Systematic Bias in Genomic Classification Due to Contaminating Non-neoplastic Tissue in Breast Tumor Samples

Abstract Background Genomic tests are available to predict breast cancer recurrence and to guide clinical decision making. These predictors provide recurrence risk scores along with a measure of uncertainty, usually a confidence interval. The confidence interval conveys random error and not systematic bias. Standard tumor sampling methods make this problematic, as it is common to have a substantial proportion (typically 30-50%) of a tumor sample comprised of histologically benign tissue. This "normal" tissue could represent a source of non-random error or systematic bias in genomic classification. Methods To assess the performance characteristics of genomic classification to systematic error from normal contamination, we collected 55 tumor samples and paired tumor-adjacent normal tissue. Using genomic signatures from the tumor and paired normal, we evaluated how increasing normal contamination altered recurrence risk scores for various genomic predictors. Results Simulations of normal tissue contamination caused misclassification of tumors in all predictors evaluated, but different breast cancer predictors showed different types of vulnerability to normal tissue bias. While two predictors had unpredictable direction of bias (either higher or lower risk of relapse resulted from normal contamination), one signature showed predictable direction of normal tissue effects. Due to this predictable direction of effect, this signature (the PAM50) was adjusted for normal tissue contamination and these corrections improved sensitivity and negative predictive value. For all three assays quality control standards and/or appropriate bias adjustment strategies can be used to improve assay reliability. Conclusions Normal tissue sampled concurrently with tumor is an important source of bias in breast genomic predictors. All genomic predictors show some sensitivity to normal tissue contamination and ideal strategies for mitigating this bias vary depending upon the particular genes and computational methods used in the predictor

Step-Wise Loss of Bacterial Flagellar Torsion Confers Progressive Phagocytic Evasion

Phagocytosis of bacteria by innate immune cells is a primary method of bacterial clearance during infection. However, the mechanisms by which the host cell recognizes bacteria and consequentially initiates phagocytosis are largely unclear. Previous studies of the bacterium Pseudomonas aeruginosa have indicated that bacterial flagella and flagellar motility play an important role in colonization of the host and, importantly, that loss of flagellar motility enables phagocytic evasion. Here we use molecular, cellular, and genetic methods to provide the first formal evidence that phagocytic cells recognize bacterial motility rather than flagella and initiate phagocytosis in response to this motility. We demonstrate that deletion of genes coding for the flagellar stator complex, which results in non-swimming bacteria that retain an initial flagellar structure, confers resistance to phagocytic binding and ingestion in several species of the gamma proteobacterial group of Gram-negative bacteria, indicative of a shared strategy for phagocytic evasion. Furthermore, we show for the first time that susceptibility to phagocytosis in swimming bacteria is proportional to mot gene function and, consequently, flagellar rotation since complementary genetically- and biochemically-modulated incremental decreases in flagellar motility result in corresponding and proportional phagocytic evasion. These findings identify that phagocytic cells respond to flagellar movement, which represents a novel mechanism for non-opsonized phagocytic recognition of pathogenic bacteria

RNA-Seq Differentiates Tumour and Host mRNA Expression Changes Induced by Treatment of Human Tumour Xenografts with the VEGFR Tyrosine Kinase Inhibitor Cediranib.

Pre-clinical models of tumour biology often rely on propagating human tumour cells in a mouse. In order to gain insight into the alignment of these models to human disease segments or investigate the effects of different therapeutics, approaches such as PCR or array based expression profiling are often employed despite suffering from biased transcript coverage, and a requirement for specialist experimental protocols to separate tumour and host signals. Here, we describe a computational strategy to profile transcript expression in both the tumour and host compartments of pre-clinical xenograft models from the same RNA sample using RNA-Seq. Key to this strategy is a species-specific mapping approach that removes the need for manipulation of the RNA population, customised sequencing protocols, or prior knowledge of the species component ratio. The method demonstrates comparable performance to species-specific RT-qPCR and a standard microarray platform, and allowed us to quantify gene expression changes in both the tumour and host tissue following treatment with cediranib, a potent vascular endothelial growth factor receptor tyrosine kinase inhibitor, including the reduction of multiple murine transcripts associated with endothelium or vessels, and an increase in genes associated with the inflammatory response in response to cediranib. In the human compartment, we observed a robust induction of hypoxia genes and a reduction in cell cycle associated transcripts. In conclusion, the study establishes that RNA-Seq can be applied to pre-clinical models to gain deeper understanding of model characteristics and compound mechanism of action, and to identify both tumour and host biomarkers

A systematic review of the diagnostic accuracy of physical examination for the detection of cirrhosis

BACKGROUND: We conducted a review of the diagnostic accuracy of clinical examination for the diagnosis of cirrhosis. The objectives were: to identify studies assessing the accuracy of clinical examination in the detection of cirrhosis; to summarize the diagnostic accuracy of reported physical examination findings; and to define the effects of study characteristics on estimates of diagnostic accuracy. METHODS: Studies were identified through electronic literature search of MEDLINE (1966 to 2000), search of bibliographic references, and contact with authors. Studies that evaluated indicants from physical examination of patients with known or suspected liver disease undergoing liver biopsy were included. Qualitative data on study characteristics were extracted. Two-by-two tables of presence or absence of physical findings for patients with and without cirrhosis were created from study data. Data for physical findings reported in each study were combined using Summary Receiver Operating Characteristic (SROC) curves or random effects modeling, as appropriate. RESULTS: Twelve studies met inclusion criteria, including a total of 1895 patients, ranging in age from 3 to 90 years. Most studies were conducted in referral populations with elevated aminotransferase levels. Ten physical signs were reported in three or more studies and ten signs in only a single study. Signs for which there was more study data were associated with high specificity (range 75–98%), but low sensitivity (range 15–68%) for histologically-proven cirrhosis. CONCLUSIONS: Physical findings are generally of low sensitivity for the diagnosis of cirrhosis, and signs with higher specificity represent decompensated disease. Most studies have been undertaken in highly selected populations

Possible Role of Horizontal Gene Transfer in the Colonization of Sea Ice by Algae

Diatoms and other algae not only survive, but thrive in sea ice. Among sea ice diatoms, all species examined so far produce ice-binding proteins (IBPs), whereas no such proteins are found in non-ice-associated diatoms, which strongly suggests that IBPs are essential for survival in ice. The restricted occurrence also raises the question of how the IBP genes were acquired. Proteins with similar sequences and ice-binding activities are produced by ice-associated bacteria, and so it has previously been speculated that the genes were acquired by horizontal transfer (HGT) from bacteria. Here we report several new IBP sequences from three types of ice algae, which together with previously determined sequences reveal a phylogeny that is completely incongruent with algal phylogeny, and that can be most easily explained by HGT. HGT is also supported by the finding that the closest matches to the algal IBP genes are all bacterial genes and that the algal IBP genes lack introns. We also describe a highly freeze-tolerant bacterium from the bottom layer of Antarctic sea ice that produces an IBP with 47% amino acid identity to a diatom IBP from the same layer, demonstrating at least an opportunity for gene transfer. Together, these results suggest that the success of diatoms and other algae in sea ice can be at least partly attributed to their acquisition of prokaryotic IBP genes

Nasopharyngeal Colonization and Invasive Disease Are Enhanced by the Cell Wall Hydrolases LytB and LytC of Streptococcus pneumoniae

Background: Streptococcus pneumoniae is a common colonizer of the human nasopharynx and one of the major pathogens causing invasive disease worldwide. Dissection of the molecular pathways responsible for colonization, invasion, and evasion of the immune system will provide new targets for antimicrobial or vaccine therapies for this common pathogen. Methodology/Principal Findings: We have constructed mutants lacking the pneumococcal cell wall hydrolases (CWHs) LytB and LytC to investigate the role of these proteins in different phases of the pneumococcal pathogenesis. Our results show that LytB and LytC are involved in the attachment of S. pneumoniae to human nasopharyngeal cells both in vitro and in vivo. The interaction of both proteins with phagocytic cells demonstrated that LytB and LytC act in concert avoiding pneumococcal phagocytosis mediated by neutrophils and alveolar macrophages. Furthermore, C3b deposition was increased on the lytC mutant confirming that LytC is involved in complement evasion. As a result, the lytC mutant showed a reduced ability to successfully cause pneumococcal pneumonia and sepsis. Bacterial mutants lacking both LytB and LytC showed a dramatically impaired attachment to nasopharyngeal cells as well as a marked degree of attenuation in a mouse model of colonization. In addition, C3b deposition and phagocytosis was more efficient for the double lytB lytC mutant and its virulence was greatly impaired in both systemic and pulmonary models of infection. Conclusions/Significance: This study confirms that the CWHs LytB and LytC of S. pneumoniae are essential virulence factor

Streptococcus pneumoniae in Biofilms Are Unable to Cause Invasive Disease Due to Altered Virulence Determinant Production

It is unclear whether Streptococcus pneumoniae in biofilms are virulent and contribute to development of invasive pneumococcal disease (IPD). Using electron microscopy we confirmed the development of mature pneumococcal biofilms in a continuous-flow-through line model and determined that biofilm formation occurred in discrete stages with mature biofilms composed primarily of dead pneumococci. Challenge of mice with equal colony forming units of biofilm and planktonic pneumococci determined that biofilm bacteria were highly attenuated for invasive disease but not nasopharyngeal colonization. Biofilm pneumococci of numerous serotypes were hyper-adhesive and bound to A549 type II pneumocytes and Detroit 562 pharyngeal epithelial cells at levels 2 to 11-fold greater than planktonic counterparts. Using genomic microarrays we examined the pneumococcal transcriptome and determined that during biofilm formation S. pneumoniae down-regulated genes involved in protein synthesis, energy production, metabolism, capsular polysaccharide (CPS) production, and virulence. We confirmed these changes by measuring CPS by ELISA and immunoblotting for the toxin pneumolysin and the bacterial adhesins phosphorylcholine (ChoP), choline-binding protein A (CbpA), and Pneumococcal serine-rich repeat protein (PsrP). We conclude that biofilm pneumococci were avirulent due to reduced CPS and pneumolysin production along with increased ChoP, which is known to bind C-reactive protein and is opsonizing. Likewise, biofilm pneumococci were hyper-adhesive due to selection for the transparent phase variant, reduced CPS, and enhanced production of PsrP, CbpA, and ChoP. These studies suggest that biofilms do not directly contribute to development of IPD and may instead confer a quiescent mode of growth during colonization