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BackgroundSeveral studies have documented the significant impact of methodological choices in microbiome analyses. The myriad of methodological options available complicate the replication of results and generally limit the comparability of findings between independent studies that use differing techniques and measurement pipelines. Here we describe the Mosaic Standards Challenge (MSC), an international interlaboratory study designed to assess the impact of methodological variables on the results. The MSC did not prescribe methods but rather asked participating labs to analyze 7 shared reference samples (5x human stool samples and 2x mock communities) using their standard laboratory methods. To capture the array of methodological variables, each participating lab completed a metadata reporting sheet that included 100 different questions regarding the details of their protocol. The goal of this study was to survey the methodological landscape for microbiome metagenomic sequencing (MGS) analyses and the impact of methodological decisions on metagenomic sequencing results.ResultsA total of 44 labs participated in the MSC by submitting results (16S or WGS) along with accompanying metadata; thirty 16S rRNA gene amplicon datasets and 14 WGS datasets were collected. The inclusion of two types of reference materials (human stool and mock communities) enabled analysis of both MGS measurement variability between different protocols using the biologically-relevant stool samples, and MGS bias with respect to ground truth values using the DNA mixtures. Owing to the compositional nature of MGS measurements, analyses were conducted on the ratio of Firmicutes: Bacteroidetes allowing us to directly apply common statistical methods. The resulting analysis demonstrated that protocol choices have significant effects, including both bias of the MGS measurement associated with a particular methodological choices, as well as effects on measurement robustness as observed through the spread of results between labs making similar methodological choices. In the analysis of the DNA mock communities, MGS measurement bias was observed even when there was general consensus among the participating laboratories.ConclusionThis study was the result of a collaborative effort that included academic, commercial, and government labs. In addition to highlighting the impact of different methodological decisions on MGS result comparability, this work also provides insights for consideration in future microbiome measurement study design.

Background:We have previously shown that the long non-coding (lnc)RNA prostate cancer associated 3 (PCA3; formerly prostate cancer antigen 3) functions as a trans-dominant negative oncogene by targeting the previously unrecognized prostate cancer suppressor gene PRUNE2 (a homolog of the Drosophila prune gene), thereby forming a functional unit within a unique allelic locus in human cells. Here, we investigated the PCA3/PRUNE2 regulatory axis from early (tumorigenic) to late (biochemical recurrence) genetic events during human prostate cancer progression.Methods:The reciprocal PCA3 and PRUNE2 gene expression relationship in paired prostate cancer and adjacent normal prostate was analyzed in two independent retrospective cohorts of clinically annotated cases post-radical prostatectomy: a single-institutional discovery cohort (n=107) and a multi-institutional validation cohort (n=497). We compared the tumor gene expression of PCA3 and PRUNE2 to their corresponding expression in the normal prostate. We also serially examined clinical/pathological variables including time to disease recurrence.Results:We consistently observed increased expression of PCA3 and decreased expression of PRUNE2 in prostate cancer compared with the adjacent normal prostate across all tumor grades and stages. However, there was no association between the relative gene expression levels of PCA3 or PRUNE2 and time to disease recurrence, independent of tumor grades and stages.Conclusions:We concluded that upregulation of the lncRNA PCA3 and targeted downregulation of the protein-coding PRUNE2 gene in prostate cancer could be early (rather than late) molecular events in the progression of human prostate tumorigenesis but are not associated with biochemical recurrence. Further studies of PCA3/PRUNE2 dysregulation are warranted.Funding:We received support from the Human Tissue Repository and Tissue Analysis Shared Resource from the Department of Pathology of the University of New Mexico School of Medicine and a pilot award from the University of New Mexico Comprehensive Cancer Center. RP and WA were supported by awards from the Levy-Longenbaugh Donor-Advised Fund and the Prostate Cancer Foundation. EDN reports research fellowship support from the Brazilian National Council for Scientific and Technological Development (CNPq), Brazil, and the Associação Beneficente Alzira Denise Hertzog Silva (ABADHS), Brazil. This work has been funded in part by the NCI Cancer Center Support Grants (CCSG; P30) to the University of New Mexico Comprehensive Cancer Center (CA118100) and the Rutgers Cancer Institute of New Jersey (CA072720).

En noviembre del 2019 inicia en Wuhan, lo que sería una nueva pandemia, por un coronavirus que no había sido identificado previamente, el SARS-CoV-2. Posteriormente fue reportado en Paraguay el primer caso de covid-19 en marzo del 2020. A pesar de que se trata de un virus de manifestación preferentemente respiratoria, sabemos hasta la fecha que pueden presentar otros síntomas, incluso ser totalmente asintomáticos. Se ha visto que las lesiones en piel, al igual que en muchas otras enfermedades infecciosas, también son causadas por este nuevo virus, por lo que el dermatólogo juega un rol importante en esta batalla. Reportamos el primer caso registrado por el servicio de Dermatología del Hospital Nacional de Itaugua, de manifestaciones cutáneas en un lactante, secundarias a la enfermedad de COVID-19. Correspondencia: Lenny Gutierrez Correo: lennygutigmail.com Conflicto de interés: Los autores declaran no poseer conflicto de interés. Recibido: 02/07/2020 Aceptado:13/07/2020

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.

Extracellular vesicles (EVs) have been recognized as relevant players in cell-cell communication. To fully explore their potential as carriers of biological information in clinical settings, protocols capable of dealing with minute amounts of proteins, lipids, and nucleic acids present in their cargo are a requirement. Here we delve into a protocol to decipher the total transcriptome of EVs, from undetectable amounts of EVs-derived RNA from clinical samples.

ImportanceSARS-CoV-2 genomic variants impacts the overall sensitivity of COVID-19 diagnosis, leading to false-negative diagnosis and the continued spread of the virus.ObjectiveTo evaluate how nucleotide variability in target primer binding sites of the SARS-CoV-2 genomes may impact diagnosis using different recommended primer/probe sets, as well as to suggest the best primer/probes for diagnosis.DesignWe downloaded 105,118 public SARS-CoV-2 genomes from GISAID (Sept, 25th, 2020), removed genomes of apparent worst quality (genome length 5% ambiguous bases) and missing metadata, and performed an analysis of complementarity for the 13 most used diagnostic primers/probe sets for RT-PCR detection. We calculated the N rate and % of genome recovery, with all primer/probe-sets considering viral origin and clade. Results: Our findings indicate that currently, the Paris_nCoV-IP2, -IP4 and WHO|E_Sarbeco primer/probe sets for COVID-19, to perform the best diagnostically worldwide, recovering >99.5% of the good quality SARS-CoV-2 genomes from GISAID, with no mismatches. The Chinese_CDC|2019-nCoV-NP primer/probe set, among the first to be designed during the pandemic, was the most susceptible to currently most abundant SARS-CoV-2 variants. Mismatches encompassing the binding sites for this set are more frequent in Clade-GR and are highly prevalent in over 30 countries globally, including Brazil and India, two of the hardest hit countries. Conclusions: Detection of SARS-CoV-2 in patients may be hampered by significant variability in parts of the viral genome that are targeted by some widely used primer sets. The geographic distribution of different viral clades indicates that continuous assessment of primer sets via sequencing-based surveillance and viral evolutionary analysis is critical to accurate diagnostics. This study highlights sequence variance in target regions that may reduce the efficiency of primer:target hybridization that in turn may lead to the undetected spread of the virus. As such, due to this variance, the Chinese_CDC|2019-nCoV-NP-set should be used with caution, or avoided, especially in countries with high prevalence of the GR clade.Key PointsQuestionHow variable are the binding-sites of primers/probes used for COVID-19 diagnosis?FindingsWe investigated nucleotide variations in primer-binding sites used for COVID-19 diagnosis, in 93,143 SARS-CoV-2 genomes, and found primer sets targeting regions of increasingly nucleotide variance over time, such as the Chinese_CDC|2019-nCoV-NP. The frequency of these variations is higher in Clade-GR whose frequency is increasing worldwide. Paris_nCoV-IP2, IP4 and WHO|E_Sarbeco performed best.MeaningWe suggest the use of some sets to be halted and reinforce the importance of a continuous surveillance of SARS-CoV-2 variations to prompt the use of the best primers.

IntroductionTherapies that target activatingEgfr, Alk, Ros1and other mutations have become first-line treatments that improve NSCLC patient’s life expectancy. Latin-American patients are poorly represented in clinical trials and in genomic databases, thus little is known about the prevalence of actionable mutations in this population. This study characterizes, for the first time, the somatic mutations found in 52 actionable genes, and describe a novel set of potentially actionable mutations, in NSCLC patients from Chile, Brazil and Peru, while correlating these genomic occurrences with relevant clinical, demographic and pathology aspects.Methods1732 subjects diagnosed with NSCLC were analyzed. DNA and RNA were sequenced using a 52 genes NGS panel. Mutations were annotated using the Variant Effect Predictor, COSMIC, OncoKB and the Cancer Genome Interpreter to categorize somatic mutations.ResultsWe found a total of 1713 mutations with 626 (36.5%) novel, potentially driver mutations. 66.1% of these novel mutations were predicted as Tier 1 driver mutations. Actionable mutations forRet and Alkwere more prevalent in Brazil than in Chile, whereasMetexon-14 skipping was significantly enriched in Chile. In Peru,Egfris higher whileKrasis lower. A high number of novels potentially driver mutations in know NSCLC actionable genes, such asAlk, Erbb2, Ret, Met, andRos1, was found.ConclusionsThe analysis of many Latin America subjects revealed a significant number of clinically actionable but also novel somatic mutations in cancer genes highlighting the importance of including less-represented populations in clinical trials and molecular studies.

Extracellular vesicles (EVs) have been recognized as relevant players in cell-cell communication. To fully explore their potential as carriers of biological information in clinical settings, protocols capable of dealing with minute amounts of proteins, lipids, and nucleic acids present in their cargo are a requirement. Here we delve into a protocol to decipher the total transcriptome of EVs, from undetectable amounts of EVs-derived RNA from clinical samples.

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.

Gastric cancer (GC) is the fifth most common type of cancer worldwide with high incidences in Asia, Central, and South American countries. This patchy distribution means that GC studies are neglected by large research centers from developed countries. The need for further understanding of this complex disease, including the local importance of epidemiological factors and the rich ancestral admixture found in Brazil, stimulated the implementation of the GE4GAC project. GE4GAC aims to embrace epidemiological, clinical, molecular and microbiological data from Brazilian controls and patients with malignant and pre-malignant gastric disease. In this letter, we summarize the main goals of the project, including subject and sample accrual and current findings.

Inflammatory breast carcinoma (IBC) is one of the most lethal forms of human breast cancer, and effective treatment for IBC is an unmet clinical need in contemporary oncology. Tumor-targeted theranostic approaches are emerging in precision medicine, but only a few specific biomarkers are available. Here we report up-regulation of the 78-kDa glucose-regulated protein (GRP78) in two independent discovery and validation sets of specimens derived from IBC patients, suggesting translational promise for clinical applications. We show that a GRP78-binding motif displayed on either bacteriophage or adeno-associated virus/phage (AAVP) particles or loop-grafted onto a human antibody fragment specifically targets orthotopic IBC and other aggressive breast cancer models in vivo. To evaluate the theranostic value, we used GRP78-targeting AAVP particles to deliver the human Herpes simplex virus thymidine kinase type-1 (HSVtk) transgene, obtaining simultaneous in vivo diagnosis through PET imaging and tumor treatment by selective activation of the prodrug ganciclovir at tumor sites. Translation of this AAVP system is expected simultaneously to image, monitor, and treat the IBC phenotype and possibly other aggressive (e.g., invasive and/or metastatic) subtypes of breast cancer, based on the inducible cell-surface expression of the stress-response chaperone GRP78, and possibily other cell-surface receptors in human tumors.

Simple Summary Mutational signatures due to DNA mismatch repair deficiency (dMMR) is common in many cancers. However, the prognostic value of dMMR-associated mutational signatures remains to be assessed. Here, we performed a de novo extraction of mutational signatures in a cohort of 787 patients with gastric cancer. We detected three dMMR-related signatures, one of which clearly discriminates tumors with MLH1 gene silencing through hypermethylation of its promoter. We showed evidence that classification based on mutational signature exposure can be used to identify groups of patients with common clinical, immunological, and mutational features related directly to better prognosis. Abstract DNA mismatch repair deficiency (dMMR) is associated with the microsatellite instability (MSI) phenotype and leads to increased mutation load, which in turn may impact anti-tumor immune responses and treatment effectiveness. Various mutational signatures directly linked to dMMR have been described for primary cancers. To investigate which mutational signatures are associated with prognosis in gastric cancer, we performed a de novo extraction of mutational signatures in a cohort of 787 patients. We detected three dMMR-related signatures, one of which clearly discriminates tumors with MLH1 gene silencing caused by promoter hypermethylation (area under the curve = 98%). We then demonstrated that samples with the highest exposure of this signature share features related to better prognosis, encompassing clinical and molecular aspects and altered immune infiltrate composition. Overall, the assessment of the prognostic value and of the impact of modifications in MMR-related genes on shaping specific dMMR mutational signatures provides evidence that classification based on mutational signature exposure enables prognosis stratification.

Os autores do resumo nao possuem vinculo com a Fiocruz, entretanto, ao submeterem o trabalho no evento organizado por Bio-Manguinhos, cedem automaticamente os direitos a unidade.

Although numerous studies have shown the relevance of the gut microbiome in several diseases, underlying questions remain concerning the stomach microbiome and the establishment of a causal link between the microbiota and the development of gastric diseases, much beyond Helicobacter pylori and Epstein Barr virus. In this study, we aimed to characterize the bacterial composition of the stomach of subjects undergoing upper endoscopy, including gastric cancer (GC) individuals, aiming to identify fluctuations in bacterial populations that might be associated with stomach health. During endoscopic examination at A.C. Camargo Cancer Center (Sao Paulo, Brazil), gastric fluids (GF) were recovered from either GC patients (113) or individuals with gastric-related complaints, such as superficial gastritis (SG; 79), atrophic gastritis (AG; 12), and intestinal metaplasia (IM; 33). For eubacteria identification, the V3-V4 region of the 16S rRNA gene was amplified and paired-end sequenced (Illumina MiSeq). Analyses were carried out using Qiime2 and phyloseq packages. On average, we identified between 14 and 104 OTUs per subject, evidencing the potential of GFs for determining the stomach microbial composition and the interindividual variation. Testing of sample richness between GC and controls showed significant differences between the number of OTUs observed in each group (an average of 44 and 52 OTUs, respectively—Mann-Whitney test, p2) in AG samples as compared to SG patients, which are also increased in the IM, and GC. These results indicate these bacteria to be potentially associated with the stomach dysbiosis that may lead to the carcinogenesis cascade. Besides the regular turnover of the gastric epithelial tissue and pouring of cells onto the gastric cavity, GF certainly contains a high proportion of transient oral-derived bacteria, while stomach-resident microbiota is expected to be in close contact with the gastric epithelia. Preliminary data analyzing the bacterial content of the saliva x GF in a subset of patients showed no significant beta-diversity differences, but both fluids differ significantly from their biopsies’ composition. We are currently performing culturomics and transcriptomics to identify bacteria that are alive in the stomach. As GC is a complex malignancy with limited treatment options, these results may contribute to developing new interventions to treat and to better understand this disease. Citation Format: Thais F. Bartelli, Gabriela E. Albuquerque, Alexandre Defelicibus, Marianna S. Serpa, Lais L.S. Abrantes, Rodrigo Borges, Felipe Coimbra, Adriane G. Pelosof, Israel T. Silva, Diana N. Nunes, Emmanuel Dias-Neto. Unravelling the gastric microbiome in health and disease: Gastric cancer beyond Helicobacter pylori in a Brazilian cohort [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B09.

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.

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 The standard treatment for patients (pts) with localized squamous cell carcinoma of the anal canal (SCCAC) is chemoradiation (ChRT). Yet nearly 30% of patients are not cured and undergo salvage anorectal amputation. SCCAC is mostly a virus-associated tumor and thus potentially immunogenic. In fact, immune checkpoint inhibitors are promising in trials of metastatic SCCAC. Recently, studies have shown that the composition of the intestinal microbiota influences response to immunotherapy in some solid tumors, and the replacement of the intestinal "carcinogenic" by a protective microbiota has led to investigations with prebiotics and probiotics (PreProbiotics). Yet, there are no studies on the use of these agents in SCCAC. Thus we are conducting a randomized phase II study to test the efficacy of PreProbiotics during definitive ChRT aiming to improve the cure rate of pts with localized SCCAC. Trial design Randomized open label parallel phase II trial, where eligible pts will be randomized 1:1 to receive PreProbiotics starting one week prior to ChRT and throughout treatment until response evaluation at 6 to 8 weeks post ChRT or conventional ChRT. Eligible pts are ≥ 18 years, with histologically confirmed SCCAC, localized disease (≥ T2N0M0), indication to start definitive ChRT; HIV seropositive pts are eligible. Pts with active infection requiring antibiotics will be excluded. Primary endpoint: complete clinical and radiological response (CR) at 6 to 8 weeks post ChRT. Secondary endpoints: CR at 6 months, progression free survival, colostomy-free survival, metabolic response measured by 18-FDG PET-CT (baseline and at 6-8 weeks), toxicity, incidence of HPV in tumor tissues. All pts will have the following biological samples collected for correlative studies at baseline, 6-8 weeks and 6 months post ChRT: blood samples for circulating tumor DNA, inflammatory cytokines, anal/rectal swabs and feces to evaluate microbiota. Sample size assumptions: the H0 is CR at week 6 - 8 of 70% and H1, 90%; with a type I error of 10%, power of 80% and attrition rate of 10%, the final sample size is 75 patients. Clinical trial identification NCT03870607. Legal entity responsible for the study Rachel Riechelmann. Funding AC Camargo Cancer Center. Disclosure All authors have declared no conflicts of interest.

e15585 Background: Loss of expression of ARID1A, a tumor suppressor gene involved in chromatin remodeling and transcription activation, has been associated with worse prognosis in Asian GC patients (Yang et al. 2016). Mutations in ARID1A have been found in 8-27% of GC, usually leading to gene/protein inactivation. Here we evaluated the possible involvement of ARID1A mutations and clinical characteristics, while considering genomic ancestry and survival, in a cohort of Brazilian GC patients. Methods: We included 112 pts diagnosed with GC and treated at AC Camargo Cancer Center before 2013. The study was approved by local IRB. Genomic DNA was used for capture-based enrichment of a customized gene panel including 99 genes. Libraries were sequenced in the NextSeq 500 platform (Illumina), using paired-end reads (2x75bp). For ancestry inference we used a set of ancestry informative markers, covered by target and off-target reads, described by Elhaik et al. (2014). Results: Median age was 64y (37-91), 63% were male, M:F ratio was 1.73. Most cases were classified as Diffuse (47.3%) followed by Intestinal (41.1%), Mixed (2.7%) and 8.9% were deemed unclassifiable by Lauren´s classification. 22.3% were stage I, 20.5% stage II, 42.9% stage III and 14.3% stage IV. 11.6% were in the GEJ and 80.4% were in corpus/antrum. Five patients were EBV positive (4.5%). Genomic ancestry was as follows: 55.4% European, 27.7% Asian, 8.9% African and 8% were highly admixed ( < 50% of any ancestry). ARID1A was mutated in 19% of cases and showed no association with age at diagnosis (p = 0.21), gender (p = 0.76), tumor location (p = 0.55), staging (p = 0.42), Lauren (p = 0.14) or EBV (p = 0.42). ARID1A had no impact on overall survival (OS) (HR 1.17; 95%CI 0.59-2.31; p = 0.6) or disease-free survival (DFS) (HR 1.24; 95%CI 0.66-2.32; p = 0.5), including the subgroup with Asian genomic background: OS-Asia (HR 1.08; 95%CI 0.31-3.81; p = 0.9), DFS-Asia (HR 1.15; 95%CI 0.32-4.12; p = 0.8). Conclusions: ARID1A is a common driver in GC among Brazilian patients. Unlike in Asians, ARID1A was not prognostic in this Brazilian cohort even in the subgroup with a predominant ( > 50%) Asian genomic ancestry.

e15588 Background: Clinical characteristics, treatment response and overall survival of GC patients differ between Asian and non-Asian countries. Here we evaluated the possible associations between PGA, clinical characteristics and survival in an admixed GC Brazilian cohort. Methods: We included 112 GC pts diagnosed and treated at AC Camargo Cancer Center (São Paulo, Brazil) before 2013. The study was approved by local IRB. Genomic DNA was used for capture-based enrichment of a customized gene panel including 99 genes. Libraries were sequenced in the NextSeq 500 platform (Illumina), using paired-end reads (2x75bp). Ancestries were determined through a set of ancestry informative markers (AIMs), covered by target and off-target reads, described by Elhaik et al. (2014). Results: An average of 406 AIMs were recovered from the available samples, revealing average ancestries was as follows: 55.4% European, 27.7% Asian, 8.9% African; 8% of subjects were highly admixed (HA; < 50% of any ancestry). We found no association between PGA and age at diagnosis (p = 0.58), tumor location (p = 0.34), Lauren (p = 0.24) and staging (p = 0.68). There was an association between PGA and gender (p = 0.04) and a marginal association between PGA and EBV (p = 0.056). BRCA2 was the only gene enriched in the Asian subgroup, compared to the other groups combined (p = 0.009). The median follow-up time was 95 months. We found no differences in median overall survival (OS) (p = 0.4) or disease-free survival (DFS) (p = 0.6) according to PGA. The HA subgroup presented worst survival outcomes compared to the other groups aggregated (mOS 34m; 95%CI 5-80 x mOS 71m; 95% CI 44-85, respectively), but the difference was not statistically significant (HR 1,73; 95% CI 0.74-4.05; p = 0.2). Even for patients with > 75% AIMs for any given ancestry, we found no differences in OS between Europeans (n = 25; mOS 86m; 95%CI 80-NA), Asians (n = 22; mOS 83m; 95%CI 51-NA) and Africans (n = 5; mOS 67m; 95%CI 22-NA), p = 0.4. Conclusions: The most prevalent ancestries in this Brazilian GC cohort were European, followed by Asian and African. Although we found associations between ancestry and a few clinical aspects, PGA was not associated with survival.

Background: Whereas the pathological aspects of Gastric Adenocarcinomas (GAC) are well defined, there is still an important lack of biomarkers predictive of its occurrence, aggressiveness and response to neoadjuvant treatment. Nowadays, it is known that isolated or clustered Circulating Tumor Cells (respectively CTCs or CTMs, for Microemboli) can be found in the blood of patients many months before tumor detection. In this sense, the identification of CTCs/CTMs in GAC patients has a potential value as biomarkers for tumor growth and invasiveness. Objective: to study the predictive value of CTCs and CTMs from GAC patients, evaluating their presence and correlation with HER2-status and clinical-pathological characteristics. Methods: Blood was prospectively collected from 72 GAC patients (62.5% men) recruited at the Department of Abdominal Surgery of the A.C.Camargo Cancer Center, São Paulo, Brazil, from March 2016 to March 2017, after the signature of informed consent forms previously approved by the institutional review board. Blood was first collected before the first neoadjuvant cycle (at diagnosis) and also before adjuvant treatment (diagnosis of metastasis). The samples were processed and filtered in ISET (Rarecells, France) allowing the isolation and quantification of CTCs and CTMs, and immunocytochemistry (ICC) with anti-HER2. Results: The most common tumor status were T1b (15/20.8%) and N1 (14/19.4%) and diffuse tumors (Lauren's) was the most common subtype (37/51.4%); 55 patients had non-metastatic disease (76.4%) at moment of inclusion. CTCs were found in 61/72 patients (84.7%) with a median of 2.5 CTCs/mL (0-39.6 CTCs/mL). We found more CTC/mL in non-metastatic disease (2.8 CTCs/mL versus 0.9 CTCs/mL, p Conclusions: CTCs were more prevalent in patients with initial disease, reinforcing their possible role in the growth of the primary tumor maybe in a self-seeding mechanism. Also, the good HER2-correlation found between primary tumors and CTCs suggest the use of the later as surrogates of the primary tumor for determining targeted therapies with anti-HER2 antibodies. Citation Format: Emne Ali Abdallah, Alexcia Camila Braun, Bianca Troncarelli Flores, Lais Lie Senda, Maria Dirlei Ferreira de Souza Begnami, Felipe José Coimbra, Emmanuel Dias-Neto, Diana Nunes, Wilson Luiz Costa Junior, Ludmilla T. Chinen. Circulating tumor cells and circulating tumor microemboli in the context of gastric adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1569.

Background: There is a complex interplay between smoking and patient demographics in the emergence and development of cancer. We vetted the possible relationship between gender, race and age with mutational processes operating on aerodigestive tract cancers, known to be highly associated to tobacco. Methods: We used an empirical Bayesian approach to identify genomic patterns that lead to mutational processes in head and neck, esophageal, and lung cancers from TCGA database followed by confirmation in independent cohorts (cBioPortal). Results: After confronting clinical information and smoking-habits to molecular findings in those cancers, we identified imprints of different mutational processes in a disease-, gender- and race-specific fashion. Black and female patients - despite their lower tobacco consumption in comparison to whites or males - had tumors with the highest enrichment for tobacco-induced DNA damage. The relevance of mutational signatures related to aging varied across race and gender and were independent from chronological age. The activity of smoking signature was different according to disease histotype, and stage. tumors of never-smoker lung cancer patients showed tobacco-specific mutational signatures, which could suggest the influence of second-hand smoking. Patients with tumors enriched for the smoking mutational signature presented longer survival, whereas those with ageing-enriched signature had poorer prognosis. Conclusions: Mutational processes may unravel connections between patient features and tumorigenesis, particular to race, gender, and tumor type. Therefore, it is paramount to assess those details with greater granularity in every effort of personalized medicine either in preventive or therapeutic approaches. Citation Format: João Paulo da Silveira Nogueira Lima, Rodrigo Couto Duarte Drummond, Monize Nakamoto Provisor Santos, Renan Valieris, Rafael Andres Rosales Mitrowsky, Vladmir Cordeiro Lima, Diana Noronha Nunes, Emmanuel Dias-Neto, Israel Tojal da Silva. The interplay between patient´s features and mutational processes in tobacco related cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3245.

e16578 Background: Approved by the FDA as a diagnostic test, the long non-coding RNA PCA3 is the most specific human prostate cancer biomarker identified to date. The biological function of PCA3, which involves downregulating the tumor suppressor gene PRUNE2, has important therapeutic implications. Here we investigated RNA expression levels of these genes to better understand the dynamics of this regulatory process during cancer progression and its relevance for tumor aggressiveness. Methods: We downloaded clinical data, as well as PCA3 and PRUNE2 gene expression values (log2 RSEM), from 497 TCGA prostate cancer patients (totaling 550 samples) and their matched normal tissues using the Xena Browser. Expression values for both genes, and their ratios, were evaluated in terms of tumor vs non-tumor tissue, biochemical recurrence, pathologic T stage, Gleason scores and age at diagnosis. All p-values were obtained by the Wilcoxon rank Sum (2 groups) or the Kruskal-Wallis Test (3+ groups). Results: Using 52 malignant/non-malignant tissue pairs from our cohort, we confirmed previous findings that showed simultaneous PCA3 upregulation and PRUNE2 reduction in malignant samples when compared to non-malignant ( p7) and in younger subjects (

Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of all highly and moderately expressed, and between 40% and 50% of rarely expressed, human genes. In our most thoroughly sequenced tissue, the breast, the 130,000 ORESTES generated are derived from transcripts from an estimated 70% of all genes expressed in that tissue, with an equally efficient representation of both highly and poorly expressed genes. In this respect, we find that the capacity of the ORESTES strategy both for gene discovery and shotgun transcript sequence generation significantly exceeds that of conventional ESTs. The distribution of ORESTES is such that many human transcripts are now represented by a scaffold of partial sequences distributed along the length of each gene product. The experimental joining of the scaffold components, by reverse transcription–PCR, represents a direct route to transcript finishing that may represent a useful alternative to full-length cDNA cloning.

A series of eight microsatellite loci were assayed for both loss of heterozygosity and new mutated alleles in 91 head and neck squamous cell carcinomas. In 58 cases, alterations were detected and used as markers for assaying the presence of circulating tumor-derived DNA in the patients' plasma. This was unambiguously detected in 17 cases. The probability of detecting circulating DNA was independent of tumor stage and was found to be present even in some individuals with stage I tumors. The presence of such DNA, however, could not be correlated with disease outcome or other significant clinical parameters, suggesting that it has no prognostic significance. The results indicate that circulating tumor-derived DNA could be used as a means of early diagnosis of head and neck tumors.

The intermediate hosts of S. mansoni in South America, B. glabrata, B. tenagophila, and B. straminea, were identified by restriction fragment length polymorphism (RFLP) analysis of the internal transcribed spacer region of the rRNA gene. The restriction patterns obtained with DdeI were the most informative of the eight enzymes that were tried. The RFLP profiles obtained using this enzyme are highly distinctive and exhibit low levels of intraspecific polymorphism even between specimens collected from diverse regions of Brazil, Argentine, Paraguay, and Uruguay. The method proved useful for the identification of DNA extracted from eggs, permitting species identification while preserving the living adult specimens for further studies.

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.

TP53 protein is one of the most important and studied human tumor suppressors. Whereas TP53 drives the malignant transformation and is the most frequently mutated gene in human cancers, the location and the type of its mutations for specific tumor types in ethnically admixed populations have not been evaluated. In this study we employed an amplicon panel to capture and to deep-sequence all the exons of TP53 in a series of 29 tumor biopsy samples derived from Brazilian individuals diagnosed with gastric adenocarcinomas (GACs), which were compared to Asian and Europeans. TP53 sequences were obtained from Brazilian samples with the Ion Torrent AmpliSeq TP53-panel and determined in the Ion PGM Torrent and Ion Proton platforms. We generated a mean of 7.25 million reads per patient, resulting in a median coverage >13,000X. All samples were covered above 6,400X. The mutations observed here were compared to those described in patients from different ethnic groups obtained from TCGA composed for 103 Asians patients (Japan, Hong-Kong, South Korea, Vietnam) and 78 cases of Europeans. The Asian and European populations showed higher prevalence of intestinal type GAC (53.7% and 61.6%, respectively) in contrast to our cohort, in which 69% of the patients had diffuse-type GACs (pA p.Arg282Trp in the DNA binding domain in exon 8, was found in all three sample groups studied, suggesting a possible role for this mutation in GAC pathogenesis. Although TP53 is the most frequently mutated gene in GACs we found no specific patterns that could explain the differences in the clinical behavior of this tumor between Asian and non-Asian patients. Further analyses are being performed trying to correlate mutations in TP53 and other genes with the different clinical outcomes observed in GACs from Asians and non-Asians, and in a setting of high genetic admixture, as observed in Brazil. Citation Format: Melissa Pool Pizzi, Helano Carioca Freitas, Maria Galli Amorim, Bruna Durães de Figueiredo Barros, Frederico Omar Gleber-Netto, Ana Flávia Mattos Costa, Maria Dirlei Begnami, Adriane Graicer Pelosof, Diana Noronha Nunes, Emmanuel Dias-Neto. Gastric adenocarcinoma TP53 mutations in an ethnically admixed population. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3697.

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.

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.

Background: Cancer stem cells (CSCs) contribute to tumor metastasis, chemotherapy resistance, and are highly tumorigenic when injected into immunodeficient mice. High levels of aldehyde dehydrogenase (ALDH1) are characteristic of stem cells and allow the isolation of subpopulations of CSCs, including those derived from breast cancer. HER2+ tumors, which are more aggressive than tumors with no amplification of this oncogene, are enriched in CSCs, a finding that suggests the participation of this oncogene in the generation and maintenance of CSCs. miR-4728-3p is a poorly investigated miRNA, which is located inside an intron of HER2. Its expression is increased in HER2+ tumors and cell lines, but its role in CSC formation and tumor behavior has not been investigated. Aim: to contribute to the better understanding of the role of miRNA-4728-3p in the biology of CSCs in HER2+ breast cancer cell lines. Methods: miR-4728-3p levels were evaluated in breast cancer cell lines MCF-7, BT-474 and SKBR3 grown in adherent conditions and in conditions that favor the formation of mammospheres for 7, 14, 21 and 28 days. Overexpression of miR-4728-3p was performed using lentiviral transduction with plasmids for the overexpression of this miRNA. Flow cytometry was performed to analyze ALDH1 expression in adherent cells and cells derived from mammospheres and also in cells overexpressing miRNA-4728-3p. Results: Mammospheres in culture for 7 days showed high levels of ALDH1 for all cells lines evaluated. The overexpression of miR-4728-3p induced a significant increment (>10-fold) of ALDH1+ subpopulations in MCF-7 and SKBR3, reinforcing its possible role in the biology of CSCs. Conclusion: HER2-amplification is an event that intrinsically leads to the amplification of miR-4728-3p. The effects of the amplification of this oncogene and this miRNA have to be evaluated together as both may have important consequences for tumor behavior and CSC formation. This increased expression of ALDH1 after miR-4728-3p overexpression suggest the importance of this miRNA in the biology of CSC formation. Note: This abstract was not presented at the meeting. Citation Format: Juliana Laino do Val Carneiro, Diana Noronha Nunes, Emmanuel Dias-Neto. Evaluation of the role of miRNA-4728-3p, a miRNA located in an intron of HER2 gene, in breast cancer stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 532. doi:10.1158/1538-7445.AM2014-532

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.