Contrived Materials and a Data Set for the Evaluation of Liquid Biopsy Tests: A Blood Profiling Atlas in Cancer (BLOODPAC) Community Study.

The Blood Profiling Atlas in Cancer (BLOODPAC) Consortium is a collaborative effort involving stakeholders from the public, industry, academia, and regulatory agencies focused on developing shared best practices on liquid biopsy. This report describes the results from the JFDI (Just Freaking Do It) study, a BLOODPAC initiative to develop standards on the use of contrived materials mimicking cell-free circulating tumor DNA, to comparatively evaluate clinical laboratory testing procedures. Nine independent laboratories tested the concordance, sensitivity, and specificity of commercially available contrived materials with known variant-allele frequencies (VAFs) ranging from 0.

Postoperative Circulating Tumor DNA Can Predict High Risk Patients with Colorectal Cancer Based on Next-Generation Sequencing.

The objective of this study was to characterize circulating tumor DNA (ctDNA) mutations in colorectal cancer (CRC) patients and evaluate their prognostic values during treatment. Forty-nine patients with CRC planned for operation were enrolled. A total of 115 plasma samples were collected pre-operation, post-operation, and post-chemotherapy.

Evaluating the analytical validity of circulating tumor DNA sequencing assays for precision oncology.

Circulating tumor DNA (ctDNA) sequencing is being rapidly adopted in precision oncology, but the accuracy, sensitivity and reproducibility of ctDNA assays is poorly understood. Here we report the findings of a multi-site, cross-platform evaluation of the analytical performance of five industry-leading ctDNA assays. We evaluated each stage of the ctDNA sequencing workflow with simulations, synthetic DNA spike-in experiments and proficiency testing on standardized, cell-line-derived reference samples.

Liquid Biopsy Enables Quantification of the Abundance and Interindividual Variability of Hepatic Enzymes and Transporters.

Variability in individual capacity for hepatic elimination of therapeutic drugs is well recognized and is associated with variable expression and activity of liver enzymes and transporters. Although genotyping offers some degree of stratification, there is often large variability within the same genotype. Direct measurement of protein expression is impractical due to limited access to tissue biopsies.

Evaluation of NGS and RT-PCR Methods for ALK Rearrangement in European NSCLC Patients: Results from the European Thoracic Oncology Platform Lungscape Project.

Introduction: The reported prevalence of ALK receptor tyrosine kinase gene (ALK) rearrangement in NSCLC ranges from 2% to 7%. The primary standard diagnostic method is fluorescence in situ hybridization (FISH). Recently, immunohistochemistry (IHC) has also proved to be a reproducible and sensitive technique.

Collaborating to Compete: Blood Profiling Atlas in Cancer (BloodPAC) Consortium.

The cancer community understands the value of blood profiling measurements in assessing and monitoring cancer. We describe an effort among academic, government, biotechnology, diagnostic, and pharmaceutical companies called the Blood Profiling Atlas in Cancer (BloodPAC) Project. BloodPAC will aggregate, make freely available, and harmonize for further analyses, raw datasets, relevant associated clinical data (e.

Development and validation of a scalable next-generation sequencing system for assessing relevant somatic variants in solid tumors.

Next-generation sequencing (NGS) has enabled genome-wide personalized oncology efforts at centers and companies with the specialty expertise and infrastructure required to identify and prioritize actionable variants. Such approaches are not scalable, preventing widespread adoption. Likewise, most targeted NGS approaches fail to assess key relevant genomic alteration classes.

Analysis of the RNA content of the exosomes derived from blood serum and urine and its potential as biomarkers.

Exosomes are tiny vesicles (30-150 nm) constantly secreted by all healthy and abnormal cells, and found in abundance in all body fluids. These vesicles, loaded with unique RNA and protein cargo, have a wide range of biological functions, including cell-to-cell communication and signalling. As such, exosomes hold tremendous potential as biomarkers and could lead to the development of minimally invasive diagnostics and next generation therapies within the next few years.

Dissecting childhood asthma with nasal transcriptomics distinguishes subphenotypes of disease.

Background: Bronchial airway expression profiling has identified inflammatory subphenotypes of asthma, but the invasiveness of this technique has limited its application to childhood asthma.

Objectives: We sought to determine whether the nasal transcriptome can proxy expression changes in the lung airway transcriptome in asthmatic patients. We also sought to determine whether the nasal transcriptome can distinguish subphenotypes of asthma.

The complete exosome workflow solution: from isolation to characterization of RNA cargo.

Exosomes are small (30-150 nm) vesicles containing unique RNA and protein cargo, secreted by all cell types in culture. They are also found in abundance in body fluids including blood, saliva, and urine. At the moment, the mechanism of exosome formation, the makeup of the cargo, biological pathways, and resulting functions are incompletely understood.

Methods for the extraction and RNA profiling of exosomes.

Aim: To develop protocols for isolation of exosomes and characterization of their RNA content.

Methods: Exosomes were extracted from HeLa cell culture media and human blood serum using the Total exosome isolation (from cell culture media) reagent, and Total exosome isolation (from serum) reagent respectively. Identity and purity of the exosomes was confirmed by Nanosight(®) analysis, electron microscopy, and Western blots for CD63 marker.

miR-337-3p and its targets STAT3 and RAP1A modulate taxane sensitivity in non-small cell lung cancers.

NSCLC (non-small cell lung cancer) often exhibits resistance to paclitaxel treatment. Identifying the elements regulating paclitaxel response will advance efforts to overcome such resistance in NSCLC therapy. Using in vitro approaches, we demonstrated that over-expression of the microRNA miR-337-3p sensitizes NCI-H1155 cells to paclitaxel, and that miR-337-3p mimic has a general effect on paclitaxel response in NSCLC cell lines, which may provide a novel adjuvant strategy to paclitaxel in the treatment of lung cancer.

MicroRNA expression distinguishes SCLC from NSCLC lung tumor cells and suggests a possible pathological relationship between SCLCs and NSCLCs.

Background: Recent studies have shown that microRNAs (miRNAs) play roles in tumorigenesis and are reliable classifiers of certain cancer types and subtypes. However, the role of miRNAs in the pathogenesis and diagnosis of small cell carcinoma (SCLC), the majority of which represent the most aggressive lung tumors, has not been investigated.

Methods: In order to explore miRNA involvement in the pathogenesis of small cell lung carcinoma (SCLC) and the potential role of miRNAs in SCLC diagnosis, we compared the miRNA expression profile of a set of SCLC cell lines to that of a set of non-small cell lung cancer (NSCLC) cell lines and normal immortalized human bronchial epithelial cells (HBECs) using microarray analysis.

miR-93, miR-98, and miR-197 regulate expression of tumor suppressor gene FUS1.

FUS1 is a tumor suppressor gene located on human chromosome 3p21, and expression of Fus1 protein is highly regulated at various levels, leading to lost or greatly diminished tumor suppressor function in many lung cancers. Here we show that selected microRNAs (miRNA) interact with the 3'-untranslated region (3'UTR) of FUS1, leading to down-regulation of protein expression. Using computational methods, we first predicted that FUS1 is a target of three miRNAs, miR-93, miR-98, and miR-197, and then showed that exogenous overexpression of these miRNAs inhibited Fus1 protein expression.

Analysis of the regulation of lin-41 during chick and mouse limb development.

We have cloned the chicken and mouse orthologues of the Caenorhabditis elegans heterochronic gene lin-41. During limb development, lin-41 is expressed in three phases over developmental time and most notably is associated with the developing autopod. Using chicken and mouse mutants and bead implantations, we report that lin-41 is genetically and biochemically downstream of both the Shh and Fgf signaling pathways.

Selective identification of secreted and transmembrane breast cancer markers using Escherichia coli ampicillin secretion trap.

Secreted and cell surface proteins play important roles in cancer and are potential drug targets and tumor markers. Here, we describe a large-scale analysis of the genes encoding secreted and cell surface proteins in breast cancer. To identify these genes, we developed a novel signal sequence trap method called Escherichia coli ampicillin secretion trap (CAST).

ELXR: a resource for rapid exon-directed sequence analysis.

ELXR (Exon Locator and Extractor for Resequencing) streamlines the process of determining exon/intron boundaries and designing PCR and sequencing primers for high-throughput resequencing of exons. We have pre-computed ELXR primer sets for all exons identified from the human, mouse, and rat mRNA reference sequence (RefSeq) public databases curated by the National Center for Biotechnology Information. The resulting exon-flanking PCR primer pairs have been compiled into a system called ELXRdb, which may be searched by keyword, gene name or RefSeq accession number.

Transcriptional mapping and genomic analysis of the cardiac atria and ventricles.

The atria and ventricles of the heart have distinct development, structure, and physiology. However, only a few of the genes that underlie the differences between these tissues are known. We used a murine cardiac cDNA microarray to identify genes differentially expressed in the atria and ventricles.

Highly coordinated gene regulation in mouse skeletal muscle regeneration.

Mammalian skeletal muscles are capable of regeneration after injury. Quiescent satellite cells are activated to reenter the cell cycle and to differentiate for repair, recapitulating features of myogenesis during embryonic development. To understand better the molecular mechanism involved in this process in vivo, we employed high density cDNA microarrays for gene expression profiling in mouse tibialis anterior muscles after a cardiotoxin injection.

MarC-V: a spreadsheet-based tool for analysis, normalization, and visualization of single cDNA microarray experiments.

The comprehensive analysis and visualization of data extracted from cDNA microarrays can be a time-consuming and error-prone process that becomes increasingly tedious with increased number of gene elements on a particular microarray. With the increasingly large number of gene elements on today's microarrays, analysis tools must be developed to meet this challenge. Here, we present MarC-V, a Microsoft Excel spreadsheet tool with Visual Basic macros to automate much of the visualization and calculation involved in the analysis process while providing the familiarity and flexibility of Excel.

All human genes of the uteroglobin family are localized on chromosome 11q12.2 and form a dense cluster.

Rabbit uteroglobin is the founder member of a family of mammalian proteins that has expanded to more than 20 members within the last few years. All members are small, secretory, rarely glycosylated dimeric proteins with unclear physiological functions and are mainly expressed in mucosal tissues. A phylogenetic analysis shows that the family can be grouped into five subfamilies, A to E.

The imprinted antisense RNA at the Igf2r locus overlaps but does not imprint Mas1.

The gene encoding the insulin-like growth-factor type-2 receptor (Igf2r) is maternally expressed and imprinted. A CpG island in Igf2r intron 2 that carries a maternal-specific methylation imprint was shown in a transgenic model to be essential for Igf2r imprinting and for the production of an antisense RNA from the paternal allele. We report here that the endogenous region2 is the promoter for this antisense RNA (named Air, for antisense Igf2r RNA) and that the 3' end lies 107,796 bp distant in an intron of the flanking, but non-imprinted, gene Mas1.