR pathway members were assessed for concordance between primary and metastatic sites using standard immunohistochemistry (IHC) . Levels To date, none of the approved drugs for mRCC has an associated companion diagnostic biomarker test. A number of initial attempts have been made at developing predictive biomarkers that are primarily centered around VHL pathway markers, such as VHL mutations, HIF levels, VEGF isoforms and VEGF receptor levels. Phase II trials of axitinib or temsirolimus revealed no association between VHL mutational status and response to therapy ,31. HighSmall studies have suggested a potential role for VEGF and soluble forms of the VEGF receptors as predictors of response to VEGF pathway targeting therapies and cytokine therapies. Sabatino et al.  measuredClinical observations of discordance in response of primary and metastatic tumors suggest possible differences in biology. Alternatively, differences in response could be due to variable tumor microenvironment in the primary and metastatic sites. Primary RCC tumors do not seem to respond as well as RCC metastatic sites to VEGF pathway targeted therapies . StudiesBiomarker studies related to evolving biomarkers and experimental drugs are being done by our group and others. The clinical relevance of HIF-1α and HIF-2α are being studied as the hypoxia induced pathway is regularly aberrant in RCC. HIF-1α has been shown to be expressed in most RCC tumors while HIF-2α is relatively absent in early tumors, but is highly expressed in metastatic tumors ,41. B7-HThe tumors studied here exhibited variable intratumor heterogeneity in the four tumor cores. The degree of heterogeneity is not significantly different in primary and metastatic samples. Although our study evaluates protein expression, recent DNA sequencing studies have shown intratumor heterogeneity in primary renal cell carcinoma. The majority of somatic mutations (over 60%) were not present throughout the tumor in the 4 samples examined. Moreover, DNA signatures of both good and poor prognosis were detected in different regions of the same tumor. The authors suggest that intratumor heterogeneity is the cause of lack of reproducible predictive biomarkers . UtiliziOur studies showed good concordance between primary and metastatic samples for most of the markers studied. The biomarkers with the least concordance were FGF-R1 and VEGF-D. The discordance in levels of VEGF-D might be due to the fact that this is a secreted protein, and levels of FGF-R1 might be more influenced by the tumor microenvironment than the other markers studied. Conversely, other biomarkers showed excellent concordance between primary and metastatic samples. As predictive biomarkers are developed, careful studies are needed to define concordance versus discordance for individual biomarkers in order to determine whether primary specimen measurements can be used as surrogates for metastatic specimens and vice-versa.R.L. Camp is a co-founder, stockholder and consultant for a company called HistoRx that has licensed the technology for automated tissue analysis used in this study.SA drafted the manuscript, acquired the data, and participated in the analysis of data. JS helped with the acquisition of data and the analysis. AA performed the pathology review of specimens. FP and YK conducted statistical and computational analysis for this study. RC designed the tissue microarrays and performed pathology review. HK designed and coordinated the study and participated in the drafting of this manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6890/13/3/prepubList of Antibodies.Click here for file