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HER2 upregulation is related with poor outcome in many tumor types. Whereas anti-HER2 treatment is the standard approach as adjuvant therapy in HER2-overexpressing breast cancer, the frequent relapses reinforce the need for alternative treatments. Here we used next-generation sequencing (NGS) to evaluate miRNAs and circRNAs in the cell-lines HB4a and C5.2, where the latter is a HER2-overexpressing clone of the former, and also from two different populations of their secreted extracellular vesicles. Whereas circRNA-levels were stable, we found at least 16 miRNAs apparently modulated by HER2-expression. The miR223-3p, miR-421 and miR-21-5p were validated in an independent cohort of 431 breast cancer patients from The Cancer Genome Atlas (TCGA). The consistent modulation of these molecules and their possible involvement in the HER2-axis makes them promising new targets to overcome HER2-activation.

Despite being one of the most studied cancer-related infections, the relationship between Helicobacter pylori infection and gastric adenocarcinoma (GC) remains, in some points, obscure. Based on a critical analysis of the available literature regarding stomach microbiota, we aimed to shed light to a possible new interpretation of the current understanding about the Helicobacter pylori-related GC carcinogenesis. We analyzed data from the literature on Helicobacter pylori and other potential carcinogenic pathogens, in both benignant conditions and gastric adenocarcinoma. Helicobacter pylori is the dominant microorganism in benignant conditions as non-complicated gastritis. In atrophic gastritis, metaplasia and, mainly, in gastric adenocarcinoma, a strong reduction in Helicobacter pylori abundance, and increased occurrence of other microorganisms is strongly demonstrated by metagenomic experiments. While causing peptic disease and keeping the stomach's high acidity, Helicobacter pylori infection avoids gastric infection by carcinogenic intestinal microbiota. Nevertheless, Helicobacter pylori persistence may also provoke an atrophic gastritis, a condition that causes its own decline, due to a microenvironment modification, including reduced acidity, resulting in Helicobacter pylori substitution by a cancer-prone microbiota. This new interpretation might result in a dramatic modification on clinical management of Helicobacter pylori-related gastric disease.

Background Gastric adenocarcinoma (GAC) is the third deadliest malignant neoplasm worldwide, mostly because of late disease diagnosis, low chemotherapy response rates, and an overall lack of tumor biology understanding. Therefore, tools for prognosis and prediction of treatment response are needed. Quantification of circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) and their expression of biomarkers has potential clinical relevance. Our aim was to evaluate CTCs and CTM and their expression of HER2 and plakoglobin in patients with nonmetastatic GAC, correlating the findings to clinicopathological data. Materials and Methods CTC enrichment was performed with isolation by size of epithelial tumor cells, and the analysis was performed with immunocytochemistry and microscopy. Two collections were made: one at diagnosis (55 samples before neoadjuvant treatment) and one after surgery and before adjuvant therapy (33 samples). Results A high detection rate of CTCs (90%) was observed at baseline. We evaluated HER2 expression in 45/55 biopsy samples and in 42/55 CTC samples, with an overlap of 36 subjects. Besides the good agreement observed for HER2 expression in primary tumors and paired CTCs for 36 cases (69.4%; κ = 0.272), the analysis of HER2 in CTCs showed higher positivity (43%) compared with primary tumors (11%); 3/5 patients with disease progression had HER2-negative primary tumors but HER2-positive CTCs. A significant CTC count drop in follow-up was seen for CTC-HER2-positive cases (4.45 to 1.0 CTCs per mL) compared with CTC-HER2-negative cases (2.6 to 1.0 CTCs per mL). The same was observed for CTC-plakoglobin-positive cases (2.9 to 1.25 CTCs per mL). Conclusion CTC analysis, including their levels, plakoglobin, and HER2 expression, appears to be a promising tool in the understanding the biology and prognosis of GAC. Implications for Practice The analysis of circulating tumor cell levels from the blood of patients with gastric adenocarcinoma, before and after neoadjuvant treatment, is useful to better understand the behavior of the disease as well as the patients more likely to respond to treatment.

Summary We present a global atlas of 4,728 metagenomic samples from mass-transit systems in 60 cities over 3 years, representing the first systematic, worldwide catalog of the urban microbial ecosystem. This atlas provides an annotated, geospatial profile of microbial strains, functional characteristics, antimicrobial resistance (AMR) markers, and genetic elements, including 10,928 viruses, 1,302 bacteria, 2 archaea, and 838,532 CRISPR arrays not found in reference databases. We identified 4,246 known species of urban microorganisms and a consistent set of 31 species found in 97% of samples that were distinct from human commensal organisms. Profiles of AMR genes varied widely in type and density across cities. Cities showed distinct microbial taxonomic signatures that were driven by climate and geographic differences. These results constitute a high-resolution global metagenomic atlas that enables discovery of organisms and genes, highlights potential public health and forensic applications, and provides a culture-independent view of AMR burden in cities., Graphical abstract, Highlights • Cities possess a consistent “core” set of non-human microbes • Urban microbiomes echo important features of cities and city-life • Antimicrobial resistance genes are widespread in cities • Cities contain many novel bacterial and viral species, This systematic, worldwide catalog of urban microbiomes represents a metagenomic atlas important for understanding the ecology, virulence, and antibiotic resistance of city-specific microbial communities.

Triple-negative breast cancer (TNBC) is an aggressive tumor with limited treatment options and poor prognosis. We applied the in vivo phage display technology to isolate peptides homing to the immunosuppressive cellular microenvironment of TNBC as a strategy for non-malignant target discovery. We identified a cyclic peptide (CSSTRESAC) that specifically binds to a vitamin D receptor, protein disulfide-isomerase A3 (PDIA3) expressed on the cell surface of tumor-associated macrophages (TAM), and targets breast cancer in syngeneic TNBC, non-TNBC xenograft, and transgenic mouse models. Systemic administration of CSSTRESAC to TNBC-bearing mice shifted the cytokine profile toward an antitumor immune response and delayed tumor growth. Moreover, CSSTRESAC enabled ligand-directed theranostic delivery to tumors and a mathematical model confirmed our experimental findings. Finally, in silico analysis showed PDIA3-expressing TAM in TNBC patients. This work uncovers a functional interplay between a cell surface vitamin D receptor in TAM and antitumor immune response that could be therapeutically exploited.

Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) Gillson-Longenbaugh Foundation National Institutes of Health (NIH/NCATS) through the NIH Common Fund, Office of Strategic Coordination (OSC) Extracellular vesicles (EVs) are key mediators of intercellular communication. Part of their biological effects can be attributed to the transfer of cargos of diverse types of RNAs, which are promising diagnostic and prognostic biomarkers. EVs found in human biofluids are a valuable source for the development of minimally invasive assays. However, the total transcriptional landscape of EVs is still largely unknown. Here we develop a new method for total transcriptome profiling of plasma-derived EVs by next generation sequencing (NGS) from limited quantities of patient-derived clinical samples, which enables the unbiased characterization of the complete RNA cargo, including both small- and long-RNAs, in a single library preparation step. This approach was applied to RNA extracted from EVs isolated by ultracentrifugation from the plasma of five healthy volunteers. Among the most abundant RNAs identified we found small RNAs such as tRNAs, miRNAs and miscellaneous RNAs, which have largely unknown functions. We also identified protein-coding and long noncoding transcripts, as well as circular RNA species that were also experimentally validated. This method enables, for the first time, the full spectrum of transcriptome data to be obtained from minute patient-derived samples, and will therefore potentially allow the identification of cell-to-cell communication mechanisms and biomarkers. AC Camargo Canc Ctr, Lab Med Genom, Sao Paulo, SP, Brazil AC Camargo Canc Ctr, Lab Computat Biol, Sao Paulo, SP, Brazil Univ Sao Paulo, Inst Biomed Sci, Dept Cell & Dev Biol, Sao Paulo, SP, Brazil Univ Fed Sao Paulo, Electron Microscopy Ctr, Sao Paulo, SP, Brazil Univ Texas MD Anderson Canc Ctr, Dept Expt Therapeut, Houston, TX 77030 USA Univ Texas MD Anderson Canc Ctr, Ctr RNA Interference & Non Coding RNAs, Houston, TX 77030 USA Univ New Mexico, Comprehens Canc Ctr, Albuquerque, NM 87131 USA Univ New Mexico, Sch Med, Div Hematol Oncol, Dept Internal Med, Albuquerque, NM 87131 USA Univ New Mexico, Sch Med, Div Mol Med, Dept Internal Med, Albuquerque, NM 87131 USA Rockefeller Univ, Lab Mol Immunol, 1230 York Ave, New York, NY 10021 USA FMUSP, Lab Neurociencias Alzira Denise Hertzog Silva LIM, Inst Psiquiatria, Sao Paulo, SP, Brazil Univ Fed Sao Paulo, Electron Microscopy Ctr, Sao Paulo, SP, Brazil FAPESP: 2011/09172-3 FAPESP: 2014/26897-0 Web of Science

Prostate cancer, the most frequent type of cancer in men, is surpassed only by lung cancer in causing cancer‐related death. Despite major progress in defining the mutational landscape of this tumor, its etiology remains obscure. The intronic long‐noncoding RNA (lncRNA) PCA3 is a specific marker of prostate cancer that acts as a trans‐dominant negative oncogene to down‐regulate the tumor suppressor gene PRUNE2 . The unusual genomic organization and sequence of PCA3 leads us to hypothesize that it was introduced into the human genome by an as‐yet undefined oncogenic virus. We further suggest viral infection‐related mechanisms as functional in PCA3 overexpression and involved in prostate cancer initiation and/or progression. This is supported by known links between viral infections and prostate cancer, and the lack of this tumor in other mammals despite its high incidence in humans. Finally, we suggest that genomic sequence‐based approaches might help us uncover the potential role of viruses in prostate cancer etiology. The intronic lncRNA Prostate Cancer Antigen 3 ( PCA3 ) is a highly specific biomarker: While only present at low levels in normal or benign hyperplastic prostate, its expression is increased up to 100‐fold in about 95% of prostatic carcinomas (Schalken et al , 2003). Despite this strong correlation, the biological function of PCA3 remained elusive for almost two decades after its discovery (Bussemakers et al , 1999), until we established its role as a trans‐dominant negative oncogene that downregulates a protein‐coding tumor suppressor gene, prune homolog 2 ( PRUNE2 ) (Salameh et al , 2015). In experimental models of human prostate cancer, PCA3 overexpression as well as PRUNE2 silencing accelerates the growth of tumor xenografts in vivo , while increasing cell proliferation, adhesion, and migration in vitro (Salameh et al , 2015). In prostate cancer patients, …

The specific genes and molecules that drive physiological angiogenesis differ from those involved in pathological angiogenesis, suggesting distinct mechanisms for these seemingly related processes. Unveiling genes and pathways preferentially associated with pathologic angiogenesis is key to understanding its mechanisms, thereby facilitating development of novel approaches to managing angiogenesis-dependent diseases. To better understand these different processes, we elucidated the transcriptome of the mouse retina in the well-accepted oxygen-induced retinopathy (OIR) model of pathological angiogenesis. We identified 153 genes changed between normal and OIR retinas, which represent a molecular signature relevant to other angiogenesis-dependent processes such as cancer. These genes robustly predict the survival of breast cancer patients, which was validated in an independent 1,000-patient test cohort (40% difference in 15-year survival; p = 2.56 x 10−21). These results suggest that the OIR model reveals key genes involved in pathological angiogenesis, and these may find important applications in stratifying tumors for treatment intensification or for angiogenesis-targeted therapies., Author summary Angiogenesis is the formation of new blood vessels from pre-existing ones and it is the process for revascularization that predominates in adult life. Although angiogenesis is essential in physiological processes, such as wound healing, it also participates in pathological conditions, collectively known as angiogenesis-dependent diseases. Cancer and retinopathies are examples of such diseases. In cancer, angiogenesis is considered one of its hallmarks as tumors depend on a constant supply of oxygen and nutrients to grow. The more angiogenic the tumor, the more aggressive it is for the patient. In the present work, we used a well-suited animal model of retinopathy, an angiogenesis-dependent disease, to study the molecular signature for angiogenesis. We analyzed differences in gene expression from retinas developing under physiological and in retinopathic conditions to identify a molecular gene signature for angiogenesis. Because of the association between these two diseases, we tested our gene signature using data from human cancer. We showed that our gene signature has a robust prognostic value to predict breast cancer patient survival. Our results may lead to a better understanding of molecular mechanisms of pathological angiogenesis and its contribution to breast cancer.

About 4% to 7% of the non–small-cell lung cancer patients have anaplastic lymphoma kinase (ALK) rearrangements, and specific targeted therapies improve patients' outcomes significantly. ALK gene fusions are detected by immunohistochemistry or fluorescent in situ hybridization as gold standards in South America. Next-generation sequencing–based assays are a reliable alternative, able to perform simultaneous detection of multiple events from a single sample. We analyzed 4240 non–small-cell lung cancer samples collected in 37 hospitals from Chile, Brazil, and Peru, where ALK rearrangements were determined as part of their standard of care (SofC) using either immunohistochemistry or fluorescent in situ hybridization. A subset of 1450 samples was sequenced with the Oncomine Focus Assay (OFA), and the concordance with the SofC tests was measured. An orthogonal analysis was performed using a real-time quantitative PCR echinoderm microtubule-associated protein-like 4-ALK fusion detection kit. ALK fusion prevalence is similar for Chile (3.67%; N = 2142), Brazil (4.05%; N = 1013), and Peru (4.59%; N = 675). Although a comparison between OFA and SofC assays showed similar sensitivity, OFA had significantly higher specificity and higher positive predictive value, which opens new opportunities for a more specific determination of ALK gene rearrangements.

Mechanisms of viral oncogenesis are diverse and include the off-target activity of enzymes expressed by the infected cells, which evolved to target viral genomes for controlling their infection. Among these enzymes, the single-strand DNA editing capability of APOBECs represent a well-conserved viral infection response that can also cause untoward mutations in host DNA. Here we show, after evaluating somatic single-nucleotide variations and transcriptome data in 240 gastric cancer samples, a positive correlation between APOBEC3s mRNA-expression and the APOBEC-mutation signature, both increased in EBV+ tumors. The correlation was reinforced by the observation of APOBEC-mutations preferentially occuring in transcriptionally-active loci. The EBV-infection and APOBEC3 mutation-signature axis was confirmed in a validation cohort of 112 gastric cancer patients. Our findings suggest that APOBEC3 upregulation in EBV+ cancer may boost the mutation load, providing further clues to the mechanisms of EBV-induced gastric carcinogenesis. After further validation, this EBV-APOBEC axis may prove to be a secondary driving force in the mutational evolution of EBV+ gastric tumors, whose consequences in terms of prognosis and treatment implications should be vetted.

Whereas cancer patients have benefited from liquid biopsies, the scenario for gastric adenocarcinoma (GAC) is still dismal. We used next-generation deep sequencing of TP53-a highly mutated and informative gene in GAC-to assess mutations in tumor biopsies, plasma (PL) and stomach fluids (gastric wash-GW). We evaluated their potential to reveal tumor-derived mutations, useful for monitoring mutational dynamics at diagnosis, progression and treatment. Exon-capture libraries were constructed from 46 patients including tumor biopsies, GW and PL pre and post-treatment (196 samples), with high vertical coverage >8,000×. At diagnosis, we detected TP53 mutations in 15/46 biopsies (32.6%), 7/46 GW- (15.2%) and 6/46 PL-samples (13%). Biopsies and GW were concordant in 38/46 cases (82.6%) for the presence/absence of mutations and, furthermore, four GW-exclusive mutations were identified, suggesting tumor heterogeneity. Considering the combined analysis of GW and PL, TP53 mutations found in biopsies were also identified in 9/15 (60%) of cases, the highest detection level reported for GAC. Our study indicates that GW could be useful to track DNA alterations, especially if anchored to a comprehensive gene-panel designed for this malignancy.

Significance Prostate cancer has an unpredictable natural history: While most tumors are clinically indolent, some patients display lethal phenotypes. Serum prostate-specific antigen is the most often used test in prostate cancer but screening is controversial. Treatment options are limited for metastatic disease, hence the need for early diagnosis. Prostate cancer antigen 3 ( PCA3 ), a long noncoding RNA, is the most specific biomarker identified and approved as a diagnostic test. However, its inherent biological function (if any) has remained elusive. We uncovered a negative transdominant oncogenic role for PCA3 that down-regulates an unrecognized tumor suppressor gene, PRUNE2 (a human homolog of the Drosophila prune gene) thereby promoting malignant cell growth. This work defines a unique biological function for PCA3 in prostate cancer.

Whole-transcriptome evaluation by next-generation sequencing (NGS) has been widely applied in the investigation of diverse transcriptional scenarios. In many clinical situations, including needle biopsy samples or laser microdissected cells, limited amounts of RNA are usually available for the assessment of the whole transcriptome. Here, we describe an mRNA amplification protocol based on in vitro T7 transcription for transcriptome evaluation by NGS. Initially, we performed RNAseq from two human mammary epithelial cell lines and evaluated several aspects of the transcriptomes generated by linear amplification of Poly (A)+ mRNA species, including transcript representation, variability and abundance. Our protocol showed to be efficient with respect to full-length transcript coverage and quantitative expression levels. We then evaluated the applicability of using this protocol in a more realistic research scenario, analyzing tumor tissue samples microdissected by laser capture. In order to increase the quantification power of the libraries only the 3′ end of transcripts were sequenced. We found highly reproducible RNAseq data among amplified tumor samples, with a median Spearman's correlation of 80%, strongly suggesting that the amplification step and library protocol preparation lead to a consistent transcriptional profile. Altogether, we established a robust protocol for assessing the polyadenylated transcriptome derived from limited amounts of total RNA that is applicable to all NGS platforms.

Six members of the microRNA-17 (miR-17) family were mapped to three different chromosomes, although they share the same seed sequence and are predicted to target common genes, among which are those encoding hypoxia-inducible factor-1α (HIF1A) and VEGFA. Here, we evaluated the in vivo expression profile of the miR-17 family in the murine retinopathy of prematurity (ROP) model, whereby Vegfa expression is highly enhanced at the early stage of retinal neovascularization, and we found simultaneous reduction of all miR-17 family members at this stage. Using gene reporter assays, we observed binding of these miRs to specific sites in the 3' UTRs of Hif1a and Vegfa. Furthermore, overexpression of these miRs decreased HIF1A and VEGFA expression in vitro. Our data indicate that this miR-17 family elicits a regulatory synergistic down-regulation of Hif1a and Vegfa expression in this biological model. We propose the existence of a coordinated regulatory network, in which diverse miRs are synchronously regulated to target the Hif1a transcription factor, which in turn, potentiates and reinforces the regulatory effects of the miRs on Vegfa to trigger and sustain a significant physiological response.

Extracellular Vesicles (EVs) are gaining interest as central players in liquid biopsies, with potential applications in diagnosis, prognosis and therapeutic guidance in most pathological conditions. These nanosized particles transmit signals determined by their protein, lipid, nucleic acid and sugar content, and the unique molecular pattern of EVs dictates the type of signal to be transmitted to recipient cells. However, their small sizes and the limited quantities that can usually be obtained from patient-derived samples pose a number of challenges to their isolation, study and characterization. These challenges and some possible options to overcome them are discussed in this review.

Objectives The understanding of complex multifactorial diseases requires the availability of a variety of data for a large-number of affected individuals. In this data note here we provide whole exome sequencing data from a set of non-familiar multiple-sclerosis (MS) patients as well as their unaffected first-degree relatives. This data might help the identification of genomic alterations, including single nucleotide polymorphisms, de novo variations and structural genomic variations, such as copy-number alterations that may impact this disease. Data description This dataset comprises the full exome of 28 Brazilian subjects grouped in eight distinct families, consisting of four complete trios (mother–patient–father) plus another four complete trios with one added unaffected sibling. In total, we present the full exome data of eight patients diagnosed with recurrent remittent multiple sclerosis. Diagnoses were made by experienced neurologists and all enrolled patients had at least 5 years of follow up and specific MS treatment. Exomes were sequenced from leukocyte-derived DNA, after the capture of exons using biotinylated probes, in the Ion Proton platform. For each exome we generated an average of 66.1 million good quality mapped reads with an average length of ~ 160nt. On average, for 90% of the exome a vertical coverage above 20× was reached.

Multiple Sclerosis (MS) is an inflammatory neurodegenerative disease that affects approximately 2.5 million people globally. Even though the etiology of MS remains unknown, it is accepted that it involves a combination of genetic alterations and environmental factors. Here, after performing whole exome sequencing, we found a MS patient harboring a rare and homozygous single nucleotide variant (SNV; rs61745847) of the G-protein coupled receptor (GPCR) galanin-receptor 2 (GALR2) that alters an important amino acid in the TM6 molecular toggle switch region (W249L). Nuclear magnetic resonance imaging showed that the hypothalamus (an area rich in GALR2) of this patient exhibited an important volumetric reduction leading to an enlarged third ventricle. Ex vivo experiments with patient-derived blood cells (AKT phosphorylation), as well as studies in recombinant cell lines expressing the human GALR2 (calcium mobilization and NFAT mediated gene transcription), showed that galanin (GAL) was unable to stimulate cell signaling in cells expressing the variant GALR2 allele. Live cell confocal microscopy showed that the GALR2 mutant receptor was primarily localized to intracellular endosomes. We conclude that the W249L SNV is likely to abrogate GAL-mediated signaling through GALR2 due to the spontaneous internalization of this receptor in this patient. Although this homozygous SNV was rare in our MS cohort (1:262 cases), our findings raise the potential importance of impaired neuroregenerative pathways in the pathogenesis of MS, warrant future studies into the relevance of the GAL/GALR2 axis in MS and further suggest the activation of GALR2 as a potential therapeutic route for this disease.

Background Today there are more than 2 billion alcohol users and about 1.3 billion tobacco users worldwide. The chronic and heavy use of these two substances is at the heart of numerous diseases and may wreak havoc on the human oral microbiome. This study delves into the changes that alcohol and tobacco may cause on biofilms of the human oral microbiome. To do so, we used swabs to sample the oral biofilm of 22 subjects; including 9 control-individuals with no or very low consumption of alcohol and no consumption of tobacco, 7 who were chronic and heavy users of both substances and 6 active smokers that reported no significant alcohol consumption. DNA was extracted from swabs and the V1 region of the 16S rRNA gene was PCR amplified and sequenced using the Ion Torrent PGM platform, generating 3.7 million high quality reads. DNA sequences were clustered and OTUs were assigned using the ARB SILVA database and Qiime. Results We found no differences in species diversity and evenness among the groups. However, we found a significant decrease in species richness in only smokers and in smokers/drinkers when compared to controls. We found that Neisseria abundance was significantly decreased in both groups when compared to controls. Smokers had significant increases in Prevotella and Capnocytophaga and reductions in Granulicatella, Staphylococcus, Peptostreptococcus and Gemella when compared to the two other groups. Controls showed higher abundance of Aggregibacter, whilst smokers/drinkers had lower abundances of Fusobacteria. Samples from only smokers clustered closer together than to controls and smokers/drinkers, and also had a significant reduction in inter-group dissimilarity distances, indicating a more homogenous group than controls. Conclusions Our results indicate that the continued use of tobacco or alcohol plus tobacco significantly reduces bacterial richness, which apparently leads to a reduction in inter-group variability, turning the respective biofilms into a more homogenous microenvironment in terms of bacterial community composition, with possible consequences for human oral diseases. Electronic supplementary material The online version of this article (doi:10.1186/s12866-014-0250-2) contains supplementary material, which is available to authorized users.

DEAD-box proteins comprise a family of ATP-dependent RNA helicases involved in several aspects of RNA metabolism. Here we report the characterization of the human DEAD-box RNA helicase DDX26. The gene is composed of 14 exons distributed over an extension of 8,123 bp of genomic sequence and encodes a transcript of 1.8 kb that is expressed in all tissues evaluated. The predicted amino acid sequence shows a high similarity to a yeast DEAD-box RNA helicase (Dbp9b) involved in ribosome biogenesis. The new helicase maps to 7p12, a region of frequent chromosome amplifications in glioblastomas involving the epidermal growth factor receptor (EGFR) gene. Nevertheless, co-amplification of DDX26 with EGFR was not detected in nine tumors analyzed.

Human tumors exhibit two fundamentally important characteristics, extensive genetic alteration and clonality. Although it is still unclear to what extent tumors have an elevated mutational burden as compared with normal tissue, their clonality results in their ready detection. Thus, assaying tissues for clonal alterations at frequently mutated microsatellite loci represents a viable approach to cancer diagnosis. The most remarkable extension of this concept is that not only can cancer cells be detected in biological samples, but tumor DNA can also be directly detected in the serum or plasma of patients with some forms of cancer. This recent finding is currently being explored but may represent an important contribution to future diagnostic strategies.

ERBB2/HER2 amplification activates signaling cascades that lead to a tumor cell phenotype. However, despite its remarkable importance in oncology, the consequences of HER2 amplification over the extracellular vesicles (EVs) content have not yet been investigated. Here, we isolated EVs secreted by HB4a, a mammary luminal epithelial cell line and C5.2, its HER2-overexpressing clone. We isolated two EV sets (20 and 100 K) by ultracentrifugation and used electron microscopy and nanoparticle tracking analysis for their morphological characterization. We employed GeLC-MS/MS combined with isotope-coded protein labeling to evaluate cell-derived proteins and LC-MS/MS label free spectral counting to quantify the EVs proteome. We found higher HER2 levels in both C5.2-derived EVs when compared with C5.2 cells, suggesting its preferential shuttling. Proteins capable of inducing malignant transformation are enriched in both C5.2 EV subsets, including two HER2-related proteins involved in cell motility and invasion, cofilin and CD44. MetaCore™ analysis indicated an enrichment of cell adhesion and cytoskeleton-remodeling pathways in C5.2 EVs, as well as proteins related to HER2 signaling, such as sphingosine-1-phosphate pathway. Together, our data indicate that in terms of protein content, distinct vesicle sets reinforce and complement each other. Our results also suggest that HER2-upregulated proteins from EVs may be relevant for cellular malignancy and can be potential biomarkers for HER2(+) cancer patients.

Molecules differentially expressed in blood vessels among organs or between damaged and normal tissues, are attractive therapy targets; however, their identification within the human vasculature is challenging. Here we screened a peptide library in cancer patients to uncover ligand-receptors common or specific to certain vascular beds. Surveying ∼2.35 × 10 6 motifs recovered from biopsies yielded a nonrandom distribution, indicating that systemic tissue targeting is feasible. High-throughput analysis by similarity search, protein arrays, and affinity chromatography revealed four native ligand-receptors, three of which were previously unrecognized. Two are shared among multiple tissues (integrin α4/annexin A4 and cathepsin B/apolipoprotein E3) and the other two have a restricted and specific distribution in normal tissue (prohibitin/annexin A2 in white adipose tissue) or cancer (RAGE/leukocyte proteinase-3 in bone metastases). These findings provide vascular molecular markers for biotechnology and medical applications.

We report the first quantitative and qualitative analysis of the poly (A)+ transcriptome of two human mammary cell lines, differentially expressing (human epidermal growth factor receptor) an oncogene over-expressed in approximately 25% of human breast tumors. Full-length cDNA populations from the two cell lines were digested enzymatically, individually tagged according to a customized method for library construction, and simultaneously sequenced by the use of the Titanium 454-Roche-platform. Comprehensive bioinformatics analysis followed by experimental validation confirmed novel genes, splicing variants, single nucleotide polymorphisms, and gene fusions indicated by RNA-seq data from both samples. Moreover, comparative analysis showed enrichment in alternative events, especially in the exon usage category, in ERBB2 over-expressing cells, data indicating regulation of alternative splicing mediated by the oncogene. Alterations in expression levels of genes, such as LOX, ATP5L, GALNT3, and MME revealed by large-scale sequencing were confirmed between cell lines as well as in tumor specimens with different ERBB2 backgrounds. This approach was shown to be suitable for structural, quantitative, and qualitative assessment of complex transcriptomes and revealed new events mediated by ERBB2 overexpression, in addition to potential molecular targets for breast cancer that are driven by this oncogene.

For development of a new ligand-directed pharmacology, it is critical to measure delivery of targeted drug ligands via molecular imaging or diagnostic readouts (termed theranostics). Combinatorial peptide libraries serve as unbiased functional screens that can identify specific peptides targeting cell-surface receptors accessible to the circulation. As candidate drug leads, such peptides provide motifs likely to modify ligand-receptor interactions and downstream signal transduction pathways. This strategy is synergistic with genomic and proteomic approaches and has yielded insights into the specialized nature of the target tissue microenvironment. However, for this vision to be realized, one must look, as recent literature suggests, beyond receptor levels and critically analyze ligand accessibility as a key determinant in pharmacodelivery systems.

Microsatellite allele losses are characteristic features of head and neck squamous cell carcinoma and can be used as molecular markers for malignancy. We have investigated the detection of microsatellite allele loss in mouth washes and lesions brushings from 19 patients with squamous cell carcinoma of the oral cavity and oropharynx as a means of tumour detection. In 84% of the analysed cases, allele loss previously identified in the tumour of these patients, was detected in these easily obtained specimens. No alterations were found in material derived from 10 healthy individuals. Success of detection was independent of tumour stage, suggesting that this approach may be useful for early diagnosis as well as for follow-up.

Background Combinatorial phage display has been used in the last 20 years in the identification of protein-ligands and protein-protein interactions, uncovering relevant molecular recognition events. Rate-limiting steps of combinatorial phage display library selection are (i) the counting of transducing units and (ii) the sequencing of the encoded displayed ligands. Here, we adapted emerging genomic technologies to minimize such challenges. Methodology/Principal Findings We gained efficiency by applying in tandem real-time PCR for rapid quantification to enable bacteria-free phage display library screening, and added phage DNA next-generation sequencing for large-scale ligand analysis, reporting a fully integrated set of high-throughput quantitative and analytical tools. The approach is far less labor-intensive and allows rigorous quantification; for medical applications, including selections in patients, it also represents an advance for quantitative distribution analysis and ligand identification of hundreds of thousands of targeted particles from patient-derived biopsy or autopsy in a longer timeframe post library administration. Additional advantages over current methods include increased sensitivity, less variability, enhanced linearity, scalability, and accuracy at much lower cost. Sequences obtained by qPhage plus pyrosequencing were similar to a dataset produced from conventional Sanger-sequenced transducing-units (TU), with no biases due to GC content, codon usage, and amino acid or peptide frequency. These tools allow phage display selection and ligand analysis at >1,000-fold faster rate, and reduce costs ∼250-fold for generating 106 ligand sequences. Conclusions/Significance Our analyses demonstrates that whereas this approach correlates with the traditional colony-counting, it is also capable of a much larger sampling, allowing a faster, less expensive, more accurate and consistent analysis of phage enrichment. Overall, qPhage plus pyrosequencing is superior to TU-counting plus Sanger sequencing and is proposed as the method of choice over a broad range of phage display applications in vitro, in cells, and in vivo.