A comparison between SOLiD 5500XLand Ion Torrent PGM-derived miRNA expression profiles in two breast cell lines.

Next-generation sequencing (NGS) platforms allow the analysis of hundreds of millions of molecules in a single sequencing run, revolutionizing many research areas. NGS-based microRNA studies enable expression quantification in unprecedented scale without the limitations of closed-platforms. Yet, whereas a massive amount of data produced by these platforms is available, comparisons of quantification/discovery capabilities between platforms are still lacking. Here we compare two NGS-platforms: SOLiD and PGM, by evaluating their microRNA identification/quantification capabilities using two breast-derived cell-lines. A high expression correlation (R2 > 0.9) was achieved, encompassing 97% of the miRNAs, and the few discrepancies in miRNA counts were attributable to molecules that have very low expression. Quantification divergences indicative of artefactual representation were seen for 14 miRNAs (higher in SOLiD-reads) and another 10 miRNAs more abundant in PGM-data. An inspection of these revealed an increased and statistically significant count of uracyls and uracyl-stretches for PGM-enriched miRNAs, compared to SOLiD and to the miRBase. In parallel, adenines and adenine-stretches were enriched for SOLiDderived miRNA reads. We conclude that, whereas both platforms are overall consistent and can be used interchangeably for microRNA expression studies, particular sequence features appear to be indicative of specific platform bias, and their presence in microRNAs should be considered for database-analyses.

Exome and Tissue-Associated Microbiota as Predictive Markers of Response to Neoadjuvant Treatment in Locally Advanced Rectal Cancer.

The clinical and pathological responses to multimodal neoadjuvant therapy in locally advanced rectal cancers (LARCs) remain unpredictable, and robust biomarkers are still lacking. Recent studies have shown that tumors present somatic molecular alterations related to better treatment response, and it is also clear that tumor-associated bacteria are modulators of chemotherapy and immunotherapy efficacy, therefore having implications for long-term survivorship and a good potential as the biomarkers of outcome. Here, we performed whole exome sequencing and 16S ribosomal RNA (rRNA) amplicon sequencing from 44 pre-treatment LARC biopsies from Argentinian and Brazilian patients, treated with neoadjuvant chemoradiotherapy or total neoadjuvant treatment, searching for predictive biomarkers of response (responders, n = 17; non-responders, n = 27). In general, the somatic landscape of LARC was not capable to predict a response; however, a significant enrichment in mutational signature SBS5 was observed in non-responders (p = 0.0021), as well as the co-occurrence of APC and FAT4 mutations (p < 0.05). Microbiota studies revealed a similar alpha and beta diversity of bacteria between response groups. Yet, the linear discriminant analysis (LDA) of effect size indicated an enrichment of Hungatella, Flavonifractor, and Methanosphaera (LDA score ≥3) in the pre-treatment biopsies of responders, while non-responders had a higher abundance of Enhydrobacter, Paraprevotella (LDA score ≥3) and Finegoldia (LDA score ≥4). Altogether, the evaluation of these biomarkers in pre-treatment biopsies could eventually predict a neoadjuvant treatment response, while in post-treatment samples, it could help in guiding non-operative treatment strategies.

Evaluation of Bacteria and Fungi DNA Abundance in Human Tissues.

Whereas targeted and shotgun sequencing approaches are both powerful in allowing the study of tissue-associated microbiota, the human: microorganism abundance ratios in tissues of interest will ultimately determine the most suitable sequencing approach. In addition, it is possible that the knowledge of the relative abundance of bacteria and fungi during a treatment course or in pathological conditions can be relevant in many medical conditions. Here, we present a qPCR-targeted approach to determine the absolute and relative amounts of bacteria and fungi and demonstrate their relative DNA abundance in nine different human tissue types for a total of 87 samples. In these tissues, fungi genomes are more abundant in stool and skin samples but have much lower levels in other tissues. Bacteria genomes prevail in stool, skin, oral swabs, saliva, and gastric fluids. These findings were confirmed by shotgun sequencing for stool and gastric fluids. This approach may contribute to a more comprehensive view of the human microbiota in targeted studies for assessing the abundance levels of microorganisms during disease treatment/progression and to indicate the most informative methods for studying microbial composition (shotgun versus targeted sequencing) for various samples types.

Concordance Analysis of ALK Gene Fusion Detection Methods in Patients with Non-Small-Cell Lung Cancer from Chile, Brazil, and Peru.

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.

The impact of HER2 overexpression on the miRNA and circRNA transcriptomes in two breast cell lines and their vesicles.

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.

Genomics and epidemiology for gastric adenocarcinomas (GE4GAC): a Brazilian initiative to study gastric cancer

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